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Sample records for actin mrna levels

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

    SciTech Connect

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

    1987-05-01

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

  2. Beta actin and its mRNA are localized at the plasma membrane and the regions of moving cytoplasm during the cellular response to injury

    PubMed Central

    1991-01-01

    IgG localization is discontinuous. When injured endothelial and 3T3 cells were stained through wound closure, we localized beta actin only in motile cytoplasm at the wound edge. Staining disappeared as cells became quiescent upon monolayer restoration. Appearance of beta actin at the wound edge correlated with a two- to threefold increase in steady-state levels of beta actin mRNA, which rose within 15-60 min after injury and returned to noninjury levels during monolayer restoration. In situ hybridization revealed that transcripts encoding beta actin were localized at the wound edge in association with the repositioned protein. Results of these experiments indicate that beta actin and its encoded mRNA are polarized at the membrane-cytoskeletal interface within regions of moving cytoplasm. PMID:1993736

  3. Different motif requirements for the localization zipcode element of β-actin mRNA binding by HuD and ZBP1

    PubMed Central

    Kim, Hak Hee; Lee, Seung Joon; Gardiner, Amy S.; Perrone-Bizzozero, Nora I.; Yoo, Soonmoon

    2015-01-01

    Interactions of RNA-binding proteins (RBPs) with their target transcripts are essential for regulating gene expression at the posttranscriptional level including mRNA export/localization, stability, and translation. ZBP1 and HuD are RBPs that play pivotal roles in mRNA transport and local translational control in neuronal processes. While HuD possesses three RNA recognition motifs (RRMs), ZBP1 contains two RRMs and four K homology (KH) domains that either increase target specificity or provide a multi-target binding capability. Here we used isolated cis-element sequences of the target mRNA to examine directly protein-RNA interactions in cell-free systems. We found that both ZBP1 and HuD bind the zipcode element in rat β-actin mRNA's 3′ UTR. Differences between HuD and ZBP1 were observed in their binding preference to the element. HuD showed a binding preference for U-rich sequence. In contrast, ZBP1 binding to the zipcode RNA depended more on the structural level, as it required the proper spatial organization of a stem-loop that is mainly determined by the U-rich element juxtaposed to the 3′ end of a 5′-ACACCC-3′ motif. On the basis of this work, we propose that ZBP1 and HuD bind to overlapping sites in the β-actin zipcode, but they recognize different features of this target sequence. PMID:26152301

  4. A Novel Actin mRNA Splice Variant Regulates ACTG1 Expression

    PubMed Central

    Drummond, Meghan C.; Friderici, Karen H.

    2013-01-01

    Cytoplasmic actins are abundant, ubiquitous proteins in nucleated cells. However, actin expression is regulated in a tissue- and development-specific manner. We identified a novel cytoplasmic-γ-actin (Actg1) transcript that includes a previously unidentified exon (3a). Inclusion of this exon introduces an in-frame termination codon. We hypothesized this alternatively-spliced transcript down-regulates γ-actin production by targeting these transcripts for nonsense-mediated decay (NMD). To address this, we investigated conservation between mammals, tissue-specificity in mice, and developmental regulation using C2C12 cell culture. Exon 3a is 80% similar among mammals and varies in length from 41 nucleotides in humans to 45 in mice. Though the predicted amino acid sequences are not similar between all species, inclusion of exon 3a consistently results in the in the introduction of a premature termination codon within the alternative Actg1 transcript. Of twelve tissues examined, exon 3a is predominantly expressed in skeletal muscle, cardiac muscle, and diaphragm. Splicing to include exon 3a is concomitant with previously described down-regulation of Actg1 in differentiating C2C12 cells. Treatment of differentiated C2C12 cells with an inhibitor of NMD results in a 7-fold increase in exon 3a-containing transcripts. Therefore, splicing to generate exon 3a-containing transcripts may be one component of Actg1 regulation. We propose that this post-transcriptional regulation occurs via NMD, in a process previously described as “regulated unproductive splicing and translation” (RUST). PMID:24098136

  5. Ubiquitin ligase TRIM3 controls hippocampal plasticity and learning by regulating synaptic γ-actin levels

    PubMed Central

    Schreiber, Joerg; Végh, Marlene J.; Dawitz, Julia; Kroon, Tim; Loos, Maarten; Labonté, Dorthe; Li, Ka Wan; Van Nierop, Pim; Van Diepen, Michiel T.; De Zeeuw, Chris I.; Kneussel, Matthias; Meredith, Rhiannon M.; Smit, August B.

    2015-01-01

    Synaptic plasticity requires remodeling of the actin cytoskeleton. Although two actin isoforms, β- and γ-actin, are expressed in dendritic spines, the specific contribution of γ-actin in the expression of synaptic plasticity is unknown. We show that synaptic γ-actin levels are regulated by the E3 ubiquitin ligase TRIM3. TRIM3 protein and Actg1 transcript are colocalized in messenger ribonucleoprotein granules responsible for the dendritic targeting of messenger RNAs. TRIM3 polyubiquitylates γ-actin, most likely cotranslationally at synaptic sites. Trim3−/− mice consequently have increased levels of γ-actin at hippocampal synapses, resulting in higher spine densities, increased long-term potentiation, and enhanced short-term contextual fear memory consolidation. Interestingly, hippocampal deletion of Actg1 caused an increase in long-term fear memory. Collectively, our findings suggest that temporal control of γ-actin levels by TRIM3 is required to regulate the timing of hippocampal plasticity. We propose a model in which TRIM3 regulates synaptic γ-actin turnover and actin filament stability and thus forms a transient inhibitory constraint on the expression of hippocampal synaptic plasticity. PMID:26527743

  6. In vivo imaging of labelled endogenous β-actin mRNA during nucleocytoplasmic transport.

    PubMed

    Grünwald, David; Singer, Robert H

    2010-09-30

    Export of messenger RNA occurs via nuclear pores, which are large nanomachines with diameters of roughly 120 nm that are the only link between the nucleus and cytoplasm. Hence, mRNA export occurs over distances smaller than the optical resolution of conventional light microscopes. There is extensive knowledge on the physical structure and composition of the nuclear pore complex, but transport selectivity and the dynamics of mRNA export at nuclear pores remain unknown. Here we developed a super-registration approach using fluorescence microscopy that can overcome the current limitations of co-localization by means of measuring intermolecular distances of chromatically different fluorescent molecules with nanometre precision. With this method we achieve 20-ms time-precision and at least 26-nm spatial precision, enabling the capture of highly transient interactions in living cells. Using this approach we were able to spatially resolve the kinetics of mRNA transport in mammalian cells and present a three-step model consisting of docking (80 ms), transport (5-20 ms) and release (80 ms), totalling 180 ± 10 ms. Notably, the translocation through the channel was not the rate-limiting step, mRNAs can move bi-directionally in the pore complex and not all pores are equally active.

  7. Correlation of serum IgE levels and clinical manifestations in patients with actinic prurigo*

    PubMed Central

    Cuevas-Gonzalez, Juan Carlos; Lievanos-Estrada, Zahide; Vega-Memije, Maria Elisa; Hojyo-Tomoka, Maria Teresa; Dominguez-Soto, Luciano

    2016-01-01

    BACKGROUND: Actinic prurigo is an idiopathic photodermatosis, the pathophysiology of which has been hypothesized to involve subtype IV type b (Th2) hypersensitive response, whereby IL4, IL5, and IL13 are secreted and mediate the production of B cells, IgE, and IgG4. OBJECTIVES: To examine the association of serum IgE levels and the clinical severity of injuries. METHODS: This case-control study comprised patients with a clinical and histopathological diagnosis of actinic prurigo, as well as clinically healthy subjects, from whom 3cc of peripheral blood was taken for immunoassay. Cases were classified by lesion severity as mild, moderate, and severe. Descriptive statistics were analyzed, and chi-square test was performed. RESULTS: We included 21 actinic prurigo patients and 21 subjects without disease; 11 patients with actinic prurigo had elevated serum IgE levels, and 10 had low serum levels. Six actinic prurigo (AP) patients with elevated serum levels of IgE had moderate injuries, 4 had severe injuries, and 1 had minor injuries. Eight out of 10 patients with normal IgE levels presented with minor injuries in the clinical evaluation. The 21 controls did not have increased serum IgE levels. CONCLUSIONS: Elevated IgE levels are associated with moderate to severe clinical lesions, suggesting that actinic prurigo entails a type IV subtype b hypersensitivity response in which Th2 cells predominate. PMID:26982774

  8. BIOMARKERS OF ENDOCRINE DISRUPTION AT THE MRNA LEVEL

    EPA Science Inventory

    Denslow, Nancy D., Christopher J. Bowman, Gillian Robinson, H. Stephen Lee, Ronald J. Ferguson, Michael J. Hemmer and Leroy C. Folmar. 1999. Biomarkers of Endocrine Disruption at the mRNA Level. In: Environmental Toxicology and Risk Assessment: Standardization of Biomarkers for ...

  9. Trichosanthin-induced specific changes of cytoskeleton configuration were associated with the decreased expression level of actin and tubulin genes in apoptotic Hela cells.

    PubMed

    Wang, Ping; Li, Ji-Cheng

    2007-09-15

    Trichosanthin (TCS) possesses a broad spectrum of biological and pharmacological activities, including anti-cancer activities through apoptosis pathway. However, little is known about the effects of TCS on the cytoskeleton configuration and expression of actin and tubulin genes in Hela cell apoptosis. In the present study, apoptotic cytoskeleton structures were observed by confocal immunofluorescence microscopy, absolute amounts of actin and tubulin subunit mRNAs were determined by quantitative real-time PCR assays (QRT-PCR). Our results showed that the execution phase of cell apoptosis was a highly coordinated process of cellular reorganization, depolymerized microfilaments (MFs) accumulated in the coarsened cytoplasm and apoptotic bodies, followed by the formation of a ring microtubule (MT) structure beneath the plasma membrane. Importantly, apoptosis occurred by a suppression of actin and tubulin subunit gene expression. In particular, a rapid decrease in the amounts of gamma-actin mRNA preceded that of beta-actin; alpha- and beta-tubulin mRNAs were subsequently down-regulated in the later stage of Hela cell apoptosis. These results suggested that the execution of Hela cell apoptosis induced by TCS accompanied the specific changes of cytoskeleton configuration and, significantly, decreased the expression level of actin and tubulin subunit genes in different stages.

  10. Cardiac actin is the major actin gene product in skeletal muscle cell differentiation in vitro.

    PubMed Central

    Bains, W; Ponte, P; Blau, H; Kedes, L

    1984-01-01

    We examined the expression of alpha-skeletal, alpha-cardiac, and beta- and gamma-cytoskeletal actin genes in a mouse skeletal muscle cell line (C2C12) during differentiation in vitro. Using isotype-specific cDNA probes, we showed that the alpha-skeletal actin mRNA pool reached only 15% of the level reached in adult skeletal muscle and required several days to attain this peak, which was then stably maintained. However, these cells accumulated a pool of alpha-cardiac actin six times higher than the alpha-skeletal actin mRNA peak within 24 h of the initiation of differentiation. After cells had been cultured for an additional 3 days, this pool declined to 10% of its peak level. In contrast, over 95% of the actin mRNA in adult skeletal muscle coded for alpha-actin. This suggests that C2C12 cells express a pattern of sarcomeric actin genes typical of either muscle development or regeneration and distinct from that seen in mature, adult tissue. Concurrently in the course of differentiation the beta- and gamma-cytoskeletal actin mRNA pools decreased to less than 10% of their levels in proliferating cells. The decreases in beta- and gamma-cytoskeletal actin mRNAs are apparently not coordinately regulated. Images PMID:6493226

  11. Cellular Levels of Signaling Factors Are Sensed by β-actin Alleles to Modulate Transcriptional Pulse Intensity.

    PubMed

    Kalo, Alon; Kanter, Itamar; Shraga, Amit; Sheinberger, Jonathan; Tzemach, Hadar; Kinor, Noa; Singer, Robert H; Lionnet, Timothée; Shav-Tal, Yaron

    2015-04-21

    The transcriptional response of β-actin to extra-cellular stimuli is a paradigm for transcription factor complex assembly and regulation. Serum induction leads to a precisely timed pulse of β-actin transcription in the cell population. Actin protein is proposed to be involved in this response, but it is not known whether cellular actin levels affect nuclear β-actin transcription. We perturbed the levels of key signaling factors and examined the effect on the induced transcriptional pulse by following endogenous β-actin alleles in single living cells. Lowering serum response factor (SRF) protein levels leads to loss of pulse integrity, whereas reducing actin protein levels reveals positive feedback regulation, resulting in elevated gene activation and a prolonged transcriptional response. Thus, transcriptional pulse fidelity requires regulated amounts of signaling proteins, and perturbations in factor levels eliminate the physiological response, resulting in either tuning down or exaggeration of the transcriptional pulse.

  12. Dexamethasone increases growth hormone (GH)-releasing hormone (GRH) receptor mRNA levels in cultured rat anterior pituitary cells.

    PubMed

    Tamaki, M; Sato, M; Matsubara, S; Wada, Y; Takahara, J

    1996-06-01

    To examine the effects of glucocorticoid (GC) on growth hormone (GH)-releasing hormone (GRH) receptor gene expression, a highly-sensitive and quantitative reverse-transcribed polymerase chain reaction (RT-PCR) method was used in this study. Rat anterior pituitary cells were isolated and cultured for 4 days. The cultured cells were treated with dexamethasone for 2, 6, and 24 h. GRH receptor mRNA levels were determined by competitive RT-PCR using a recombinant RNA as the competitor. Dexamethasone significantly increased GRH receptor mRNA levels at 5 nM after 6- and 24 h-incubations, and the maximal effect was found at 25 nM. The GC receptor-specific antagonist, RU 38486 completely eliminated the dexamethasone-induced enhancement of GRH receptor mRNA levels. Dexamethasone did not alter the mRNA levels of beta-actin and prolactin at 5 nM for 24 h, whereas GH mRNA levels were significantly increased by the same treatment. The GH response to GRH was significantly enhanced by the 24-h incubation with 5 nM dexamethasone. These findings suggest that GC stimulates GRH receptor gene expression through the ligand-activated GC receptors in the rat somatotrophs. The direct effects of GC on the GRH receptor gene could explain the enhancement of GRH-induced GH secretion.

  13. Biomarkers of endocrine disruption at the mRNA level

    SciTech Connect

    Denslow, N.D.; Bowman, C.J.; Robinson, G.; Lee, H.S.; Ferguson, R.J.; Hemmmer, M.J.; Folmar, L.C.

    1999-07-01

    A large number of estrogen-mimicking, anthropogenic chemicals capable of disrupting normal reproductive function have been identified. The ubiquitous distribution of these compounds, many as components of complex industrial or municipal waste, has spurred an effort to develop methods to screen for chemicals which disrupt normal endocrine regulation of reproduction. The authors have developed assays that both allow exposure of animals in vivo and measure the response at the level of gene activation. The authors have developed a probe for measuring the induction of vitellogenin mRNA by Northern Blot in livers of sheepshead minnows treated with 17-{beta}-estradiol. The authors have also developed a strategy for using Differential Display Polymerase Chain Reaction for determining gene induction profiles following exposure to estradiol. These methods should be adaptable to a variety of structurally diverse estrogen mimics.

  14. Activity regulates the levels of acetylcholine receptor alpha-subunit mRNA in cultured chicken myotubes.

    PubMed Central

    Klarsfeld, A; Changeux, J P

    1985-01-01

    In vitro blocking the spontaneous activity of primary cultures of chicken embryo myotubes with tetrodotoxin increases approximately equal to 2-fold their content in surface acetylcholine receptor. To investigate this effect at the level of gene expression, chicken genomic DNA sequences coding for the acetylcholine receptor alpha subunit were isolated and characterized. They were shown to belong to a single-copy, polymorphic gene with at least two alleles in the chicken strain utilized. Probes derived from these genomic clones were used to quantitate levels of alpha-subunit mRNA. In culture, a 2-day exposure to tetrodotoxin increased these mRNA levels up to 13-fold, a value similar to that observed after denervation of chick leg muscle (approximately equal to 17-fold). Actin mRNA levels varied little in any of these experiments. These results support the notion that membrane electrical activity affects acetylcholine receptor expression by regulating the accumulation of the corresponding mRNAs. Images PMID:2989833

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

    NASA Astrophysics Data System (ADS)

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

    2016-11-01

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

  16. 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.

  17. Semiquantitation of human chemokine mRNA levels with a newly constructed multispecific competitor fragment.

    PubMed

    Dumoulin, F L; Altfeld, M; Rockstroh, J K; Leifeld, L; Sauerbruch, T; Spengler, U

    1999-04-22

    Chemokines are a group of inducible, locally acting proinflammatory cytokines which have been implicated in the pathogenesis of a variety of diseases. Important members of the group include monocyte chemoattractant protein (MCP)-1, -2, -3, macrophage inhibitory protein (MIP)-1alpha, -1beta and RANTES (regulated upon activation, normal T expressed and secreted). To facilitate further investigation of the human chemokines, we have constructed a novel multispecific competitor fragment containing primer binding sites for the CC-chemokines MCP-1, MCP-2, MCP-3, MIP-1alpha, MIP-1beta and RANTES, the CXC-chemokines MIP-2alpha, MIP-2beta as well as for the housekeeping gene beta-actin. Using this competitor fragment we can demonstrate reliable semiquantitation of reverse transcribed chemokine mRNAs. The assay should be useful for further studies, in particular for the semiquantitation of chemokine mRNA species from small cell or tissue specimens.

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

    PubMed Central

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

    1994-01-01

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

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

    PubMed

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

    2006-06-01

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

  20. Increased myogenic repressor Id mRNA and protein levels in hindlimb muscles of aged rats.

    PubMed

    Alway, Stephen E; Degens, Hans; Lowe, Dawn A; Krishnamurthy, Gururaj

    2002-02-01

    The objective of this study was to determine if levels of repressors to myogenic regulatory factors (MRFs) differ between muscles from young adult and aged animals. Total RNA from plantaris, gastrocnemius, and soleus muscles of Fischer 344 x Brown Norway rats aged 9 mo (young adult, n = 10) and 37 mo (aged, n = 10) was reverse transcribed and then amplified by PCR. To obtain a semiquantitative measure of the mRNA levels, PCR signals were normalized to cyclophilin or 18S signals from the corresponding reverse transcription product. Normalization to cyclophilin and 18S gave similar results. The mRNA levels of MyoD and myogenin were approximately 275-650% (P < 0.001) and approximately 500-1,100% (P < 0.001) greater, respectively, in muscles from aged compared with young adults. In contrast, the protein levels were lower in plantaris and gastrocnemius muscles and similar in the soleus muscle of aged vs. young adult rats. Id repressor mRNA levels were approximately 300-900% greater in fast and slow muscles of aged animals (P < or = 0.02), and Mist 1 mRNA was approximately 50% greater in the plantaris and gastrocnemius muscles (P < 0.01). The mRNA level of Twist mRNA was not significantly affected by aging. Id-1, Id-2, and Id-3 protein levels were approximately 17-740% greater (P < 0.05) in hindlimb muscles of aged rats compared with young adult rats. The elevated levels of Id mRNA and protein suggest that MRF repressors may play a role in gene regulation of fast and slow muscles in aged rats.

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

    PubMed

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

    1993-01-01

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

  2. Circulating RANKL is inversely related to RANKL mRNA levels in bone in osteoarthritic males

    PubMed Central

    Findlay, David; Chehade, Mellick; Tsangari, Helen; Neale, Susan; Hay, Shelley; Hopwood, Blair; Pannach, Susan; O'Loughlin, Peter; Fazzalari, Nicola

    2008-01-01

    Introduction The relationship of circulating levels of receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) with the expression of these molecules in bone has not been established. The objective of this study was to measure, in humans, the serum levels of RANKL and OPG, and the corresponding levels in bone of mRNA encoding these proteins. Methods Fasting blood samples were obtained on the day of surgery from patients presenting for hip replacement surgery for primary osteoarthritis (OA). Intraoperatively, samples of intertrochanteric trabecular bone were collected for analysis of OPG and RANKL mRNA, using real time RT-PCR. Samples were obtained from 40 patients (15 men with age range 50 to 79 years, and 25 women with age range 47 to 87 years). Serum total RANKL and free OPG levels were measured using ELISA. Results Serum OPG levels increased over the age range of this cohort. In the men RANKL mRNA levels were positively related to age, whereas serum RANKL levels were negatively related to age. Again, in the men serum RANKL levels were inversely related (r = -0.70, P = 0.007) to RANKL mRNA levels. Also in the male group, RANKL mRNA levels were associated with a number of indices of bone structure (bone volume fraction relative to bone tissue volume, specific surface of bone relative to bone tissue volume, and trabecular thickness), bone remodelling (eroded surface and osteoid surface), and biochemical markers of bone turnover (serum alkaline phosphatase and osteocalcin, and urinary deoxypyridinoline). Conclusion This is the first report to show a relationship between serum RANKL and the expression of RANKL mRNA in bone. PMID:18182105

  3. Structure, chromosome location, and expression of the human. gamma. -actin gene: Differential evolution, location, and expression of the cytoskeletal BETA- and. gamma. -actin genes

    SciTech Connect

    Erba, H.P.; Eddy, R.; Shows, T.; Kedes, L.; Gunning, P.

    1988-04-01

    The accumulation of the cytoskeletal ..beta..-and ..gamma..-actin mRNAs was determined in a variety of mouse tissues and organs. The ..beta..-iosform is always expressed in excess of the ..gamma..-isoform. However, the molar ratio of ..beta..- to ..gamma..-actin mRNA varies from 1.7 in kidney and testis to 12 in sarcomeric muscle to 114 in liver. The authors conclude that, whereas the cytoskeletal ..beta..- and ..gamma..-actins are truly coexpressed, their mRNA levels are subject to differential regulation between different cell types. The human ..gamma..-actin gene has been cloned and sequenced, and its chromosome location has been determined. The gene is located on human chromosome 17, unlike ..beta..-actin which is on chromosome 7. Thus, if these genes are also unlinked in the mouse, the coexpression of the ..beta..- and ..gamma..-actin genes in rodent tissues cannot be determined by gene linkage. Comparison of the human ..beta..- and ..gamma..-actin genes reveals that noncoding sequences in the 5'-flanking region and in intron III have been conserved since the duplication that gave rise to these two genes. In contrast, there are sequences in intron III and the 3'-untranslated region which are not present in the ..beta..-actin gene but are conserved between the human ..gamma..-actin and the Xenopus borealis type 1 actin genes. Such conserved noncoding sequences may contribute to the coexpression of ..beta..- and ..gamma..-actin or to the unique regulation and function of the ..gamma..-actin gene. Finally, the authors demonstrate that the human ..gamma..-actin gene is expressed after introduction into mouse L cells and C2 myoblasts and that, upon fusion of C2 cells to form myotubes, the human ..gamma..-actin gene is appropriately regulated.

  4. Aggregated LDL in contact with macrophages induces local increases in free cholesterol levels that regulate local actin polymerization

    PubMed Central

    Grosheva, Inna; Haka, Abigail S.; Qin, Chunbo; Pierini, Lynda M.; Maxfield, Frederick R.

    2009-01-01

    Objective Interaction of macrophages with aggregated matrix-anchored lipoprotein deposits is an important initial step in atherogenesis. Aggregated lipoproteins require different cellular uptake processes than those used for endocytosis of monomeric lipoproteins. In this study, we tested the hypothesis that engagement of aggregated LDL (agLDL) by macrophages could lead to local increases in free cholesterol levels and that these increases in free cholesterol regulate signals that control cellular actin. Methods and Results AgLDL resides for prolonged periods in surface-connected compartments. While agLDL is still extracellular, we demonstrate that an increase in free cholesterol occurs at sites of contact between agLDL and cells due to hydrolysis of agLDL-derived cholesteryl ester. This increase in free cholesterol causes enhanced actin polymerization around the agLDL. Inhibition of cholesteryl ester hydrolysis results in decreased actin polymerization. Conclusions We describe a novel process that occurs during agLDL-macrophage interactions in which local release of free cholesterol causes local actin polymerization, promoting a pathologic positive feedback loop for increased catabolism of agLDL and eventual foam cell formation. PMID:19556523

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

    PubMed

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

    2013-02-01

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

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

    PubMed

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

    2015-11-01

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

  7. Fusion of mitochondria in tobacco suspension cultured cells is dependent on the cellular ATP level but not on actin polymerization.

    PubMed

    Wakamatsu, Kairo; Fujimoto, Masaru; Nakazono, Mikio; Arimura, Shin-ichi; Tsutsumi, Nobuhiro

    2010-10-01

    Mitochondria in plant cells undergo fusion and fission frequently. Although the mechanisms and proteins of mitochondrial fusion are well known in yeast and mammalian cells, they remain poorly understood in plant cells. To clarify the physiological requirements for plant mitochondrial fusion, we investigated the fusion frequency of mitochondria in tobacco cultured cells using the photoconvertible fluorescent protein Kaede and some physiological inhibitors. The latter included two uncouplers, 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), an inhibitor of mitochondrial ATP synthase, oligomycin, and an actin polymerization inhibitor, latrunculin B (Lat B). The frequency of mitochondrial fusion was clearly reduced by DNP, CCCP and oligomycin, but not by Lat B, although Lat B severely inhibited mitochondrial movement. Moreover, DNP, CCCP and oligomycin evidently lowered the cellular ATP levels. These results indicate that plant mitochondrial fusion depends on the cellular ATP level, but not on actin polymerization.

  8. Amyloid precursor protein mRNA levels in Alzheimer's disease brain.

    PubMed

    Preece, Paul; Virley, David J; Costandi, Moheb; Coombes, Robert; Moss, Stephen J; Mudge, Anne W; Jazin, Elena; Cairns, Nigel J

    2004-03-17

    Insoluble beta-amyloid deposits in Alzheimer's disease (AD) brain are proteolytically derived from the membrane bound amyloid precursor protein (APP). The APP gene is differentially spliced to produce isoforms that can be classified into those containing a Kunitz-type serine protease inhibitor domain (K(+), APP(751), APP(770), APRP(365) and APRP(563)), and those without (K(-), APP(695) and APP(714)). Given the hypothesis that Abeta is a result of aberrant catabolism of APP, differential expression of mRNA isoforms containing protease inhibitors might play an active role in the pathology of AD. We took 513 cerebral cortex samples from 90 AD and 81 control brains and quantified the mRNA isoforms of APP with TaqMan real-time RT-PCR. After adjustment for age at death, brain pH and gender we found a change in the ratio of KPI(+) to KPI(-) mRNA isoforms of APP. Three separate probes, designed to recognise only KPI(+) mRNA species, gave increases of between 28% and 50% in AD brains relative to controls (p=0.002). There was no change in the mRNA levels of KPI-(APP 695) (p=0.898). Therefore, whilst KPI-mRNA levels remained stable the KPI(+) species increased specifically in the AD brains.

  9. Transcriptional bursting explains the noise–versus–mean relationship in mRNA and protein levels

    SciTech Connect

    Dar, Roy; Shaffer, Sydney M.; Singh, Abhyudai; Razooky, Brandon S.; Simpson, Michael L.; Raj, Arjun; Weinberger, Leor S.

    2016-07-28

    Recent analysis demonstrates that the HIV-1 Long Terminal Repeat (HIV LTR) promoter exhibits a range of possible transcriptional burst sizes and frequencies for any mean-expression level. However, these results have also been interpreted as demonstrating that cell-tocell expression variability (noise) and mean are uncorrelated, a significant deviation from previous results. Here, we re-examine the available mRNA and protein abundance data for the HIV LTR and find that noise in mRNA and protein expression scales inversely with the mean along analytically predicted transcriptional burst-size manifolds. We then experimentally perturb transcriptional activity to test a prediction of the multiple burst-size model: that increasing burst frequency will cause mRNA noise to decrease along given burst-size lines as mRNA levels increase. In conclusion, the data show that mRNA and protein noise decrease as mean expression increases, supporting the canonical inverse correlation between noise and mean.

  10. Transcriptional bursting explains the noise–versus–mean relationship in mRNA and protein levels

    DOE PAGES

    Dar, Roy; Shaffer, Sydney M.; Singh, Abhyudai; ...

    2016-07-28

    Recent analysis demonstrates that the HIV-1 Long Terminal Repeat (HIV LTR) promoter exhibits a range of possible transcriptional burst sizes and frequencies for any mean-expression level. However, these results have also been interpreted as demonstrating that cell-tocell expression variability (noise) and mean are uncorrelated, a significant deviation from previous results. Here, we re-examine the available mRNA and protein abundance data for the HIV LTR and find that noise in mRNA and protein expression scales inversely with the mean along analytically predicted transcriptional burst-size manifolds. We then experimentally perturb transcriptional activity to test a prediction of the multiple burst-size model: thatmore » increasing burst frequency will cause mRNA noise to decrease along given burst-size lines as mRNA levels increase. In conclusion, the data show that mRNA and protein noise decrease as mean expression increases, supporting the canonical inverse correlation between noise and mean.« less

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

    PubMed Central

    2012-01-01

    Background In recent years, the identification of peripheral biomarkers that are associated with psychiatric diseases, such as Major Depressive Disorder (MDD), has become relevant because these biomarkers may improve the efficiency of the differential diagnosis process and indicate targets for new antidepressant drugs. Two recent candidate genes, ErbB3 and Fgfr1, are growth factors whose mRNA levels have been found to be altered in the leukocytes of patients that are affected by bipolar disorder in a depressive state. On this basis, the aim of the study was to determine if ErbB3 and Fgfr1 mRNA levels could be a biomarkers of MDD. Methods We measured by Real Time PCR ErbB3 and Fgfr1 mRNA expression levels in leukocytes of MDD patients compared with controls. Successively, to assess whether ErbB3 mRNA levels were influenced by previous antidepressant treatment we stratified our patients sample in two cohorts, comparing drug-naive versus drug-free patients. Moreover, we evaluated the levels of the transcript in MDD patients after 12 weeks of antidepressant treatment, and in prefrontal cortex of rats stressed and treated with an antidepressant drug of the same class. Results These results showed that ErbB3 but not Fgfr1 mRNA levels were reduced in leukocytes of MDD patients compared to healthy subjects. Furthermore, ErbB3 levels were not affected by antidepressant treatment in either human or animal models Conclusions Our data suggest that ErbB3 might be considered as a biomarker for MDD and that its deficit may underlie the pathopsysiology of the disease and is not a consequence of treatment. Moreover the study supports the usefulness of leukocytes as a peripheral system for identifying biomarkers in psychiatric diseases. PMID:22989054

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

    PubMed Central

    Mündel, Toby; Pilegaard, Henriette; Hawke, Emma; Leikis, Murray; Lopez-Villalobos, Nicolas; Oliveira, Rodrigo S. F.; Bishop, David J.

    2015-01-01

    Minimizing the decrease in intracellular pH during high-intensity exercise training promotes greater improvements in mitochondrial respiration. This raises the intriguing hypothesis that pH may affect the exercise-induced transcription of genes that regulate mitochondrial biogenesis. Eight males performed 10x2-min cycle intervals at 80% V˙O2peak intensity on two occasions separated by ~2 weeks. Participants ingested either ammonium chloride (ACID) or calcium carbonate (PLA) the day before and on the day of the exercise trial in a randomized, counterbalanced order, using a crossover design. Biopsies were taken from the vastus lateralis muscle before and after exercise. The mRNA level of peroxisome proliferator-activated receptor co-activator 1α (PGC-1α), citrate synthase, cytochome c and FOXO1 was elevated at rest following ACID (P<0.05). During the PLA condition, the mRNA content of mitochondrial- and glucose-regulating proteins was elevated immediately following exercise (P<0.05). In the early phase (0–2 h) of post-exercise recovery during ACID, PGC-1α, citrate synthase, cytochome C, FOXO1, GLUT4, and HKII mRNA levels were not different from resting levels (P>0.05); the difference in PGC-1α mRNA content 2 h post-exercise between ACID and PLA was not significant (P = 0.08). Thus, metabolic acidosis abolished the early post-exercise increase of PGC-1α mRNA and the mRNA of downstream mitochondrial and glucose-regulating proteins. These findings indicate that metabolic acidosis may affect mitochondrial biogenesis, with divergent responses in resting and post-exercise skeletal muscle. PMID:26656911

  13. Upregulation of specific mRNA levels in rat brain after cell phone exposure.

    PubMed

    Yan, Ji-Geng; Agresti, Michael; Zhang, Lin-Ling; Yan, Yuhui; Matloub, Hani S

    2008-01-01

    Adult Sprague-Dawley rats were exposed to regular cell phones for 6 h per day for 126 days (18 weeks). RT-PCR was used to investigate the changes in levels of mRNA synthesis of several injury-associated proteins. Calcium ATPase, Neural Cell Adhesion Molecule, Neural Growth Factor, and Vascular Endothelial Growth Factor were evaluated. The results showed statistically significant mRNA up-regulation of these proteins in the brains of rats exposed to cell phone radiation. These results indicate that relative chronic exposure to cell phone microwave radiation may result in cumulative injuries that could eventually lead to clinically significant neurological damage.

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

    SciTech Connect

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

    1982-01-01

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

  15. Molecular cloning of tomato fruit polygalacturonase: Analysis of polygalacturonase mRNA levels during ripening.

    PubMed

    Dellapenna, D; Alexander, D C; Bennett, A B

    1986-09-01

    The expression of a gene encoding the cell wall-degrading enzyme polygalacturonase [poly(1,4-alpha-D-galacturonide) glucanohydrolase, EC 3.2.1.15] was characterized during tomato fruit ripening. Polygalacturonase was purified from ripe tomato fruit and used to produce highly specific antiserum. Immunoblot analyses detected a 45- and a 46-kDa protein in ripe fruit but immunoprecipitation of in vitro translation products of mRNA from ripe tomato fruit yielded a single 54-kDa polypeptide, suggesting post-translational processing. A plasmid cDNA library was prepared from poly(A)(+) RNA isolated from ripe tomato fruit. The cDNA library was inserted into a lambda-based expression vector, and polygalacturonase cDNA clones were identified by immunological screening. Hybrid-select translation experiments indicated that the cDNAs encode a 54-kDa in vitro translation product that is specifically immunoprecipitated with polygalacturonase antiserum. RNA-blot analysis indicated that the 1.9-kilobase polygalacturonase mRNA was virtually absent from immature-green fruit, accumulated steadily during the ripening process, and was at its highest level in red-ripe fruit. There was at least a 2000-fold increase in the level of polygalacturonase mRNA between immature-green and red-ripe tomato fruit. These studies show that the levels of polygalacturonase mRNA are developmentally regulated during tomato fruit ripening.

  16. Molecular cloning of tomato fruit polygalacturonase: Analysis of polygalacturonase mRNA levels during ripening

    PubMed Central

    DellaPenna, Dean; Alexander, Danny C.; Bennett, Alan B.

    1986-01-01

    The expression of a gene encoding the cell wall-degrading enzyme polygalacturonase [poly(1,4-α-D-galacturonide) glucanohydrolase, EC 3.2.1.15] was characterized during tomato fruit ripening. Polygalacturonase was purified from ripe tomato fruit and used to produce highly specific antiserum. Immunoblot analyses detected a 45- and a 46-kDa protein in ripe fruit but immunoprecipitation of in vitro translation products of mRNA from ripe tomato fruit yielded a single 54-kDa polypeptide, suggesting post-translational processing. A plasmid cDNA library was prepared from poly(A)+ RNA isolated from ripe tomato fruit. The cDNA library was inserted into a λ-based expression vector, and polygalacturonase cDNA clones were identified by immunological screening. Hybrid-select translation experiments indicated that the cDNAs encode a 54-kDa in vitro translation product that is specifically immunoprecipitated with polygalacturonase antiserum. RNA-blot analysis indicated that the 1.9-kilobase polygalacturonase mRNA was virtually absent from immature-green fruit, accumulated steadily during the ripening process, and was at its highest level in red-ripe fruit. There was at least a 2000-fold increase in the level of polygalacturonase mRNA between immature-green and red-ripe tomato fruit. These studies show that the levels of polygalacturonase mRNA are developmentally regulated during tomato fruit ripening. Images PMID:16593752

  17. Light-dark condition regulates sirtuin mRNA levels in the retina.

    PubMed

    Ban, Norimitsu; Ozawa, Yoko; Inaba, Takaaki; Miyake, Seiji; Watanabe, Mitsuhiro; Shinmura, Ken; Tsubota, Kazuo

    2013-11-01

    Sirtuins (Sirt1-7) are nicotinamide adenine dinucleotide (NAD)-dependent protein deacetylases/ADP-ribosyltransferases that modulate many metabolic responses affecting aging. Sirtuins expressed in tissues and organs involved in systemic metabolism have been extensively studied. However, the characteristics of sirtuins in the retina, where local energy expenditure changes dynamically in response to light stimuli, are largely unknown. Here we analyzed sirtuin mRNA levels by real-time PCR, and found that all seven sirtuins are highly expressed in the retina compared with other tissues, such as liver. We then analyzed the sirtuin mRNA profiles in the retina over time, under a 12-h light/12-h dark cycle (LD condition) and in constant darkness (DD condition). All seven sirtuins showed significant daily variation under the LD condition, with all except Sirt6 being increased in the dark phase. The expression patterns were different under the DD condition, suggesting that sirtuin mRNA levels except Sirt6 are affected by light-dark condition. These findings were not obtained in the brain and liver. In addition, the mRNA expression patterns of Nicotinamide phosphoribosyltransferase (Nampt), peroxisome proliferator-activated receptor gamma coactivator (PGC1α), and transcription factor A, mitochondrial (Tfam) in the retina, were similar to those of the sirtuins except Sirt6. Our observations provide new insights into the metabolic mechanisms of the retina and the sirtuins' regulatory systems.

  18. Rapid monitoring of mRNA levels with a molecular beacon during microbial fermentation.

    PubMed

    Dong, Dexian; Pang, Yanping; Gao, Qian; Huang, Xianqing; Xu, Yuquan; Li, Rongxiu

    2010-02-01

    In the microbial fermentation bioreactor, the processes of mRNA transcription, protein translation, and enzyme-catalyzed biosynthesis remain as "black boxes" of industrial monitoring and process control. Monitoring the kinetics of these "black boxes" is very helpful for optimizing and controlling the microbial fermentation process. This study first applied a molecular beacon (MB) to monitor the changes in the mRNA level of the phzC gene during antibiotic phenazine-1-carboxylic acid fermentation. Seven typical MB hybridization buffers were compared, and the effect of formamide on MBs was also studied. The results showed that rapid monitoring of the mRNA level using MBs was feasible. The optimal hybridization buffer for phzC MB was 100 mM Tris, 1 mM MgCl(2), pH 8.0. The optimal hybridization temperature was 35 degrees C, and formamide proved unsuitable for MB hybridization. The limit of detection of phzC MB was 1.67 nM and MB hybridization was complete by 7 min. Given that the time for RNA extraction is 12 min, it is possible that monitoring of phzC mRNA can be completed in less than 20 min. Since production of most amine acids, organic acids, wines, antibiotics, and proteins relies on microbial fermentation, our method may have some potential for application in these other microbial industries.

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

    PubMed

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

    2014-09-01

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

  20. Enhanced levels of scrapie responsive gene mRNA in BSE-infected mouse brain.

    PubMed

    Dandoy-Dron, F; Benboudjema, L; Guillo, F; Jaegly, A; Jasmin, C; Dormont, D; Tovey, M G; Dron, M

    2000-03-10

    The expression of the mRNA of nine scrapie responsive genes was analyzed in the brains of FVB/N mice infected with bovine spongiform encephalopathy (BSE). The RNA transcripts of eight genes were overexpressed to a comparable extent in both BSE-infected and scrapie-infected mice, indicating a common series of pathogenic events in the two transmissible spongiform encephalopathies (TSEs). In contrast, the serine proteinase inhibitor spi 2, an analogue of the human alpha-1 antichymotrypsin gene, was overexpressed to a greater extent in the brains of scrapie-infected animals than in animals infected with BSE, reflecting either an agent specific or a mouse strain specific response. The levels of spi 2 mRNA were increased during the course of scrapie prior to the onset of clinical signs of the disease and the increase reached 11 to 45 fold relative to uninfected controls in terminally ill mice. Spi 2, in common with four of the other scrapie responsive genes studied, is known to be associated with pro-inflammatory processes. These observations underline the importance of cell reactivity in TSE. In addition, scrg2 mRNA the level of which is enhanced in TSE-infected mouse brain, was identified as a previously unrecognized long transcript of the murine aldolase C gene. However, the level of the principal aldolase C mRNA is unaffected in TSE. The increased representation of the longer transcript in the late stage of the disease may reflect changes in mRNA processing and/or stability in reactive astrocytes or in damaged Purkinje cells.

  1. Actinic keratosis

    MedlinePlus

    Solar keratosis; Sun-induced skin changes - keratosis; Keratosis - actinic (solar); Skin lesion - actinic keratosis ... likely to develop it if you: Have fair skin, blue or green eyes, or blond or red ...

  2. Differential responses of intestinal glucose transporter mRNA transcripts to levels of dietary sugars.

    PubMed

    Miyamoto, K; Hase, K; Takagi, T; Fujii, T; Taketani, Y; Minami, H; Oka, T; Nakabou, Y

    1993-10-01

    Dietary sugars are known to stimulate intestinal glucose transport activity, but the specific signals involved are unknown. The Na(+)-dependent glucose co-transporter (SGLT1), the liver-type facilitative glucose transporter (GLUT2) and the intestinal-type facilitative glucose transporter (GLUT5) are all expressed in rat jejunum [Miyamoto, Hase, Taketani, Minami, Oka, Nakabou and Hagihira (1991) Biochem. Biophys. Res. Commun. 181, 1110-1117]. In the present study we have investigated the effects of dietary sugars on these glucose transporter genes. A high-glucose diet stimulated glucose transport activity and increased the levels of SGLT1 and GLUT2 mRNAs in rat jejunum. 3-O-Methylglucose, D-galactose, D-fructose, D-mannose and D-xylose can mimic the regulatory effect of glucose on the SGLT1 mRNA level in rat jejunum. However, only D-galactose and D-fructose increased the levels of GLUT2 mRNA. The GLUT5 mRNA level was increased significantly only by D-fructose. Our results suggest that the increase in intestinal transport activity in rats caused by dietary glucose is due to an increase in the levels of SGLT1 and GLUT2 mRNAs, and that these increases in mRNA may be caused by an enhancement of the transcriptional rate. Furthermore, for expression of the SGLT1 gene, the signal need not be a metabolizable or transportable substrate whereas, for expression of the GLUT2 gene, metabolism of the substrate in the liver may be necessary for signalling. Only D-fructose is an effective signal for expression of the GLUT5 gene.

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

    NASA Astrophysics Data System (ADS)

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

    2008-02-01

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

  4. Plasma Gelsolin Levels Decrease in Diabetic State and Increase upon Treatment with F-Actin Depolymerizing Versions of Gelsolin

    PubMed Central

    Khatri, Neeraj; Sagar, Amin; Peddada, Nagesh; Choudhary, Vikas; Chopra, Bhupinder Singh; Garg, Veena; Ashish

    2014-01-01

    The study aims to map plasma gelsolin (pGSN) levels in diabetic humans and mice models of type II diabetes and to evaluate the efficacy of gelsolin therapy in improvement of diabetes in mice. We report that pGSN values decrease by a factor of 0.45 to 0.5 in the blood of type II diabetic humans and mice models. Oral glucose tolerance test in mice models showed that subcutaneous administration of recombinant pGSN and its F-actin depolymerizing competent versions brought down blood sugar levels comparable to Sitagliptin, a drug used to manage hyperglycemic condition. Further, daily dose of pGSN or its truncated versions to diabetic mice for a week kept sugar levels close to normal values. Also, diabetic mice treated with Sitagliptin for 7 days, showed increase in their pGSN values with the decrease in blood glucose as compared to their levels at the start of treatment. Gelsolin helped in improving glycemic control in diabetic mice. We propose that gelsolin level monitoring and replacement of F-actin severing capable gelsolin(s) should be considered in diabetic care. PMID:25478578

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

    PubMed Central

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

    2016-01-01

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

  6. Anabolic androgenic steroid nandrolone decanoate reduces hypothalamic proopiomelanocortin mRNA levels.

    PubMed

    Lindblom, Jonas; Kindlundh, Anna M S; Nyberg, Fred; Bergström, Lena; Wikberg, Jarl E S

    2003-10-03

    Supratherapeutical doses of anabolic androgenic steroids (AASs) have dramatic effects on metabolism in humans, and also inhibit feeding and reduce the rate of body weight gain in rats. In order to test the hypothesis that the AAS metabolic syndrome is accompanied by alterations in the central melanocortin system, we evaluated body weight, food intake and hypothalamic agouti-related protein (AgRP) and proopiomelanocortin (POMC) mRNA levels following administration of different doses of the anabolic androgenic steroid nandrolone decanoate. In order to distinguish changes induced by the steroid treatment per se from those resulting from the reduced food intake and growth rate, we also compared the effect of nandrolone decanoate on AgRP and POMC mRNA expression with both normally fed, and food restricted control groups. We here report that administration of nandrolone specifically reduces arcuate nucleus POMC mRNA levels while not affecting the expression level of AgRP. The effect on POMC expression was not observed in the food restricted controls, excluding the possibility that the observed effect was a mere response to the reduced food intake and body weight. These results raise the possibility that some of the metabolic and behavioural consequences of AAS abuse may be the result of alterations in the melanocortin system.

  7. Enteral glutamine stimulates protein synthesis and decreases ubiquitin mRNA level in human gut mucosa.

    PubMed

    Coëffier, Moïse; Claeyssens, Sophie; Hecketsweiler, Bernadette; Lavoinne, Alain; Ducrotté, Philippe; Déchelotte, Pierre

    2003-08-01

    Effects of glutamine on whole body and intestinal protein synthesis and on intestinal proteolysis were assessed in humans. Two groups of healthy volunteers received in a random order enteral glutamine (0.8 mmol.kg body wt(-1)x h(-1)) compared either to saline or isonitrogenous amino acids. Intravenous [2H5]phenylalanine and [13C]leucine were simultaneously infused. After gas chromatography-mass spectrometry analysis, whole body protein turnover was estimated from traced plasma amino acid fluxes and the fractional synthesis rate (FSR) of gut mucosal protein was calculated from protein and intracellular phenylalanine and leucine enrichments in duodenal biopsies. mRNA levels for ubiquitin, cathepsin D, and m-calpain were analyzed in biopsies by RT-PCR. Glutamine significantly increased mucosal protein FSR compared with saline. Glutamine and amino acids had similar effects on FSR. The mRNA level for ubiquitin was significantly decreased after glutamine infusion compared with saline and amino acids, whereas cathepsin D and m-calpain mRNA levels were not affected. Enteral glutamine stimulates mucosal protein synthesis and may attenuate ubiquitin-dependent proteolysis and thus improve protein balance in human gut.

  8. Polyamines cause elevation of steroid 5α-reductase mRNA levels by suppressing mRNA degradation in C6 glioma cells.

    PubMed

    Morita, Kyoji; Lee, Mi-Sook; Her, Song; Nishibori, Naoyoshi

    2014-10-01

    Polyamines are widely distributed in living organisms, and considered to play a potential role in various cellular processes. The effects of polyamines on gene expression as well as cell proliferation have been suggested to be closely associated with the physiological and pathological functions. However, it seems necessary to investigate their potential roles in the regulation of cellular metabolism and functions. Previously, glial cells have been suggested to be involved in the protection and preservation of neuronal functions, probably through the production of neurotrophic factors in the brain. On the other hand, neuroactive 5α-reduced steroids promote glial cell differentiation, resulting in enhancement of their ability to produce brain-derived neurotrophic factor (BDNF). Based on these findings, polyamines are assumed to stimulate the expression of the gene encoding steroid 5α-reductase (5α-R), which can induce the production of neuroactive 5α-reduced steroids in glial cells. The effects of polyamines on 5α-R mRNA levels in C6 glioma cells were examined as a model experiment. In consequence, spermine (SPM) and spermidine (SPD), but not putrescine (PUT), have been shown to elevate 5α-R mRNA levels without activating the 5α-R promoter. Furthermore, SPM increased 5α-R mRNA levels under the conditions in which the mRNA biosynthesis was inhibited. Therefore, it can be speculated that polyamines increase 5α-R mRNA levels as a consequence of suppressing the degradation of mRNA.

  9. Elevated level of HSPA1L mRNA correlates with graft-versus-host disease.

    PubMed

    Atarod, Sadaf; Turner, Brie; Pearce, Kim Frances; Ahmed, Shaheda S; Norden, Jean; Bogunia-Kubik, Katarzyna; Wang, Xiao-nong; Collin, Matthew; Dickinson, Anne Mary

    2015-06-01

    Graft-versus-host disease (GVHD) can be a fatal complication of allogeneic stem cell transplantation (allo-HSCT). GVHD can be classified as acute (aGVHD: up to 100 days) or chronic (cGVHD: after 100 days) based on the time-point of disease occurrence. At present there are a limited number of biomarkers available for use in the clinic. Thus, the aim of this research was to evaluate the biomarker potential of the extensively studied Heat Shock Protein 70 family members (HSPA1A/HSPA1B and HSPA1L) at the messenger RNA (mRNA) level in acute and cGVHD patient cohorts. In the skin biopsies, HSPA1L mRNA expression was lower in patients with severe aGVHD (grades II-III) when compared to those with none or low grade aGVHD (grades 0-I) and normal controls. In whole blood, HSPA1L mRNA expression level was significantly (p = 0.008) up-regulated at 28 days post-transplant in cGVHD patients with a significant area under the curve (AUC = 0.773). In addition, HSPA1B expression in whole blood was significantly higher at 3 months post-transplant in both the aGVHD grade II-III (p = 0.012) and cGVHD (p = 0.027) patients. Our initial results in this small cohort show that quantifying HSPA1L mRNA expression in the whole blood of allo-HSCT patients at day 28 post-allo-HSCT may be a useful predictive biomarker for cGVHD.

  10. Prolonged fasting and cortisol reduce myostatin mRNA levels in tilapia larvae; short-term fasting elevates.

    PubMed

    Rodgers, Buel D; Weber, Gregory M; Kelley, Kevin M; Levine, Michael A

    2003-05-01

    Myostatin negatively regulates muscle growth and development and has recently been characterized in several fishes. We measured fasting myostatin mRNA levels in adult tilapia skeletal muscle and in whole larvae. Although fasting reduced some growth indexes in adults, skeletal muscle myostatin mRNA levels were unaffected. By contrast, larval myostatin mRNA levels were sometimes elevated after a short-term fast and were consistently reduced with prolonged fasting. These effects were specific for myostatin, as mRNA levels of glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphatase were unchanged. Cortisol levels were elevated in fasted larvae with reduced myostatin mRNA, whereas in addition immersion of larvae in 1 ppm (2.8 microM) cortisol reduced myostatin mRNA in a time-dependent fashion. These results suggest that larval myostatin mRNA levels may initially rise but ultimately fall during a prolonged fast. The reduction is likely mediated by fasting-induced hypercortisolemia, indicating divergent evolutionary mechanisms of glucocorticoid regulation of myostatin mRNA, since these steroids upregulate myostatin gene expression in mammals.

  11. Induction of human spermine oxidase SMO(PAOh1) is regulated at the levels of new mRNA synthesis, mRNA stabilization and newly synthesized protein.

    PubMed

    Wang, Yanlin; Hacker, Amy; Murray-Stewart, Tracy; Fleischer, Jennifer G; Woster, Patrick M; Casero, Robert A

    2005-03-15

    The oxidation of polyamines induced by antitumour polyamine analogues has been associated with tumour response to specific agents. The human spermine oxidase, SMO(PAOh1), is one enzyme that may play a direct role in the cellular response to the antitumour polyamine analogues. In the present study, the induction of SMO(PAOh1) enzyme activity by CPENSpm [N1-ethyl-N11-(cyclopropyl)methyl-4,8,diazaundecane] is demonstrated to be a result of newly synthesized mRNA and protein. Inhibition of new RNA synthesis by actinomycin D inhibits both the appearance of SMO(PAOh1) mRNA and enzyme activity. Similarly, inhibition of newly synthesized protein with cycloheximide prevents analogue-induced enzyme activity. Half-life determinations indicate that stabilization of SMO(PAOh1) protein does not play a significant role in analogue-induced activity. However, half-life experiments using actinomycin D indicate that CPENSpm treatment not only increases mRNA expression, but also leads to a significant increase in mRNA half-life (17.1 and 8.8 h for CPENSpm-treated cells and control respectively). Using reporter constructs encompassing the SMO(PAOh1) promoter region, a 30-90% increase in transcription is observed after exposure to CPENSpm. The present results are consistent with the hypothesis that analogue-induced expression of SMO(PAOh1) is a result of increased transcription and stabilization of SMO(PAOh1) mRNA, leading to increased protein production and enzyme activity. These data indicate that the major level of control of SMO(PAOh1) expression in response to polyamine analogues exposure is at the level of mRNA.

  12. 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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  15. Radiation-induced reductions in transporter mRNA levels parallel reductions in intestinal sugar transport

    PubMed Central

    Roche, Marjolaine; Neti, Prasad V. S. V.; Kemp, Francis W.; Agrawal, Amit; Attanasio, Alicia; Douard, Véronique; Muduli, Anjali; Azzam, Edouard I.; Norkus, Edward; Brimacombe, Michael; Howell, Roger W.

    2010-01-01

    More than a century ago, ionizing radiation was observed to damage the radiosensitive small intestine. Although a large number of studies has since shown that radiation reduces rates of intestinal digestion and absorption of nutrients, no study has determined whether radiation affects mRNA expression and dietary regulation of nutrient transporters. Since radiation generates free radicals and disrupts DNA replication, we tested the hypotheses that at doses known to reduce sugar absorption, radiation decreases the mRNA abundance of sugar transporters SGLT1 and GLUT5, prevents substrate regulation of sugar transporter expression, and causes reductions in sugar absorption that can be prevented by consumption of the antioxidant vitamin A, previously shown by us to radioprotect the testes. Mice were acutely irradiated with 137Cs gamma rays at doses of 0, 7, 8.5, or 10 Gy over the whole body. Mice were fed with vitamin A-supplemented diet (100× the control diet) for 5 days prior to irradiation after which the diet was continued until death. Intestinal sugar transport was studied at days 2, 5, 8, and 14 postirradiation. By day 8, d-glucose uptake decreased by ∼10–20% and d-fructose uptake by 25–85%. With increasing radiation dose, the quantity of heterogeneous nuclear RNA increased for both transporters, whereas mRNA levels decreased, paralleling reductions in transport. Enterocytes of mice fed the vitamin A supplement had ≥ 6-fold retinol concentrations than those of mice fed control diets, confirming considerable intestinal vitamin A uptake. However, vitamin A supplementation had no effect on clinical or transport parameters and afforded no protection against radiation-induced changes in intestinal sugar transport. Radiation markedly reduced GLUT5 activity and mRNA abundance, but high-d-fructose diets enhanced GLUT5 activity and mRNA expression in both unirradiated and irradiated mice. In conclusion, the effect of radiation may be posttranscriptional, and

  16. Increased beta-actin and tubulin polymerization in regrowing axons: relationship to the conditioning lesion effect.

    PubMed

    Lund, Linda M; Machado, Victor M; McQuarrie, Irvine G

    2002-12-01

    Spinal motor neurons of Sprague-Dawley rats were examined to determine which of the neuronal isoforms of actin (beta or gamma) upregulate following axon injury. In situ hybridization studies showed greater beta-actin mRNA levels but no change in gamma-actin mRNA levels-suggesting that axon regrowth utilizes beta-actin. We radiolabeled the newly synthesized actin and tubulin that are subsequently transported in the axon to the site of an axotomizing injury. This allowed us to evaluate changes in polymerization as new cytoskeletal elements approach the injury site. Previous studies had shown that the rate of the most rapid subcomponent of actin and tubulin transport (called SCb) accelerates following axotomy (J. Jacob and I. McQuarrie, J. Neurobiol. 22: 570-583, 1991). This rate increase is associated with an increased proportion of SCb tubulin and actin in polymer (vs monomer) form (J. Jacob and I. McQuarrie, J. Neurosci, Res. 43: 412-419, 1996). However, in that study newly synthesized proteins were radiolabeled at 7 days after axotomy-which is at the peak of increased protein synthesis. This time-course did not examine actin and tubulin that were already in transit in axons when the injury occurred. This actin and tubulin would enter the regrowing axons first. Here, we have radiolabeled newly synthesized proteins 3 days prior to axotomy. For beta-tubulin, the ratio of monomer to polymer was unaffected. For actin, the equilibrium shifted strongly toward polymerization. We conclude that the acceleration of axonal outgrowth seen after the second of two serial axotomies (the "conditioning lesion effect") is related to the ability of actin that is already in transit to polymerize in response to the first axotomy.

  17. Bacterial nucleators: actin' on actin

    PubMed Central

    Bugalhão, Joana N.; Mota, Luís Jaime; Franco, Irina S.

    2015-01-01

    The actin cytoskeleton is a key target of numerous microbial pathogens, including protozoa, fungi, bacteria and viruses. In particular, bacterial pathogens produce and deliver virulence effector proteins that hijack actin dynamics to enable bacterial invasion of host cells, allow movement within the host cytosol, facilitate intercellular spread or block phagocytosis. Many of these effector proteins directly or indirectly target the major eukaryotic actin nucleator, the Arp2/3 complex, by either mimicking nucleation promoting factors or activating upstream small GTPases. In contrast, this review is focused on a recently identified class of effector proteins from Gram-negative bacteria that function as direct actin nucleators. These effector proteins mimic functional activities of formins, WH2-nucleators and Ena/VASP assembly promoting factors demonstrating that bacteria have coopted the complete set of eukaryotic actin assembly pathways. Structural and functional analyses of these nucleators have revealed several motifs and/or mechanistic activities that are shared with eukaryotic actin nucleators. However, functional effects of these proteins during infection extend beyond plain actin polymerization leading to interference with other host cell functions such as vesicle trafficking, cell cycle progression and cell death. Therefore, their use as model systems could not only help in the understanding of the mechanistic details of actin polymerization but also provide novel insights into the connection between actin dynamics and other cellular pathways. PMID:26416078

  18. Changes in surfactant protein A mRNA levels in a rat model of insulin-treated diabetic pregnancy.

    PubMed

    Moglia, B B; Phelps, D S

    1996-02-01

    Maternal diabetes during pregnancy is associated with increased risk of neonatal respiratory distress syndrome (RDS). Previous studies using rat models for the diabetic pregnancy have documented decreased amounts of surfactant protein mRNA in the lungs of fetuses. In this study, we measured fetal lung surfactant-associated protein A (SP-A) mRNA from diabetic rats treated with insulin by daily injection or osmotic pump. Lungs were taken from fetuses on gestational d 20, and RNA was isolated and subjected to Northern blotting and densitometry to quantify SP-A mRNA. Fetal lung SP-A mRNA from untreated diabetic pregnancies was 34 +/- 2.9% of control. Insulin treatment increased levels to 55 +/- 4.2% of control values. Fetal lung SP-A mRNA levels were affected by the timing, length, and effectiveness of insulin treatment. Although levels from all treatment groups were still less than control values, insulin treatment during the last 5 or 10 d of pregnancy resulted in a substantial increase in SP-A mRNA levels over those of from untreated diabetic pregnancies. However, fetuses from the group with insulin treatment for the entire pregnancy showed decreases in fetal SP-A mRNA levels. Although the mechanism(s) responsible for the effects of diabetes and its treatment on fetal SP-A expression remain unclear, it appears unlikely that hyperglycemia is the principal cause.

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

    PubMed

    Hansen, M C; Nielsen, A K; Molin, S; Hammer, K; Kilstrup, M

    2001-08-01

    Regulation of gene expression can be analyzed by a number of different techniques. Some techniques monitor the level of specific mRNA directly, and others monitor indirectly by determining the level of enzymes encoded by the mRNA. Each method has its own inherent way of normalization. When results obtained by these techniques are compared between experiments in which differences in growth rates, strains, or stress treatments occur, the normalization procedure may have a significant impact on the results. In this report we present a solution to the normalization problem in RNA slot blotting experiments, in which mRNA levels routinely are normalized to a fixed amount of extracted total RNA. The cellular levels of specific mRNA species were estimated using a renormalization with the total RNA content per cell. By a combination of fluorescence in situ rRNA hybridization, which estimates the relative level of rRNA per cell, and slot blotting to rRNA probes, which estimates the level of rRNA per extracted total RNA, the amount of RNA per cell was calculated in a series of heat shock experiments with the gram-positive bacterium Lactococcus lactis. It was found that the level of rRNA per cell decreased to 30% in the course of the heat shock. This lowered ribosome level led to a decrease in the total RNA content, resulting in a gradually increasing overestimation of the mRNA levels throughout the experiment. Using renormalized cellular mRNA levels, the HrcA-mediated regulation of the genes in the hrcA-grpE-dnaK operon was analyzed. The hybridization data suggested a complex heat shock regulation indicating that the mRNA levels continued to rise after 30 min, but after renormalization the calculated average cellular levels exhibited a much simpler induction pattern, eventually attaining a moderately increased value.

  20. Actinous enigma or enigmatic actin

    PubMed Central

    Povarova, Olga I; Uversky, Vladimir N; Kuznetsova, Irina M; Turoverov, Konstantin K

    2014-01-01

    Being the most abundant protein of the eukaryotic cell, actin continues to keep its secrets for more than 60 years. Everything about this protein, its structure, functions, and folding, is mysteriously counterintuitive, and this review represents an attempt to solve some of the riddles and conundrums commonly found in the field of actin research. In fact, actin is a promiscuous binder with a wide spectrum of biological activities. It can exist in at least three structural forms, globular, fibrillar, and inactive (G-, F-, and I-actin, respectively). G-actin represents a thermodynamically instable, quasi-stationary state, which is formed in vivo as a result of the energy-intensive, complex posttranslational folding events controlled and driven by cellular folding machinery. The G-actin structure is dependent on the ATP and Mg2+ binding (which in vitro is typically substituted by Ca2+) and protein is easily converted to the I-actin by the removal of metal ions and by action of various denaturing agents (pH, temperature, and chemical denaturants). I-actin cannot be converted back to the G-form. Foldable and “natively folded” forms of actin are always involved in interactions either with the specific protein partners, such as Hsp70 chaperone, prefoldin, and the CCT chaperonin during the actin folding in vivo or with Mg2+ and ATP as it takes place in the G-form. We emphasize that the solutions for the mysteries of actin multifunctionality, multistructurality, and trapped unfolding can be found in the quasi-stationary nature of this enigmatic protein, which clearly possesses many features attributed to both globular and intrinsically disordered proteins.

  1. Variations in mRNA and protein levels of Ikaros family members in pediatric T cell acute lymphoblastic leukemia

    PubMed Central

    Mitchell, Julie L.

    2016-01-01

    Background Pediatric T cell acute lymphoblastic leukemia (T-ALL) is a highly heterogeneous disease in which the cells share phenotypic characteristics with normal human thymocytes. The Ikaros family of transcription factors includes five members that are required for normal T cell development and are implicated in leukemogenesis. The goal of this work was to correlate the pattern of expression of Ikaros family members with the phenotype of the T-ALL cells. Methods We obtained twenty-four samples from pediatric T-ALL patients and used multi-parameter flow cytometry to characterize each sample, comparing the phenotype of the leukemic cells with normal human thymocytes. Then, we defined the expression levels of each Ikaros family member to determine whether the mRNA levels or splicing or protein levels were similar to the normal patterns seen during human T cell development. Results Multi-parameter analysis of the phenotype of T-ALL cells revealed that each patient’s cells were unique and could not be readily correlated with stages of T cell development. Similarly, the pattern of Ikaros expression varied among patients. In most patients, Ikaros mRNA was the dominant family member expressed, but some patients’ cells contained mostly Helios, Aiolos, or Eos mRNA. Despite that most patients had elevated mRNA levels of Ikaros family members and unique patterns of mRNA splicing, most patients had significantly reduced protein levels of Ikaros and Aiolos. Conclusions Our analysis of the cell phenotype and Ikaros expression levels in T-ALL cells revealed the extent of heterogeneity among patients. While it is rarely possible to trace leukemic cells to their developmental origin, we found distinct patterns of Ikaros family mRNA levels in groups of patients. Further, mRNA and protein levels of Ikaros and Aiolos did not correlate, indicating that mRNA and protein levels are regulated via distinct mechanisms. PMID:27826566

  2. Catabolite control of the elevation of PGK mRNA levels by heat shock in Saccharomyces cerevisiae.

    PubMed

    Piper, P W; Curran, B; Davies, M W; Hirst, K; Lockheart, A; Seward, K

    1988-05-01

    Heat shock enhances the very high level of transcription of the phosphoglycerate kinase (PGK) gene in fermentative cultures of Saccharomyces cerevisiae. This response of PGK mRNA levels was not found on gluconeogenic carbon sources, and could be switched on or off subject to availability of fermentable carbon source. The addition of glucose to yeast growing on glycerol resulted in acquisition, within 30-60 min, of the ability to elevate PGK mRNA levels after heat shock. In addition, in aerobic cultures growing on glucose the exhaustion of the medium glucose coincided with a loss of the heat-shock effect on PGK mRNA and a switch-over to slower growth by aerobic respiration. Levels of hsp26 mRNA were analysed during these experiments. Contrasting with this requirement for fermentable catabolite for manifestation of a heat-shock response of PGK mRNA levels, the PGK enzyme was not synthesized at a greater level in heat-shocked fermentative than in gluconeogenic cultures. PGK is one of only a few proteins made efficiently after mild heat shock of yeast. Thus, heat-stress-induced elevation of PGK mRNA levels does not appreciably increase PGK synthesis during exposure to high temperatures and so its role may be to assist cells repressed in mitochondrial function during recovery following a heat shock.

  3. Time Course of Behavioral Alteration and mRNA Levels of Neurotrophic Factor Following Stress Exposure in Mouse.

    PubMed

    Hashikawa, Naoya; Ogawa, Takumi; Sakamoto, Yusuke; Ogawa, Mami; Matsuo, Yumi; Zamami, Yoshito; Hashikawa-Hobara, Narumi

    2015-08-01

    Stress is known to affect neurotrophic factor expression, which induces depression-like behavior. However, whether there are time-dependent changes in neurotrophic factor mRNA expression following stress remains unclear. In the present study, we tested whether chronic stress exposure induces long-term changes in depression-related behavior, serum corticosterone, and hippocampal proliferation as well as neurotrophic factor family mRNA levels, such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), and ciliary neurotrophic factor (CNTF), in the mouse hippocampus. The mRNA level of neurotrophic factors (BDNF, NGF, NT-3, and CNTF) was measured using the real-time PCR. The serum corticosterone level was evaluated by enzyme-linked immunosorbent assay, and, for each subject, the hippocampal proliferation was examined by 5-bromo-2-deoxyuridine immunostaining. Mice exhibited depression-like behavior in the forced-swim test (FST) and decreased BDNF mRNA and hippocampal proliferation in the middle of the stress exposure. After 15 days of stress exposure, we observed increased immobility in the FST, serum corticosterone levels, and BDNF mRNA levels and degenerated hippocampal proliferation, maintained for at least 2 weeks. Anhedonia-like behavior in the sucrose preference test and NGF mRNA levels were decreased following 15 days of stress. NGF mRNA levels were significantly higher 1 week after stress exposure. The current data demonstrate that chronic stress exposure induces prolonged BDNF and NGF mRNA changes and increases corticosterone levels and depression-like behavior in the FST, but does not alter other neurotrophic factors or performance in the sucrose preference test.

  4. Dietary copper can regulate the level of mRNA for dopamine B-hydroxylase in rat adrenal gland

    SciTech Connect

    Sabban, E.L.; Failla, M.L.; McMahon, A.; Seidel, K.E. Dept. of Agriculture, Beltsville, MD )

    1991-03-15

    Recent studies have shown that Cu deficiency markedly alters the levels of dopamine (DA) and norepinephrine (NE) in several peripheral tissues of rodents. Conversion of DA to NE is mediated by dopamine B-hydroxylase (DBM). Here the authors examined the effect of dietary Cu deficiency on the levels of DA, NE and DBM mRNA in rat adrenal gland. Severe Cu deficiency was induced by feeding low Cu diet to dams beginning at 17d gestation and weaning pups to the same diet. At 7 wks of age rats fed {minus}Cu diet were characterized by depressed growth, low tissue Cu, enlarged hearts and moderate anemia. Concentrations of DA were higher in adrenals and hearts of {minus}Cu rats compared to +Cu controls. While cardiac level of NE in {minus}Cu rats were reduced to 17% that of controls, adrenal NE was unchanged by Cu deficiency. To investigate possible mechanisms responsible for the response of adrenal gland to Cu deficiency, RNA was isolated and the levels of DBH mRNA and tyrosine hydroxylase (TH) mRNA were analyzed by Northern blots. Steady state levels of adrenal DBH mRNA was increased 2-3 fold in {minus}Cu rats, whereas TH mRNA were unchanged by dietary Cu status. Upon feeding the {minus}Cu rats the Cu adequate diet overnight, there was a further increase in DBH mRNA and a slight elevation of TH mRNA levels. The results indicate that dietary copper can markedly affect the level of DBH mRNA in rat adrenal gland.

  5. Cofilin-mediated actin dynamics promotes actin bundle formation during Drosophila bristle development

    PubMed Central

    Wu, Jing; Wang, Heng; Guo, Xuan; Chen, Jiong

    2016-01-01

    The actin bundle is an array of linear actin filaments cross-linked by actin-bundling proteins, but its assembly and dynamics are not as well understood as those of the branched actin network. Here we used the Drosophila bristle as a model system to study actin bundle formation. We found that cofilin, a major actin disassembly factor of the branched actin network, promotes the formation and positioning of actin bundles in the developing bristles. Loss of function of cofilin or AIP1, a cofactor of cofilin, each resulted in increased F-actin levels and severe defects in actin bundle organization, with the defects from cofilin deficiency being more severe. Further analyses revealed that cofilin likely regulates actin bundle formation and positioning by the following means. First, cofilin promotes a large G-actin pool both locally and globally, likely ensuring rapid actin polymerization for bundle initiation and growth. Second, cofilin limits the size of a nonbundled actin-myosin network to regulate the positioning of actin bundles. Third, cofilin prevents incorrect assembly of branched and myosin-associated actin filament into bundles. Together these results demonstrate that the interaction between the dynamic dendritic actin network and the assembling actin bundles is critical for actin bundle formation and needs to be closely regulated. PMID:27385345

  6. Regulation of type I (epidermal) transglutaminase mRNA levels during squamous differentiation: down regulation by retinoids.

    PubMed Central

    Floyd, E E; Jetten, A M

    1989-01-01

    Squamous differentiation of rabbit tracheal epithelial cells is accompanied by an approximately 50-fold increase in the activity of type I (epidermal) transglutaminase, while the levels of type II (tissue) transglutaminase remain almost undetectable. To identify a cDNA encoding type I transglutaminase, we screened a library of cDNA clones prepared from poly(A)+ RNA isolated from squamous-differentiated rabbit tracheal epithelial cells. Four overlapping clones (represented by clone pTG-7) which span a range of 2.8 kilobases were identified; partial sequencing of pTG-7 indicated that it encodes a transglutaminaselike protein. pTG-7 hybridized to a 3.6-kilobase mRNA which is distinct from that for type II transglutaminase. pTG-7 mRNA levels were low in proliferative cells, increased dramatically in squamous-differentiated cells, and could be further enhanced by growth of the cells in high concentrations (2 mM) of calcium ions. Retinoic acid, which blocks the expression of the squamous phenotype, prevented this increase in pTG-7 mRNA levels. These changes in levels of pTG-7 mRNA parallel the changes in type I transglutaminase activity observed under similar culture conditions. These data indicate that pTG-7 encodes the mRNA for transglutaminase type I and that expression of this mRNA is negatively regulated by retinoic acid. Images PMID:2574824

  7. Evaluation of diagnostic relevance of mRNA levels in peripheral blood: predictive value for mortality in hemodialysis patients.

    PubMed

    Füth, Reiner; Herder, Christian; Förster, Stefan; Müller-Scholze, Sylvia; Kruse, Niels; Rieckmann, Peter; Heinig, Antonia; Koenig, Wolfgang; Scherbaum, Werner A; Kolb, Hubert; Martin, Stephan

    2004-09-21

    In clinical practice, diagnosis and risk prediction are usually based on the analysis of serum or plasma proteins whereas gene expression analysis is not used on a routine basis. In order to compare the diagnostic and predictive relevance of serum protein and peripheral blood mRNA levels, we determined cytokine levels of end-stage renal failure patients undergoing hemodialysis. These patients face a high mortality mainly due to acceleration of atherosclerosis and subsequent severe vascular events. mRNA expression of the pro-inflammatory cytokine TNF alpha was significantly elevated in hemodialysis patients and further increased after 2 h of dialysis treatment. In contrast, gene expression of the anti-inflammatory cytokine TGF beta was significantly decreased. Patients who died during the observation period of 36 months had significantly increased mRNA levels of TNF alpha and decreased TGF beta mRNA expression at baseline. Survival analysis indicated that increased TNF alpha mRNA levels (P < 0.02) and TNF alpha/TGF beta mRNA ratios (P < 0.001) predict mortality. The corresponding cytokines in serum showed some association with disease, but serum concentrations neither changed during hemodialysis nor predicted mortality. This study shows that gene expression patterns of circulating leukocytes may present an important new diagnostic tool to predict clinical outcome in patients with inflammatory vascular diseases.

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

    PubMed

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

    2017-02-16

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

  9. Quantitation of the mRNA levels of Epo and EpoR in various tissues in the ovine fetus.

    PubMed

    David, R Bruce; Lim, Gaik Bee; Moritz, Karen M; Koukoulas, Irene; Wintour, E Marelyn

    2002-02-25

    A partial cDNA of the sheep erythropoietin receptor (EpoR) was obtained and used in real-time PCR to quantitate mRNA levels in placenta, liver and kidney throughout development (term=150 days). This was compared with Epo mRNA levels in the same tissues. Both Epo and EpoR mRNA were present in the placenta throughout gestation at low levels from 66 days onwards and these did not vary throughout gestation. Compared with the expression levels in the placenta, the levels of EpoR gene expression in the liver at 66, 99 and 140 days were, median (range)-288 (120-343), 278 (63-541) and 7 (3-15), respectively, reflecting the disappearance of erythropoiesis after 130 days. Low levels of EpoR gene expression were seen in the kidney at 3 (2-5), 5 (2-7), and 7 (2-10) times that in the placenta at 66, 99, and 140 days, respectively. By hybridization histochemistry the EpoR mRNA was located in the proximal tubular cells of the mesonephros and metanephros at 42 days. Epo mRNA levels in the kidney were 215 (116-867), 528 (113-765) and 46 (15-204) times those in the placenta at 69, 99, and 140 days, respectively. In the liver at the same ages the concentrations of mRNA were lower than in the kidney, the liver/placenta ratios being 50 (11-90), 17 (3-39), 9 (5-14). At 130 days Epo/EpoR levels in the hippocampus were 6+/-3 and 8+/-3 times that in the term placenta, respectively. These studies demonstrate that the ovine placenta expresses the Epo gene from at least 66 days of gestation. However, gene expression levels are very low compared with those in the liver and kidney, and even the hippocampus.

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

    PubMed

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

    2010-01-01

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

  11. Comparing mRNA levels using in situ hybridization of a target gene and co-stain.

    PubMed

    Wunderlich, Zeba; Bragdon, Meghan D; DePace, Angela H

    2014-06-15

    In situ hybridization is an important technique for measuring the spatial expression patterns of mRNA in cells, tissues, and whole animals. However, mRNA levels cannot be compared across experiments using typical protocols. Here we present a semi-quantitative method to compare mRNA levels of a gene across multiple samples. This method yields an estimate of the error in the measurement to allow statistical comparison. Our method uses a typical in situ hybridization protocol to stain for a target gene and an internal standard, which we refer to as a co-stain. As a proof of concept, we apply this method to multiple lines of transgenic Drosophila embryos, harboring constructs that express reporter genes to different levels. We generated this test set by mutating enhancer sequences to contain different numbers of binding sites for Zelda, a transcriptional activator. We demonstrate that using a co-stain with in situ hybridization is an effective method to compare mRNA levels across samples. This method requires only minor modifications to existing in situ hybridization protocols and uses straightforward analysis techniques. This strategy can be broadly applied to detect quantitative, spatially resolved changes in mRNA levels.

  12. Comparing mRNA levels using in situ hybridization of a target gene and co-stain

    PubMed Central

    Wunderlich, Zeba; Bragdon, Meghan D; DePace, Angela H

    2014-01-01

    In situ hybridization is an important technique for measuring the spatial expression patterns of mRNA in cells, tissues, and whole animals. However, mRNA levels cannot be compared across experiments using typical protocols. Here we present a semi-quantitative method to compare mRNA levels of a gene across multiple samples. This method yields an estimate of the error in the measurement to allow statistical comparison. Our method uses a typical in situ hybridization protocol to stain for a target gene and an internal standard, which we refer to as a co-stain. As a proof of concept, we apply this method to multiple lines of transgenic Drosophila embryos, harboring constructs that express reporter genes to different levels. We generated this test set by mutating enhancer sequences to contain different numbers of binding sites for Zelda, a transcriptional activator. We demonstrate that using a co-stain with in situ hybridization is an effective method to compare mRNA levels across samples. This method requires only minor modifications to existing in situ hybridization protocols and uses straightforward analysis techniques. This strategy can be broadly applied to detect quantitative, spatially resolved changes in mRNA levels. PMID:24434507

  13. TDP-43 regulates its mRNA levels through a negative feedback loop

    PubMed Central

    Ayala, Youhna M; De Conti, Laura; Avendaño-Vázquez, S Eréndira; Dhir, Ashish; Romano, Maurizio; D'Ambrogio, Andrea; Tollervey, James; Ule, Jernej; Baralle, Marco; Buratti, Emanuele; Baralle, Francisco E

    2011-01-01

    TAR DNA-binding protein (TDP-43) is an evolutionarily conserved heterogeneous nuclear ribonucleoprotein (hnRNP) involved in RNA processing, whose abnormal cellular distribution and post-translational modification are key markers of certain neurodegenerative diseases, such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We generated human cell lines expressing tagged forms of wild-type and mutant TDP-43 and observed that TDP-43 controls its own expression through a negative feedback loop. The RNA-binding properties of TDP-43 are essential for the autoregulatory activity through binding to 3′ UTR sequences in its own mRNA. Our analysis indicated that the C-terminal region of TDP-43, which mediates TDP-43–hnRNP interactions, is also required for self-regulation. TDP-43 binding to its 3′ UTR does not significantly change the pre-mRNA splicing pattern but promotes RNA instability. Moreover, blocking exosome-mediated degradation partially recovers TDP-43 levels. Our findings demonstrate that cellular TDP-43 levels are under tight control and it is likely that disease-associated TDP-43 aggregates disrupt TDP-43 self-regulation, thus contributing to pathogenesis. PMID:21131904

  14. Nck adaptors, besides promoting N-WASP mediated actin-nucleation activity at pedestals, influence the cellular levels of enteropathogenic Escherichia coli Tir effector

    PubMed Central

    Nieto-Pelegrin, Elvira; Kenny, Brendan; Martinez-Quiles, Narcisa

    2014-01-01

    Enteropathogenic Escherichia coli (EPEC) binding to human intestinal cells triggers the formation of disease-associated actin rich structures called pedestals. The latter process requires the delivery, via a Type 3 secretion system, of the translocated Intimin receptor (Tir) protein into the host plasma membrane where binding of a host kinase-modified form to the bacterial surface protein Intimin triggers pedestal formation. Tir-Intimin interaction recruits the Nck adaptor to a Tir tyrosine phosphorylated residue where it activates neural Wiskott-Aldrich syndrome protein (N-WASP); initiating the major pathway to actin polymerization mediated by the actin-related protein (Arp) 2/3 complex. Previous studies with Nck-deficient mouse embryonic fibroblasts (MEFs) identified a key role for Nck in pedestal formation, presumed to reflect a lack of N-WASP activation. Here, we show the defect relates to reduced amounts of Tir within Nck-deficient cells. Indeed, Tir delivery and, thus, pedestal formation defects were much greater for MEFs than HeLa (human epithelial) cells. Crucially, the levels of two other effectors (EspB/EspF) within Nck-deficient MEFs were not reduced unlike that of Map (Mitochondrial associated protein) which, like Tir, requires CesT chaperone function for efficient delivery. Interestingly, drugs blocking various host protein degradation pathways failed to increase Tir cellular levels unlike an inhibitor of deacetylase activity (Trichostatin A; TSA). Treatments with TSA resulted in significant recovery of Tir levels, potentiation of actin polymerization and improvement in bacterial attachment to cells. Our findings have important implications for the current model of Tir-mediated actin polymerization and opens new lines of research in this area. PMID:25482634

  15. Actinic reticuloid

    SciTech Connect

    Marx, J.L.; Vale, M.; Dermer, P.; Ragaz, A.; Michaelides, P.; Gladstein, A.H.

    1982-09-01

    A 58-year-old man has his condition diagnosed as actinic reticuloid on the basis of clinical and histologic findings and phototesting data. He had clinical features resembling mycosis fungoides in light-exposed areas. Histologic findings disclosed a bandlike infiltrate with atypical mononuclear cells in the dermis and scattered atypical cells in the epidermis. Electron microscopy disclosed mononuclear cells with bizarre, convoluted nuclei, resembling cerebriform cells of Lutzner. Phototesting disclosed a diminished minimal erythemal threshold to UV-B and UV-A. Microscopic changes resembling actinic reticuloid were reproduced in this patient 24 and 72 hours after exposure to 15 minimal erythemal doses of UV-B.

  16. Genetic diversity of Cryptosporidium in fish at the 18S and actin loci and high levels of mixed infections.

    PubMed

    Yang, Rongchang; Palermo, Cindy; Chen, Linda; Edwards, Amanda; Paparini, Andrea; Tong, Kaising; Gibson-Kueh, Susan; Lymbery, Alan; Ryan, Una

    2015-12-15

    Cryptosporidium is an enteric parasite that infects humans and a wide range of animals. Relatively little is known about the epidemiology and taxonomy of Cryptosporidium in fish. In the present study, a total of 775 fish, belonging to 46 species and comprising ornamental fish, marine fish and freshwater fish were screened for the prevalence of Cryptosporidium by PCR. The overall prevalence of Cryptosporidium in fish was 5.3% (41/775), with prevalences ranging from 1.5 to 100% within individual host species. Phylogenetic analysis of these Cryptosporidium isolates as well as 14 isolates from previous studies indicated extensive genetic diversity as well as evidence for mixed infections. At the 18S locus the following species were identified; Cryptosporidium molnari-like genotype (n=14), Cryptosporidium huwi (n=8), piscine genotype 2 (n=4), piscine genotype 3-like (n=1), piscine genotype 4 (n=2), piscine genotype 5 (n=13), piscine genotype 5-like (n=1) and five novel genotypes (n=5). At the actin locus, species identification agreed with the 18S locus for only 52.3% of isolates sequenced, indicating high levels of mixed infections. Future studies will need to employ both morphological characterization and deep sequencing amplicon-based technologies to better understand the epidemiological and phylogenetic relationships of piscine-derived Cryptosporidium species and genotypes, particularly when mixed infections are detected.

  17. Lack of effect of antipsychotic and antidepressant drugs on glutamate receptor mRNA levels in rat brains.

    PubMed

    Oretti, R G; Spurlock, G; Buckland, P R; McGuffin, P

    1994-08-15

    By employing multiprobe oligonucleotide solution hybridisation (MOSH) we have measured the levels of mRNA encoding the NMDA receptor subtypes (R1, R2A, R2B and R2C) and the non-NMDA glutamate receptor subtypes (GluR1, 2, 3, and 4) within rat brain following, 1-32 days of antipsychotic or antidepressant drug administration. The results suggest that the drugs studied do not significantly alter rat glutamatergic system mRNA levels when compared to controls.

  18. Thyroid hormone regulates Ca(2+)-ATPase mRNA levels of sarcoplasmic reticulum during neonatal development of fast skeletal muscle.

    PubMed

    van der Linden, G C; Simonides, W S; van Hardeveld, C

    1992-12-01

    In gastrocnemius muscle from newborn rats the mRNA for the fast sarcoplasmic reticulum (SR) Ca(2+)-ATPase isoform (SERCA1) comprised over 90% of total SR Ca(2+)-ATPase mRNA content and increased 5-fold between day 5 and 20 after birth, whereas in hypothyroid muscle the SERCA1 message level remained constant. Triiodothyronine (T3) treatment of 2-day-old euthyroid rats induced a precocious stimulation of SERCA1 mRNA levels, indicating that T3 is the determining factor in the stimulation of SERCA1 message levels and that this stimulation underlies the previously reported effect of the thyroid status on the neonatal development of SR Ca(2+)-ATPase activity. The low mRNA level for the slow SR Ca(2+)-ATPase isoform (SERCA2) was constant in both euthyroid and hypothyroid muscle development. Nevertheless, T3 treatment of hypothyroid neonates induced a transient stimulation of SERCA2 message levels, indicating that SERCA2 is responsive to higher levels of T3.

  19. Limited availability of ZBP1 restricts axonal mRNA localization and nerve regeneration capacity.

    PubMed

    Donnelly, Christopher J; Willis, Dianna E; Xu, Mei; Tep, Chhavy; Jiang, Chunsu; Yoo, Soonmoon; Schanen, N Carolyn; Kirn-Safran, Catherine B; van Minnen, Jan; English, Arthur; Yoon, Sung Ok; Bassell, Gary J; Twiss, Jeffery L

    2011-09-30

    Subcellular localization of mRNAs is regulated by RNA-protein interactions. Here, we show that introduction of a reporter mRNA with the 3'UTR of β-actin mRNA competes with endogenous mRNAs for binding to ZBP1 in adult sensory neurons. ZBP1 is needed for axonal localization of β-actin mRNA, and introducing GFP with the 3'UTR of β-actin mRNA depletes axons of endogenous β-actin and GAP-43 mRNAs and attenuates both in vitro and in vivo regrowth of severed axons. Consistent with limited levels of ZBP1 protein in adult neurons, mice heterozygous for the ZBP1 gene are haploinsufficient for axonal transport of β-actin and GAP-43 mRNAs and for regeneration of peripheral nerve. Exogenous ZBP1 can rescue the RNA transport deficits, but the axonal growth deficit is only rescued if the transported mRNAs are locally translated. These data support a direct role for ZBP1 in transport and translation of mRNA cargos in axonal regeneration in vitro and in vivo.

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

    PubMed Central

    Ikeda, Hitoshi; Kurano, Makoto; Sato, Masaya; Kudo, Hiroki; Maki, Harufumi; Koike, Kazuhiko; Hasegawa, Kiyoshi; Kokudo, Norihiro; Yatomi, Yutaka

    2016-01-01

    Hepatocellular carcinoma (HCC) commonly develops in patients with liver fibrosis; in these patients, the blood levels of lysophosphatidic acid (LPA) and its generating enzyme autotaxin (ATX) increase with the liver fibrosis stage. We aimed to examine the potential relevance of ATX and LPA in HCC. Fifty-eight HCC patients who underwent surgical treatment were consecutively enrolled in the study. Among the LPA receptors in HCC, higher LPA2 mRNA levels correlated with poorer differentiation, and higher LPA6 mRNA levels correlated with microvascular invasion, which suggested a higher malignant potential of HCC with increased LPA2 and LPA6 expression. In patients with primary HCC, neither LPA2 nor LPA6 mRNA levels were associated with recurrence. However, when serum ATX levels were combined for analysis as a surrogate for plasma LPA levels, the cumulative intra-hepatic recurrence rate was higher in patients in whom both serum ATX levels and LPA2 or LPA6 mRNA levels were higher than the median. However, the mRNA level of phosphatidic acid-selective phospholipase A1ɑ, another LPA-generating enzyme, in HCC patients was not associated with pathological findings or recurrence, even in combination with the expression of LPA receptors. Higher LPA2 mRNA levels were associated with poorer differentiation, and higher LPA6 levels were associated with microvascular invasion in HCC; both became a risk factor for recurrence after surgical treatment when combined with increased serum ATX levels. ATX and LPA receptors merit consideration as therapeutic targets of HCC. PMID:27583415

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

    PubMed

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

    2009-12-01

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

  2. Recombinant interleukin 2 regulates levels of c-myc mRNA in a cloned murine T lymphocyte.

    PubMed Central

    Reed, J C; Sabath, D E; Hoover, R G; Prystowsky, M B

    1985-01-01

    The cellular oncogene c-myc has been implicated in the regulation of growth of normal and neoplastic cells. Recently, it was suggested that c-myc gene expression may control the G0----G1-phase transition in normal lymphocytes that were stimulated to enter the cell cycle by the lectin concanavalin A (ConA). Here we describe the effects of purified recombinant interleukin 2 (rIL2) and of ConA on levels of c-myc mRNA in the noncytolytic murine T-cell clone L2. In contrast to resting (G0) primary cultures of lymphocytes, quiescent L2 cells have a higher RNA content than resting splenocytes and express receptors for interleukin 2 (IL2). Resting L2 cells are therefore best regarded as early G1-phase cells. Purified rIL2 was found to stimulate the rapid accumulation of c-myc mRNA in L2 cells. Levels of c-myc mRNA became maximal within 1 h and declined gradually thereafter. In contrast, ConA induced slower accumulation of c-myc mRNA in L2 cells, with increased levels of c-myc mRNA becoming detectable 4 to 8 h after stimulation. Experiments with the protein synthesis inhibitor cycloheximide demonstrated that the increase in levels of c-myc mRNA that were induced by ConA was a direct effect of this lectin and not secondary to IL2 production. Cyclosporin A, an immunosuppressive agent, markedly reduced the accumulation of c-myc mRNA that was induced by ConA but only slightly diminished the accumulation of c-myc mRNA that was induced by rIL2. Taken together, these data provide evidence that (i) c-myc gene expression can be regulated by at least two distinct pathways in T lymphocytes, only one of which is sensitive to cyclosporine A, and (ii) the accumulation of c-myc mRNA can be induced in T cells by IL2 during the G1 phase of the cell cycle. Images PMID:3879814

  3. Fascin regulates nuclear actin during Drosophila oogenesis

    PubMed Central

    Kelpsch, Daniel J.; Groen, Christopher M.; Fagan, Tiffany N.; Sudhir, Sweta; Tootle, Tina L.

    2016-01-01

    Drosophila oogenesis provides a developmental system with which to study nuclear actin. During Stages 5–9, nuclear actin levels are high in the oocyte and exhibit variation within the nurse cells. Cofilin and Profilin, which regulate the nuclear import and export of actin, also localize to the nuclei. Expression of GFP-tagged Actin results in nuclear actin rod formation. These findings indicate that nuclear actin must be tightly regulated during oogenesis. One factor mediating this regulation is Fascin. Overexpression of Fascin enhances nuclear GFP-Actin rod formation, and Fascin colocalizes with the rods. Loss of Fascin reduces, whereas overexpression of Fascin increases, the frequency of nurse cells with high levels of nuclear actin, but neither alters the overall nuclear level of actin within the ovary. These data suggest that Fascin regulates the ability of specific cells to accumulate nuclear actin. Evidence indicates that Fascin positively regulates nuclear actin through Cofilin. Loss of Fascin results in decreased nuclear Cofilin. In addition, Fascin and Cofilin genetically interact, as double heterozygotes exhibit a reduction in the number of nurse cells with high nuclear actin levels. These findings are likely applicable beyond Drosophila follicle development, as the localization and functions of Fascin and the mechanisms regulating nuclear actin are widely conserved. PMID:27535426

  4. Effect of the increased stability of the penicillin amidase mRNA on the protein expression levels.

    PubMed

    Viegas, Sandra C; Schmidt, Dorothea; Kasche, Volker; Arraiano, Cecília M; Ignatova, Zoya

    2005-09-12

    Several factors at transcriptional, post-transcriptional or post-translational level determine the fate of a target protein and can severely restrict its yield. Here, we focus on the post-transcriptional regulation of the biosynthesis of the periplasmic protein, penicillin amidase (PA). The PA mRNA stability was determined under depleted RNase conditions in strains carrying single or multiple RNase deletions. Single deletion of the endonuclease RNase E yielded, as the highest, a fourfold stabilization of the PA mRNA. This effect, however, was reduced twice at post-translational level. The RNase II, generating secondary exonucleolytic cleavages in the mRNA, although not significantly influencing the PA mRNA decay, led also to an increase of the amount of mature PA. The non-proportional correlation between increased mRNA longevity and amount of active enzyme propose that the rational strategies for yield improvement must be based on a simultaneous tuning of more than one yield restricting factor.

  5. Spaceflight has compartment- and gene-specific effects on mRNA levels for bone matrix proteins in rat femur

    NASA Technical Reports Server (NTRS)

    Evans, G. L.; Morey-Holton, E.; Turner, R. T.

    1998-01-01

    In the present study, we evaluated the possibility that the abnormal bone matrix produced during spaceflight may be associated with reduced expression of bone matrix protein genes. To test this possibility, we investigated the effects of a 14-day spaceflight (SLS-2 experiment) on steady-state mRNA levels for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), osteocalcin, osteonectin, and prepro-alpha(1) subunit of type I collagen in the major bone compartments of rat femur. There were pronounced site-specific differences in the steady-state levels of expression of the mRNAs for the three bone matrix proteins and GAPDH in normal weight-bearing rats, and these relationships were altered after spaceflight. Specifically, spaceflight resulted in decreases in mRNA levels for GAPDH (decreased in proximal metaphysis), osteocalcin (decreased in proximal metaphysis), osteonectin (decreased in proximal and distal metaphysis), and collagen (decreased in proximal and distal metaphysis) compared with ground controls. There were no changes in mRNA levels for matrix proteins or GAPDH in the shaft and distal epiphysis. These results demonstrate that spaceflight leads to site- and gene-specific decreases in mRNA levels for bone matrix proteins. These findings are consistent with the hypothesis that spaceflight-induced decreases in bone formation are caused by concomitant decreases in expression of genes for bone matrix proteins.

  6. Hypoxia may increase rat insulin mRNA levels by promoting binding of the polypyrimidine tract-binding protein (PTB) to the pyrimidine-rich insulin mRNA 3'-untranslated region.

    PubMed Central

    Tillmar, Linda; Welsh, Nils

    2002-01-01

    BACKGROUND: Recent reports identify the 3'-UTR of insulin mRNA as crucial for control of insulin messenger stability. This region contains a pyrimidine-rich sequence, which is similar to the hypoxia-responsive mRNA-stabilizing element of tyrosine hydroxylase. This study aimed to determine whether hypoxia affects insulin mRNA levels. MATERIALS AND METHODS: Rat islets were incubated at normoxic or hypoxic conditions and with or without hydrogen peroxide and a nitric oxide donor. Insulin mRNA was determined by Northern hybridization. Islet homogenates were used for electrophoretic mobility shift assay with an RNA-oligonucleotide, corresponding to the pyrimidine-rich sequence of the 3'-UTR of rat insulin I mRNA. The expression of reporter gene mRNA, in islets transfected with reporter gene constructs containing the wild-type or mutated insulin mRNA pyrimidine-rich sequences, was measured by semiquantitive RT-PCR. RESULTS: Insulin mRNA was increased in response to hypoxia. This was paralleled by increased binding of the polypyrimidine tract-binding protein (PTB) to the pyrimidine-rich sequence of the 3'-UTR of insulin mRNA, which was counteracted by hydrogen peroxide. The reporter gene mRNA level containing the wild-type binding site was not increased in response to hypoxia, but mutation of the site resulted in a destabilization of the mRNA. CONCLUSIONS: The complete understanding of different diabetic conditions requires the elucidation of mechanisms that control insulin gene expression. Our data show that hypoxia may increase insulin mRNA levels by promoting the binding of PTB to the insulin mRNA 3'-UTR. Hydrogen peroxide abolishes the hypoxic effect indicating involvement of reactive oxygen species and/or the redox potential in the oxygen-signaling pathway. PMID:12359957

  7. Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts

    PubMed Central

    1993-01-01

    Granulation tissue fibroblasts (myofibroblasts) develop several ultrastructural and biochemical features of smooth muscle (SM) cells, including the presence of microfilament bundles and the expression of alpha-SM actin, the actin isoform typical of vascular SM cells. Myofibroblasts have been proposed to play a role in wound contraction and in retractile phenomena observed during fibrotic diseases. We show here that the subcutaneous administration of transforming growth factor- beta 1 (TGF beta 1) to rats results in the formation of a granulation tissue in which alpha-SM actin expressing myofibroblasts are particularly abundant. Other cytokines and growth factors, such as platelet-derived growth factor and tumor necrosis factor-alpha, despite their profibrotic activity, do not induce alpha-SM actin in myofibroblasts. In situ hybridization with an alpha-SM actin probe shows a high level of alpha-SM actin mRNA expression in myofibroblasts of TGF beta 1-induced granulation tissue. Moreover, TGF beta 1 induces alpha-SM actin protein and mRNA expression in growing and quiescent cultured fibroblasts and preincubation of culture medium containing whole blood serum with neutralizing antibodies to TGF beta 1 results in a decrease of alpha-SM actin expression by fibroblasts in replicative and non-replicative conditions. These results suggest that TGF beta 1 plays an important role in myofibroblast differentiation during wound healing and fibrocontractive diseases by regulating the expression of alpha-SM actin in these cells. PMID:8314838

  8. The nonlinear dynamics and fluctuations of mRNA levels in cross-talking pathway activated transcription.

    PubMed

    Yu, Jianshe; Sun, Qiwen; Tang, Moxun

    2014-12-21

    Gene transcription is a stochastic process, and is often activated by multiple signal transduction pathways. In this work, we study gene transcription activated randomly by two cross-talking pathways, with the messenger RNA (mRNA) molecules being produced in a simple birth and death process. We derive the analytical formulas for the mean and the second moment of mRNA copy numbers and characterize the nature of transcription noise. We find that the stationary noise strength Φ is close to its baseline limit 1 when the mRNA level is high due to strong activation or stable transcription, or the mRNA level is low due to unstable transcription or ineffective mRNA production. If Φ stays well above 1, then the gene is infrequently active but mRNAs are accumulated rapidly once it is active. In this case, the system generates a transcriptional bursting, and the mean mRNA level peaks at a finite time. By examining the nonlinear dependance of Φ on transcriptional efficiency, we show that the maximum noise strength is attained only when the gene is silent in the majority of cells as observed in recent experiments. By comparing the current findings with our previous results in sequential pathway model, we come up with a profound conclusion that parallel, cross-talking pathways tend to increase transcription noise, whereas sequential pathways tend to reduce transcription noise. A further study on gene transcription activated by entangling pathways may help us reveal the subtle connection between the characteristics of transcription noise and the topology of genetic network.

  9. Effects of selenium-enriched probiotics on heat shock protein mRNA levels in piglet under heat stress conditions.

    PubMed

    Gan, Fang; Ren, Fei; Chen, Xingxiang; Lv, Chenhui; Pan, Cuiling; Ye, Gengping; Shi, Jun; Shi, Xiuli; Zhou, Hong; Shituleni, Shituleni Andreas; Huang, Kehe

    2013-03-13

    The effects of selenium-enriched probiotics (SP) on tissue selenium (Se) deposition, glutathione peroxidase-1 (GPx1) activity and mRNA level, and heat shock protein (Hsp) mRNA levels of piglets under heat stress conditions were investigated. A total of 48 crossbred ([Landrace × Yorkshire] × Duroc) piglets were randomly divided into 4 groups and fed a basal diet (Con, 0.16 mg Se/kg) or basal diets with added probiotics (P, 0.16 mg Se/kg), sodium selenite (SS, 0.46 mg Se/kg), or SP (0.46 mg Se/kg), respectively, for 42 days. Three piglets were randomly selected from each group for blood sample collection at days 0, 14, 28, and 42 and for liver, kidney, and spleen sample collection at day 42. The results showed that P, SS, and SP could significantly down-regulate the average mRNA levels of Hsp70 (17.3, 23.7, and 40.1%) and Hsp27 (22.4, 24.4, and 44.7%) of the tissues, respectively (P < 0.05), whereas SS and SP could significantly elevate Se concentration, GPx1 activity and mRNA level (P < 0.05). The maximal effects of these parameters were observed in SP. It was concluded that SP is a feasible dietary supplementation of piglets under heat stress conditions during the summer season.

  10. 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.

  11. Elevated Glucose Levels Promote Contractile and Cytoskeletal Gene Expression in Vascular Smooth Muscle via Rho/Protein Kinase C and Actin Polymerization*

    PubMed Central

    Hien, Tran Thi; Turczyńska, Karolina M.; Dahan, Diana; Ekman, Mari; Grossi, Mario; Sjögren, Johan; Nilsson, Johan; Braun, Thomas; Boettger, Thomas; Garcia-Vaz, Eliana; Stenkula, Karin; Swärd, Karl; Gomez, Maria F.; Albinsson, Sebastian

    2016-01-01

    Both type 1 and type 2 diabetes are associated with increased risk of cardiovascular disease. This is in part attributed to the effects of hyperglycemia on vascular endothelial and smooth muscle cells, but the underlying mechanisms are not fully understood. In diabetic animal models, hyperglycemia results in hypercontractility of vascular smooth muscle possibly due to increased activation of Rho-kinase. The aim of the present study was to investigate the regulation of contractile smooth muscle markers by glucose and to determine the signaling pathways that are activated by hyperglycemia in smooth muscle cells. Microarray, quantitative PCR, and Western blot analyses revealed that both mRNA and protein expression of contractile smooth muscle markers were increased in isolated smooth muscle cells cultured under high compared with low glucose conditions. This effect was also observed in hyperglycemic Akita mice and in diabetic patients. Elevated glucose activated the protein kinase C and Rho/Rho-kinase signaling pathways and stimulated actin polymerization. Glucose-induced expression of contractile smooth muscle markers in cultured cells could be partially or completely repressed by inhibitors of advanced glycation end products, L-type calcium channels, protein kinase C, Rho-kinase, actin polymerization, and myocardin-related transcription factors. Furthermore, genetic ablation of the miR-143/145 cluster prevented the effects of glucose on smooth muscle marker expression. In conclusion, these data demonstrate a possible link between hyperglycemia and vascular disease states associated with smooth muscle contractility. PMID:26683376

  12. Activity and mRNA Levels of Enzymes Involved in Hepatic Fatty Acid Synthesis in Rats Fed Naringenin.

    PubMed

    Hashimoto, Toru; Ide, Takashi

    2015-11-04

    We investigated the physiological activity of naringenin in affecting hepatic lipogenesis and serum and liver lipid levels in rats. Rats were fed diets containing 0, 1, or 2.5 g/kg naringenin for 15 d. Naringenin at a dietary level of 2.5 g/kg significantly decreased the activities and the mRNA levels of various lipogenic enzymes and sterol regulatory element binding protein-1c (SREBP-1c) mRNA level. The activities and the mRNA levels were also 9-22% and 12-38% lower, respectively, in rats fed a 1 g/kg naringenin diet than in the animals fed a naringenin-free diet, although the differences were not significant in many cases. Naringenin at 2.5 g/kg significantly lowered serum triacylglycerol, cholesterol, and phospholipid and hepatic triacylglycerol and cholesterol. This flavonoid at 1.0 g/kg also significantly lowered these parameters except for serum triacylglycerol. Naringenin levels in serum and liver dose-dependently increased, and hepatic concentrations reached levels that can affect various signaling pathways.

  13. Increased glutamate decarboxylase mRNA levels in the striatum and pallidum of MPTP-treated primates.

    PubMed

    Soghomonian, J J; Pedneault, S; Audet, G; Parent, A

    1994-10-01

    The mRNA levels encoding for the enzyme glutamate decarboxylase (GAD67) were measured by computerized image analysis after in situ hybridization histochemistry and radioautography in the striatum and pallidum of normal squirrel monkeys (Saimiri sciureus), or after treatment with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). All MPTP-injected monkeys exhibited profound motor deficits including akinesia. The dopaminergic innervation, as visualized and quantified on x-ray films after 3H-mazindol binding on tissue sections, was uniformly lost throughout the striatum of MPTP-treated monkeys. Brain sections processed with a probe synthesized from a feline or human GAD67 cDNA exhibited intense radioautographic labeling throughout the striatum. When measured on x-ray films, the intensity of GAD67 mRNA labeling was increased in the striatum of MPTP-treated versus control monkeys. Increased labeling reached statistical significance in the dorsolateral sector of the rostral putamen and throughout the putamen and the caudate at the caudal, postcommissural, level. Analysis of emulsion radioautographs demonstrated that the increase in GAD67 mRNA labeling in MPTP-treated monkeys occurred in individual neurons of the striatum. In the external and internal segments of the pallidum, numerous neurons labeled with the GAD67 cRNA probe were visualized on emulsion radioautographs. The intensity of GAD67 mRNA labeling in single neurons of both pallidal segments was increased in MPTP-treated versus control monkeys. Construction of the histograms of frequency distribution of labeling indicated that this increase occurred in a majority of labeled neurons. The present study demonstrates that GAD67 mRNA levels are significantly altered in the striatum and pallidum of parkinsonian monkeys. The preferential increase of GAD67 mRNA labeling in the dorsolateral putamen, which receives afferents from the sensorimotor cortex, provides further evidence of the involvement of

  14. Influence of dietary lipids on hepatic mRNA levels of proteins regulating plasma lipoproteins in baboons with high and low levels of large high density lipoproteins.

    PubMed

    Kushwaha, R S; McMahan, C A; Mott, G E; Carey, K D; Reardon, C A; Getz, G S; McGill, H C

    1991-12-01

    Selective breeding of baboons has produced families with increased plasma levels of large high density lipoproteins (HDL1) and very low (VLDL) and low (LDL) density lipoproteins when the animals consume a diet enriched in cholesterol and saturated fat. High HDL1 baboons have a slower cholesteryl ester transfer, which may account for the accumulation of HDL1, but not of VLDL and LDL. To investigate the mechanism of accumulation of VLDL + LDL in plasma of the high HDL1 phenotype, we selected eight half-sib pairs of baboons, one member of each pair with high HDL1, the other member with little or no HDL1 on the same high cholesterol, saturated fat diet. Baboons were fed a chow diet and four experimental diets consisting of high and low cholesterol with corn oil, and high and low cholesterol with lard, each for 6 weeks, in a crossover design. Plasma lipids and lipoproteins and hepatic mRNA levels were measured on each diet. HDL1 phenotype, type of dietary fat, and dietary cholesterol affected plasma cholesterol and apolipoprotein (apo) B concentrations, whereas dietary fat alone affected plasma triglyceride and apoA-I concentrations. HDL1 phenotype and dietary cholesterol alone did not influence hepatic mRNA levels, whereas dietary lard, compared to corn oil, significantly increased hepatic apoE mRNA levels and decreased hepatic LDL receptor and HMG-CoA synthase mRNA levels. Hepatic apoA-I message was associated with cholesterol concentration in HDL fractions as well as with apoA-I concentrations in the plasma or HDL. However, hepatic apoB message level was not associated with plasma or LDL apoB levels. Total plasma cholesterol, including HDL, was negatively associated with hepatic LDL receptor and HMG-CoA synthase mRNA levels. However, compared with low HDL1 baboons, high HDL1 baboons had higher concentrations of LDL and HDL cholesterol at the same hepatic mRNA levels. These studies suggest that neither overproduction of apoB from the liver nor decreased hepatic LDL

  15. Changes in actin and tubulin expression in osteogenic cells cultured on bioactive glass-based surfaces.

    PubMed

    Martins, Carolina Scanavez; Ferraz, Emanuela Prado; De Castro-Raucci, Larissa Moreira Spinola; Teixeira, Lucas Novaes; Maximiano, William Marcatti Amarú; Rosa, Adalberto Luiz; De Oliveira, Paulo Tambasco

    2015-11-01

    The present study evaluated whether the changes in the labeling pattern of cytoskeletal proteins in osteogenic cells cultured on bioactive glass-based materials are due to altered mRNA and protein levels. Primary rat-derived osteogenic cells were plated on Bioglass® 45S5, Biosilicate®, and borosilicate (bioinert control). The following parameters were assayed: (i) qualitative epifluorescence analysis of actin and tubulin; (ii) quantitative mRNA and protein expression for actin and tubulin by real-time PCR and ELISA, respectively, and (iii) qualitative analysis of cell morphology by scanning electron microscopy (SEM). At days 3 and 7, the cells grown on borosilicate showed typical actin and tubulin labeling patterns, whereas those on the bioactive materials showed roundish areas devoid of fluorescence signals. The cultures grown on bioactive materials showed significant changes in actin and tubulin mRNA expression that were not reflected in the corresponding protein levels. A positive correlation between the mRNA and protein as well as an association between epifluorescence imaging and quantitative data were only detected for the borosilicate. SEM imaging of the cultures on the bioactive surfaces revealed cells partly or totally coated with material aggregates, whose characteristics resembled the substrate topography. The culturing of osteogenic cells on Bioglass® 45S5 and Biosilicate® affect actin and tubulin mRNA expression but not the corresponding protein levels. Changes in the labeling pattern of these proteins should then be attributed, at least in part, to the presence of a physical barrier on the cell surface as a result of the material surface reactions, thus limiting fluorescence signals.

  16. Effects of seawater acclimation on mRNA levels of corticosteroid receptor genes in osmoregulatory and immune systems in trout

    USGS Publications Warehouse

    Yada, T.; Hyodo, S.; Schreck, C.B.

    2008-01-01

    Influence of environmental salinity on expression of distinct corticosteroid receptor (CR) genes, glucocorticoid receptor (GR)-1 and -2, and mineralcorticoid receptor (MR), was examined in osmoregulatory and hemopoietic organs and leucocytes of steelhead trout (Oncorhynchus mykiss). There was no significant difference in plasma cortisol levels between freshwater (FW)- or seawater (SW)-acclimated trout, whereas Na+, K+-ATPase was activated in gill of SW fish. Plasma lysozyme levels also showed a significant increase after acclimation to SW. In SW-acclimated fish, mRNA levels of GR-1, GR-2, and MR were significantly higher in gill and body kidney than those in FW. Head kidney and spleen showed no significant change in these CR mRNA levels after SW-acclimation. On the other hand, leucocytes isolated from head kidney and peripheral blood showed significant decreases in mRNA levels of CR in SW-acclimated fish. These results showed differential regulation of gene expression of CR between osmoregulatory and immune systems. ?? 2008 Elsevier Inc. All rights reserved.

  17. Induction of the SHARP-2 mRNA level by insulin is mediated by multiple signaling pathways.

    PubMed

    Kanai, Yukiko; Asano, Kosuke; Komatsu, Yoshiko; Takagi, Katsuhiro; Ono, Moe; Tanaka, Takashi; Tomita, Koji; Haneishi, Ayumi; Tsukada, Akiko; Yamada, Kazuya

    2017-02-01

    The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is an insulin-inducible transcription factor which represses transcription of the rat phosphoenolpyruvate carboxykinase gene. In this study, a regulatory mechanism of the SHARP-2 mRNA level by insulin was analyzed. Insulin rapidly induced the level of SHARP-2 mRNA. This induction was blocked by inhibitors for phosphoinositide 3-kinase (PI 3-K), protein kinase C (PKC), and mammalian target of rapamycin (mTOR), actinomycin D, and cycloheximide. Whereas an adenovirus infection expressing a dominant negative form of atypical PKC lambda (aPKCλ) blocked the insulin-induction of the SHARP-2 mRNA level, insulin rapidly activated the mTOR. Insulin did not enhance transcriptional activity from a 3.7 kb upstream region of the rat SHARP-2 gene. Thus, we conclude that insulin induces the expression of the rat SHARP-2 gene at the transcription level via both a PI 3-K/aPKCλ- and a PI 3-K/mTOR- pathways and that protein synthesis is required for this induction.

  18. Wastewater treatment plant effluent alters pituitary gland gonadotropin mRNA levels in juvenile coho salmon (Oncorhynchus kisutch).

    PubMed

    Harding, Louisa B; Schultz, Irvin R; da Silva, Denis A M; Ylitalo, Gina M; Ragsdale, Dave; Harris, Stephanie I; Bailey, Stephanie; Pepich, Barry V; Swanson, Penny

    2016-09-01

    It is well known that endocrine disrupting compounds (EDCs) present in wastewater treatment plant (WWTP) effluents interfere with reproduction in fish, including altered gonad development and induction of vitellogenin (Vtg), a female-specific egg yolk protein precursor produced in the liver. As a result, studies have focused on the effects of EDC exposure on the gonad and liver. However, impacts of environmental EDC exposure at higher levels of the hypothalamic-pituitary-gonad axis are less well understood. The pituitary gonadotropins, follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) are involved in all aspects of gonad development and are subject to feedback from gonadal steroids making them a likely target of endocrine disruption. In this study, the effects of WWTP effluent exposure on pituitary gonadotropin mRNA expression were investigated to assess the utility of Lh beta-subunit (lhb) as a biomarker of estrogen exposure in juvenile coho salmon (Oncorhynchus kisutch). First, a controlled 72-h exposure to 17α-ethynylestradiol (EE2) and 17β-trenbolone (TREN) was performed to evaluate the response of juvenile coho salmon to EDC exposure. Second, juvenile coho salmon were exposed to 0, 20 or 100% effluent from eight WWTPs from the Puget Sound, WA region for 72h. Juvenile coho salmon exposed to 2 and 10ng EE2L(-1) had 17-fold and 215-fold higher lhb mRNA levels relative to control fish. Hepatic vtg mRNA levels were dramatically increased 6670-fold, but only in response to 10ng EE2L(-1) and Fsh beta-subunit (fshb) mRNA levels were not altered by any of the treatments. In the WWTP effluent exposures, lhb mRNA levels were significantly elevated in fish exposed to five of the WWTP effluents. In contrast, transcript levels of vtg were not affected by any of the WWTP effluent exposures. Mean levels of natural and synthetic estrogens in fish bile were consistent with pituitary lhb expression, suggesting that the observed lhb induction may be due to

  19. Inverse relationship between estrogen receptor and epidermal growth factor receptor mRNA levels in human breast cancer cell lines.

    PubMed

    Lee, C S; Hall, R E; Alexander, I E; Koga, M; Shine, J; Sutherland, R L

    1990-01-01

    Epidermal growth factor receptors (EGF-R) are present in a number of human breast cancer cell lines and tumor biopsies. Furthermore, it has been suggested that EGF-R levels are higher in estrogen receptor negative (ER-) than in ER+ human breast tumors and that EGF-R status may be a prognostic indicator in breast cancer. The present study was undertaken to establish whether there is a quantitative relationship between EGF-R and ER mRNA concentrations in a series of 10 well-characterized human breast cancer cell lines. All cell lines expressed detectable quantities of EGF-R mRNA by Northern analysis but the relative abundance of EGF-R mRNA varied more than 50-fold. Two transcripts corresponding to the 10.5- and 5.8-kb mRNAs described in other cell types were present but in different relative proportions in different cell lines. When these lines were divided into an ER+ and an ER- group based on their ability to bind estradiol, ER- cell lines were shown to express significantly higher concentrations of EGF-R mRNA than did ER+ cell lines (p less than 0.005). Furthermore, linear-regression analysis revealed a significant inverse relationship between ER and EGF-R mRNA concentrations both within the group of 10 human breast cancer cell lines as a whole (r = 0.66) and within the 6 functionally ER + lines (r = 0.77). This demonstration of a significant (p less than 0.005) inverse relationship between the concentrations of ER and EGF-R mRNAs in ER + cell lines raises the possibility of reciprocal regulation of the expression of these genes in human breast cancer.

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

    SciTech Connect

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

    2015-04-03

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

  1. Rat uterine oxytocin receptor and estrogen receptor α and β mRNA levels are regulated by estrogen through multiple estrogen receptors.

    PubMed

    Murata, Takuya; Narita, Kazumi; Ichimaru, Toru

    2014-03-07

    Estrogen action is mediated through several types of receptors (ERs), such as ERα, ERβ and putative membrane ERs. Oxytocin receptor (OTR) and ER expression levels in the rat uterus are regulated by estrogen; however, which types of ERs are involved has not been elucidated. This study examined OTR, ERα and ERβ levels in ovariectomized rats treated with 17β-estradiol (E2), an ERα agonist (PPT), an ERβ agonist (DPN) or estren (Es). E2 and PPT increased OTR mRNA levels and decreased ERα and ERβ mRNA levels 3 and 6 h posttreatment. DPN decreased ERα and ERβ mRNA levels at 3 and 6 h, while OTR mRNA levels increased at 3 h and decreased at 6 h. OTR mRNA levels increased 3 h after the Es treatment and then declined until 6 h. ERα and ERβ mRNA levels decreased by 3 h and remained low until 6 h posttreatment with Es. The ER antagonist ICI182,780 (ICI) suppressed the increases in OTR mRNA levels induced 3 h after the Es treatment. However, ICI and tamoxifen (Tam) had no significant effect on ERα and ERβ mRNA levels in the Es-treated or vehicle-treated group. In intact rats, proestrus-associated increases in OTR mRNA levels were antagonized by both ICI and Tam. However, decreases in ERα and ERβ mRNA levels were not antagonized by Tam and ICI, respectively. Therefore, uterine OTR gene expression is upregulated by estrogen through the classical nuclear (or non-nuclear) ERs, ERα and ERβ, while the levels of these ERs are downregulated by estrogen through multiple pathways including Es-sensitive nonclassical ERs.

  2. Frameshift mutations in the v-src gene of avian sarcoma virus act in cis to specifically reduce v-src mRNA levels.

    PubMed Central

    Simpson, S B; Stoltzfus, C M

    1994-01-01

    A portion of the avian sarcoma virus (ASV) primary RNA transcripts is alternatively spliced in chicken embryo fibroblast cells to two different messages, the src and env mRNAs. Frameshift mutations of the viral genome causing premature translation termination within the src gene result in a decreased steady-state level of the src mRNA. In marked contrast, frameshift mutations at various positions of the env gene do not decrease the level of the env mRNA. We show that the src gene product is not required in trans for splicing and accumulation of src mRNA. Conversely, the truncated Src proteins do not act negatively in trans to decrease specifically the levels of src mRNA. Taken together, these results indicate that the frameshift mutations act in cis to reduce src mRNA levels. A double mutant with a lesion in the src initiator AUG and a frameshift within the src gene demonstrated wild-type RNA levels, indicating that the src mRNA must be recognized as a translatable mRNA for the effect on src mRNA levels to occur. Our results indicate that the reduced levels do not result from decreased cytoplasmic stability of the mature src mRNA. We also show that the src gene frameshift mutations affect src mRNA levels when expressed from intronless src cDNA clones. We conclude that the reduction of src mRNA levels triggered by the presence of frameshift mutations within the src gene occurs while it is associated with the nucleus. Our data also strongly suggest that this occurs at a step of RNA processing or transport independent of RNA splicing. Images PMID:8114716

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

    PubMed

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

    2002-03-01

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

  4. Voluntary exercise and clomipramine treatment elevate prepro-galanin mRNA levels in the locus coeruleus in rats.

    PubMed

    Holmes, Philip V; Yoo, Ho Sang; Dishman, Rod K

    2006-11-06

    Exercise exerts antidepressant effects in humans and rodent models of affective disorders. These effects may be mediated by the upregulation of endogenous factors that exert antidepressant actions. The physiological functions and behavioral actions of the neuropeptide galanin (GAL) suggest antidepressant activity. Previous studies have shown that various modes of exercise elevate GAL gene expression in the locus coeruleus (LC) in rats. The present experiments examined the interaction between voluntary exercise and antidepressant pharmacotherapy. Male Sprague-Dawley rats were provided access to activity wheels (exercise condition) or inoperative wheels (sedentary condition) for 28 days. Rats in each group were injected with clomipramine (10mg/kg/day) or vehicle throughout this period (for 3 weeks). Prepro-GAL mRNA in the LC was measured by in situ hybridization histochemistry. Exercise and clomipramine treatment significantly elevated GAL gene expression, though prepro-GAL mRNA levels in rats receiving both interventions did not differ from sedentary controls that received vehicle. Prepro-GAL mRNA levels were significantly correlated with running distance. The results further implicate a role for GAL in the antidepressant effects of exercise and pharmacotherapy, though the mechanisms through which these treatments influence GAL gene expression appear to differ significantly.

  5. Acetylcholine receptor-inducing factor from chicken brain increases the level of mRNA encoding the receptor. alpha. subunit

    SciTech Connect

    Harris, D.A.; Falls, D.L.; Dill-Devor, R.M.; Fischbach, G.D. )

    1988-03-01

    A 42-kDa glycoprotein isolated from chicken brain, referred to as acetylcholine receptor-inducing activity (ARIA), that stimulates the rate of incorporation of acetylcholine receptors into the surface of chicken myotubes may play a role in the nerve-induced accumulation of receptors at developing neuromuscular synapses. Using nuclease-protection assays, the authors have found that ARIA causes a 2- to 16-fold increase in the level of mRNA encoding the {alpha} subunit of the receptor, with little or no change in the levels of {gamma}- and {delta}-subunit messengers. ARIA also increases the amount of a putative nuclear precursor of {alpha}-subunit mRNA, consistent with an activation of gene transcription. These results suggest that the concentration of {alpha} subunit may limit the rate of biosynthesis of the acetylcholine receptors in chicken myotubes. They also indicate that neuronal factors can regulate the expression of receptor subunit genes in a selective manner. Tetrodotoxin, 8-bromo-cAMP, and forskolin also increase the amount of {alpha}-subunit mRNA, with little change in the amount of {gamma}- and {delta}-subunit mRNAs. Unlike ARIA, however, these agents have little effect on the concentration of the {alpha}-subunit nuclear precursor.

  6. A Nascent Peptide Signal Responsive to Endogenous Levels of Polyamines Acts to Stimulate Regulatory Frameshifting on Antizyme mRNA.

    PubMed

    Yordanova, Martina M; Wu, Cheng; Andreev, Dmitry E; Sachs, Matthew S; Atkins, John F

    2015-07-17

    The protein antizyme is a negative regulator of cellular polyamine concentrations from yeast to mammals. Synthesis of functional antizyme requires programmed +1 ribosomal frameshifting at the 3' end of the first of two partially overlapping ORFs. The frameshift is the sensor and effector in an autoregulatory circuit. Except for Saccharomyces cerevisiae antizyme mRNA, the frameshift site alone only supports low levels of frameshifting. The high levels usually observed depend on the presence of cis-acting stimulatory elements located 5' and 3' of the frameshift site. Antizyme genes from different evolutionary branches have evolved different stimulatory elements. Prior and new multiple alignments of fungal antizyme mRNA sequences from the Agaricomycetes class of Basidiomycota show a distinct pattern of conservation 5' of the frameshift site consistent with a function at the amino acid level. As shown here when tested in Schizosaccharomyces pombe and mammalian HEK293T cells, the 5' part of this conserved sequence acts at the nascent peptide level to stimulate the frameshifting, without involving stalling detectable by toe-printing. However, the peptide is only part of the signal. The 3' part of the stimulator functions largely independently and acts at least mostly at the nucleotide level. When polyamine levels were varied, the stimulatory effect was seen to be especially responsive in the endogenous polyamine concentration range, and this effect may be more general. A conserved RNA secondary structure 3' of the frameshift site has weaker stimulatory and polyamine sensitizing effects on frameshifting.

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

    SciTech Connect

    Tin-Tin-Win-Shwe Mitsushima, Dai; Yamamoto, Shoji; Fukushima, Atsushi; Funabashi, Toshiya; Kobayashi, Takahiro; Fujimaki, Hidekazu

    2008-01-15

    Recently, there have been increasing reports that nano-sized component of particulate matter can reach the brain and may be associated with neurodegenerative diseases. Previously, our laboratory has studied the effect of intranasal instillation of nano-sized carbon black (CB) (14 nm and 95 nm) on brain cytokine and chemokine mRNA expressions and found that 14-nm CB increased IL-1{beta}, TNF-{alpha}, CCL2 and CCL3 mRNA expressions in the olfactory bulb, not in the hippocampus of mice. To investigate the effect of a single administration of nanoparticles on neurotransmitters and proinflammatory cytokines in a mouse olfactory bulb, we performed in vivo microdialysis and real-time PCR methods. Ten-week-old male BALB/c mice were implanted with guide cannula in the right olfactory bulb and, 1 week later, were instilled vehicle or CB (14 nm, 250 {mu}g) intranasally. Six hours after the nanoparticle instillation, the mice were intraperitoneally injected with normal saline or 50 {mu}g of bacteria cell wall component lipoteichoic acid (LTA), which may potentiate CB-induced neurologic effect. Extracellular glutamate and glycine levels were significantly increased in the olfactory bulb of CB-instilled mice when compared with vehicle-instilled control mice. Moreover, we found that LTA further increased glutamate and glycine levels. However, no alteration of taurine and GABA levels was observed in the olfactory bulb of the same mice. We also detected immunological changes in the olfactory bulb 11 h after vehicle or CB instillation and found that IL-1{beta} mRNA expression was significantly increased in CB- and LTA-treated mice when compared with control group. However, TNF-{alpha} mRNA expression was increased significantly in CB- and saline-treated mice when compared with control group. These findings suggest that nanoparticle CB may modulate the extracellular amino acid neurotransmitter levels and proinflammatory cytokine IL-1 {beta} mRNA expressions synergistically with LTA

  8. The arabidopsis ACT7 actin gene is expressed in rapidly developing tissues and responds to several external stimuli.

    PubMed Central

    McDowell, J M; An, Y Q; Huang, S; McKinney, E C; Meagher, R B

    1996-01-01

    ACT7 encodes one of the six distinct and ancient subclasses of actin protein in the complex Arabidopsis actin gene family. We determined the sequence and structure of the Arabidopsis thaliana ACT7 actin gene and investigated its tissue-specific expression and regulation. The ACT7 mRNA levels varied by 128-fold among several different tissues and organs. The highest levels of aCT7 mRNA were found in rapidly expanding vegetative organs, the lowest in pollen. A translational fusion with the 5' end of ACT 7 (1.9 kb) joined to the beta-glucuronidase reporter gene was strongly and preferentially expressed in all young, developing vegetative tissues of transgenic Arabidopsis plants. ACT7 was the only Arabidopsis actin gene strongly expressed in the hypocotyl and seed coat. Although no beta-glucuronidase expression was seen in developing ovules or immature seeds, strong expression was seen in dry seeds and immediately after imbibition in the entire seedling. ACT7 was the only Arabidopsis actin gene to respond strongly to auxin, other hormone treatments, light regime, and wounding, and may be the primary actin gene responding to external stimuli. The ACT7 promoter sequence contains a remarkable number of motifs with sequence similarity to putative phytohormone response elements. PMID:8754679

  9. Actinic Prurigo.

    PubMed

    Rodríguez-Carreón, Alma Angélica; Rodríguez-Lobato, Erika; Rodríguez-Gutiérrez, Georgina; Cuevas-González, Juan Carlos; Mancheno-Valencia, Alexandra; Solís-Arias, Martha Patricia; Vega-Memije, María Elisa; Hojyo-Tomoka, María Teresa; Domínguez-Soto, Luciano

    2015-01-01

    Actinic prurigo is an idiopathic photodermatosis that affects the skin, as well as the labial and conjunctival mucosa in indigenous and mestizo populations of Latin America. It starts predominantly in childhood, has a chronic course, and is exacerbated with solar exposure. Little is known of its pathophysiology, including the known mechanisms of the participation of HLA-DR4 and an abnormal immunologic response with increase of T CD4+ lymphocytes. The presence of IgE, eosinophils, and mast cells suggests that it is a hypersensitivity reaction (likely type IVa or b). The diagnosis is clinical, and the presence of lymphoid follicles in the mucosal histopathologic study of mucosa is pathognomonic. The best available treatment to date is thalidomide, despite its secondary effects.

  10. Nonsense-mediated mRNA decay modulates immune receptor levels to regulate plant antibacterial defense.

    PubMed

    Gloggnitzer, Jiradet; Akimcheva, Svetlana; Srinivasan, Arunkumar; Kusenda, Branislav; Riehs, Nina; Stampfl, Hansjörg; Bautor, Jaqueline; Dekrout, Bettina; Jonak, Claudia; Jiménez-Gómez, José M; Parker, Jane E; Riha, Karel

    2014-09-10

    Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic RNA surveillance mechanism that degrades aberrant mRNAs. NMD impairment in Arabidopsis is linked to constitutive immune response activation and enhanced antibacterial resistance, but the underlying mechanisms are unknown. Here we show that NMD contributes to innate immunity in Arabidopsis by controlling the turnover of numerous TIR domain-containing, nucleotide-binding, leucine-rich repeat (TNL) immune receptor-encoding mRNAs. Autoimmunity resulting from NMD impairment depends on TNL signaling pathway components and can be triggered through deregulation of a single TNL gene, RPS6. Bacterial infection of plants causes host-programmed inhibition of NMD, leading to stabilization of NMD-regulated TNL transcripts. Conversely, constitutive NMD activity prevents TNL stabilization and impairs plant defense, demonstrating that host-regulated NMD contributes to disease resistance. Thus, NMD shapes plant innate immunity by controlling the threshold for activation of TNL resistance pathways.

  11. TCF4 sequence variants and mRNA levels are associated with neurodevelopmental characteristics in psychotic disorders.

    PubMed

    Wirgenes, K V; Sønderby, I E; Haukvik, U K; Mattingsdal, M; Tesli, M; Athanasiu, L; Sundet, K; Røssberg, J I; Dale, A M; Brown, A A; Agartz, I; Melle, I; Djurovic, S; Andreassen, O A

    2012-05-08

    TCF4 is involved in neurodevelopment, and intergenic and intronic variants in or close to the TCF4 gene have been associated with susceptibility to schizophrenia. However, the functional role of TCF4 at the level of gene expression and relationship to severity of core psychotic phenotypes are not known. TCF4 mRNA expression level in peripheral blood was determined in a large sample of patients with psychosis spectrum disorders (n = 596) and healthy controls (n = 385). The previously identified TCF4 risk variants (rs12966547 (G), rs9960767 (C), rs4309482 (A), rs2958182 (T) and rs17512836 (C)) were tested for association with characteristic psychosis phenotypes, including neurocognitive traits, psychotic symptoms and structural magnetic resonance imaging brain morphometric measures, using a linear regression model. Further, we explored the association of additional 59 single nucleotide polymorphisms (SNPs) covering the TCF4 gene to these phenotypes. The rs12966547 and rs4309482 risk variants were associated with poorer verbal fluency in the total sample. There were significant associations of other TCF4 SNPs with negative symptoms, verbal learning, executive functioning and age at onset in psychotic patients and brain abnormalities in total sample. The TCF4 mRNA expression level was significantly increased in psychosis patients compared with controls and positively correlated with positive- and negative-symptom levels. The increase in TCF4 mRNA expression level in psychosis patients and the association of TCF4 SNPs with core psychotic phenotypes across clinical, cognitive and brain morphological domains support that common TCF4 variants are involved in psychosis pathology, probably related to abnormal neurodevelopment.

  12. C3a Increases VEGF and Decreases PEDF mRNA Levels in Human Retinal Pigment Epithelial Cells.

    PubMed

    Long, Qin; Cao, Xiaoguang; Bian, Ailing; Li, Ying

    2016-01-01

    Complement activation, specifically complement 3 (C3) activation and C3a generation, contributes to an imbalance between angiogenic stimulation by vascular endothelial growth factor (VEGF) and angiogenic inhibition by pigment epithelial derived factor (PEDF), leading to pathological angiogenesis. This study aimed to investigate the effects of C3a and small interfering RNA (siRNA) targeting C3 on the levels of VEGF and PEDF mRNAs in human retinal pigment epithelial (RPE) cells. ARPE-19 cells were cultured in the presence of exogenous C3a at 0.1 μM and 0.3 μM C3a for 24, 48, and 72 hours. 0.1 pmol/μL duplexes of siRNA targeting C3 were applied for C3a inhibition by transfecting ARPE-19 cells for 48 hours. RT-PCR was performed to examine the level of VEGF and PEDF mRNA. A random siRNA duplex was set for control siRNA. Results demonstrated that exogenous C3a significantly upregulated VEGF and downregulated PEDF mRNA levels in cultured ARPE-19 cells, and siRNA targeting C3 transfection reversed the above changes, significantly reducing VEGF and enhancing PEDF mRNAs level in ARPE-19 cells compared to the control. The present data provided evidence that reducing C3 activation can decreases VEGF and increase PEDF mRNA level in RPE and may serve as a potential therapy in pathological angiogenesis.

  13. C3a Increases VEGF and Decreases PEDF mRNA Levels in Human Retinal Pigment Epithelial Cells

    PubMed Central

    Long, Qin; Cao, Xiaoguang; Bian, Ailing

    2016-01-01

    Complement activation, specifically complement 3 (C3) activation and C3a generation, contributes to an imbalance between angiogenic stimulation by vascular endothelial growth factor (VEGF) and angiogenic inhibition by pigment epithelial derived factor (PEDF), leading to pathological angiogenesis. This study aimed to investigate the effects of C3a and small interfering RNA (siRNA) targeting C3 on the levels of VEGF and PEDF mRNAs in human retinal pigment epithelial (RPE) cells. ARPE-19 cells were cultured in the presence of exogenous C3a at 0.1 μM and 0.3 μM C3a for 24, 48, and 72 hours. 0.1 pmol/μL duplexes of siRNA targeting C3 were applied for C3a inhibition by transfecting ARPE-19 cells for 48 hours. RT-PCR was performed to examine the level of VEGF and PEDF mRNA. A random siRNA duplex was set for control siRNA. Results demonstrated that exogenous C3a significantly upregulated VEGF and downregulated PEDF mRNA levels in cultured ARPE-19 cells, and siRNA targeting C3 transfection reversed the above changes, significantly reducing VEGF and enhancing PEDF mRNAs level in ARPE-19 cells compared to the control. The present data provided evidence that reducing C3 activation can decreases VEGF and increase PEDF mRNA level in RPE and may serve as a potential therapy in pathological angiogenesis. PMID:27747237

  14. Amyloid precursor protein mRNA levels in the mononuclear blood cells of Alzheimer's and Down's patients.

    PubMed

    Buckland, P; Tidmarsh, S; Spurlock, G; Kaiser, F; Yates, M; O'Mahony, G; McGuffin, P

    1993-06-01

    Amyloid precursor protein (APP) is expressed by many non-neural tissues and it is possible that over-expression of the APP gene in non-neural tissue is responsible for the deposition of amyloid beta-protein in the brain and elsewhere. One possible source of beta-protein is circulating mononuclear blood cells which have previously been shown to express APP. To test this hypothesis, RNA was isolated from the mononuclear blood cells of patients suffering from Alzheimer's disease (n = 27), Down's syndrome (n = 13), senile dementia non-Alzheimer type (n = 14) and from normal individuals (n = 48). The relative abundance of mRNA coding for different splicing variants of the amyloid precursor protein (APP) mRNA was measured using multiprobe oligonucleotide solution hybridisation (MOSH). There was no significant difference in APP mRNA levels between any of the groups. This indicates that Alzheimer's disease is not characterised by an increase in production of APP in circulating mononuclear blood cells.

  15. Actin Dynamics: From Nanoscale to Microscale

    PubMed Central

    Carlsson, Anders E.

    2010-01-01

    The dynamic nature of actin in cells manifests itself in many ways: Polymerization near the cell edge is balanced by depolymerization in the interior, externally induced actin polymerization is followed by depolymerization, and spontaneous oscillations of the cell periphery are frequently seen. I discuss how mathematical modeling relates quantitative measures of actin dynamics to the rates of underlying molecular level processes. The rate of actin incorporation at the leading edge of a moving cell is roughly consistent with existing theories, and the factors determining the characteristic time of actin polymerization are fairly well understood. However, our understanding of actin disassembly is limited, in particular the interplay between severing and depolymerization and the role of specific combinations of proteins in implementing disassembly events. The origins of cell-edge oscillations, and their possible relation to actin waves, are a fruitful area of future research. PMID:20462375

  16. Overexpression of tau downregulated the mRNA levels of Kv channels and improved proliferation in N2A cells.

    PubMed

    Li, Xiantao; Hu, Ximu; Li, Xiaoqing; Hao, Xuran

    2015-01-01

    Microtubule binding protein tau has a crucial function in promoting the assembly and stabilization of microtubule. Besides tuning the action potentials, voltage-gated K+ channels (Kv) are important for cell proliferation and appear to play a role in the development of cancer. However, little is known about the possible interaction of tau with Kv channels in various tissues. In the present study, tau plasmids were transiently transfected into mouse neuroblastoma N2A cells to explore the possible linkages between tau and Kv channels. This treatment led to a downregulation of mRNA levels of several Kv channels, including Kv2.1, Kv3.1, Kv4.1, Kv9.2, and KCNH4, but no significant alteration was observed for Kv5.1 and KCNQ4. Furthermore, the macroscopic currents through Kv channels were reduced by 36.5% at +60 mV in tau-transfected N2A cells. The proliferation rates of N2A cells were also improved by the induction of tau expression and the incubation of TEA (tetraethylammonium) for 48 h by 120.9% and 149.3%, respectively. Following the cotransfection with tau in HEK293 cells, the mRNA levels and corresponding currents of Kv2.1 were significantly declined compared with single Kv2.1 transfection. Our data indicated that overexpression of tau declined the mRNA levels of Kv channels and related currents. The effects of tau overexpression on Kv channels provided an alternative explanation for low sensitivity to anti-cancer chemicals in some specific cancer tissues.

  17. Overexpression of Tau Downregulated the mRNA Levels of Kv Channels and Improved Proliferation in N2A Cells

    PubMed Central

    Li, Xiantao; Hu, Ximu; Li, Xiaoqing; Hao, Xuran

    2015-01-01

    Microtubule binding protein tau has a crucial function in promoting the assembly and stabilization of microtubule. Besides tuning the action potentials, voltage-gated K+ channels (Kv) are important for cell proliferation and appear to play a role in the development of cancer. However, little is known about the possible interaction of tau with Kv channels in various tissues. In the present study, tau plasmids were transiently transfected into mouse neuroblastoma N2A cells to explore the possible linkages between tau and Kv channels. This treatment led to a downregulation of mRNA levels of several Kv channels, including Kv2.1, Kv3.1, Kv4.1, Kv9.2, and KCNH4, but no significant alteration was observed for Kv5.1 and KCNQ4. Furthermore, the macroscopic currents through Kv channels were reduced by 36.5% at +60 mV in tau-tranfected N2A cells. The proliferation rates of N2A cells were also improved by the induction of tau expression and the incubation of TEA (tetraethylammonium) for 48 h by 120.9% and 149.3%, respectively. Following the cotransfection with tau in HEK293 cells, the mRNA levels and corresponding currents of Kv2.1 were significantly declined compared with single Kv2.1 transfection. Our data indicated that overexpression of tau declined the mRNA levels of Kv channels and related currents. The effects of tau overexpression on Kv channels provided an alternative explanation for low sensitivity to anti-cancer chemicals in some specific cancer tissues. PMID:25590133

  18. Impact of estrogen receptor gene polymorphisms and mRNA levels on obesity and lipolysis – a cohort study

    PubMed Central

    Nilsson, Maria; Dahlman, Ingrid; Jiao, Hong; Gustafsson, Jan-Åke; Arner, Peter; Dahlman-Wright, Karin

    2007-01-01

    Background The estrogen receptors α and β (ESR1, ESR2) have been implicated in adiposity, lipid metabolism and feeding behaviour. In this report we analyse ESR1 and ESR2 gene single nucleotide polymorphisms (SNPs) for association with obesity. We also relate adipose tissue ESR1 mRNA levels and ESR1 SNPs to adipocyte lipolysis and lipogenesis phenotypes. Methods 23 ESR1 and 11 ESR2 tag-SNPs, covering most of the common haplotype variation in each gene according to HAPMAP data, were analysed by Chi2 for association with obesity in a cohort comprising 705 adults with severe obesity and 402 lean individuals. Results were replicated in a cohort comprising 837 obese and 613 lean subjects. About 80% of both cohorts comprised women and 20% men. Adipose tissue ESR1 mRNA was quantified in 122 women and related to lipolysis and lipogenesis by multiple regression. ESR1 SNPs were analysed for association with adipocyte lipolysis and lipogenesis phenotypes in 204 obese women by simple regression. Results No ESR1 SNP was associated with obesity. Five ESR2 SNPs displayed nominal significant allelic association with obesity in women and one in men. The two ESR2 SNPs associated with obesity with nominal P value < 0.01 were genotyped in a second cohort where no association with obesity was observed. There was an inverse correlation between ESR1 mRNA levels in abdominal subcutaneous (sc) adipose tissue and basal lipolysis, as well as responsiveness to adrenoceptor agonists independent of age and BMI (P value 0.009–0.045). ESR1 rs532010 was associated with lipolytic sensitivity to noradrenaline (nominal P value 0.012), and ESR1 rs1884051 with responsiveness to the non-selective beta-adrenoceptor agonist isoprenaline (nominal P value 0.05). These associations became non-significant after Bonferroni correction. Conclusion ESR1 gene alleles are unlikely to be a major cause of obesity in women. A minor importance of ESR2 on severe obesity cannot be excluded. The inverse correlation

  19. Regional brain-derived neurotrophic factor mRNA and protein levels following transient forebrain ischemia in the rat.

    PubMed

    Kokaia, Z; Nawa, H; Uchino, H; Elmér, E; Kokaia, M; Carnahan, J; Smith, M L; Siesjö, B K; Lindvall, O

    1996-05-01

    Levels of BDNF mRNA and protein were measured in the rat brain using in situ hybridization and a two-site enzyme immunoassay. Under basal conditions, the highest BDNF concentration was found in the dentate gyrus (88 ng/g), while the levels in CA3 (50 ng/g), CA1 (18 ng/g) and parietal cortex (8 ng/g) were markedly lower. Following 10 min of forebrain ischemia, BDNF protein increased transiently in the dentate gyrus (to 124% of control at 6 h after the insult) and CA3 region (to 131% of control, at 1 week after the insult). In CA1 and parietal cortex, BDNF protein decreased to 73-75% of control at 24 h. In contrast, BDNF mRNA expression in dentate granule cells and CA3 pyramidal layer was transiently elevated to 287 and 293% of control, respectively, at 2 h, whereas no change was detected in CA1 or neocortex. The regional BDNF protein levels shown here correlate at least partly with regional differences in cellular resistance to ischemic damage, which is consistent with the hypothesis of a neuroprotective role of BDNF.

  20. Methylation of bone SOST, its mRNA, and serum sclerostin levels correlate strongly with fracture risk in postmenopausal women.

    PubMed

    Reppe, Sjur; Noer, Agate; Grimholt, Runa M; Halldórsson, Bjarni V; Medina-Gomez, Carolina; Gautvik, Vigdis T; Olstad, Ole Kristoffer; Berg, Jens Petter; Datta, Harish; Estrada, Karol; Hofman, Albert; Uitterlinden, André G; Rivadeneira, Fernando; Lyle, Robert; Collas, Philippe; Gautvik, Kaare M

    2015-02-01

    Inhibition of sclerostin, a glycoprotein secreted by osteocytes, offers a new therapeutic paradigm for treatment of osteoporosis (OP) through its critical role as Wnt/catenin signaling regulator. This study describes the epigenetic regulation of SOST expression in bone biopsies of postmenopausal women. We correlated serum sclerostin to bone mineral density (BMD), fractures, and bone remodeling parameters, and related these findings to epigenetic and genetic disease mechanisms. Serum sclerostin and bone remodeling biomarkers were measured in two postmenopausal groups: healthy (BMD T-score > -1) and established OP (BMD T-score < -2.5, with at least one low-energy fracture). Bone specimens were used to analyze SOST mRNAs, single nucleotide polymorphisms (SNPs), and DNA methylation changes. The SOST gene promoter region showed increased CpG methylation in OP patients (n = 4) compared to age and body mass index (BMI) balanced controls (n = 4) (80.5% versus 63.2%, p = 0.0001) with replication in independent cohorts (n = 27 and n = 36, respectively). Serum sclerostin and bone SOST mRNA expression correlated positively with age-adjusted and BMI-adjusted total hip BMD (r = 0.47 and r = 0.43, respectively; both p < 0.0005), and inversely to serum bone turnover markers. Five SNPs, one of which replicates in an independent population-based genomewide association study (GWAS), showed association with serum sclerostin or SOST mRNA levels under an additive model (p = 0.0016 to 0.0079). Genetic and epigenetic changes in SOST influence its bone mRNA expression and serum sclerostin levels in postmenopausal women. The observations suggest that increased SOST promoter methylation seen in OP is a compensatory counteracting mechanism, which lowers serum sclerostin concentrations and reduces inhibition of Wnt signaling in an attempt to promote bone formation.

  1. A Nascent Peptide Signal Responsive to Endogenous Levels of Polyamines Acts to Stimulate Regulatory Frameshifting on Antizyme mRNA*

    PubMed Central

    Yordanova, Martina M.; Wu, Cheng; Andreev, Dmitry E.; Sachs, Matthew S.; Atkins, John F.

    2015-01-01

    The protein antizyme is a negative regulator of cellular polyamine concentrations from yeast to mammals. Synthesis of functional antizyme requires programmed +1 ribosomal frameshifting at the 3′ end of the first of two partially overlapping ORFs. The frameshift is the sensor and effector in an autoregulatory circuit. Except for Saccharomyces cerevisiae antizyme mRNA, the frameshift site alone only supports low levels of frameshifting. The high levels usually observed depend on the presence of cis-acting stimulatory elements located 5′ and 3′ of the frameshift site. Antizyme genes from different evolutionary branches have evolved different stimulatory elements. Prior and new multiple alignments of fungal antizyme mRNA sequences from the Agaricomycetes class of Basidiomycota show a distinct pattern of conservation 5′ of the frameshift site consistent with a function at the amino acid level. As shown here when tested in Schizosaccharomyces pombe and mammalian HEK293T cells, the 5′ part of this conserved sequence acts at the nascent peptide level to stimulate the frameshifting, without involving stalling detectable by toe-printing. However, the peptide is only part of the signal. The 3′ part of the stimulator functions largely independently and acts at least mostly at the nucleotide level. When polyamine levels were varied, the stimulatory effect was seen to be especially responsive in the endogenous polyamine concentration range, and this effect may be more general. A conserved RNA secondary structure 3′ of the frameshift site has weaker stimulatory and polyamine sensitizing effects on frameshifting. PMID:25998126

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

    PubMed

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

    2010-01-01

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

  3. Actin dynamics and cofilin-actin rods in Alzheimer disease

    PubMed Central

    Bamburg, James R.; Bernstein, Barbara W.

    2017-01-01

    Cytoskeletal abnormalities and synaptic loss, typical of both familial and sporadic Alzheimer disease (AD), are induced by diverse stresses such as neuroinflammation, oxidative stress, and energetic stress, each of which may be initiated or enhanced by proinflammatory cytokines or amyloid-β (Aβ) peptides. Extracellular Aβ-containing plaques and intracellular phospho-tau-containing neurofibrillary tangles are postmortem pathologies required to confirm AD and have been the focus of most studies. However, AD brain, but not normal brain, also have increased levels of cytoplasmic rod-shaped bundles of filaments composed of ADF/cofilin-actin in a 1:1 complex (rods). Cofilin, the major ADF/cofilin isoform in mammalian neurons, severs actin filaments at low cofilin/actin ratios and stabilizes filaments at high cofilin/actin ratios. It binds cooperatively to ADP-actin subunits in F-actin. Cofilin is activated by dephosphorylation and may be oxidized in stressed neurons to form disulfide-linked dimers, required for bundling cofilin-actin filaments into stable rods. Rods form within neurites causing synaptic dysfunction by sequestering cofilin, disrupting normal actin dynamics, blocking transport, and exacerbating mitochondrial membrane potential loss. Aβ and proinflammatory cytokines induce rods through a cellular prion protein-dependent activation of NADPH oxidase and production of reactive oxygen species. Here we review recent advances in our understanding of cofilin biochemistry, rod formation, and the development of cognitive deficits. We will then discuss rod formation as a molecular pathway for synapse loss that may be common between all three prominent current AD hypotheses, thus making rods an attractive therapeutic target. PMID:26873625

  4. Effects of dietary medium-chain triacylglycerol on mRNA level of gluconeogenic enzymes in malnourished rats.

    PubMed

    Kojima, Keiichi; Kasai, Michio

    2008-12-01

    We have reported previously that dietary medium-chain triacylglycerol (MCT) improved serum albumin concentration and protein balance in malnourished rats. To clarify the mechanisms for this effect of MCT, hepatic messenger RNA levels of gluconeogenic enzymes, pyruvate dehydrogenase (PDH) and alanine aminotransferase (ALT) were measured in rats fed low-protein diets containing either MCT or isocaloric long-chain triacylglycerol (LCT) for 2 wk. The serum albumin concentration in rats fed the MCT diet was significantly higher compared with those fed the LCT diet. Serum free fatty acids and ketone body fraction were higher in rats fed MCT compared with those fed the LCT diet. The hepatic mRNA level of PDH was significantly lower in rats fed MCT than those fed LCT. But, there was no significant difference between the two groups in mRNA of gluconeogenic enzymes or ALT. These results suggest that ketone bodies, which are an alternative energy source and might spare blood glucose, increase by MCT feeding, and the reason for the PEM (protein-energy malnutrition)-improving effect of MCT is not caused by suppression of gluconeogenesis.

  5. The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis.

    PubMed

    Spracklen, Andrew J; Fagan, Tiffany N; Lovander, Kaylee E; Tootle, Tina L

    2014-09-15

    Dynamic remodeling of the actin cytoskeleton is required for both development and tissue homeostasis. While fixed image analysis has provided significant insight into such events, a complete understanding of cytoskeletal dynamics requires live imaging. Numerous tools for the live imaging of actin have been generated by fusing the actin-binding domain from an actin-interacting protein to a fluorescent protein. Here we comparatively assess the utility of three such tools--Utrophin, Lifeact, and F-tractin--for characterizing the actin remodeling events occurring within the germline-derived nurse cells during Drosophila mid-oogenesis or follicle development. Specifically, we used the UAS/GAL4 system to express these tools at different levels and in different cells, and analyzed these tools for effects on fertility, alterations in the actin cytoskeleton, and ability to label filamentous actin (F-actin) structures by both fixed and live imaging. While both Utrophin and Lifeact robustly label F-actin structures within the Drosophila germline, when strongly expressed they cause sterility and severe actin defects including cortical actin breakdown resulting in multi-nucleate nurse cells, early F-actin filament and aggregate formation during stage 9 (S9), and disorganized parallel actin filament bundles during stage 10B (S10B). However, by using a weaker germline GAL4 driver in combination with a higher temperature, Utrophin can label F-actin with minimal defects. Additionally, strong Utrophin expression within the germline causes F-actin formation in the nurse cell nuclei and germinal vesicle during mid-oogenesis. Similarly, Lifeact expression results in nuclear F-actin only within the germinal vesicle. F-tractin expresses at a lower level than the other two labeling tools, but labels cytoplasmic F-actin structures well without causing sterility or striking actin defects. Together these studies reveal how critical it is to evaluate the utility of each actin labeling tool

  6. The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis

    PubMed Central

    Spracklen, Andrew J.; Fagan, Tiffany N.; Lovander, Kaylee E.; Tootle, Tina L.

    2015-01-01

    Dynamic remodeling of the actin cytoskeleton is required for both development and tissue homeostasis. While fixed image analysis has provided significant insight into such events, a complete understanding of cytoskeletal dynamics requires live imaging. Numerous tools for the live imaging of actin have been generated by fusing the actin-binding domain from an actin-interacting protein to a fluorescent protein. Here we comparatively assess the utility of three such tools – Utrophin, Lifeact, and F-tractin – for characterizing the actin remodeling events occurring within the germline-derived nurse cells during Drosophila mid-oogenesis or follicle development. Specifically, we used the UAS/GAL4 system to express these tools at different levels and in different cells, and analyzed these tools for effects on fertility, alterations in the actin cytoskeleton, and ability to label filamentous actin (F-actin) structures by both fixed and live imaging. While both Utrophin and Lifeact robustly label F-actin structures within the Drosophila germline, when strongly expressed they cause sterility and severe actin defects including cortical actin breakdown resulting in multi-nucleate nurse cells, early F-actin filament and aggregate formation during stage 9 (S9), and disorganized parallel actin filament bundles during stage 10B (S10B). However, by using a weaker germline GAL4 driver in combination with a higher temperature, Utrophin can label F-actin with minimal defects. Additionally, strong Utrophin expression within the germline causes F-actin formation in the nurse cell nuclei and germinal vesicle during mid-oogenesis. Similarly, Lifeact expression results in nuclear F-actin only within the germinal vesicle. F-tractin expresses at a lower level than the other two labeling tools, but labels cytoplasmic F-actin structures well without causing sterility or striking actin defects. Together these studies reveal how critical it is to evaluate the utility of each actin labeling

  7. Effects of first exogenous nutrients on the mRNA levels of atrogin-1/MAFbx and GLUT1 in the skeletal muscles of newly hatched chicks.

    PubMed

    Ijiri, Daichi; Shimamoto, Saki; Kawaguchi, Mana; Furukawa, Airi; Nakashima, Kazuki; Tada, Osamu; Ohtsuka, Akira

    2017-03-01

    The aim of this study was to examine the effects of first exogenous nutrients on the mRNA levels of muscle atrophy F-box (atrogin-1/MAFbx) and glucose transporters (GLUTs) in the skeletal muscles of newly hatched chicks with no feed experience. In experiment 1, newly hatched chicks had free access to feed or were fasted for the first 24h. The chicks having free access to feed for the first 24h increased their body weight and had decreased atrogin-1/MAFbx mRNA levels in their sartorius and pectoralis major muscles compared with the fasted chicks. In experiment 2, newly hatched chicks received a single feed via intubation into the crop. Three hours after intubation, levels of atrogin-1/MAFbx mRNA in the sartorius muscle were decreased whereas the plasma insulin concentration and phosphorylated AKT levels in the sartorius muscle were increased. In addition, the mRNA levels of GLUT1 and GLUT8 were increased in the sartorius muscle after the intubation. However, in the pectoralis major muscle, AKT phosphorylation and levels of atrogin-1/MAFbx, GLUT1 and GLUT8 mRNA were not affected 3h after intubation. The first exogenous nutrients increased the level of phosphorylated AKT in the sartorius muscle of newly hatched chicks, possibly because of the decrease in atrogin-1/MAFbx mRNA levels. Furthermore, the sartorius muscle in newly hatched chicks appeared to be more susceptible to the first feed compared with the pectoralis major muscle.

  8. Time-course of SKF-81297-induced increase in glutamic acid decarboxylase 65 and 67 mRNA levels in striatonigral neurons and decrease in GABA(A) receptor alpha1 subunit mRNA levels in the substantia nigra, pars reticulata, in adult rats with a unilateral 6-hydroxydopamine lesion.

    PubMed

    Yamamoto, N; Soghomonian, J-J

    2008-06-26

    Striatal projection neurons use GABA as their neurotransmitter and express the rate-limiting synthesizing enzyme glutamic acid decarboxylase (GAD) and the vesicular GABA transporter vGAT. The chronic systemic administration of an agonist of dopamine D1/D5-preferring receptors is known to alter GAD mRNA levels in striatonigral neurons in intact and dopamine-depleted rats. In the present study, the effects of a single or subchronic systemic administration of the dopamine D1/D5-preferring receptor agonist SKF-81297 on GAD65, GAD67, PPD and vGAT mRNA levels in the striatum and GABA(A) receptor alpha1 subunit mRNA levels in the substantia nigra, pars reticulata, were measured in rats with a unilateral 6-hydroxydopamine (6-OHDA) lesion. After a single injection of SKF-81297, striatal GAD65 mRNA levels were significantly increased at 3 but not 72 h. In contrast, striatal GAD67 mRNA levels were increased and nigral alpha1 mRNA levels were decreased at 72 but not 3 h. Single cell analysis on double-labeled sections indicated that increased GAD or vGAT mRNA levels after acute SKF-81297 occurred in striatonigral neurons identified by their lack of preproenkephalin expression. Subchronic SKF-81297 induced significant increases in striatal GAD67, GAD65, preprodynorphin and vGAT mRNA levels and decreases in nigral alpha1 mRNA levels. In the striatum contralateral to the 6-OHDA lesion, subchronic but not acute SKF-81297 induced a significant increase in GAD65 mRNA levels. The other mRNA levels were not significantly altered. Finally, striatal GAD67 mRNA levels were negatively correlated with nigral alpha1 mRNA levels in the dopamine-depleted but not dopamine-intact side. The results suggest that different signaling pathways are involved in the modulation by dopamine D1/D5 receptors of GAD65 and GAD67 mRNA levels in striatonigral neurons. They also suggest that the down-regulation of nigral GABA(A) receptors is linked to the increase in striatal GAD67 mRNA levels in the dopamine

  9. Effect of ozone on degradation and mRNA levels of Rubisco in relation to potato leaf age

    SciTech Connect

    Eckardt, N.A.; Pell, E.J. )

    1993-05-01

    Leaf senescence is characterized by loss of the major photosynthetic enzyme, Ribulose bisphosphate carboxylase (Rubisco). Exposure to ozone (O[sub 3]) is often associated with a premature decline in the quantity of this enzyme. Declines in Rubisco quantity could arise through inhibition of synthesis or enhancement of degradation. Several experiments were conducted to investigate the effect of O[sub 3] on these events in immature and mature leaves of potato. The effect of O[sub 3] on Rubisco synthesis was investigated indirectly by measuring the relative quantities of mRNA for the rubisco large (rbcL) and small (rbcS) subunits following a 5 hour exposure to 0.309 [mu]L L[sup [minus]1] O[sup 3] or charcoal-filtered air. O[sup 3] treatment was associated with a significant loss in rbcS mRNA in immature and mature potato leaves sampled immediately following the exposure. After the O[sup 3] exposure, a set of plants was placed in the dark at 30 C for two days. Levels of rbcS mRNA declined rapidly during the first twelve hours of dark incubation, thus declines in Rubisco quantity following two days of dark incubation were ascribed to degradation. Enhanced degradation due to O[sub 3] during the dark incubation was observed in the mature leaves, but not in the immature leaves. We conclude that O[sub 3] can cause both inhibited synthesis and enhanced degradation of Rubisco, and the response in dependent on leaf age.

  10. NOX4 activity is determined by mRNA levels and reveals a unique pattern of ROS generation

    PubMed Central

    Serrander, Lena; Cartier, Laetitia; Bedard, Karen; Banfi, Botond; Lardy, Bernard; Plastre, Olivier; Sienkiewicz, Andrzej; Fórró, Lászlo; Schlegel, Werner; Krause, Karl-Heinz

    2007-01-01

    NOX4 is an enigmatic member of the NOX (NADPH oxidase) family of ROS (reactive oxygen species)-generating NADPH oxidases. NOX4 has a wide tissue distribution, but the physiological function and activation mechanisms are largely unknown, and its pharmacology is poorly understood. We have generated cell lines expressing NOX4 upon tetracycline induction. Tetracycline induced a rapid increase in NOX4 mRNA (1 h) followed closely (2 h) by a release of ROS. Upon tetracycline withdrawal, NOX4 mRNA levels and ROS release decreased rapidly (<24 h). In membrane preparations, NOX4 activity was selective for NADPH over NADH and did not require the addition of cytosol. The pharmacological profile of NOX4 was distinct from other NOX isoforms: DPI (diphenyleneiodonium chloride) and thioridazine inhibited the enzyme efficiently, whereas apocynin and gliotoxin did not (IC50>100 μM). The pattern of NOX4-dependent ROS generation was unique: (i) ROS release upon NOX4 induction was spontaneous without need for a stimulus, and (ii) the type of ROS released from NOX4-expressing cells was H2O2, whereas superoxide (O2−) was almost undetectable. Probes that allow detection of intracellular O2− generation yielded differential results: DHE (dihydroethidium) fluorescence and ACP (1-acetoxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine) ESR measurements did not detect any NOX4 signal, whereas a robust signal was observed with NBT. Thus NOX4 probably generates O2− within an intracellular compartment that is accessible to NBT (Nitro Blue Tetrazolium), but not to DHE or ACP. In conclusion, NOX4 has a distinct pharmacology and pattern of ROS generation. The close correlation between NOX4 mRNA and ROS generation might hint towards a function as an inducible NOX isoform. PMID:17501721

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

    PubMed

    Ortíz-Martinez, David Mizael; Rivas-Morales, Catalina; de la Garza-Ramos, Myriam Angelica; Verde-Star, Maria Julia; Nuñez-Gonzalez, Maria Adriana; Leos-Rivas, Catalina

    2016-01-01

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

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

    PubMed Central

    Ortíz-Martinez, David Mizael; Rivas-Morales, Catalina; de la Garza-Ramos, Myriam Angelica; Verde-Star, Maria Julia; Nuñez-Gonzalez, Maria Adriana

    2016-01-01

    Diabetes mellitus is a public health problem worldwide. For this reason, ethanolic extract of Miconia sp. from Oaxaca, Mexico, was selected in search of an alternative against this disease. The effect of Miconia sp. on mRNA expression of PPARγ on cell line 3T3-L1, its effect on alpha amylase and alpha glucosidase, lipid accumulation during adipogenesis, and cell viability on VERO cells were evaluated. The mRNA levels of PPARγ increased on 1.393 ± 0.008 folds, lipid accumulation was increased by 29.55% with Miconia sp. extract and 34.57% with rosiglitazone, and α-amylase and α-glycosidase were inhibited with IC50 values from 28.23 ± 2.15 μg/mL and 1.95 ± 0.15 μg/mL, respectively; the IC50 on antiproliferative activity on VERO cells was 314.54 ± 45.40 μg/mL. In case of α-amylase and α-glycosidase assays, IC50 (inhibitory concentration 50) refers to necessary extract amounts to inhibit 50% of enzymatic activity. On the other hand, on antiproliferative activity, IC50 (inhibitory concentration 50) refers to necessary extract amounts to inhibit 50% of cell proliferation. It was concluded that the compounds present in Miconia sp. ethanolic extract increase mRNA expression of PPARγ, inhibit α-amylase and α-glucosidase, and increase lipid accumulation. It constitutes an alternative as adjuvant in diabetes mellitus treatment; therefore, we recommend continuing identifying the compounds responsible for its promising in vivo antidiabetic activity. PMID:27478477

  13. The Potential Roles of Actin in The Nucleus

    PubMed Central

    Falahzadeh, Khadijeh; Banaei-Esfahani, Amir; Shahhoseini, Maryam

    2015-01-01

    Over the past few decades, actin’s presence in the nucleus has been demonstrated. Actin is a key protein necessary for different nuclear processes. Although actin is well known for its functional role in dynamic behavior of the cytoskeleton, emerging studies are now highlighting new roles for actin. At the present time there is no doubt about the presence of actin in the nucleus. A number of studies have uncovered the functional involvement of actin in nuclear processes. Actin as one of the nuclear components has its own structured and functional rules, such as nuclear matrix association, chromatin remodeling, transcription by RNA polymerases I, II, III and mRNA processing. In this historical review, we attempt to provide an overview of our current understanding of the functions of actin in the nucleus. PMID:25870830

  14. Uncoupling protein-3 mRNA levels are increased in white adipose tissue and skeletal muscle of bezafibrate-treated rats.

    PubMed

    Cabrero, A; Llaverías, G; Roglans, N; Alegret, M; Sánchez, R; Adzet, T; Laguna, J C; Vázquez, M

    1999-07-05

    Fibrates are hypolipidemic drugs that are also able to improve glucose tolerance in animals and diabetic patients through an unknown mechanism. Since uncoupling proteins (UCP) seem to play an important role in the pathogenesis of non-insulin-dependent diabetes mellitus (NIDDM), we examined whether treatment of rats with bezafibrate for 3, 7, or 15 days modified UCP mRNA levels. Using RT-PCR, we observed a weak ectopic expression of UCP-1 and a 2-fold increase in UCP-3 mRNA levels in white adipose tissue after 7 and 15 days of treatment. Moreover, bezafibrate administration caused a 1. 7-fold induction in UCP-3 mRNA levels in skeletal muscle on day 7. Since UCP-3 mRNA levels are reduced in skeletal muscle of diabetic patients, this effect may be involved in the improvement of insulin sensitivity caused by bezafibrate in NIDDM.

  15. Rapid upregulation of the hippocampal connexins 36 and 45 mRNA levels during memory consolidation.

    PubMed

    Beheshti, Siamak; Zeinali, Reyhaneh; Esmaeili, Abolghasem

    2017-03-01

    Gap junction channels are implicated in learning and memory process. However, their role on each of the particular stages of memory formation has been studied less. In this study, the time profile of the expression levels of hippocampal connexins 36 and 45 (Cx36 and Cx45) mRNAs was measured during memory consolidation, in a passive avoidance paradigm. Totally 30 adult male rats were distributed into 5 groups of each 6. At different times profiles (30min, 3, 6 and 24h) following training, rats were decapitated and their hippocampi were immediately removed and frozen in liquid nitrogen. Total RNA was extracted and cDNA was synthesized, using oligo-dt primers. A quantitative real-time PCR was used to measure the levels of each of Cx36 and Cx45 mRNAs. Both connexins showed a rapid upregulation (30min) at the transcriptional level, which declined in later times and reached to the control level at 24h. The rapid up-regulation of Cx36 and Cx45 mRNAs might be accompanied with increasing intercellular coupling via gap junction channels and neuronal oscillatory activities required for memory consolidation. The results highlight the role of gap junctional coupling between hippocampal neurons during memory consolidation in the physiological conditions.

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

    NASA Technical Reports Server (NTRS)

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

    1993-01-01

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

  17. Toxaphene affects the levels of mRNA transcripts that encode antioxidant enzymes in Hydra.

    PubMed

    Woo, Seonock; Lee, Aekyung; Won, Hyokyoung; Ryu, Jae-Chun; Yum, Seungshic

    2012-06-01

    We evaluated toxaphene-induced acute toxicity in Hydra magnipapillata. The median lethal concentrations of the animals (LC(50)) were determined to be 34.5 mg/L, 25.0 mg/L and 12.0 mg/L after exposure to toxaphene for 24 h, 48 h and 72 h, respectively. Morphological responses of hydra polyps to a range of toxaphene concentrations suggested that toxaphene negatively affects the nervous system of H. magnipapillata. We used real-time quantitative PCR of RNA extracted from polyps exposed to two concentrations of toxaphene (0.3 mg/L and 3 mg/L) for 24 h to evaluate the differential regulation of levels of transcripts that encode six antioxidant enzymes (CAT, G6PD, GPx, GR, GST and SOD), two proteins involved in detoxification and molecular stress responses (CYP1A and UB), and two proteins involved in neurotransmission and nerve cell differentiation (AChE and Hym-355). Of the genes involved in antioxidant responses, the most striking changes were observed for transcripts that encode GPx, G6PD, SOD, CAT and GST, with no evident change in levels of transcripts encoding GR. Levels of UB and CYP1A transcripts increased in a dose-dependent manner following exposure to toxaphene. Given that toxaphene-induced neurotoxicity was not reflected in the level of AChE transcripts and only slight accumulation of Hym-355 transcript was observed only at the higher of the two doses of toxaphene tested, there remains a need to identify transcriptional biomarkers for toxaphene-mediated neurotoxicity in H. magnipapillata. Transcripts that respond to toxaphene exposure could be valuable biomarkers for stress levels in H. magnipapillata and may be useful for monitoring the pollution of aquatic environments.

  18. A clone screening method using mRNA levels to determine specific productivity and product quality for monoclonal antibodies.

    PubMed

    Lee, Christina J; Seth, Gargi; Tsukuda, Joni; Hamilton, Robert W

    2009-03-01

    To meet increasing demands for efficient and streamlined production processes of therapeutic antibodies, improved methods of screening clones are required. In this article, we examined the potential of using antibody transcript levels as criteria for clone screening. We evaluated the QuantiGene Plex, a commercially available, high-throughput assay for simultaneously measuring multiple transcripts from cell lysate. Using the development of stable Chinese hamster ovary cell lines as examples, we investigated the relationship between transcript and antibody levels through several rounds of screening. First, we observed that measured heavy chain transcript levels are generally correlated with specific productivity, enabling the identification of high-producing clones from mRNA. Second, we observed that low ratios (< 1.5) of light to heavy chain transcript levels may be indicative of high antibody aggregation levels, allowing for the rapid identification and elimination of clones of questionable product quality. Therefore, an efficient process of identifying high-producing clones of desirable product quality is possible by using QuantiGene Plex assay to measure antibody transcript levels.

  19. RNAi effects on actin mRNAs in Homalodisca vitripennis cells

    PubMed Central

    Rosa, Cristina; Kamita, Shizuo G; Dequine, Haley; Wuriyanghan, Ha; Lindbo, John A; Falk, Bryce W

    2010-01-01

    The xylem feeding leafhopper Homalodisaca vitripennis (H. vitripennis) is an unusually robust and efficient vector of Xylella fastidiosa, a Gram-negative bacterium which causes several very important plant diseases. Here we investigated RNA interference (RNAi) to target actin, a key component of insect cells and whole bodies, in H. vitripennis cells. RNAi effectors were delivered via lipid based transfection and real-time RT-PCR, RNA hybridization, and microscopic analyses were employed to verify RNAi effects. When actin dsRNAs were used, a 10-fold decrease in the target H. vitripennis actin mRNA level was seen in cells. Altered phenotypic effects also were evident in transfected cells, as were small interfering RNAs, hallmarks of RNAi. The use of H. vitripennis cells and RNAi offers new opportunities to research hemipterans, the most important insect vectors of plant pathogens. PMID:20628496

  20. Ultra-fast alterations in mRNA levels uncover multiple players in light stress acclimation in plants.

    PubMed

    Suzuki, Nobuhiro; Devireddy, Amith R; Inupakutika, Madhuri A; Baxter, Aaron; Miller, Gad; Song, Luhua; Shulaev, Elena; Azad, Rajeev K; Shulaev, Vladimir; Mittler, Ron

    2015-11-01

    The acclimation of plants to changes in light intensity requires rapid responses at several different levels. These include biochemical and biophysical responses as well as alterations in the steady-state level of different transcripts and proteins. Recent studies utilizing promoter::reporter constructs suggested that transcriptional responses to changes in light intensity could occur within seconds, rates for which changes in mRNA expression are not routinely measured or functionally studied. To identify and characterize rapid changes in the steady-state level of different transcripts in response to light stress we performed RNA sequencing analysis of Arabidopsis thaliana plants subjected to light stress. Here we report that mRNA accumulation of 731 transcripts occurs as early as 20-60 sec following light stress application, and that at least five of these early response transcripts play an important biological role in the acclimation of plants to light stress. More than 20% of transcripts accumulating in plants within 20-60 sec of initiation of light stress are H2 O2 - and ABA-response transcripts, and the accumulation of several of these transcripts is inhibited by transcriptional inhibitors. In accordance with the association of rapid response transcripts with H2 O2 and ABA signaling, a mutant impaired in ABA sensing (abi-1) was found to be more tolerant to light stress, and the response of several of the rapid response transcripts was altered in mutants impaired in reactive oxygen metabolism. Our findings reveal that transcriptome reprogramming in plants could occur within seconds of initiation of abiotic stress and that this response could invoke known as well as unknown proteins and pathways.

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

    PubMed

    Volkoff, Hélène

    2014-06-01

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

  2. Effect of copy number and mRNA processing and stabilization on transcript and protein levels from an engineered dual-gene operon.

    PubMed

    Smolke, Christina D; Keasling, Jay D

    2002-05-20

    To study the effect of mRNA stability and DNA copy number on protein production from a dual-gene operon, a synthetic operon containing the reporter genes gfp and lacZ under the control of the araBAD promoter was placed in pMB1-based (approximately 100 copies/cell) and F plasmid-based (approximately 1 copy/cell) vectors. DNA cassettes encoding secondary structures were placed at the 5' and 3' ends of the genes and a putative RNase E site was placed between the two genes. Although the copy number of the pMB1-based vectors was approximately 100-fold greater than the copy number of the F plasmid-based vectors, transcript and protein levels from the pMB1-based vector were not 100-fold greater than from the F plasmid-based vectors. In identical plasmid backbones, different combinations of mRNA control elements were used to alter steady-state levels of transcripts. Control elements that amplified the stability of one coding region relative to another amplified the ratio of protein produced from those transcripts. The effects of mRNA stability control elements were greater at low inducer concentrations, where mRNA levels limit protein production, than at high inducer concentrations. Although we can alter mRNA and protein levels through copy number, induction level, and mRNA stability control elements, some aspect of gene expression remains dependent on inherent characteristics of the coding region.

  3. Manganese exposure alters extracellular GABA, GABA receptor and transporter protein and mRNA levels in the developing rat brain.

    PubMed

    Anderson, Joel G; Fordahl, Steve C; Cooney, Paula T; Weaver, Tara L; Colyer, Christa L; Erikson, Keith M

    2008-11-01

    Unlike other essential trace elements (e.g., zinc and iron) it is the toxicity of manganese (Mn) that is more common in human populations than its deficiency. Data suggest alterations in dopamine biology may drive the effects associated with Mn neurotoxicity, though recently gamma-aminobutyric acid (GABA) has been implicated. In addition, iron deficiency (ID), a common nutritional problem, may cause disturbances in neurochemistry by facilitating accumulation of Mn in the brain. Previous data from our lab have shown decreased brain tissue levels of GABA as well as decreased (3)H-GABA uptake in synaptosomes as a result of Mn exposure and ID. These results indicate a possible increase in the concentration of extracellular GABA due to alterations in expression of GABA transport and receptor proteins. In this study weanling-male Sprague-Dawley rats were randomly placed into one of four dietary treatment groups: control (CN; 35mg Fe/kg diet), iron-deficient (ID; 6mg Fe/kg diet), CN with Mn supplementation (via the drinking water; 1g Mn/l) (CNMn), and ID with Mn supplementation (IDMn). Using in vivo microdialysis, an increase in extracellular GABA concentrations in the striatum was observed in response to Mn exposure and ID although correlational analysis reveals that extracellular GABA is related more to extracellular iron levels and not Mn. A diverse effect of Mn exposure and ID was observed in the regions examined via Western blot and RT-PCR analysis, with effects on mRNA and protein expression of GAT-1, GABA(A), and GABA(B) differing between and within the regions examined. For example, Mn exposure reduced GAT-1 protein expression by approximately 50% in the substantia nigra, while increasing mRNA expression approximately four-fold, while in the caudate putamen mRNA expression was decreased with no effect on protein expression. These data suggest that Mn exposure results in an increase in extracellular GABA concentrations via altered expression of transport and

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

    PubMed Central

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

    2016-01-01

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

  5. Arginase-1 mRNA expression correlates with myeloid-derived suppressor cell levels in peripheral blood of NSCLC patients.

    PubMed

    Heuvers, Marlies E; Muskens, Femke; Bezemer, Koen; Lambers, Margaretha; Dingemans, Anne-Marie C; Groen, Harry J M; Smit, Egbert F; Hoogsteden, Henk C; Hegmans, Joost P J J; Aerts, Joachim G J V

    2013-09-01

    Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature and progenitor myeloid cells with immunosuppressive activity that are increased in cancer patients. Until now, the characterization of MDSC in humans was very challenging. The aim of this study was to determine the characterization and optimal assessment of MDSC and to investigate their presence and function in blood of advanced-stage NSCLC patients. We determined MDSC and lymphocyte populations in peripheral blood mononuclear cells (PBMC) samples of 185 treatment-naïve NSCLC patients and 20 healthy controls (HC). NSCLC patients had an increased population of PMN-MDSC compared to HC (p < 0.0001). Frequencies of CD4(+) and CD8(+) T-cells were significantly decreased in NSCLC patients (p < 0.0001 and p = 0.05). We found that PMN-MDSC were able to suppress T-cell proliferation in vitro. qRT-PCR showed that arginase-1 (Arg-1) mRNA is mainly expressed by MDSC and that the level of Arg-1 in PBMC correlates with the frequency of MDSC in PBMC (Spearman's rho: 0.797). There were significant differences in MDSC and lymphocyte populations between NSCLC patients and HC. We found that MDSC frequencies are stable up to six hours at room temperature after blood was drawn and that cryopreservation leads to a strong decrease of MDSC in PBMC. We show that Arg-1 mRNA expression is a valuable method to determine the levels of MDSC in peripheral blood of cancer patients. This method is therefore a useful alternative for the complex flowcytometric analysis in large multicenter patient studies.

  6. Tetrodotoxin administration in the suprachiasmatic nucleus prevents NMDA-induced reductions in pineal melatonin without influencing Per1 and Per2 mRNA levels.

    PubMed

    Paul, Ketema N; Gamble, Karen L; Fukuhara, Chiaki; Novak, Colleen M; Tosini, Gianluca; Albers, H Elliott

    2004-05-01

    The suprachiasmatic nucleus (SCN) of the anterior hypothalamus contains a light-entrainable circadian pacemaker. Neurons in the SCN are part of a circuit that conveys light information from retinal efferents to the pineal gland. Light presented during the night acutely increases mRNA levels of the circadian clock genes Per1 and Per2 in the SCN, and acutely suppresses melatonin levels in the pineal gland. The present study investigated whether the ability of light to increase Per1 and Per2 mRNA levels and suppress pineal melatonin levels requires sodium-dependent action potentials in the SCN. Per1 and Per2 mRNA levels in the SCN and pineal melatonin levels were measured in Syrian hamsters injected with tetrodotoxin (TTX) prior to light exposure or injection of N-methyl-D-aspartate (NMDA). TTX inhibited the ability of light to increase Per1 and Per2 mRNA levels and suppress pineal melatonin levels. TTX did not, however, influence the ability of NMDA to increase Per1 and Per2 mRNA levels, though it did inhibit the ability of NMDA to suppress pineal melatonin levels. These results demonstrate that action potentials in the SCN are not necessary for NMDA receptor activation to increase Per1 and Per2 mRNA levels, but are necessary for NMDA receptor activation to decrease pineal melatonin levels. Taken together, these data support the hypothesis that the mechanism through which light information is conveyed to the pacemaker in the SCN is separate from and independent of the mechanism through which light information is conveyed to the SCN cells whose efferents suppress pineal melatonin levels.

  7. Changes in dihydrofolate reductase (DHFR) mRNA levels can account fully for changes in DHFR synthesis rates during terminal differentiation in a highly amplified myogenic cell line.

    PubMed Central

    Schmidt, E E; Merrill, G F

    1991-01-01

    Dihydrofolate reductase (DHFR) enzyme is preferentially synthesized in proliferative cells. A mouse muscle cell line resistant to 300 microM methotrexate was developed to investigate the molecular levels at which DHFR is down-regulated during myogenic withdrawal from the cell cycle. H- alpha R300T cells contained 540 copies of the endogenous DHFR gene and overexpressed DHFR mRNA and DHFR protein. Despite DHFR gene amplification, the cells remained diploid. As H- alpha R300T myoblasts withdrew from the cell cycle and committed to terminal differentiation, DHFR mRNA levels and DHFR synthesis rates decreased with closely matched kinetics. After 15 to 24 h, committed cells contained 5% the proliferative level of DHFR mRNA (80 molecules per committed cell) and synthesized DHFR protein at 6% the proliferative rate. At no point during the commitment process did the decrease in DHFR synthesis rate exceed the decrease in DHFR message. The decrease in DHFR mRNA levels during commitment was sufficient to account fully for the decrease in rates of DHFR synthesis. Furthermore, DHFR mRNA remained polysomal, and the average number of ribosomes per message remained constant (five to six ribosomes per DHFR mRNA). The constancy of polysome size, along with the uniform rate of DHFR synthesis per message, indicated that DHFR mRNA was efficiently translated in postreplicative cells. The results support a model wherein replication-dependent changes in DHFR synthesis rates are determined exclusively by changes in DHFR mRNA levels. Images PMID:2046674

  8. β-glucuronidase mRNA levels are correlated with gait and working memory in premutation females: understanding the role of FMR1 premutation alleles

    PubMed Central

    Kraan, C. M.; Cornish, K. M.; Bui, Q. M.; Li, X.; Slater, H. R.; Godler, D. E.

    2016-01-01

    Fragile X tremor ataxia syndrome (FXTAS) is a late-onset disorder manifesting in a proportion of FMR1 premutation individuals (PM: 55-199 CGG triplet expansions). FXTAS is associated with elevated levels of FMR1 mRNA which are toxic. In this study, relationships between neurocognitive and intra-step gait variability measures with mRNA levels, measured in blood samples, were examined in 35 PM and 35 matched control females. The real-time PCR assays measured FMR1 mRNA, and previously used internal control genes: β-Glucuronidase (GUS), Succinate Dehydrogenase 1 (SDHA) and Eukaryotic Translation Initiation Factor 4A (EI4A2). Although there was significant correlation of gait variability with FMR1 mRNA levels (p = 0.004) when normalized to GUS (FMR1/GUS), this was lost when FMR1 was normalized to SDHA and EI4A2 (2IC). In contrast, GUS mRNA level normalized to 2IC showed a strong correlation with gait variability measures (p < 0.007), working memory (p = 0.001) and verbal intelligence scores (p = 0.008). PM specific changes in GUS mRNA were not mediated by FMR1 mRNA. These results raise interest in the role of GUS in PM related disorders and emphasise the importance of using appropriate internal control genes, which have no significant association with PM phenotype, to normalize FMR1 mRNA levels. PMID:27387142

  9. Age, sex, and lactating status regulate ghrelin secretion and GOAT mRNA levels from isolated rat stomach.

    PubMed

    Al-Massadi, O; Crujeiras, A B; González, R C; Pardo, M; Diéguez, C; Casanueva, F F; Seoane, L M

    2010-09-01

    Ghrelin is a stomach derivate peptide involved in energy homeostasis regulation, and ghrelin O-acyltransferase (GOAT) is the enzyme responsible for ghrelin acylation. Puberty is a period characterized by profound changes in the metabolic requirements and notable variations of sexual hormone levels. On the other hand, the weaning process is a fundamental modification of the diet, which implicates several adaptations of the gastrointestinal tract physiology. Until now the direct secretion of ghrelin by the stomach in these conditions, without interferences from other organs, has never been studied. The main objective of this article was to investigate how the stomach modulates ghrelin production and secretion as well as GOAT expression on these periods of life. Gastric ghrelin secretion is regulated through postnatal life in an independent way of gastric expression and circulating levels of this hormone. The present work shows a strong regulation of gastric ghrelin secretion by estrogens. The weaning strongly regulates gastric ghrelin secretion. Animals subjected to delayed weaning present a lower body weight than the corresponding controls. For the first time, it is shown that a noticeable decrease in circulating levels of testosterone and estrogens is associated with delay of weaning. GOAT mRNA levels in the stomach are strongly regulated by age, breastfeeding, and testosterone. In conclusion, the stomach itself regulates ghrelin and GOAT production to adapt the organism to the metabolic requirements demanded through each stage of life.

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

    PubMed Central

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

    2016-01-01

    Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by functional defects in mismatch repair (MMR) genes, including mutL homolog 1 (MLH1) and mutS homolog 2 (MSH2). This study aimed to assess whether the mRNA expression of MLH1 in peripheral blood could be used as a biomarkers for the diagnosis of HNPCC. The mRNA level of MLH1 was determined in 19 HNPCC families (46 members) using real-time quantitative polymerase chain reaction (qPCR). The mRNA levels of MLH1 in HNPCC were significantly lower than controls (P < 0.001). Receiver operating characteristic (ROC) curve showed a high diagnostic value of the mRNA level of MLH1 for the diagnosis of HNPCC with the area under curve of 0.858. At an optimal cut-off value (0.511), the mRNA level of MLH1 had a sensitivity of 81.3% and a specificity of 86.7% for distinguishing HNPCC from controls. In conclusion, the mRNA expression of MLH1 in peripheral blood may serve as a biomarker for the diagnosis of HNPCC. PMID:27294005

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

    PubMed

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

    2009-11-01

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

  12. The MYC mRNA 3'-UTR couples RNA polymerase II function to glutamine and ribonucleotide levels.

    PubMed

    Dejure, Francesca R; Royla, Nadine; Herold, Steffi; Kalb, Jacqueline; Walz, Susanne; Ade, Carsten P; Mastrobuoni, Guido; Vanselow, Jens T; Schlosser, Andreas; Wolf, Elmar; Kempa, Stefan; Eilers, Martin

    2017-04-13

    Deregulated expression of MYC enhances glutamine utilization and renders cell survival dependent on glutamine, inducing "glutamine addiction". Surprisingly, colon cancer cells that express high levels of MYC due to WNT pathway mutations are not glutamine-addicted but undergo a reversible cell cycle arrest upon glutamine deprivation. We show here that glutamine deprivation suppresses translation of endogenous MYC via the 3'-UTR of the MYC mRNA, enabling escape from apoptosis. This regulation is mediated by glutamine-dependent changes in adenosine-nucleotide levels. Glutamine deprivation causes a global reduction in promoter association of RNA polymerase II (RNAPII) and slows transcriptional elongation. While activation of MYC restores binding of MYC and RNAPII function on most promoters, restoration of elongation is imperfect and activation of MYC in the absence of glutamine causes stalling of RNAPII on multiple genes, correlating with R-loop formation. Stalling of RNAPII and R-loop formation can cause DNA damage, arguing that the MYC 3'-UTR is critical for maintaining genome stability when ribonucleotide levels are low.

  13. Differential changes in vascular mRNA levels between rat iliac and renal arteries produced by cessation of voluntary running.

    PubMed

    Padilla, Jaume; Jenkins, Nathan T; Roberts, Michael D; Arce-Esquivel, Arturo A; Martin, Jeffrey S; Laughlin, M Harold; Booth, Frank W

    2013-01-01

    Early vascular changes at the molecular level caused by adoption of a sedentary lifestyle are incompletely characterized. Herein, we employed the rodent wheel-lock model to identify mRNAs in the arterial wall that are responsive to the acute transition from higher to lower levels of daily physical activity. Specifically, we evaluated whether short-term cessation of voluntary wheel running alters vascular mRNA levels in rat conduit arteries previously reported to have marked increases (i.e. iliac artery) versus marked decreases (i.e. renal artery) in blood flow during running. We used young female Wistar rats with free access to voluntary running wheels. Following 23 days of voluntary running (average distance of ∼15 km per night; ∼4.4 h per night), rats in one group were rapidly transitioned to a sedentary state by locking the wheels for 7 days (n = 9; wheel-lock 7 day rats) or remained active in a second group for an additional 7 days (n = 9; wheel-lock 0 day rats). Real-time PCR was conducted on total RNA isolated from iliac and renal arteries to evaluate expression of 25 pro-atherogenic and anti-atherogenic genes. Compared with the iliac arteries of wheel-lock 0 day rats, iliac arteries of wheel-lock 7 day rats exhibited increased expression of TNFR1 (+19%), ET1 (+59%) and LOX-1 (+31%; all P < 0.05). Moreover, compared with renal arteries of wheel-lock 0 day rats, renal arteries of wheel-lock 7 day rats exhibited decreased expression of ETb (-23%), p47phox (-32%) and p67phox (-19%; all P < 0.05). These data demonstrate that cessation of voluntary wheel running for 7 days produces modest, but differential changes in mRNA levels between the iliac and renal arteries of healthy rats. This heterogeneous influence of short-term physical inactivity could be attributed to the distinct alteration in haemodynamic forces between arteries.

  14. Prognostic significance of preoperative and postoperative CK19 and CEA mRNA levels in peripheral blood of patients with gastric cardia cancer

    PubMed Central

    Qiao, Yu-Feng; Chen, Chuan-Gui; Yue, Jie; Ma, Ming-Quan; Ma, Zhao; Yu, Zhen-Tao

    2017-01-01

    AIM To evaluate the clinical and prognostic significance of preoperative and postoperative cytokeratin 19 (CK19) and carcinoembryonic antigen (CEA) mRNA levels in peripheral blood of patients with gastric cardia cancer (GCC). METHODS We detected the preoperative and postoperative mRNA levels of CK19 and CEA in peripheral blood of 129 GCC patients by using reverse transcription-polymerase chain reaction and evaluated their clinical and prognostic significance by univariate Kaplan-Meier survival analysis and multivariate Cox proportional hazard analysis. A new prognostic model which stratified patients into three different risk groups was established based on the independent prognostic factors. RESULTS Elevated preoperative and postoperative CK19 and CEA mRNA levels in peripheral blood of GCC patients were associated with lymph node metastasis. Univariate analysis showed that tumor size, histological grade, depth of tumor invasion, lymph node metastasis, preoperative CK19 mRNA, and preoperative and postoperative CEA mRNA levels were correlated with the prognosis of GCC patients. The multivariate analysis showed that lymph node status (P = 0.018), preoperative CK19 (P = 0.035) and CEA (P = 0.011) mRNA levels were independent prognostic factors for overall survival (OS). The 5-year OS rates for the low-, intermediate-, and high-risk groups were 48.3%, 22.6%, and 4.6%, respectively (P < 0.001). CONCLUSION Elevated preoperative CK19 and CEA mRNA levels may be regarded as promising biomarkers for predicting lymph node metastasis and poor prognosis in patients with GCC. This new prognostic model may help us identify the subpopulations of GCC patients with the highest risk. PMID:28293089

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

    PubMed Central

    2016-01-01

    Purpose. This study aimed to evaluate the clinical efficacy of using expression levels of CCL24 (eotaxin-2) mRNA on the ocular surface as a biomarker in patients with vernal keratoconjunctivitis (VKC) and atopic keratoconjunctivitis (AKC). Methods. Eighteen patients with VKC or AKC (VKC/AKC group) and 12 control subjects (control group) were enrolled in this study. The VKC/AKC clinical score was determined by objective findings in patients by using the 5-5-5 exacerbation grading scale. All subjects underwent modified impression cytology and specimens were obtained from the upper tarsal conjunctiva. Expression levels of CCL24 (eotaxin-2) mRNA on the ocular surface were determined using real-time reverse transcription polymerase chain reaction. Results. The VKC group was divided into two subgroups, depending on the clinical score: the active stage subgroup with 100 points or more of clinical scores and the stable stage subgroup with 100 points or less. CCL24 (eotaxin-2) mRNA expression levels in the active VKC/AKC stage subgroup were significantly higher than those in the stable VKC/AKC subgroup and the control group. Clinical scores correlated significantly with CCL24 (eotaxin-2) mRNA expression levels in the VKC group. Conclusions. CCL24 (eotaxin-2) mRNA expression levels on the ocular surface are a useful biomarker for clinical severity of VKC/AKC. PMID:27721987

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

    PubMed

    Bukowski, R

    2014-01-01

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

  17. Why is Actin Patchy?

    NASA Astrophysics Data System (ADS)

    Carlsson, Anders

    2009-03-01

    The intracellular protein actin, by reversibly polymerizing into filaments, generates forces for motion and shape changes of many types of biological cells. Fluorescence imaging studies show that actin often occurs in the form of localized patches of size roughly one micrometer at the cell membrane. Patch formation is most prevalent when the free-actin concentration is low. I investigate possible mechanisms for the formation of actin patches by numerically simulating the ``dendritic nucleation'' model of actin network growth. The simulations include filament growth, capping, branching, severing, and debranching. The attachment of membrane-bound activators to actin filaments, and subsequent membrane diffusion of unattached activators, are also included. It is found that as the actin concentration increases from zero, the actin occurs in patches at lower actin concentrations, and the size of the patches increases with increasing actin concentration. At a critical value of the actin concentration, the system undergoes a transition to complete coverage. The results are interpreted within the framework of reaction-diffusion equations in two dimensions.

  18. Effects of Acupuncture on mRNA Levels of Apoptotic Factors in Perihematomal Brain Tissue During the Acute Phase of Cerebral Hemorrhage.

    PubMed

    Li, Zuowei; Zheng, Xiaonan; Li, Ping; Itoua, Eudes Saturnin Régis; Moukassa, Donatien; Ndinga Andely, Françoise

    2017-03-30

    BACKGROUND To explore the time-dependent effects of acupuncture on mRNA levels of the apoptotic factors BCL-2 and BAX in a rat cerebral hemorrhage model, slow injection of autologous blood to the caudate nucleus was used to generate the cerebral hemorrhage model. MATERIAL AND METHODS A sham surgery control group, groups with acupuncture applied 3, 9, 24, and 48 hours after model induction, and time-matched model-only control groups were used. In situ hybridization was used to detect BCL-2 and BAX mRNA expression, and semi-quantitative RT-PCR was used to measure the expression. RESULTS The number of BCL-2 and BAX mRNA-positive cells significantly increased during the acute phase of cerebral hemorrhage. BCL-2 mRNA was significantly upregulated in acupuncture groups compared to other groups, whereas BAX mRNA levels in the acupuncture groups were lower in the other groups, except for the sham surgery group. Additionally, earlier acupuncture intervention was associated with a lower ratio of expression between the two genes. Changes in BCL-2 and BAX mRNA expression were consistent with changes in the number of cells positive for BCL-2 and BAX mRNA; however, the change in the expression ratio was consistent with the change in the number of cells positive for BCL-2 mRNA, but opposite to the change in the number of cells positive for BAX mRNA. CONCLUSIONS Acupuncture ameliorated changes in expression of apoptotic factors in the brain induced by acute cerebral hemorrhage and may thus protect the brain, with greater efficacy when the delay before acupuncture was minimized.

  19. Effects of Acupuncture on mRNA Levels of Apoptotic Factors in Perihematomal Brain Tissue During the Acute Phase of Cerebral Hemorrhage

    PubMed Central

    Li, Zuowei; Zheng, Xiaonan; Li, Ping; Itoua, Eudes Saturnin Régis; Moukassa, Donatien; Andely, Françoise Ndinga

    2017-01-01

    Background To explore the time-dependent effects of acupuncture on mRNA levels of the apoptotic factors BCL-2 and BAX in a rat cerebral hemorrhage model, slow injection of autologous blood to the caudate nucleus was used to generate the cerebral hemorrhage model. Material/Methods A sham surgery control group, groups with acupuncture applied 3, 9, 24, and 48 hours after model induction, and time-matched model-only control groups were used. In situ hybridization was used to detect BCL-2 and BAX mRNA expression, and semi-quantitative RT-PCR was used to measure the expression. Results The number of BCL-2 and BAX mRNA-positive cells significantly increased during the acute phase of cerebral hemorrhage. BCL-2 mRNA was significantly upregulated in acupuncture groups compared to other groups, whereas BAX mRNA levels in the acupuncture groups were lower in the other groups, except for the sham surgery group. Additionally, earlier acupuncture intervention was associated with a lower ratio of expression between the two genes. Changes in BCL-2 and BAX mRNA expression were consistent with changes in the number of cells positive for BCL-2 and BAX mRNA; however, the change in the expression ratio was consistent with the change in the number of cells positive for BCL-2 mRNA, but opposite to the change in the number of cells positive for BAX mRNA. Conclusions Acupuncture ameliorated changes in expression of apoptotic factors in the brain induced by acute cerebral hemorrhage and may thus protect the brain, with greater efficacy when the delay before acupuncture was minimized. PMID:28357997

  20. FGF15/19 protein levels in the portal blood do not reflect changes in the ileal FGF15/19 or hepatic CYP7A1 mRNA levels.

    PubMed

    Shang, Quan; Guo, Grace L; Honda, Akira; Saumoy, Monica; Salen, Gerald; Xu, Guorong

    2013-10-01

    It has been proposed that bile acid suppression of CYP7A1 gene expression is mediated through a gut-liver signaling pathway fibroblast growth factor (FGF)15/19-fibroblast growth factor receptor 4 which is initiated by activation of farnesoid X receptor in the ileum but not in the liver. This study evaluated whether FGF15/19 protein levels in the portal blood reflected changes in FGF15/19 mRNA in the ileum. Studies were conducted in Sprague Dawley rats and New Zealand white rabbits fed regular chow (controls), supplemented with cholesterol (Ch) or cholic acid (CA). After feeding CA, ileal FGF15 mRNA increased 8.5-fold in rats and FGF19 rose 16-fold in rabbits associated with 62 and 75% reduction of CYP7A1 mRNA, respectively. Neither FGF15 nor FGF19 protein levels changed in the portal blood to correspond with the marked increase of FGF15/19 mRNA levels in the ileum or inhibited CYP7A1 expression in the liver. Further, in Ch-fed rats, CYP7A1 mRNA increased 1.9-fold (P < 0.001) although FGF15 mRNA levels in the ileum and portal blood FGF15 protein levels were not decreased. In Ch-fed rabbits, although FGF19 mRNA levels in the ileum and liver did not increase significantly, CYP7A1 mRNA declined 49% (P < 0.05). We were unable to find corresponding changes of FGF15/19 protein levels in the portal blood in rats and rabbits where the mRNA levels of FGF15/19 in the ileum and CYP7A1 in the liver change significantly.

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

    PubMed Central

    Blom, Elin S.; Wang, Yijing; Skoglund, Lena; Hansson, Anita C.; Ubaldi, Massimo; Lourdusamy, Anbarasu; Sommer, Wolfgang H.; Mielke, Matthew; Hyman, Bradley T.; Heilig, Markus; Lannfelt, Lars; Nilsson, Lars N. G.; Ingelsson, Martin

    2011-01-01

    Several components in the Wnt pathway, including β-catenin and glycogen synthase kinase 3 beta, have been implied in AD pathogenesis. Here, mRNA brain levels from five-month-old tg-ArcSwe and nontransgenic mice were compared using Affymetrix microarray analysis. With surprisingly small overall changes, Wnt signaling was the most affected pathway with altered expression of nine genes in tg-ArcSwe mice. When analyzing mRNA levels of these genes in human brain, transcription factor 7-like 2 (TCF7L2) and v-myc myelocytomatosis viral oncogene homolog (MYC), were increased in Alzheimer's disease (AD) (P < .05). Furthermore, no clear differences in TCF7L2 and MYC mRNA were found in brains with frontotemporal lobar degeneration, suggesting that altered regulation of these Wnt-related genes could be specific to AD. Finally, mRNA levels of three neurogenesis markers were analyzed. Increased mRNA levels of dihydropyrimidinase-like 3 were observed in AD brain, suggesting that altered Wnt pathway regulation may signify synaptic rearrangement or neurogenesis. PMID:21234373

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  3. Serum IL8 and mRNA level of CD11b in circulating neutrophils are increased in clinically amyopathic dermatomyositis with active interstitial lung disease.

    PubMed

    Zou, Jing; Chen, Jie; Yan, Qingran; Guo, Qiang; Bao, Chunde

    2016-01-01

    The objective of this study is to assess serum IL8 and the potential activity of circulating neutrophils on relative messenger RNA (mRNA) levels and their relationship with disease activity in clinically amyopathic dermatomyositis (CADM) associated with interstitial lung disease (ILD). We studied 18 CADM patients and compared them with 18 classic dermatomyositis (DM) patients and 18 healthy control subjects. Serum IL8 level and mRNA expressions of neutrophils (chemokine (C-X-C motif) receptor 1 (CXCR1), cluster of differentiation molecule 11b (CD11b), cluster of differentiation 64 (CD64), myeloid cell leukemia 1 (MCL1), interleukin-18 (IL18)) were detected. The overproduction of serum IL8 level was most significant in the CADM group with active period. The mRNA expressions of CD11b, IL18, and MCL1 were greatly increased in the neutrophils in patients with CADM compared with DM or healthy controls. Up-expressions of CD11b, IL18, and MCL1 were detected in the neutrophils in CADM patients of active period compared with remission period. A positive correlation was found between CD11b mRNA level and high-resolution computed tomography (HRCT) score, in CADM associated with ILD. Serum IL8 level and mRNA levels of CD11b, MCL1, and IL18 in circulating neutrophils are related with the disease activity of CADM-ILD. The mRNA level of CD11b is positively correlated with HRCT score in CADM-ILD.

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

    PubMed Central

    TAMADA, Hiromichi; ADACHI, Nahoko; KAWATE, Noritoshi; INABA, Toshio; HATOYA, Shingo; SAWADA, Tsutomu

    2015-01-01

    Factors involved in patency of uterine cervices in the bitch with pyometra remain to be clarified. This study examined relationship between patency and mRNA levels for inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-1, COX-2 and prostaglandin E synthase (PGES) in the uterine cervix of bitches with pyometra. Cervical patency was measured by inserting the stainless steel rods with different diameter into cervical canals. Levels of mRNA expression were determined by semi-quantitative reverse transcription-polymerase chain reaction. The cervical patency was positively correlated with mRNA levels for COX-2 and PGES, but not those for iNOS and COX-1. The results suggest that gene expression of COX-2 and PGES may be involved in the regulation of patency in the uterine cervix of bitches with pyometra. PMID:26596635

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

    PubMed

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

    2016-03-01

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

  6. Rice Stripe Virus Infection Alters mRNA Levels of Sphingolipid-Metabolizing Enzymes and Sphingolipids Content in Laodelphax striatellus

    PubMed Central

    Li, Fei-Qiang; Bai, Yue-Liang; Shi, Xiao-Xiao; Zhu, Mu-Fei; Zhang, Min-Jing; Mao, Cun-Gui; Zhu, Zeng-Rong

    2017-01-01

    Sphingolipids and their metabolites have been implicated in viral infection and replication in mammal cells but how their metabolizing enzymes in the host are regulated by viruses remains largely unknown. Here we report the identification of 12 sphingolipid genes and their regulation by Rice stripe virus in the small brown planthopper (Laodelphax striatellus Fallén), a serious pest of rice throughout eastern Asia. According to protein sequence similarity, we identified 12 sphingolipid enzyme genes in L. striatellus. By comparing their mRNA levels in viruliferous versus nonviruliferous L. striatellus at different life stages by qPCR, we found that RSV infection upregulated six genes (LsCGT1, LsNAGA1, LsSGPP, LsSMPD4, LsSMS, and LsSPT) in most stages of L. striatellus. Especially, four genes (LsCGT1, LsSMPD2, LsNAGA1, and LsSMS) and another three genes (LsNAGA1, LsSGPP, and LsSMS) were significantly upregulated in viruliferous third-instar and fourth-instar nymphs, respectively. HPLC-MS/MS results showed that RSV infection increased the levels of various ceramides, such as Cer18:0, Cer20:0, and Cer22:0 species, in third and fourth instar L. striatellus nymphs. Together, these results demonstrate that RSV infection alters the transcript levels of various sphingolipid enzymes and the contents of sphingolipids in L. striatellus, indicating that sphingolipids may be important for RSV infection or replication in L. striatellus. PMID:28130458

  7. Effects of epidermal growth factor and platelet-derived growth factor on c-fos and c-myc mRNA levels in normal human fibroblasts

    SciTech Connect

    Paulsson, Y.; Bywater, M.; Westermark, B. ); Heldin, C.H. )

    1987-07-01

    The mRNA levels of two proto-oncogenes, c-fos and c-myc, were determined in human foreskin fibroblasts exposed to epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) in a serum-free, defined medium (MCDB 104). Untreated, quiescent cells were found to have low or undetectable levels of c-fos and c-myc mRNA. Within 10 min after the addition of EGF or PDGF the c-fos mRNA level increased, reached a peak at 30 min, and then declined to the control level after 60 min. The level of c-myc mRNA increased somewhat later and peaked after 8 h in cultures treated with either of the growth factors. The c-myc mRNA level remained elevated throughout the 24 h of investigation. The concentrations of EGF and PDGF required for a maximal effect on c-fos or c-myc expression were found to be similar to those that give maximal effect on cell proliferation. Both c-fos and c-myc mRNA expression were superinduced by the addition of cycloheximide. The present results conform to the view that the c-fos and c-myc proto-oncogenes may be important (or necessary) but not sufficient for the initiation of DNA synthesis. Moreover, the finding that both EGF and PDGF increase c-fos and c-myc expression supports the previous suggestion that these two growth factors may in part act via a common intracellular pathway in the prereplicative phase of human fibroblasts.

  8. Gallium nitrate increases type I collagen and fibronectin mRNA and collagen protein levels in bone and fibroblast cells.

    PubMed

    Bockman, R S; Guidon, P T; Pan, L C; Salvatori, R; Kawaguchi, A

    1993-08-01

    Gallium is a Group IIIa transitional element with therapeutic efficacy in the treatment of metabolic bone disorders. Previously described antiresorptive effects of gallium on osteoclasts are not sufficient to account for the full range of effects of gallium on bone structure and metabolism. We have recently shown that gallium nitrate inhibits osteocalcin gene expression and the synthesis of osteocalcin protein, an osteoblast-specific bone matrix protein that is thought to serve as a signal to trigger osteoclastic resorption. Here we present evidence for an additional mechanism by which gallium may function to augment bone mass by altering matrix protein synthesis by osteoblastic and fibroblastic cells. Rat calvarial explants exposed to gallium nitrate for 48 h showed increased incorporation of 3H-proline into hydroxyproline and collagenase digestible protein. In addition, gallium treatment increased steady-state mRNA levels for fibronectin and type I procollagen chains in primary rat calvarial osteoblast-enriched cultures, the ROS 17/2.8 osteoblastic osteosarcoma line, and nontransformed human dermal fibroblasts. These findings suggest that the exposure of mesenchymally-derived cells to gallium results in an altered pattern of matrix protein synthesis that would favor increased bone formation.

  9. ∆(9)-Tetrahydrocannabinol decreases NOP receptor density and mRNA levels in human SH-SY5Y cells.

    PubMed

    Cannarsa, Rosalia; Carretta, Donatella; Lattanzio, Francesca; Candeletti, Sanzio; Romualdi, Patrizia

    2012-02-01

    Several studies demonstrated a cross-talk between the opioid and cannabinoid system. The NOP receptor and its endogenous ligand nociceptin/orphanin FQ represent an opioid-related functional entity that mediates some non-classical opioid effects. The relationship between cannabinoid and nociceptin/NOP system is yet poorly explored. In this study, we used the neuroblastoma SH-SY5Y cell line to investigate the effect of delta-9-tetrahydrocannabinol (∆(9)-THC) on nociceptin/NOP system. Results revealed that the exposure to ∆(9)-THC (100, 150, and 200 nM) for 24 h produces a dose-dependent NOP receptor B (max) down-regulation. Moreover, ∆(9)-THC caused a dose-dependent decrease in NOP mRNA levels. The selective cannabinoid receptor CB1 antagonist AM251 (1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide) reduces both effects, suggesting that ∆(9)-THC activation of CB1 receptor is involved in the observed effects. These data show evidence of a cross-talk between NOP and CB1 receptors, thus suggesting a possible interplay between cannabinoid and nociceptin/NOP system.

  10. mRNA and Protein levels of rat pancreas specific protein disulphide isomerase are downregulated during Hyperglycemia.

    PubMed

    Gupta, Rajani; Bhar, Kaushik; Sen, Nandini; Bhowmick, Debajit; Mukhopadhyay, Satinath; Panda, Koustubh; Siddhanta, Anirban

    2016-02-01

    Diabetes (Type I and Type II) which affects nearly every organ in the body is a multi-factorial non-communicable disorder. Hyperglycemia is the most characteristic feature of this disease. Loss of beta cells is common in both types of diabetes whose detailed cellular and molecular mechanisms are yet to be elucidated. As this disease is complex, identification of specific biomarkers for its early detection, management and devising new therapies is challenging. Based on the fact that functionally defective proteins provide the biochemical basis for many diseases, in this study, we tried to identify differentially expressed proteins during hyperglycemia. For that, hyperglycemia was induced in overnight fasted rats by intra-peritoneal injection of streptozotocin (STZ). The pancreas was isolated from control and treated rats for subsequent analyses. The 2D-gel electrophoresis followed by MALDI-TOF-MS-MS analyses revealed several up- and down-regulated proteins in hyperglycemic rat pancreas including the downregulation of a pancreas specific isoform of protein disulphide isomerase a2 (Pdia2).This observation was validated by western blot. Quantitative PCR experiments showed that the level of Pdia2 mRNA is also proportionally reduced in hyperglycemic pancreas.

  11. Genome level analysis of rice mRNA 3′-end processing signals and alternative polyadenylation

    PubMed Central

    Shen, Yingjia; Ji, Guoli; Haas, Brian J.; Wu, Xiaohui; Zheng, Jianti; Reese, Greg J.; Li, Qingshun Quinn

    2008-01-01

    The position of a poly(A) site of eukaryotic mRNA is determined by sequence signals in pre-mRNA and a group of polyadenylation factors. To reveal rice poly(A) signals at a genome level, we constructed a dataset of 55 742 authenticated poly(A) sites and characterized the poly(A) signals. This resulted in identifying the typical tripartite cis-elements, including FUE, NUE and CE, as previously observed in Arabidopsis. The average size of the 3′-UTR was 289 nucleotides. When mapped to the genome, however, 15% of these poly(A) sites were found to be located in the currently annotated intergenic regions. Moreover, an extensive alternative polyadenylation profile was evident where 50% of the genes analyzed had more than one unique poly(A) site (excluding microheterogeneity sites), and 13% had four or more poly(A) sites. About 4% of the analyzed genes possessed alternative poly(A) sites at their introns, 5′-UTRs, or protein coding regions. The authenticity of these alternative poly(A) sites was partially confirmed using MPSS data. Analysis of nucleotide profile and signal patterns indicated that there may be a different set of poly(A) signals for those poly(A) sites found in the coding regions. Based on the features of rice poly(A) signals, an updated algorithm termed PASS-Rice was designed to predict poly(A) sites. PMID:18411206

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

    PubMed

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

    2016-08-01

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

  13. Changes in mRNA levels for brain-derived neurotrophic factor after wheel running in rats selectively bred for high- and low-aerobic capacity

    PubMed Central

    Groves-Chapman, Jessica L.; Murray, Patrick S.; Stevens, Kristin L.; Monroe, Derek; Koch, Lauren G.; Britton, Steven L.; Holmes, Philip V.

    2012-01-01

    We evaluated levels of exercise-induced brain-derived neurotrophic factor (BDNF) messenger RNA (mRNA) within the hippocampal formation in rats selectively bred for 1) high intrinsic (i.e., untrained) aerobic capacity (High Capacity Runners, HCR), 2) low intrinsic aerobic capacity (Low Capacity Runners, LCR), and 3) unselected Sprague-Dawley (SD) rats with or without free access to running wheels for three weeks. The specific aim of the study was to determine whether a dose-response relationship exists between cumulative running distance and levels of BDNF mRNA. No additional treatments or behavioral manipulations were used. HCR, LCR, and SD rats were grouped by strain and randomly assigned to sedentary or activity (voluntary access to activity wheel) conditions. Animals were killed after 21 days of exposure to the assigned conditions. Daily running distances (mean ± standard deviation meters/d) during week three were: HCR (4726 ± 3220), SD (2293 ± 3461), LCR (672 ± 323). Regardless of strain, levels of BDNF mRNA in CA1 were elevated in wheel runners compared to sedentary rats and this difference persisted after adjustment for age (p=0.040). BDNF mRNA was not affected by intrinsic aerobic capacity and was not related to total running distance. The results support that BDNF mRNA expression is increased by unlimited access to activity wheel running for 3 weeks but is not dependent upon accumulated running distance. PMID:22024546

  14. Changes in mRNA levels for brain-derived neurotrophic factor after wheel running in rats selectively bred for high- and low-aerobic capacity.

    PubMed

    Groves-Chapman, Jessica L; Murray, Patrick S; Stevens, Kristin L; Monroe, Derek C; Koch, Lauren G; Britton, Steven L; Holmes, Philip V; Dishman, Rod K

    2011-11-24

    We evaluated levels of exercise-induced brain-derived neurotrophic factor (BDNF) messenger RNA (mRNA) within the hippocampal formation in rats selectively bred for 1) high intrinsic (i.e., untrained) aerobic capacity (High Capacity Runners, HCR), 2) low intrinsic aerobic capacity (Low Capacity Runners, LCR), and 3) unselected Sprague-Dawley (SD) rats with or without free access to running wheels for 3 weeks. The specific aim of the study was to determine whether a dose-response relationship exists between cumulative running distance and levels of BDNF mRNA. No additional treatments or behavioral manipulations were used. HCR, LCR, and SD rats were grouped by strain and randomly assigned to sedentary or activity (voluntary access to activity wheel) conditions. Animals were killed after 21 days of exposure to the assigned conditions. Daily running distances (mean ± standard deviation meters/day) during week three were: HCR (4726 ± 3220), SD (2293 ± 3461), LCR (672 ± 323). Regardless of strain, levels of BDNF mRNA in CA1 were elevated in wheel runners compared to sedentary rats and this difference persisted after adjustment for age (p=0.040). BDNF mRNA was not affected by intrinsic aerobic capacity and was not related to total running distance. The results support that BDNF mRNA expression is increased by unlimited access to activity wheel running for 3 weeks but is not dependent upon accumulated running distance.

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

    PubMed

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

    2015-11-01

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

  16. Activation of gene expression by herpes simplex virus type 1 ICP0 occurs at the level of mRNA synthesis.

    PubMed Central

    Jordan, R; Schaffer, P A

    1997-01-01

    ICP0 is a nuclear phosphoprotein involved in the activation of herpes simplex virus type 1 (HSV-1) gene expression during lytic infection and reactivation from viral latency. Although available evidence suggests that ICP0 acts at the level of transcription, definitive studies specifically addressing this issue have not been reported. In the present study we measured the ability of ICP0 to activate gene expression (i) from promoters representing the major kinetic classes of viral genes in transient expression assays and (ii) from the same promoters during viral infection at multiplicities of infection ranging from 0.1 to 5.0 PFU/cell. The levels of synthesis and steady-state accumulation of mRNA, mRNA stability, and levels of protein synthesis were compared in cells transfected with a reporter plasmid in the presence and absence of ICP0 and in cells infected with wild-type HSV-1 or an ICP0 null mutant, n212. In transient expression assays and during viral infection at all multiplicities tested, the levels of steady-state mRNA and protein were significantly lower in the absence of ICP0, indicating that ICP0 activates gene expression at the level of mRNA accumulation. In transient expression assays and during infection at low multiplicities (< 1 PFU/cell) in the presence or absence of ICP0, marked increases in the levels of viral mRNAs accompanied by proportional increases in the levels of protein synthesis were observed with increasing multiplicity. At a high multiplicity (5 PFU/cell) in the presence or absence of ICP0, mRNA levels did not increase as a function of multiplicity and changes in the levels of protein were no longer related to changes in the levels of mRNA. Collectively, these tests indicate that transcription of viral genes is rate limiting at low multiplicities and that translation is rate limiting at high multiplicities, independent of ICP0. Consistent with the lower levels of mRNA detected in the absence of ICP0, the rates of transcription initiation

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

    PubMed

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

    2012-03-01

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

  18. Vanadate and selenium inhibit the triiodothyronine induced enzyme activity and mRNA level for both fatty acid synthase and malic enzyme

    SciTech Connect

    Zhu, Y.; Mirmiran, R.; Goodridge, A.G.; Stapleton, S.R. Western Michigan Univ., Kalamazoo )

    1991-03-15

    In chick-embryo hepatocytes in culture, triiodothyronine stimulates enzyme activity, mRNA level and transcription rate for both fatty acid synthase (FAS) and malic enzyme (ME). Insulin alone has no effect but amplifies the induction by T3. Recent evidence has demonstrated the insulin-mimicking action of vanadate and selenium on various physiological processes. Little information, however, is available on the affects of vanadate and selenium on the expression of genes that are regulated by insulin. These studies were initiated to test the potential of vanadate and selenium to mimic the amplification affect of insulin on the T3 induction of FAS and ME. In chick-embryo hepatocytes incubated in a chemically defined medium, addition of T3 for 48h causes an increase in the enzyme activity and mRNA level for both FAS and ME. Addition of sodium vanadate or sodium selenate (20 {mu}M) coincident with the T3 almost completely inhibited the stimulation of FAS and ME activity and accumulation of their respective mRNA's. Fifty percent maximal inhibition occurred at about 3-40{mu}M vanadate or 5-10{mu}M selenium. Vanadate and selenium similarity inhibited FAS and ME enzyme activity and mRNA level when the cells were incubated in the presence of insulin and T3. The effect of these metals was selective; isocitrate dehydrogenase activity as well as the level of glyceraldehyde 3-phosphate mRNA were not affected by any of the additions made to the cells in culture. This effect by vanadate and selenium also does not appear to be a generalized effect of metals on lipogenic enzymes as molydate under similar experimental conditions has no effect on either the enzyme activity or mRNA level of FAS or ME. Studies are continuing to determine the mechanism of action of these agents on the regulation of lipogenic enzymes.

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

    PubMed

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

    2013-03-01

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

  20. Starvation and diet composition affect mRNA levels of the high density-lipoprotein-beta glucan binding protein in the shrimp Litopenaeus vannamei.

    PubMed

    Muhlia-Almazán, Adriana; Sánchez-Paz, Arturo; García-Carreño, Fernando; Peregrino-Uriarte, Alma Beatriz; Yepiz-Plascencia, Gloria

    2005-10-01

    A high density lipoprotein-beta glucan binding protein (HDL-BGBP) is synthesized in the hepatopancreas of the white shrimp Litopenaeus vannamei and secreted to the hemolymph. Recently, we reported the HDL-BGBP full length cDNA sequence and found that the predicted polypeptide is larger than the mature protein and also, that it contains a long 5'- and 3'-UTRs that may be involved in transcript level regulation. To test whether starvation and feeding may play a role in regulating HDL-BGBP mRNA levels, two different stimuli were evaluated: starvation and composition of diets. After 24 h, the steady state HDL-BGBP mRNA levels of starved shrimp decreased, suggesting that synthesis of the lipoprotein is less required in the absence of food. When shrimp were fed with diets containing different concentrations of protein and lipids, changes in HDL-BGBP mRNA levels were also detected. Shrimp fed the lower concentration of protein and lipid feed accumulated higher levels of HDL-BGBP mRNA. These results indicate that feeding influences HDL-BGBP transcript levels in the hepatopancreas.

  1. Expression of cardiac alpha-actin spares extraocular muscles in skeletal muscle alpha-actin diseases--quantification of striated alpha-actins by MRM-mass spectrometry.

    PubMed

    Ravenscroft, Gianina; Colley, Stephen M J; Walker, Kendall R; Clement, Sophie; Bringans, Scott; Lipscombe, Richard; Fabian, Victoria A; Laing, Nigel G; Nowak, Kristen J

    2008-12-01

    As with many skeletal muscle diseases, the extraocular muscles (EOMs) are spared in skeletal muscle alpha-actin diseases, with no ophthalmoplegia even in severely affected patients. We hypothesised that the extraocular muscles sparing in these patients was due to significant expression of cardiac alpha-actin, the alpha-actin isoform expressed in heart and foetal skeletal muscle. We have shown by immunochemistry, Western blotting and a novel MRM-mass spectrometry technique, comparable levels of cardiac alpha-actin in the extraocular muscles of human, pig and sheep to those in the heart. The sparing of extraocular muscles in skeletal muscle alpha-actin disease is thus probably due to greater levels of cardiac alpha-actin, than the negligible amounts in skeletal muscles, diluting out the effects of the mutant skeletal muscle alpha-actin.

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

    PubMed

    Ohno, Misa; Tsuda, Kyoko; Sakaguchi, Masayoshi; Sugahara, Yasusato; Oyama, Fumitaka

    2012-01-01

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

  3. p53 missense but not truncation mutations are associated with low levels of p21CIP1/WAF1 mRNA expression in primary human sarcomas

    PubMed Central

    Mousses, S; Gokgoz, N; Wunder, J S; Ozcelik, H; Bull, S; Bell, R S; Andrulis, I L

    2001-01-01

    Many growth-suppressing signals converge to control the levels of the CDK inhibitor p21CIP1/WAF1. Some human cancers exhibit low levels of expression of p21CIP1/WAF1and mutations in p53 have been implicated in this down-regulation. To evaluate whether the presence of p53 mutations was related to the in vivo expression of p21CIP1/WAF1 mRNA in sarcomas we measured the p21CIP1/WAF1 mRNA levels for a group of 71 primary bone and soft tissue tumours with known p53 status. As expected, most tumours with p53 mutations expressed low levels of p21CIP1/WAF1 mRNA. However, we identified a group of tumours with p53 gene mutations that exhibited normal or higher levels of p21CIP1/WAF1 mRNA. The p53 mutations in the latter group were not the common missense mutations in exons 4–9, but were predominantly nonsense mutations predicted to result in truncation of the p53 protein. The results of this study suggest that different types of p53 mutations can have different effects on the expression of downstream genes such as p21CIP1/WAF1 in human sarcomas. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11401317

  4. IMPACT OF GESTATIONAL COCAINE TREATMENT OR PRENATAL COCAINE EXPOSURE ON EARLY POSTPARTUM OXYTOCIN mRNA LEVELS AND RECEPTOR BINDING IN THE RAT

    PubMed Central

    McMurray, M.S.; Cox, E.T.; Jarrett, T.M.; Williams, S.K.; Walker, C.H.; Johns, J.M.

    2008-01-01

    Prior research reported decreased oxytocin levels in specific brain regions correlated with disruptions in maternal care following gestational cocaine treatment in rats. Similarly, prenatal exposure to cocaine impaired subsequent maternal behavior in adulthood, but behavioral alterations were not associated with decreases in oxytocin levels in the same brain regions as were found in their cocaine-treated rat dams. To determine if other aspects of the oxytocin system are disrupted by cocaine treatment or prenatal exposure to cocaine during critical time points associated with maternal care, oxytocin mRNA transcription and receptor binding were examined on postpartum day two in relevant brain regions following gestational treatment with, or prenatal exposure to, either cocaine or saline. We hypothesized that oxytocin mRNA levels and receptor binding would be differentially affected by cocaine in the early postpartum period of dams and their offspring. Our findings indicate that gestational cocaine treatment resulted in significant increases in oxytocin mRNA levels in only the paraventricular nucleus of cocaine-treated dams, with almost significant increases in both generations in the supraoptic nucleus, but no significant effects of cocaine on receptor binding in either generation of dams. These findings indicate that in addition to oxytocin levels, cocaine treatment or prenatal exposure primarily affects oxytocin mRNA synthesis, with little effect on receptor binding in specific brain regions associated with maternal behavior in the early postpartum period of the rat. PMID:18579201

  5. ROCK/actin/MRTF signaling promotes the fibrogenic phenotype of fibroblast-like synoviocytes derived from the temporomandibular joint

    PubMed Central

    Yokota, Seiji; Chosa, Naoyuki; Kyakumoto, Seiko; Kimura, Hitomichi; Ibi, Miho; Kamo, Masaharu; Satoh, Kazuro; Ishisaki, Akira

    2017-01-01

    Malocclusion caused by abnormal jaw development or muscle overuse during mastication results in abnormal mechanical stress to the tissues surrounding the temporomandibular joint (TMJ). Excessive mechanical stress against soft and hard tissues around the TMJ is involved in the pathogenesis of inflammatory diseases, including osteoarthritis (OA). OA-related fibrosis is a possible cause of joint stiffness in OA. However, cellular and molecular mechanisms underlying fibrosis around the TMJ remain to be clarified. Here, we established a cell line of fibroblast-like synoviocytes (FLSs) derived from the mouse TMJ. Then, we examined whether the Rho-associated coiled-coil forming kinase (ROCK)/actin/myocardin-related transcription factor (MRTF) gene regulatory axis positively regulates the myofibroblast (MF) differentiation status of FLSs. We found that i) FLSs extensively expressed the MF markers α-smooth muscle actin (α-SMA) and type I collagen; and ii) an inhibitor against the actin-polymerizing agent ROCK, Y-27632; iii) an actin-depolymerizing agent cytochalasin B; iv) an inhibitor of the MRTF/serum response factor-regulated transcription, CCG-100602, clearly suppressed the mRNA levels of α-SMA and type I collagen in FLSs; and v) an MF differentiation attenuator fibroblast growth factor-1 suppressed filamentous actin formation and clearly suppressed the mRNA levels of α-SMA and type I collagen in FLSs. These results strongly suggest that the ROCK/actin/MRTF axis promotes the fibrogenic activity of synoviocytes around the TMJ. Our findings partially clarify the molecular mechanisms underlying the emergence of TMJ-OA and may aid in identifying drug targets for treating this condition at the molecular level. PMID:28259960

  6. Levels of BMP-6 mRNA in goat ovarian follicles and in vitro effects of BMP-6 on secondary follicle development.

    PubMed

    Frota, Isana M A; Leitão, Cintia C F; Costa, José J N; van den Hurk, Robert; Saraiva, Márcia V A; Figueiredo, José R; Silva, José R V

    2013-08-01

    Expression of BMP-6 mRNA was quantified by real-time polymerase chain reaction (PCR) and the BMP-6 protein was demonstrated by immunohistochemistry in the primordial, primary, secondary, small and large antral follicles of goat. Furthermore, the influence of BMP-6 on increase in diameter, antrum formation and expression of BMP-6 and FSH-R in in vitro cultured secondary follicles was studied. Therefore, goat primordial, primary and secondary follicles, as well as small and large antral follicles were obtained and the mRNA levels of BMP-6 were quantified by PCR in real time. Expression of BMP-6 protein in goat follicles was demonstrated by immunohistochemistry. The influence of BMP-6 in the presence or absence of follicle-stimulating hormone (FSH) on both the development of secondary follicles and the expression of mRNA for BMP-6 and FSH-R was evaluated after 6 days of culture. Furthermore, the follicular diameter and the formation of the antrum were evaluated before and after 6 days of culture and compared by Kruskal-Wallis and chi-squared tests (P < 0.05), respectively. The results show that the level of mRNA for BMP-6 in primary and secondary follicles was significantly higher than in the primordial follicles (P < 0.05). Similar levels of BMP-6 mRNA were observed in cumulus-oocyte complexes and mural granulosa/theca cells from small and large antral follicles, respectively. BMP-6 protein was expressed in oocytes of all categories of follicles and in granulosa cells from secondary follicles onwards. Addition of BMP-6 to the culture medium increased the diameter of secondary follicles mainly by antrum formation after 6 days' culture, in the presence or absence of FSH (P < 0.05). Furthermore, addition of FSH resulted in increased levels of BMP-6 mRNA in these follicles (P < 0.05). Simultaneous administration of FSH and BMP-6 enhanced the levels of FSH receptor (FSH-R) mRNA (P < 0.05). It is concluded that BMP-6 mRNA is increased during transition from primordial to

  7. Structural features and gene-expression profiles of actin homologs in Porphyra yezoensis (Rhodophyta).

    PubMed

    Kitade, Yukihiro; Nakamura, Michiko; Uji, Toshiki; Fukuda, Satoru; Endo, Hirotoshi; Saga, Naotsune

    2008-10-15

    The marine red alga Porphyra yezoensis contains an actin gene family consisting of at least four isoforms (PyACT1, 2, 3 and 4). The amino acid identity between isoforms exceeds 83%, and each contains a putative nuclear export signal (NES). We scanned the sequences for amino acids in regions homologous to the intermonomeric interface of actin filaments. Few residues expected to engage in cross-linking were conserved between the four isoforms. The results of the sequence analyses suggest that PyACT2 probably functions in the nucleus as a monomer (G-actin) or in other unconventional forms. In addition, the distribution and position of the introns were different from those in florideophycean actin genes. The expression level of PyACT3 in matured gametophytes was significantly higher than in those in a vegetative state, although the mRNA was detected at similar levels in both apical and basal parts of thalli. The expression levels of PyACT2 and 4, on the other hand, did not change significantly between the matured and vegetative gametophytes. The PyACT3 may serve as a molecular marker for monitoring thallus maturation in this species.

  8. MEIS1 intronic risk haplotype associated with restless legs syndrome affects its mRNA and protein expression levels

    PubMed Central

    Xiong, Lan; Catoire, Hélène; Dion, Patrick; Gaspar, Claudia; Lafrenière, Ronald G.; Girard, Simon L.; Levchenko, Anastasia; Rivière, Jean-Baptiste; Fiori, Laura; St-Onge, Judith; Bachand, Isabelle; Thibodeau, Pascale; Allen, Richard; Earley, Christopher; Turecki, Gustavo; Montplaisir, Jacques; Rouleau, Guy A.

    2009-01-01

    Restless legs syndrome (RLS) is a common neurological disorder characterized by an irresistible urge to move the legs at night, which is often accompanied by unpleasant sensations. A recent genomewide association study identified an association between RLS and intronic markers from the MEIS1 gene. Comparative genomic analysis indicates that MEIS1 is the only gene encompassed in this evolutionarily conserved chromosomal segment, i.e. a conservation synteny block, from mammals to fish. We carried out a series of experiments to delineate the role of MEIS1 in RLS pathogenesis and the underlying genetic mechanism. We sequenced all 13 MEIS1 exons and their splice junctions in 285 RLS probands with confirmed clinical diagnosis and did not identify any causative coding or exon–intron junction mutations. We found no evidence of structural variation or disease-associated haplotype differential splicing. However, sequencing of conserved regions of MEIS1 introns 8 and 9 identified a novel single nucleotide polymorphism (C13B_2) significantly associated with RLS (allelic association, P = 1.81E−07). We detected a significant decrease in MEIS1 mRNA expression by quantitative real-time polymerase chain reaction in lymphoblastoid cell lines (LCLs) and brain tissues from RLS patients homozygous for the intronic RLS risk haplotype, compared with those homozygous for the non-risk haplotype. Finally, we found significantly decreased MEIS1 protein levels in the same batch of LCLs and brain tissues from the homozygous carriers of the risk haplotype, compared with the homozygous non-carriers. Therefore, these data suggest that reduced expression of the MEIS1 gene, possibly through intronic cis-regulatory element(s), predisposes to RLS. PMID:19126776

  9. Effect of Danshen aqueous extract on serum hs-CRP, IL-8, IL-10, TNF-α levels, and IL-10 mRNA, TNF-α mRNA expression levels, cerebral TGF-β1 positive expression level and its neuroprotective mechanisms in CIR rats.

    PubMed

    Liang, Xue-Yun; Li, Hai-Ning; Yang, Xiao-Yan; Zhou, Wen-Yan; Niu, Jian-Guo; Chen, Ben-Dong

    2013-04-01

    To observe the effects of Danshen aqueous extract (DSAE) on the cerebral tissue and nerve stem cells in cerebral ischemia reperfusion (CIR) rats. The model rats were prepared by occlusion of the middle cerebral artery for 2 h and then by reperfusion. They were randomly divided into five groups: a control group, an CIR group and three DSAE-treated groups. As compared with the sham control group, there was significant increase (P < 0.05, P < 0.01) in the serum high-sensitivity C-reactive protein (hs-CRP) and interleukin-8 (IL-8) levels, interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-α) levels, and IL-10 mRNA, TNF-α mRNA expression levels, function score, Infarct size, TUNEL + cell counts, cerebral transforming growth factor beta 1 (TGF-β1) positive expression and cerebral neuron specific enolase (NSE) levels, and decrease in fas-associated protein with death domain (FADD) and death-associated protein (Daxx) positive expression levels in the CIR group. Compared with CIR group, DSAE treatment dose-dependently significantly decreased serum hs-CRP, IL-8, IL-10, TNF-α levels, and IL-10 mRNA, TNF-α mRNA expression levels, function score, Infarct size, TUNEL + cell counts, cerebral TGF-β1 positive expression and cerebral NSE levels, and increase FADD and Daxx positive expression levels in the CIR + DSAE groups. Taken together, these results suggest that DSAE has a neuroprotective role in the CIR rats, which may be related to improvement of immunity function, proteins and genes expression.

  10. High STAT1 mRNA levels but not its tyrosine phosphorylation are associated with macrophage infiltration and bad prognosis in breast cancer

    PubMed Central

    2014-01-01

    Background STAT1 has been attributed a function as tumor suppressor. However, in breast cancer data from microarray analysis indicated a predictive value of high mRNA expression levels of STAT1 and STAT1 target genes belonging to the interferon-related signature for a poor response to therapy. To clarify this issue we have determined STAT1 expression levels and activation by different methods, and investigated their association with tumor infiltration by immune cells. Additionally, we evaluated the interrelationship of these parameters and their significance for predicting disease outcome. Methods Expression of STAT1, its target genes SOCS1, IRF1, CXCL9, CXCL10, CXCL11, IFIT1, IFITM1, MX1 and genes characteristic for immune cell infiltration (CD68, CD163, PD-L1, PD-L2, PD-1, CD45, IFN-γ, FOXP3) was determined by RT-PCR in two independent cohorts comprising 132 breast cancer patients. For a subset of patients, protein levels of total as well as serine and tyrosine-phosphorylated STAT1 were ascertained by immunohistochemistry or immunoblotting and protein levels of CXCL10 by ELISA. Results mRNA expression levels of STAT1 and STAT1 target genes, as well as protein levels of total and serine-phosphorylated STAT1 correlated with each other in neoplastic tissue. However, there was no association between tumor levels of STAT1 mRNA and tyrosine-phosphorylated STAT1 and between CXCL10 serum levels and CXCL10 expression in the tumor. Tumors with increased STAT1 mRNA amounts exhibited elevated expression of genes characteristic for tumor-associated macrophages and immunosuppressive T lymphocytes. Survival analysis revealed an association of high STAT1 mRNA levels and bad prognosis in both cohorts. A similar prognostically relevant correlation with unfavorable outcome was evident for CXCL10, MX1, CD68, CD163, IFN-γ, and PD-L2 expression in at least one collective. By contrast, activation of STAT1 as assessed by the level of STAT1-Y701 phosphorylation was linked to positive

  11. The Expression Level of mRNA, Protein, and DNA Methylation Status of FOSL2 of Uyghur in XinJiang in Type 2 Diabetes

    PubMed Central

    Cao, Guolei; Wang, Xiaoli

    2016-01-01

    Objective. We investigated the expression levels of both FOSL2 mRNA and protein as well as evaluating DNA methylation in the blood of type 2 diabetes mellitus (T2DM) Uyghur patients from Xinjiang. This study also evaluated whether FOSL2 gene expression had demonstrated any associations with clinical and biochemical indicators of T2DM. Methods. One hundred Uyghur subjects where divided into two groups, T2DM and nonimpaired glucose tolerance (NGT) groups. DNA methylation of FOSL2 was also analyzed by MassARRAY Spectrometry and methylation data of individual units were generated by the EpiTyper v1.0.5 software. The expression levels of FOS-like antigen 2 (FOSL2) and the protein expression levels were analyzed. Results. Significant differences were observed in mRNA and protein levels when compared with the NGT group, while methylation rates of eight CpG units within the FOSL2 gene were higher in the T2DM group. Methylation of CpG sites was found to inversely correlate with expression of other markers. Conclusions. Results show that a correlation between mRNA, protein, and DNA methylation of FOSL2 gene exists among T2DM patients from Uyghur. FOSL2 protein and mRNA were downregulated and the DNA became hypermethylated, all of which may be involved in T2DM pathogenesis in this population. PMID:28050569

  12. Effect of sulfide, osmotic, and thermal stresses on taurine transporter mRNA levels in the gills of the hydrothermal vent-specific mussel Bathymodiolus septemdierum.

    PubMed

    Nakamura-Kusakabe, Ikumi; Nagasaki, Toshihiro; Kinjo, Azusa; Sassa, Mieko; Koito, Tomoko; Okamura, Kei; Yamagami, Shosei; Yamanaka, Toshiro; Tsuchida, Shinji; Inoue, Koji

    2016-01-01

    Hydrothermal vent environmental conditions are characterized by high sulfide concentrations, fluctuating osmolality, and irregular temperature elevations caused by vent effluents. These parameters represent potential stressors for organisms that inhabit the area around hydrothermal vents. Here, we aimed to obtain a better understanding of the adaptation mechanisms of marine species to hydrothermal vent environments. Specifically, we examined the effect of sulfide, osmolality, and thermal stress on the expression of taurine transporter (TAUT) mRNA in the gill of the deep-sea mussel Bathymodiolus septemdierum, which is a dominant species around hydrothermal vent sites. We analyzed TAUT mRNA levels by quantitative real-time polymerase chain reaction (PCR) in the gill of mussels exposed to sulfide (0.1 or 1mg/L Na2S·9H2O), hyper- (115% seawater) and hypo- (97.5%, 95.5%, and 85% seawater) osmotic conditions, and thermal stresses (12°C and 20°C) for 24 and 48h. The results showed that mussels exposed to relatively low levels of sulfide (0.1mg/L) and moderate heat stress (12°C) exhibited higher TAUT mRNA levels than the control. Although hyper- and hypo-osmotic stress did not significantly change TAUT mRNA levels, slight induction was observed in mussels exposed to low osmolality. Our results indicate that TAUT is involved in the coping mechanism of mussels to various hydrothermal vent stresses.

  13. Schistocephalus solidus infections increase gonadotropins and gonadotropin releasing hormone (GnRH3) mRNA levels in the three-spined stickleback, Gasterosteus aculeatus.

    PubMed

    Shao, Yi Ta; Tseng, Yung Che; Trombley, Susanne; Hwang, Pung Pung; Schmitz, Monika; Borg, Bertil

    2012-09-01

    Parasites often impair the reproduction of their hosts, one well known case being the cestode Schistocephalus solidus which is a common parasite in three-spined sticklebacks, Gasterosteus aculeatus. One of the possible ways that this could be exerted is by suppression on the brain-pituitary-gonadal (BPG) axis. In this study, mRNA levels of FSH-β and LH-β and of GnRH2 (cGnRH II) and GnRH3 (sGnRH) were measured via Q-PCR in infected and uninfected fish sampled from the field a few weeks before the onset of breeding. The pituitary mRNA levels of both FSH-β and LH-β were higher in infected males than in uninfected males. Also in females, FSH-β mRNA levels were higher in infected individuals than in others, whereas there was no significant difference found in LH-β expression. Brain mRNA levels of GnRH3 were higher in infected fish than in uninfected fish in both sexes, but no difference was found in GnRH2 mRNA levels. Thus, infection by S. solidus was able to alter the expressions not only of gonadotropins (GtHs), but also of GnRH which has not been observed previously. However, the effects are opposite to what should be expected if the parasite suppressed reproduction via actions on the brain-pituitary level. The gonads are perhaps more likely to be impaired by the parasites in other ways, and changed feedbacks on the BPG axis could then lead to the increases in GtHs and GnRH.

  14. Zip1, Zip2, and Zip8 mRNA expressions were associated with growth hormone level during the growth hormone provocation test in children with short stature.

    PubMed

    Sun, Ping; Wang, Shifu; Jiang, Yali; Tao, Yanting; Tian, Yuanyuan; Zhu, Kai; Wan, Haiyan; Zhang, Lehai; Zhang, Lianying

    2013-10-01

    Short stature of children is affected by multiple factors. One of them is growth hormone (GH) deficiency. Growth hormone therapy can increase the final height of children with growth hormone deficiency. Zinc is found to induce dimerization and to enhance the bioactivity of human GH. Two gene families have been identified involved in zinc homeostasis. Previous studies in our laboratory have shown that Zip1, Zip2, Zip6, and ZnT1 mRNA were associated with zinc level in established human breast cancer in nude mice model; Zip8 was significantly lower in zinc-deficient Wistar rats in kidney. In this study, five zinc transporters: Zip1, Zip2, Zip6, Zip8, and ZnT1 were chosen. We aimed to investigate the mRNA expression of zinc transporters and to explore the relationship between zinc transporters and growth hormone in short stature children. Growth hormone provocation test is used to confirm the diagnosis of growth hormone deficiency. Six short children for the test were enrolled. At the same time, 15 sex- and age-matched normal children were enrolled as control. The expression levels of zinc transporters in peripheral blood mononuclear cells were determined by quantitative real-time PCR. Zip1 and Zip2 mRNA expression positively correlated with growth hormone level (r = 0.5133, P = 0.0371; r = 0.6719, P = 0.0032); Zip8 mRNA expression negatively correlated with growth hormone level (r = -0.5264, P = 0.0285) during the test in short stature children. The average expression level of Zip2 was significantly higher and Zip6, Zip8 mRNA levels were significantly lower in short stature children than in health controls at 0 min (P < 0.05, P < 0.05).

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

    PubMed Central

    Staroń, Robert; Van Swelm, Rachel P. L.; Lipiński, Paweł; Gajowiak, Anna; Lenartowicz, Małgorzata; Bednarz, Aleksandra; Gajewska, Małgorzata; Pieszka, Marek; Laarakkers, Coby M. M.; Swinkels, Dorine W.; Starzyński, Rafał R.

    2015-01-01

    Among livestock, domestic pig (Sus scrofa) is a species, in which iron metabolism has been most intensively examined during last decade. The obvious reason for studying the regulation of iron homeostasis especially in young pigs is neonatal iron deficiency anemia commonly occurring in these animals. Moreover, supplementation of essentially all commercially reared piglets with iron entails a need for monitoring the efficacy of this routine practice followed in the swine industry for several decades. Since the discovery of hepcidin many studies confirmed its role as key regulator of iron metabolism and pointed out the assessment of its concentrations in biological fluids as diagnostic tool for iron-related disorder. Here we demonstrate that urine hepcidin-25 levels measured by a combination of weak cation exchange chromatography and time-of-flight mass spectrometry (WCX-TOF MS) are highly correlated with mRNA hepcidin expression in the liver and plasma hepcidin-25 concentrations in anemic and iron-supplemented 28-day old piglets. We also found a high correlation between urine hepcidin level and hepatic non-heme iron content. Our results show that similarly to previously described transgenic mouse models of iron disorders, young pigs constitute a convenient animal model to explore accuracy and relationship between indicators for assessing systemic iron status. PMID:26323096

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

    PubMed

    Staroń, Robert; Van Swelm, Rachel P L; Lipiński, Paweł; Gajowiak, Anna; Lenartowicz, Małgorzata; Bednarz, Aleksandra; Gajewska, Małgorzata; Pieszka, Marek; Laarakkers, Coby M M; Swinkels, Dorine W; Starzyński, Rafał R

    2015-01-01

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

  17. Actin dynamics shape microglia effector functions.

    PubMed

    Uhlemann, Ria; Gertz, Karen; Boehmerle, Wolfgang; Schwarz, Tobias; Nolte, Christiane; Freyer, Dorette; Kettenmann, Helmut; Endres, Matthias; Kronenberg, Golo

    2016-06-01

    Impaired actin filament dynamics have been associated with cellular senescence. Microglia, the resident immune cells of the brain, are emerging as a central pathophysiological player in neurodegeneration. Microglia activation, which ranges on a continuum between classical and alternative, may be of critical importance to brain disease. Using genetic and pharmacological manipulations, we studied the effects of alterations in actin dynamics on microglia effector functions. Disruption of actin dynamics did not affect transcription of genes involved in the LPS-triggered classical inflammatory response. By contrast, in consequence of impaired nuclear translocation of phospho-STAT6, genes involved in IL-4 induced alternative activation were strongly downregulated. Functionally, impaired actin dynamics resulted in reduced NO secretion and reduced release of TNFalpha and IL-6 from LPS-stimulated microglia and of IGF-1 from IL-4 stimulated microglia. However, pathological stabilization of the actin cytoskeleton increased LPS-induced release of IL-1beta and IL-18, which belong to an unconventional secretory pathway. Reduced NO release was associated with decreased cytoplasmic iNOS protein expression and decreased intracellular arginine uptake. Furthermore, disruption of actin dynamics resulted in reduced microglia migration, proliferation and phagocytosis. Finally, baseline and ATP-induced [Ca(2+)]int levels were significantly increased in microglia lacking gelsolin, a key actin-severing protein. Together, the dynamic state of the actin cytoskeleton profoundly and distinctly affects microglia behaviours. Disruption of actin dynamics attenuates M2 polarization by inhibiting transcription of alternative activation genes. In classical activation, the role of actin remodelling is complex, does not relate to gene transcription and shows a major divergence between cytokines following conventional and unconventional secretion.

  18. Tryptophan hydroxylase mRNA levels are elevated by repeated immobilization stress in rat raphe nuclei but not in pineal gland.

    PubMed

    Chamas, F; Serova, L; Sabban, E L

    1999-06-04

    Repeated stress triggers a wide range of adaptive changes in the central nervous system including the elevation of serotonin (5-HT) metabolism and an increased susceptibility to affective disorders. To begin to examine whether these changes are mediated by alterations in gene expression for tryptophan hydroxylase (TPH), the rate-limiting enzyme in 5-HT biosynthesis, we quantitated its mRNA levels by competitive reverse transcription-polymerase chain reaction (RT-PCR). Repeated immobilization stress (2 h, 7 days) elicited a six- or ten-fold rise in TPH mRNA in median raphe nucleus (MRN) and dorsal raphe nucleus (DRN), respectively, without significantly altering TPH mRNA levels in the pineal gland. In contrast, there was little change in mRNA levels for GTP cyclohydrolase I (GTPCH), the rate limiting enzyme in synthesis of the tetrahydrobiopterin (BH4), the obligate cofactor for TPH. This is the first study to reveal stress-elicited activation of TPH gene expression.

  19. Etomoxir, sodium 2-[6-(4-chlorophenoxy)hexyl]oxirane-2-carboxylate, up-regulates uncoupling protein-3 mRNA levels in primary culture of rat preadipocytes.

    PubMed

    Cabrero, A; Alegret, M; Sánchez, R; Adzet, T; Laguna, J C; Vázquez, M

    1999-09-16

    Uncoupling proteins (UCPs) are mitochondrial membrane proton transporters that uncouple respiration from oxidative phosphorylation by dissipating the proton gradient across the membrane. Treatment of primary culture of rat preadipocytes for 24 h with 40 microM etomoxir, an irreversible inhibitor of carnitine palmitoyltransferase I (CPT-I), up-regulated UCP-3 mRNA levels (3. 6-fold induction), whereas changes in UCP-2 mRNA levels were not significant. As a consequence of increased UCP-3 expression, a fall in the mitochondrial membrane potential was detected by flow cytometry. Etomoxir treatment modified neither L-CPT-I (liver-type) nor PPARalpha mRNA levels in preadipocytes. In contrast, mRNA expression of acyl-CoA oxidase (ACO), the rate-limiting enzyme of peroxisomal fatty acid beta-oxidation, whose transcription is controlled by PPARalpha, was significantly induced (1.3-fold induction, P = 0.015). These findings suggest that the effects of etomoxir were mediated by PPARalpha. Since it has been reported that the intracellular accumulation of lipids following the inhibition of CPT-I by etomoxir leads to a PPARalpha-mediated metabolic response that increases the expression of genes involved in alternate fatty acid oxidation pathways, these results seem to implicate UCP-3 in this protective metabolic response. It remains to be studied whether reductions in the expression of UCP-3 could compromise this response, giving rise to lipotoxic effects on cells.

  20. Prostaglandins temporally regulate cytoplasmic actin bundle formation during Drosophila oogenesis.

    PubMed

    Spracklen, Andrew J; Kelpsch, Daniel J; Chen, Xiang; Spracklen, Cassandra N; Tootle, Tina L

    2014-02-01

    Prostaglandins (PGs)--lipid signals produced downstream of cyclooxygenase (COX) enzymes--regulate actin dynamics in cell culture and platelets, but their roles during development are largely unknown. Here we define a new role for Pxt, the Drosophila COX-like enzyme, in regulating the actin cytoskeleton--temporal restriction of actin remodeling during oogenesis. PGs are required for actin filament bundle formation during stage 10B (S10B). In addition, loss of Pxt results in extensive early actin remodeling, including actin filaments and aggregates, within the posterior nurse cells of S9 follicles; wild-type follicles exhibit similar structures at a low frequency. Hu li tai shao (Hts-RC) and Villin (Quail), an actin bundler, localize to all early actin structures, whereas Enabled (Ena), an actin elongation factor, preferentially localizes to those in pxt mutants. Reduced Ena levels strongly suppress early actin remodeling in pxt mutants. Furthermore, loss of Pxt results in reduced Ena localization to the sites of bundle formation during S10B. Together these data lead to a model in which PGs temporally regulate actin remodeling during Drosophila oogenesis by controlling Ena localization/activity, such that in S9, PG signaling inhibits, whereas at S10B, it promotes Ena-dependent actin remodeling.

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

    PubMed

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

    2012-12-01

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

  2. Lower levels of cannabinoid 1 receptor mRNA in female eating disorder patients: association with wrist cutting as impulsive self-injurious behavior.

    PubMed

    Schroeder, Marc; Eberlein, Christian; de Zwaan, Martina; Kornhuber, Johannes; Bleich, Stefan; Frieling, Helge

    2012-12-01

    The cannabinoid 1 (CB 1) receptor as the primary mediator of the endocannabinoid (EC) system was found to play a role in eating disorders (EDs), depression, anxiety, and suicidal behavior. The CB 1 receptor is assumed to play a crucial role in the central reward circuitry with impact on body weight and personality traits like novelty-seeking behavior. In a previous study we found higher levels of CB 1 receptor mRNA in patients with anorexia nervosa (AN) and bulimia nervosa (BN) compared to healthy control women (HCW). The aim of the present study was to investigate the possible influence of the EC and the CB 1 receptor system on wrist cutting as self-injurious behavior (SIB) in women with EDs (n=43; AN: n=20; BN: n=23). Nine ED patients with repetitive wrist cutting (AN, n=4; BN, n=5) were compared to 34 ED patients without wrist cutting and 26 HCW. Levels of CB 1 receptor mRNA were determined in peripheral blood samples using quantitative real-time PCR. ED patients with self-injurious wrist cutting exhibited significantly lower CB 1 receptor mRNA levels compared with ED patients without wrist cutting and HCW. No significant differences were found between ED patients without a history of wrist cutting and HCW. Furthermore, a negative association was detected between CB 1 receptor mRNA levels and Beck Depression Inventory (BDI) scores. To our knowledge, this is the first study reporting a down-regulation of CB 1 receptor mRNA in patients with EDs and wrist cutting as SIB. Due to the small sample size, our results should be regarded as preliminary and further studies are warranted to reveal the underlying mechanisms.

  3. Metabolic hormones regulate basal and growth hormone-dependent igf2 mRNA level in primary cultured coho salmon hepatocytes: effects of insulin, glucagon, dexamethasone, and triiodothyronine.

    PubMed

    Pierce, A L; Dickey, J T; Felli, L; Swanson, P; Dickhoff, W W

    2010-03-01

    Igf1 and Igf2 stimulate growth and development of vertebrates. Circulating Igfs are produced by the liver. In mammals, Igf1 mediates the postnatal growth-promoting effects of growth hormone (Gh), whereas Igf2 stimulates fetal and placental growth. Hepatic Igf2 production is not regulated by Gh in mammals. Little is known about the regulation of hepatic Igf2 production in nonmammalian vertebrates. We examined the regulation of igf2 mRNA level by metabolic hormones in primary cultured coho salmon hepatocytes. Gh, insulin, the glucocorticoid agonist dexamethasone (Dex), and glucagon increased igf2 mRNA levels, whereas triiodothyronine (T(3)) decreased igf2 mRNA levels. Gh stimulated igf2 mRNA at physiological concentrations (0.25x10(-9) M and above). Insulin strongly enhanced Gh stimulation of igf2 at low physiological concentrations (10(-11) M and above), and increased basal igf2 (10(-8) M and above). Dex stimulated basal igf2 at concentrations comparable to those of stressed circulating cortisol (10(-8) M and above). Glucagon stimulated basal and Gh-stimulated igf2 at supraphysiological concentrations (10(-7) M and above), whereas T(3) suppressed basal and Gh-stimulated igf2 at the single concentration tested (10(-7) M). These results show that igf2 mRNA level is highly regulated in salmon hepatocytes, suggesting that liver-derived Igf2 plays a significant role in salmon growth physiology. The synergistic regulation of igf2 by insulin and Gh in salmon hepatocytes is similar to the regulation of hepatic Igf1 production in mammals.

  4. Cholera toxin treatment of vascular smooth muscle cells decreases smooth muscle α-actin content and abolishes the platelet-derived growth factor-BB-stimulated DNA synthesis

    PubMed Central

    Sachinidis, Agapios; Seul, Claudia; Gouni-Berthold, Ioanna; Seewald, Stefan; Ko, Yon; Vetter, Hans; Fingerle, Jürgen; Hoppe, Jürgen

    2000-01-01

    The second messenger cyclic AMP regulates diverse biological processes such as cell morphology and cell growth. We examined the role of the second messenger cyclic AMP on rat aortic vascular smooth muscle cell (VSMC) morphology and the intracellular transduction pathway mediated by platelet-derived growth factor β-receptor (PDGF-Rβ). The effect of PDGF-BB on VSMCs growth was assessed by [3H]-thymidine incorporation. Tyrosine phosphorylation of PDGF-Rβ, PLC-γ1, ERK1 and ERK2, p125FAK and paxillin as well as Sm α-actin was examined by the chemiluminescence Western blotting method. Actin mRNA level was quantitated by Northern blotting. Visualization of Sm α-actin filaments, paxillin and PDGF-Rβ was performed by immunfluorescence microscopy. Cholera toxin (CTX; 10 nM) treatment lead to a large and sustained increase in the cyclic AMP concentration after 2 h which correlated with change of VSMC morphology including complete disruption of the Sm α-actin filament array and loss of focal adhesions. Treatment of VSMCs with CTX did not influence tyrosine phosphorylation of p125FAK and paxillin but decreased the content of a Sm α-actin protein. Maximal decrease of 70% was observed after 24 h of treatment. CTX also caused a 90% decrease of the actin mRNA level. CTX treatment completely abolished PDGF-BB stimulated DNA-synthesis although PDGF-Rβ level and subcellular distribution and translocation was not altered. Furthermore CTX attenuated the PDGF-BB-induced tyrosine phosphorylation of the PDGF-Rβ, PI 3′-K, PLC-γ1 and ERK1/2 indicating an action of cyclic AMP on PDGF-β receptor. We conclude that although cyclic AMP attenuates the PDGF-Rβ mediated intracellular transduction pathway, an intact actin filament may be required for the PDGF-BB-induced DNA synthesis in VSMCs. PMID:10928958

  5. CAP1 was associated with actin and involved in Schwann cell differentiation and motility after sciatic nerve injury.

    PubMed

    Zhu, Xinhui; Yao, Li; Guo, Aisong; Li, Aihong; Sun, Huiqing; Wang, Ning; Liu, Hanzhang; Duan, Zhiqin; Cao, Jianhua

    2014-06-01

    Adenylate cyclase-associated protein 1 (CAP1), a member of cyclase-associated proteins that regulating actin dynamics, was shown to regulate actin filaments, localize to dynamic actin structures and mediate such processes as establishment of cell polarity, motility, morphogenesis, receptor-mediated endocytosis and mRNA location. But little is known about the role of CAP1 during peripheral nervous system injury. Here, we found the spatiotemporal protein expression of CAP1 after sciatic nerve crush. After crush, CAP1 had an increased protein expression level, reached a peak at about day 5 and then returned to the normal level at 4 weeks, similar to Oct-6. Besides, in 5-day injured tissue, using double immunofluorescent staining we found CAP1 had a colocalization with S100 and Oct-6. In vitro, during the process of cAMP-induced Schwann cells differentiation, we observed enhanced expression of CAP1 and P0. Specially, CAP1-specific siRNA-tranfected SCs did not show significant actin structure which form cellure surface tension and protrusion shape after cAMP treatment. And we observed the interaction of CAP1 with actin and that CAP1-specific siRNA-transfected SCs had a decreased motility and migration. Together, all these data indicated that the change of CAP1 protein expression was associated with Schwann cells motility and differentiation after the crush of sciatic nerve.

  6. 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.

  7. Ghrelin modulates fatty acid synthase and related transcription factor mRNA levels in a tissue-specific manner in neonatal broiler chicks.

    PubMed

    Buyse, Johan; Janssen, Sara; Geelissen, Sofie; Swennen, Quirine; Kaiya, Hiroyuki; Darras, Veerle M; Dridi, Sami

    2009-07-01

    The endogenous ligand for the growth hormone (GH) secretagogue receptor ghrelin is a peptide secreted by the stomach of mammals and stimulates food intake and enhances adiposity. In avian species, ghrelin is mainly produced by the proventriculus but reduces food intake whereas its effect on lipogenesis in different tissues is unknown. We therefore investigated the effects of a single intravenous injection of 2.8 microg (1 nmol per chick) recombinant chicken ghrelin in neonatal broiler chicks. Besides food intake and plasma corticosterone levels, mRNA levels of the key lipogenic enzyme fatty acid synthase (FAS) and its related transcription factors sterol regulatory element binding protein-1 (SREBP-1) and peroxisome proliferator-activated receptor-gamma (PPARgamma) were determined in diencephalon, liver and quadriceps femoris muscle before, and 15, 30, and 60 min after injection. Chicken ghrelin administration induced a significant short-term (<30 min) reduction in food intake and markedly elevated plasma corticosterone levels. In diencephalon, FAS, SREBP-1 and PPARgamma mRNA levels were significantly increased within 15 min after ghrelin injection. These observations suggest that central fatty acid metabolism is involved in the anorectic effects of ghrelin. In contrast, hepatic mRNA levels of FAS and both transcription factors were significantly reduced within 30 min after ghrelin injection. In muscle, FAS and transcription factor gene expression was very low and not affected by ghrelin. Overall, our results indicate that ghrelin has opposite effects on FAS and transcription factor mRNA amounts with increased levels in diencephalon (central anorectic effect) and decreased levels in liver (peripheral anti-lipogenic effect) in chickens.

  8. Gene expression in scrapie. Cloning of a new scrapie-responsive gene and the identification of increased levels of seven other mRNA transcripts.

    PubMed

    Dandoy-Dron, F; Guillo, F; Benboudjema, L; Deslys, J P; Lasmézas, C; Dormont, D; Tovey, M G; Dron, M

    1998-03-27

    To define genes associated with or responsible for the neurodegenerative changes observed in transmissible spongiform encephalopathies, we analyzed gene expression in scrapie-infected mouse brain using "mRNA differential display." The RNA transcripts of eight genes were increased 3-8-fold in the brains of scrapie-infected animals. Five of these genes have not previously been reported to exhibit increased expression in this disease: cathepsin S, the C1q B-chain of complement, apolipoprotein D, and two previously unidentified genes denominated scrapie-responsive gene (ScRG)-1 and ScRG-2, which are preferentially expressed in brain tissue. Increased expression of the three remaining genes, beta2 microglobulin, F4/80, and metallothionein II, has previously been reported to occur in experimental scrapie. Kinetic analysis revealed a concomitant increase in the levels of ScRG-1, cathepsin S, the C1q B-chain of complement, and beta2 microglobulin mRNA as well as glial fibrillary acidic protein and F4/80 transcripts, markers of astrocytosis and microglial activation, respectively. In contrast, the level of ScRG-2, apolipoprotein D, and metallothionein II mRNA was only increased at the terminal stage of the disease. ScRG-1 mRNA was found to be preferentially expressed in glial cells and to code for a short protein of 47 amino acids with a strong hydrophobic N-terminal region.

  9. Changes in levels of argininosuccinate lyase mRNA during induction by glucagon and cyclic AMP in cultured foetal-rat hepatocytes.

    PubMed Central

    Renouf, S; Buquet, C; Fairand, A; Benamar, M; Husson, A

    1993-01-01

    During the perinatal period, the activity of the urea-cycle enzyme argininosuccinate lyase (ASL) is regulated by glucocorticoids, glucagon and insulin. In this study, the effects of glucagon and cyclic AMP (cAMP) analogues were examined on the synthesis of ASL and on the level of its corresponding mRNA in cultured foetal hepatocytes. Northern-blot analysis revealed that these agents only gave a transient induction of ASL mRNA amount, which reached a peak at 6 h and declined thereafter. This induction preceded the increase in enzyme activity and amount which could be observed for 2 or 3 days of culture. Stimulation of ASL mRNA accumulation by a combination of cAMP analogues and dexamethasone was additive, indicating that glucocorticoids and cAMP are both necessary to promote hepatocyte differentiation and that inductions could occur via independent pathways. Induction by cAMP analogues could be abolished by actinomycin D, suggesting a control mechanism at the transcriptional level. Puromycin was without effect on ASL mRNA induction by cAMP, indicating that no ongoing protein synthesis was required in the stimulation process. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8387274

  10. Regulation of interleukin 3 mRNA expression in mast cells occurs at the posttranscriptional level and is mediated by calcium ions

    SciTech Connect

    Wodnar-Filipowicz, A.; Moroni, C. )

    1990-01-01

    Interleukin 3 (IL-3) is transiently produced by murine bone marrow-derived mast cells in response to antigen stimulation of the high-affinity immunoglobulin E receptors. The authors have studied the postreceptor signaling pathways involved in regulating expression of the IL-3 gene in the murine mass cell PB-3c. Large amounts of IL-3 mRNA accumulated after exposure of cells to calcium ionophore A23187, a reagent that increases intracellular Ca{sup 2+}. Phorbol 12-myristate 13-acetate, which stimulates protein kinase C, did not induce IL-3 mRNA accumulation, although it did potentiate the effect of A23187. Nuclear run-on analysis showed that the IL-3 gene is constitutively transcribed in unstimulated cells and that treatment with A23187 and/or phorbol ester has no influence on its transcription rate. The effect of A23187 was found to be due to stabilization of the IL-3 mRNA. In cells maintained in the presence of A23187 the IL-3 mRNA was stable during 3 hr of incubation with actinomycin D, whereas removal of A23187 under the same conditions resulted in rapid degradation of the mRNA. These results indicate that control of expression of the IL-3 gene in mast cells is primarily at the posttranscriptional level and that the Ca{sup 2+}-dependent signal-transduction pathway plays an important role in this process. Synthesis of granulocyte/macrophage colony-stimulating factor mRNA in response to A23187 and phorbol ester was found to be subject to both transcriptional and posttranscriptional regulation.

  11. Contraction-induced increases in Na+-K+-ATPase mRNA levels in human skeletal muscle are not amplified by activation of additional muscle mass.

    PubMed

    Nordsborg, Nikolai; Thomassen, Martin; Lundby, Carsten; Pilegaard, Henriette; Bangsbo, Jens

    2005-07-01

    The present study tested the hypothesis that exercise with a large compared with a small active muscle mass results in a higher contraction-induced increase in Na(+)-K(+)-ATPase mRNA expression due to greater hormonal responses. Furthermore, the relative abundance of Na(+)-K(+)-ATPase subunit alpha(1), alpha(2), alpha(3), alpha(4), beta(1), beta(2), and beta(3) mRNA in human skeletal muscle was investigated. On two occasions, eight subjects performed one-legged knee extension exercise (L) or combined one-legged knee extension and bilateral arm cranking (AL) for 5.00, 4.25, 3.50, 2.75, and 2.00 min separated by 3 min of rest. Leg exercise power output was the same in AL and L, but heart rate at the end of each exercise interval was higher in AL compared with L. One minute after exercise, arm venous blood lactate was higher in AL than in L. A higher level of blood epinephrine and norepinephrine was evident 3 min after exercise in AL compared with L. Nevertheless, none of the exercise-induced increases in alpha(1), alpha(2), beta(1), and beta(3) mRNA expression levels were higher in AL compared with L. The most abundant Na(+)-K(+)-ATPase subunit at the mRNA level was beta(1), which was expressed 3.4 times than alpha(2). Expression of alpha(1), beta(2), and beta(3) was less than 5% of the alpha(2) expression, and no reliable detection of alpha(3) and alpha(4) was possible. In conclusion, activation of additional muscle mass does not result in a higher exercise-induced increase in Na(+)-K(+)-ATPase subunit-specific mRNA.

  12. Transduction of light in the suprachiasmatic nucleus: evidence for two different neurochemical cascades regulating the levels of Per1 mRNA and pineal melatonin.

    PubMed

    Paul, K N; Fukuhara, C; Tosini, G; Albers, H E

    2003-01-01

    The suprachiasmatic nucleus (SCN) contains a circadian clock and regulates melatonin synthesis in the pineal gland. Light exposure during the subjective night acutely increases the mRNA levels of the Period (Per)1 gene in the SCN and acutely suppresses melatonin levels in the pineal gland. Activation of N-methyl-D-aspartate (NMDA) receptors in the SCN has been demonstrated to phase-shift the circadian clock in a manner similar to light. We tested the hypothesis that activation of excitatory amino acid (EAA) receptors in the SCN mediates the acute effects of light on Per1 mRNA levels and pineal melatonin. NMDA, injected into the SCN of Syrian hamsters during the night, acutely suppressed melatonin levels in the pineal gland. Both the NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (AP5) and the alpha-amino-3-hydroxy-5-methylisoxazoleproprionic acid (AMPA)/kainate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) inhibited the light-induced increase of Per1 mRNA levels in the SCN. In the same animals, however, these antagonists had no effect on the ability of light to suppress pineal melatonin. These results support the hypothesis that EAA receptor activation in the SCN is necessary for the acute effects of light on Per1 mRNA levels. They also indicate that NMDA receptor activation in the SCN is sufficient but may not be necessary for the acute effects of light on pineal melatonin. These data suggest that there may be at least two different neurochemical cascades that transduce the effects of light in the SCN

  13. Regulation of progastricsin mRNA levels in sea bass (Dicentrarchus labrax) in response to fluctuations in food availability.

    PubMed

    Terova, Genciana; Rimoldi, Simona; Larghi, Stefano; Bernardini, Giovanni; Gornati, Rosalba; Saroglia, Marco

    2007-11-23

    In this study the sea bass (Dicentrarchus labrax) pepsinogen C gene was isolated. The nucleotide sequences of all exons are presented. The organization of the gene is compatible with that of other aspartic proteinases. The predicted 388-residue amino acid (aa) sequence of sea bass pepsinogen C consists of a signal sequence of 16 amino acid residues, an activation peptide of 43 residues, and the mature pepsin of 329 residues containing the two characteristic active-site aspartic acids. We also analyzed fasting-induced changes in the expression of progastricsin mRNA, using real-time RT-PCR absolute quantification. Progastricsin mRNA copy number was downregulated under conditions of negative energy balance, such as starvation, and upregulated during positive energy balance, such as refeeding. These findings offer new information about the sea bass progastricsin gene and support a role of this gastric digestive enzyme in the regulation of food intake in sea bass.

  14. Atrophy of myoepithelial cells in parotid glands of diabetic mice; detection using skeletal muscle actin, a novel marker☆

    PubMed Central

    Nashida, Tomoko; Yoshie, Sumio; Haga-Tsujimura, Maiko; Imai, Akane; Shimomura, Hiromi

    2013-01-01

    In mouse parotid glands, we found expression of skeletal muscle actin (actin-α1) protein and mRNA. We isolated myoepithelial cells from the mouse parotid glands and investigated their actin-α1 expression because smooth muscle actin (actin-α2) has been used as a marker for myoepithelial cells. We used actin-α1 expression to identify pathological changes in diabetic non-obese diabetic (NOD; NOD/ShiJcl) mice—a mouse model for Sjögren's syndrome—and found myoepithelial cells to be decreased or atrophied in the diabetic state. PMID:23772384

  15. Different Relationship between hsp70 mRNA and hsp70 Levels in the Heat Shock Response of Two Salmonids with Dissimilar Temperature Preference

    PubMed Central

    Lewis, Mario; Götting, Miriam; Anttila, Katja; Kanerva, Mirella; Prokkola, Jenni M.; Seppänen, Eila; Kolari, Irma; Nikinmaa, Mikko

    2016-01-01

    The heat shock response (HSR) refers to the rapid production of heat shock proteins (hsps) in response to a sudden increase in temperature. Its regulation by heat shock factors is a good example of how gene expression is transcriptionally regulated by environmental stresses. In contrast, little is known about post-transcriptional regulation of the response. The heat shock response is often used to characterize the temperature tolerance of species with the rationale that whenever the response sets on, a species is approaching its lethal temperature. It has commonly been considered that an increase in hsp mRNA gives an accurate indication that the same happens to the protein level, but this need not be the case. With climate change, understanding the effects of temperature on gene expression of especially polar organisms has become imperative to evaluate how both biodiversity and commercially important species respond, since temperature increases are expected to be largest in polar areas. Here we studied the HSR of two phylogenetically related Arctic species, which differ in their temperature tolerance with Arctic charr having lower maximally tolerated temperature than Atlantic salmon. Arctic charr acclimated to 15°C and exposed to 7°C temperature increase for 30 min showed both an increase in hsp70 mRNA and hsp70 whereas in salmon only hsp70 mRNA increased. Our results indicate that the temperature for transcriptional induction of hsp can be different from the one required for a measurable change in inducible hsp level. The species with lower temperature tolerance, Arctic charr, are experiencing temperature stress already at the higher acclimation temperature, 15°C, as their hsp70 mRNA and hsp70 levels were higher, and they grow less than fish at 8°C (whereas for salmon the opposite is true). Consequently, charr experience more drastic heat shock than salmon. Although further studies are needed to establish the temperature range and length of exposure where hsp

  16. Different Relationship between hsp70 mRNA and hsp70 Levels in the Heat Shock Response of Two Salmonids with Dissimilar Temperature Preference.

    PubMed

    Lewis, Mario; Götting, Miriam; Anttila, Katja; Kanerva, Mirella; Prokkola, Jenni M; Seppänen, Eila; Kolari, Irma; Nikinmaa, Mikko

    2016-01-01

    The heat shock response (HSR) refers to the rapid production of heat shock proteins (hsps) in response to a sudden increase in temperature. Its regulation by heat shock factors is a good example of how gene expression is transcriptionally regulated by environmental stresses. In contrast, little is known about post-transcriptional regulation of the response. The heat shock response is often used to characterize the temperature tolerance of species with the rationale that whenever the response sets on, a species is approaching its lethal temperature. It has commonly been considered that an increase in hsp mRNA gives an accurate indication that the same happens to the protein level, but this need not be the case. With climate change, understanding the effects of temperature on gene expression of especially polar organisms has become imperative to evaluate how both biodiversity and commercially important species respond, since temperature increases are expected to be largest in polar areas. Here we studied the HSR of two phylogenetically related Arctic species, which differ in their temperature tolerance with Arctic charr having lower maximally tolerated temperature than Atlantic salmon. Arctic charr acclimated to 15°C and exposed to 7°C temperature increase for 30 min showed both an increase in hsp70 mRNA and hsp70 whereas in salmon only hsp70 mRNA increased. Our results indicate that the temperature for transcriptional induction of hsp can be different from the one required for a measurable change in inducible hsp level. The species with lower temperature tolerance, Arctic charr, are experiencing temperature stress already at the higher acclimation temperature, 15°C, as their hsp70 mRNA and hsp70 levels were higher, and they grow less than fish at 8°C (whereas for salmon the opposite is true). Consequently, charr experience more drastic heat shock than salmon. Although further studies are needed to establish the temperature range and length of exposure where hsp

  17. Involvement of the Ras/extracellular signal-regulated kinase signalling pathway in the regulation of ERCC-1 mRNA levels by insulin.

    PubMed Central

    Lee-Kwon, W; Park, D; Bernier, M

    1998-01-01

    Expression of DNA repair enzymes, which includes ERCC-1, might be under the control of hormonal and growth factor stimulation. In the present study it was observed that insulin increased ERCC-1 mRNA levels both in Chinese hamster ovary cells overexpressing human insulin receptors (HIRc cells) and in fully differentiated 3T3-L1 adipocytes. To investigate the mechanisms underlying the increase in ERCC-1 gene expression in HIRc cells, we used a variety of pharmacological tools known to inhibit distinct signalling pathways. None of these inhibitors affected the amount of ERCC-1 mRNA in unstimulated cells. The pretreatment of cells with two chemically unrelated phosphatidylinositol 3'-kinase inhibitors, wortmannin and LY294002, failed to block the doubling of ERCC-1 mRNA content by insulin. Similarly, inhibition of pp70 S6 kinase by rapamycin had no apparent effects on this insulin response. In contrast, altering the p21(ras)-dependent pathway with either manumycin, an inhibitor of Ras farnesylation, or PD98059, an inhibitor of the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK) kinase, suppressed the induction of ERCC-1 mRNA by insulin (P<0.001). Furthermore inhibition of RNA and protein synthesis negatively regulated the expression of this insulin-regulated gene (P<0.005). These results suggest that insulin enhances ERCC-1 mRNA levels by the activation of the Ras-ERK-dependent pathway without the involvement of the phosphatidylinositol 3'-kinase/pp70 S6 kinase. PMID:9531502

  18. RNA/DNA ratio and LPL and MyoD mRNA expressions in muscle of Oreochromis niloticus fed with elevated levels of palm oil

    NASA Astrophysics Data System (ADS)

    Ayisi, Christian Larbi; Zhao, Jinliang

    2016-02-01

    Palm oil is of great potential as one of the sustainable alternatives to fish oil (FO) in aquafeeds. In this present study, five isonitrogenous diets (32% crude protein) with elevated palm oil levels of 0%, 2%, 4%, 6% and 8% were used during an 8-week feeding trial to evaluate its effects on RNA/DNA ratio and lipoprotein lipase (LPL) and MyoD mRNA expressions in muscle of Oreochromis niloticus. The results showed that RNA, DNA content as well as ratio of RNA to DNA were significantly affected ( P < 0.05), in each case the highest was recorded in fish group subjected to 6% palm oil level. There was a strong positive correlation between nucleic acid concentration (RNA concentration and RNA: DNA ratio) and specific growth rate (SGR), protein efficiency ratio (PER), while a negative correlation existed between nucleic acid concentration (RNA concentration and RNA: DNA ratio) and feed conversion ratio (FCR). The mRNA expressions of LPL and MyoD in muscle were not significantly affected by the different palm oil levels, although the highest expression was observed in fish fed with 6% palm oil level. There also existed a strong positive correlation between the mRNA expression of LPL, MyoD and SGR, PER, while their correlation with FCR was negative. In conclusion, elevated palm oil affected the RNA, DNA concentration as well as RNA/DNA ratio significantly, although the mRNA expression of LPL and MyoD were not affected significantly by elevated palm oil levels.

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

    PubMed

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

    2016-02-01

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

  20. Actin Mechanics and Fragmentation*

    PubMed Central

    De La Cruz, Enrique M.; Gardel, Margaret L.

    2015-01-01

    Cell physiological processes require the regulation and coordination of both mechanical and dynamical properties of the actin cytoskeleton. Here we review recent advances in understanding the mechanical properties and stability of actin filaments and how these properties are manifested at larger (network) length scales. We discuss how forces can influence local biochemical interactions, resulting in the formation of mechanically sensitive dynamic steady states. Understanding the regulation of such force-activated chemistries and dynamic steady states reflects an important challenge for future work that will provide valuable insights as to how the actin cytoskeleton engenders mechanoresponsiveness of living cells. PMID:25957404

  1. Increased TRPV1 and PAR2 mRNA expression levels are associated only with the esophageal reflux symptoms, but not with the extraesophageal reflux symptoms

    PubMed Central

    Kim, Jin Joo; Kim, Nayoung; Choi, Yoon Jin; Kim, Joo Sung; Jung, Hyun Chae

    2016-01-01

    Abstract Transient receptor potential vanilloid-1 (TRPV1) receptor and proteinase-activated receptor 2 (PAR2) have been implicated in the mechanism of acid-induced inflammation in gastroesophageal reflux disease (GERD). We aimed to evaluate TRPV1 and PAR2 mRNA expression levels in the GERD patients and their relationship with endoscopic findings and reflux symptoms. Sixteen healthy controls, 45 patients with erosive reflux disease (ERD), and 14 nonerosive reflux disease (NERD) patients received endoscopy and completed questionnaires. Quantitative real-time polymerase chain reactions (qPCR) of TRPV1, glial cell line-derived neurotrophic factor (GDNF), nerve growth factor (NGF), PAR2, and interleukin (IL)-8 were performed in the distal esophagus specimen. The levels of TRPV1, GDNF, NGF, PAR2, and IL-8 mRNA expression were highest in the ERD group followed by NERD and control groups and the differences between control and ERD groups were statistically significant. Within the ERD group, patients with grade B in Los Angeles (LA) classification showed significantly higher levels of TRPV1, GDNF, and NGF mRNA expression than those with grade A. Presence of reflux symptoms was associated with significant higher levels of TRPV1, PAR2, and IL-8. Notably not extraesophageal but esophageal reflux symptoms were significantly associated with them. Upregulation of TRPV1 and PAR2 pathways might play a role in the development of distal esophageal inflammation and reflux symptoms. And extraesophageal reflux symptoms might not be associated with these processes. PMID:27512850

  2. Increased TRPV1 and PAR2 mRNA expression levels are associated only with the esophageal reflux symptoms, but not with the extraesophageal reflux symptoms.

    PubMed

    Kim, Jin Joo; Kim, Nayoung; Choi, Yoon Jin; Kim, Joo Sung; Jung, Hyun Chae

    2016-08-01

    Transient receptor potential vanilloid-1 (TRPV1) receptor and proteinase-activated receptor 2 (PAR2) have been implicated in the mechanism of acid-induced inflammation in gastroesophageal reflux disease (GERD). We aimed to evaluate TRPV1 and PAR2 mRNA expression levels in the GERD patients and their relationship with endoscopic findings and reflux symptoms.Sixteen healthy controls, 45 patients with erosive reflux disease (ERD), and 14 nonerosive reflux disease (NERD) patients received endoscopy and completed questionnaires. Quantitative real-time polymerase chain reactions (qPCR) of TRPV1, glial cell line-derived neurotrophic factor (GDNF), nerve growth factor (NGF), PAR2, and interleukin (IL)-8 were performed in the distal esophagus specimen.The levels of TRPV1, GDNF, NGF, PAR2, and IL-8 mRNA expression were highest in the ERD group followed by NERD and control groups and the differences between control and ERD groups were statistically significant. Within the ERD group, patients with grade B in Los Angeles (LA) classification showed significantly higher levels of TRPV1, GDNF, and NGF mRNA expression than those with grade A. Presence of reflux symptoms was associated with significant higher levels of TRPV1, PAR2, and IL-8. Notably not extraesophageal but esophageal reflux symptoms were significantly associated with them.Upregulation of TRPV1 and PAR2 pathways might play a role in the development of distal esophageal inflammation and reflux symptoms. And extraesophageal reflux symptoms might not be associated with these processes.

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

    PubMed

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

    2016-01-01

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

  4. Actin Polymerization is Stimulated by Actin Crosslinking Protein Palladin

    PubMed Central

    Gurung, Ritu; Yadav, Rahul; Brungardt, Joseph G.; Orlova, Albina; Egelman, Edward H.; Beck, Moriah R.

    2016-01-01

    The actin scaffold protein palladin regulates both normal cell migration and invasive cell motility, processes that require the coordinated regulation of actin dynamics. However, the potential effect of palladin on actin dynamics has remained elusive. Here we show that the actin binding immunoglobulin-like domain of palladin, which is directly responsible for both actin binding and bundling, also stimulates actin polymerization in vitro. Palladin eliminated the lag phase that is characteristic of the slow nucleation step of actin polymerization. Furthermore, palladin dramatically reduced depolymerization, slightly enhanced the elongation rate, and did not alter the critical concentration. Microscopy and in vitro crosslinking assays reveal differences in actin bundle architecture when palladin is incubated with actin before or after polymerization. These results suggest a model whereby palladin stimulates a polymerization-competent form of G-actin, akin to metal ions, either through charge neutralization or conformational changes. PMID:26607837

  5. High level of full-length cereblon mRNA in lower risk myelodysplastic syndrome with isolated 5q deletion is implicated in the efficacy of lenalidomide.

    PubMed

    Jonasova, Anna; Bokorova, Radka; Polak, Jaroslav; Vostry, Martin; Kostecka, Arnost; Hajkova, Hana; Neuwirtova, Radana; Siskova, Magda; Sponerova, Dana; Cermak, Jaroslav; Mikulenkova, Dana; Cervinek, Libor; Brezinova, Jana; Michalova, Kyra; Fuchs, Ota

    2015-07-01

    Downregulation of cereblon (CRBN) gene expression is associated with resistance to the immunomodulatory drug lenalidomide and poor survival outcomes in multiple myeloma (MM) patients. However, the importance of CRBN gene expression in patients with myelodysplastic syndrome (MDS) and its impact on lenalidomide therapy are not clear. In this study, we evaluate cereblon expression in mononuclear cells isolated from bone marrow [23 lower risk MDS patients with isolated 5q deletion (5q-), 37 lower risk MDS patients with chromosome 5 without the deletion of long arms (non-5q-), and 24 healthy controls] and from peripheral blood (38 patients with 5q-, 52 non-5q- patients and 25 healthy controls) to gain insight into, firstly, the role of cereblon in lower risk MDS patients with or without 5q deletion and, secondly, into the mechanisms of lenalidomide action. Patients with 5q- lower risk MDS have the highest levels of CRBN mRNA in comparison with both lower risk MDS without the deletion of long arms of chromosome 5 and healthy controls. CRBN gene expression was measured using the quantitative TaqMan real-time PCR. High levels of CRBN mRNA were detected in all lenalidomide responders during the course of therapy. A significant decrease of the CRBN mRNA level during lenalidomide treatment is associated with loss of response to treatment and disease progression. These results suggest that, similar to the treatment of MM, high levels of full-length CRBN mRNA in lower risk 5q- patients are necessary for the efficacy of lenalidomide.

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

    SciTech Connect

    Egloff, Caroline; Crump, Doug; Porter, Emily; Williams, Kim L.; Letcher, Robert J.; Gauthier, Lewis T.; Kennedy, Sean W.

    2014-09-15

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

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

    PubMed

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

    2016-07-01

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

  8. Effect of gsp oncogene on somatostatin receptor subtype 1 and 2 mRNA levels in GHRH-responsive GH3 cells.

    PubMed

    Kim, Eunhee; Sohn, Sookjin; Lee, Mina; Park, Cheolyoung; Jung, Jeechang; Park, Seungjoon

    2005-01-01

    Growth hormone releasing hormone (GHRH) signals via G protein-coupled receptors (GHRH-R) to enhance intracellular Galphas/adenylyl cyclase/cAMP signaling, which in turn has positive effects on GH synthesis and release, as well as proliferation of the GH-producing cells of the anterior pituitary gland. Some GH-producing pituitary tumors express a constitutively active mutant form of Galphas (gsp oncogene). It has been reported that these tumors are more responsive to octreotide therapy. In this study we used a rat GH-producing cell line (GH3) stably transfected with the human GHRH-R cDNA (GH3-GHRHR cells) as a model to study the effects of gsp oncogene on somatostatin (SRIH) receptor subtype 1 and 2 (sst1 and sst2) mRNA levels. Transient transfection of gsp oncogene in GH3-GHRHR cells for 48 h increased intracellular cAMP levels and GH release. Phosphodiesterase (PDE) 4, sst1 and sst2 mRNA levels were increased by G protein mutation as assessed by real-time RT-PCR. Increased PDE mRNA levels in gsp-transfected cells may be a compensatory mechanism to the constitutive activation of cAMP-dependent pathway by G protein mutation and is consistent with reports of higher PDE expression in human pituitary tumor that express gsp. Our data suggest that higher expression of sst1 and sst2 mRNA induced by the gsp oncogene may be a mechanism by which gsp-positive tumors show a greater response to SRIH. GH3 cells permanently transfected with GHRH-R can be used for in vitro studies of actions of GHRH.

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

    SciTech Connect

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

    2010-11-01

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

  10. Noise Stress-Induced Changes in mRNA Levels of Corticotropin-Releasing Hormone Family Molecules and Glucocorticoid Receptors in the Rat Brain.

    PubMed

    Eraslan, E; Akyazi, İ; Ergül-Ekiz, E; Matur, E

    2015-01-01

    Noise is a widespread stress resource that may lead to detrimental effects on the health. However, the molecular basis of the stress response caused by noise remains elusive. We have studied the effects of acute and chronic noise stress on stress-related molecules in the hypothalamus and hippocampus and also corticosterone responses. Sprague Dawley rats were randomized into control, acute and chronic noise stress groups. While the chronic noise stress group animals were exposed to 100 dB white noise for 4 h/a day during 30 days, the acute noise stress group of animals was exposed to the same level of stress once for 4 h. The expression profiles of corticotropin releasing hormone (CRH), CRH1, CRH2 receptors and glucocorticoid receptor (GR) mRNAs were analysed by RT-PCR. Chronic noise stress upregulated CRH mRNA levels in the hypothalamus. Both acute and chronic noise increased CRH-R1 mRNA in the hypothalamus but decreased it in the hippocampus. GR mRNA levels were decreased by chronic noise stress in the hippocampus. The present results suggest that while corticosterone responses have habituated to continuous noise stress, the involvement of CRH family molecules and glucocorticoid receptors in the noise stress responses are different and structure specific.

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

    PubMed

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

    2016-11-01

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

  12. Axonal actin in action: Imaging actin dynamics in neurons.

    PubMed

    Ladt, Kelsey; Ganguly, Archan; Roy, Subhojit

    2016-01-01

    Actin is a highly conserved, key cytoskeletal protein involved in numerous structural and functional roles. In neurons, actin has been intensively investigated in axon terminals-growth cones-and dendritic spines, but details about actin structure and dynamics in axon shafts have remained obscure for decades. A major barrier in the field has been imaging actin. Actin exists as soluble monomers (G-actin) as well as actin filaments (F-actin), and labeling actin with conventional fluorescent probes like GFP/RFP typically leads to a diffuse haze that makes it difficult to discern kinetic behaviors. In a recent publication, we used F-actin selective probes to visualize actin dynamics in axons, resolving striking actin behaviors that have not been described before. However, using these probes to visualize actin dynamics is challenging as they can cause bundling of actin filaments; thus, experimental parameters need to be strictly optimized. Here we describe some practical methodological details related to using these probes for visualizing F-actin dynamics in axons.

  13. [Actin in the wound healing process].

    PubMed

    Nowak, Dorota; Popow-Woźniak, Agnieszka; Raźnikiewicz, Linda; Malicka-Błaszkiewicz, Maria

    2009-01-01

    Wound healing is an important biological process of crucial value for organisms survival and retention of its proper functions. The recognition of molecular mechanisms of these phenomenon is still under investigation. The transition of mesenchymal fibroblasts to myofibroblasts is a key point in wound healing. The contraction ability of myofibroblast enables the shrinkage of a wound and closes its edges. Alpha smooth muscle actin (alpha-SMA), one of six actin isoforms, is a marker of compeletely differentiated myofibroblast. The regulation of differentiation process depends on many growth factors (especially TGF beta 1), the level of active thymosin beta 4, extracellular matrix proteins--including fibronectin, and also on specificity of microenvironment. Thymosin beta 4 is responsible for maintenance of pool of monomeric actin and actin filaments depolymerization. It can also act as a transcription factor, migration stimulator and immunomodulator, so this protein deserves for more attention in wound healing research field.

  14. Dietary soya protein intake and exercise training have an additive effect on skeletal muscle fatty acid oxidation enzyme activities and mRNA levels in rats.

    PubMed

    Morifuji, Masashi; Sanbongi, Chiaki; Sugiura, Katsumi

    2006-09-01

    Exercise training and regular physical activity increase oxidation of fat. Enhanced oxidation of fat is important for preventing lifestyle diseases such as hypertension and obesity. The aim of the present study in rats was to determine whether intake of dietary soya protein and exercise training have an additive effect on the activity and mRNA expression of enzymes involved in skeletal muscle fatty acid oxidation. Male Sprague-Dawley rats (n 32) were assigned randomly into four groups (eight rats per group) and then divided further into sedentary or exercise-trained groups fed either casein or soya protein diets. Rats in the exercise groups were trained for 2 weeks by swimming for 120 min/d, 6 d/week. Exercise training decreased hepatic triacylglycerol levels and retroperitoneal adipose tissue weight and increased skeletal muscle carnitine palmitoyltransferase 1 (CPT1) activity and mRNA expression of CPT1, beta-hydroxyacyl-CoA dehydrogenase (HAD), acyl-CoA oxidase, PPARgamma coactivator 1alpha (PGC1alpha) and PPARalpha. Soya protein significantly decreased hepatic triacylglycerol levels and epididymal adipose tissue weight and increased skeletal muscle CPT1 activity and CPT1, HAD, acyl-CoA oxidase, medium-chain acyl-CoA dehydrogenase, PGC1alpha and PPARalpha mRNA levels. Furthermore, skeletal muscle HAD activity was the highest in exercise-trained rats fed soya protein. We conclude that exercise training and soya protein intake have an important additive role on induction of PPAR pathways, leading to increased activity and mRNA expression of enzymes involved in fatty acid oxidation in skeletal muscle and reduced accumulation of body fat.

  15. MHC2TA mRNA levels and human herpesvirus 6 in multiple sclerosis patients treated with interferon beta along two-year follow-up

    PubMed Central

    2012-01-01

    Background In previous studies we found that MHC2TA +1614 genotype frequency was very different when MS patients with and without human herpesvirus 6 (HHV-6) in serum samples were compared; a different clinical behavior was also described. The purpose of the study was: 1. To evaluate if MHC2TA expression in MS patients was influenced by interferon beta (IFN-beta) treatment. 2. To study MHC2TA expression in MS patients with and without minor allele C. 3. To analyze the relation between MHC2TA mRNA levels and HHV-6 active infection in MS patients. Methods Blood and serum samples of 154 MS patients were collected in five programmed visits: basal (prior to beginning IFN-beta treatment), six, twelve, eighteen and twenty-four months later. HHV-6 in serum and MHC2TA mRNA levels were evaluated by PCR and RT-PCR, respectively. Neutralizing antibodies (NAbs) against IFN-beta were analyzed by the cytopathic effect assay. Results We found that MHC2TA mRNA levels were significantly lower among MS patients with HHV-6 active infection at the basal visit (without treatment) than in those MS patients without HHV-6 active infection at the basal visit (p = 0.012); in all the positive samples we only found variant A. Furthermore, 58/99 (58.6%) MS patients without HHV-6 along the five programmed visits and an increase of MHC2TA expression after two-years of IFN-beta treatment were clinical responders vs. 5/21 (23.8%) among those MS patients with HHV-6 and a decrease of MHC2TA mRNA levels along the two-years with IFN-beta treatment (p = 0.004); no differences were found between patients with and without NAbs. Conclusions MHC2TA mRNA levels could be decreased by the active replication of HHV-6; the absence of HHV-6 in serum and the increase of MHC2TA expression could be further studied as markers of good clinical response to IFN-beta treatment. PMID:23009575

  16. Simultaneous exposure of excess fluoride and calcium deficiency alters VDR, CaR, and calbindin D 9 k mRNA levels in rat duodenal mucosa.

    PubMed

    Tiwari, S; Gupta, S K; Kumar, K; Trivedi, R; Godbole, M M

    2004-10-01

    Fluoride ingestion reduces intestinal calcium absorption; its molecular basis has not been studied. We studied the mRNA expression of calcium-sensing receptor (CaR), vitamin D receptor (VDR) and calbindin D 9 k (D 9 k) by northern blot analysis in the duodenal mucosa of rats. Weanling pups fed with chow diet containing adequate calcium (0.5% w/w) and drinking water (NaF < 1 ppm) served as controls (Group I) and were studied at 9 and 15 weeks. The pups, born to rats fed with a calcium-deficient diet (0.03%) and excess fluoride water (NaF 50 ppm), were continued on the same diet and water (Group II) until 9 weeks of age. Subsequently, Group II rats were divided into 4 subgroups; 3 subgroups with fluoride free water [II-A adequate calcium, II-B excess calcium (Ca 2%) and II-D calcium deficient], whereas II-C received fluorinated water and adequate calcium diet until 15 weeks. At 9 weeks, as compared to group-I, group-II had decreased VDR (P < 0.001) and D 9 k mRNA (P < 0.001), whereas CaR mRNA levels increased (P < 0.05). At 15 weeks, as compared to group-I, VDR mRNA further reduced in group II-D (P < 0.001) and II-C (P < 0.001), whereas it increased in group II-A. Removal of fluoride ingestion and calcium replenishment increased D 9 k mRNA expression, maximally in adequate calcium group (P < 0.001), while it was further reduced in group II-C (P < 0.001). CaR expression decreased significantly in all the groups. We conclude that excess fluoride reduces the mRNA levels of VDR and D 9 k in the duodenal mucosa of rats, thereby possibly reducing calcium absorption. Calcium supplementation with simultaneous fluoride removal improves their expression.

  17. Effects of high environmental ammonia on branchial ammonia excretion rates and tissue Rh-protein mRNA expression levels in seawater acclimated Dungeness crab Metacarcinus magister.

    PubMed

    Martin, Michael; Fehsenfeld, Sandra; Sourial, Mary M; Weihrauch, Dirk

    2011-10-01

    In the present study of the marine Dungeness crabs Metacarcinus magister, the long term effects of high environmental ammonia (HEA) on hemolymph ammonia and urea concentrations, branchial ammonia excretion rates and mRNA expression levels of the crustacean Rh-like ammonia transporter (RhMM), H(+)-ATPase (subunit B), Na(+)/K(+)-ATPase (α-subunit) and Na(+)/H(+)-exchanger (NHE) were investigated. Under control conditions, the crabs' hemolymph exhibited a total ammonia concentration of 179.3±14.5μmol L(-1), while urea accounted for 467.2±33.5μmol L(-1), respectively. Both anterior and posterior gills were capable of excreting ammonia against a 16-fold inwardly directed gradient. Under control conditions, mRNA expression levels of RhMM were high in the gills in contrast to very low expression levels in all other tissues investigated, including the antennal gland, hepatopancreas, and skeletal muscle. After exposure to 1mmol L(-1) NH(4)Cl, hemolymph ammonia increased within the first 12h to ca. 500µmol L(-1) and crabs were able the keep this hemolymph ammonia level for at least 4 days. During this initial period, branchial RhMM and H(+)-ATPase (subunit B) mRNA expression levels roughly doubled. After 14 days of HEA exposure, hemolymph ammonia raised up to environmental levels, whereas urea levels increased by ca. 30%. At the same time, whole animal ammonia and urea excretion vanished. Additionally, branchial RhMM, H(+)-ATPase, Na(+)/K(+)-ATPase and NHE mRNA levels decreased significantly after long term HEA exposure, whereas expression levels of RhMM in the internal tissues increased substantially. Interestingly, crabs acclimated to HEA showed no mortality even after 4 weeks of HEA exposure. This suggests that M. magister possesses a highly adaptive mechanism to cope with elevated ammonia concentrations in its body fluids, including an up-regulation of an Rh-like ammonia transporter in the internal tissues and excretion or storage of waste nitrogen in a so far

  18. Effects of estrogen on food intake, serum leptin levels and leptin mRNA expression in adipose tissue of female rats.

    PubMed

    Fungfuang, Wirasak; Terada, Misao; Komatsu, Noriyuki; Moon, Changjong; Saito, Toru R

    2013-09-01

    The integration of metabolism and reproduction involves complex interactions of hypothalamic neuropeptides with metabolic hormones, fuels, and sex steroids. Of these, estrogen influences food intake, body weight, and the accumulation and distribution of adipose tissue. In this study, the effects of estrogen on food intake, serum leptin levels, and leptin mRNA expression were evaluated in ovariectomized rats. Seven-week-old female Wistar-Imamichi rats were ovariectomized and divided into three treatment groups: group 1 (the control group) received sesame oil, group 2 was given 17β-estradiol benzoate, and group 3 received 17β-estradiol benzoate plus progesterone. The body weight and food consumption of each rat were determined daily. Serum leptin levels and leptin mRNA expression were measured by ELISA and quantitative RT-PCR, respectively. Food consumption in the control group was significantly higher (P<0.05) than that in groups 2 and 3, although body weight did not significantly differ among the three groups. The serum leptin concentration and leptin mRNA expression were significantly higher (P<0.05) in groups 2 and 3 than in group 1, but no significant difference existed between groups 2 and 3. In conclusion, estrogen influenced food intake via the modulation of leptin signaling pathway in ovariectomized rats.

  19. Effects of antiepileptic drugs on mRNA levels of BDNF and NT-3 and cell neogenesis in the developing rat brain.

    PubMed

    Shi, Xiu-Yu; Wang, Ji-Wen; Cui, Hong; Li, Bao-Min; Lei, Ge-Fei; Sun, Ruo-Peng

    2010-03-01

    Epilepsy is a common neurological disorder that occurs more frequently in childhood than in adulthood. Antiepileptic drugs (AEDs) which are used to treat seizures in pregnant women, infants, and young children may cause cognitive impairment or other uncertain injury. However, the exact mechanisms responsible for adverse effects of AEDs in the developing brain are still not clear. In the present study, we investigate the effects of AEDs on mRNA levels of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), cell neogenesis and mossy fiber sprouting (MFS) in the developing rat brain. Long-term treatment with Phenobarbital (40mg/kg), valproate (100mg/kg) and topiramate (40mg/kg) reduces BDNF and NT-3 mRNA expression in the developing brain, while lamotrigine reduces mRNA expression only at high dose level (80mg/kg). Cell neogenesis only increases in the rats treated with valproate and lamotrigine. And no differences are observed between the control group and the AEDs-treated groups in the Timm scores of the CA3 region and supragranular region. Our findings present some possible mechanisms to explain why different AEDs cause different cognitive impairment.

  20. Chemotaxis and Actin Oscillations

    NASA Astrophysics Data System (ADS)

    Bodenschatz, Eberhard; Hsu, Hsin-Fang; Negrete, Jose; Beta, Carsten; Pumir, Alain; Gholami, Azam; Tarantola, Marco; Westendorf, Christian; Zykov, Vladimir

    Recently, self-oscillations of the cytoskeletal actin have been observed in Dictyostelium, a model system for studying chemotaxis. Here we report experimental results on the self-oscillation mechanism and the role of regulatory proteins and myosin II. We stimulate cells rapidly and periodically by using photo un-caging of the chemoattractant in a micro-fluidic device and measured the cellular responses. We found that the response amplitude grows with stimulation strength only in a very narrow region of stimulation, after which the response amplitude reaches a plateau. Moreover, the frequency-response is not constant but rather varies with the strength of external stimuli. To understand the underlying mechanism, we analyzed the polymerization and de-polymerization time in the single cell level. Despite of the large cell-to-cell variability, we found that the polymerization time is independent of external stimuli and the de-polymerization time is prolonged as the stimulation strength increases. Our conclusions will be summarized and the role of noise in the signaling network will be discussed. German Science Foundation CRC 937.

  1. Alterations in trace element levels and mRNA expression of Hsps and inflammatory cytokines in livers of duck exposed to molybdenum or/and cadmium.

    PubMed

    Cao, Huabin; Gao, Feiyan; Xia, Bing; Zhang, Mengmeng; Liao, Yilin; Yang, Zhi; Hu, Guoliang; Zhang, Caiying

    2016-03-01

    To evaluate the effects of dietary Molybdenum (Mo) or/and Cadmium (Cd) on trace elements and the mRNA expression levels of heat shock proteins (Hsps) and inflammatory cytokines in duck livers. 240 healthy 11-day-old ducks were randomly divided into six groups with 40 ducks in each group, which were treated with Mo or/and Cd at different doses on the basal diet for 120 days. On days 30, 60, 90 and 120, 10 birds in each group were randomly selected and euthanized and then the livers were collected to determine the contents of Mo, Cd, copper (Cu), iron (Fe), zine (Zn), Selenium (Se) and the mRNA expression levels of Hsps, inflammatory cytokines. In addition, liver tissues at 120 days were subjected to histopathological analysis with the optical microscope. The results showed that the mRNA expression of Hsp60, Hsp70, Hsp90, tumor necrosis factor-α (TNF-α), nuclear factor-kappa B (NF-κB), and cyclooxygenase-2 (COX-2) were significantly (P<0.01) upregulated in combination groups; Contents of Cu, Fe, Zn, and Se decreased in combined groups (P<0.05) in the later period of the test while contents of Mo and Cd significantly increased (P<0.01); Furthermore severe hepatocyte diffuse fatty, hepatic cords swelling, hepatic sinusoid disappeared, and inflammatory cells infiltrated around the hepatic central vein were observed in Mo combined with Cd groups. The results indicated that dietary Mo or/and Cd might lead to stress, inflammatory response, tissue damage and disturb homeostasis of trace elements in duck livers. Moreover the two elements showed a possible synergistic relationship. And the high mRNA expression of HSPs and inflammatory cytokines may play a role in the resistance of liver toxicity induced by Mo and Cd.

  2. 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.

  3. Polycation induced actin bundles.

    PubMed

    Muhlrad, Andras; Grintsevich, Elena E; Reisler, Emil

    2011-04-01

    Three polycations, polylysine, the polyamine spermine and the polycationic protein lysozyme were used to study the formation, structure, ionic strength sensitivity and dissociation of polycation-induced actin bundles. Bundles form fast, simultaneously with the polymerization of MgATP-G-actins, upon the addition of polycations to solutions of actins at low ionic strength conditions. This indicates that nuclei and/or nascent filaments bundle due to attractive, electrostatic effect of polycations and the neutralization of repulsive interactions of negative charges on actin. The attractive forces between the filaments are strong, as shown by the low (in nanomolar range) critical concentration of their bundling at low ionic strength. These bundles are sensitive to ionic strength and disassemble partially in 100 mM NaCl, but both the dissociation and ionic strength sensitivity can be countered by higher polycation concentrations. Cys374 residues of actin monomers residing on neighboring filaments in the bundles can be cross-linked by the short span (5.4Å) MTS-1 (1,1-methanedyl bismethanethiosulfonate) cross-linker, which indicates a tight packing of filaments in the bundles. The interfilament cross-links, which connect monomers located on oppositely oriented filaments, prevent disassembly of bundles at high ionic strength. Cofilin and the polysaccharide polyanion heparin disassemble lysozyme induced actin bundles more effectively than the polylysine-induced bundles. The actin-lysozyme bundles are pathologically significant as both proteins are found in the pulmonary airways of cystic fibrosis patients. Their bundles contribute to the formation of viscous mucus, which is the main cause of breathing difficulties and eventual death in this disorder.

  4. Biological implications of estrogen and androgen effects on androgen receptor and its mRNA levels in human uterine endometrium.

    PubMed

    Fujimoto, J; Nishigaki, M; Hori, M; Ichigo, S; Itoh, T; Tamaya, T

    1995-06-01

    It has been shown that some effects of testosterone are different from those of its 5 alpha-reduced metabolite, dihydrotestosterone. Briefly, activities of testosterone might be related to cellular differentiation, whereas dihydrotestosterone acts on cellular proliferation. The number of testosterone binding sites in the uterine endometrium was increased by estradiol dipropionate, and this increase was down-regulated by testosterone cypionate. Dihydrotestosterone-specific binding sites in the endometrium were not modulated by estradiol dipropionate and testosterone cypionate. The dissociation constants of the binding sites for testosterone and dihydrotestosterone were not altered by these steroids. Estradiol dipropionate with or without testosterone cypionate induced androgen receptor mRNA expression in the endometrium. In conclusion, testosterone might predominantly affect cellular differentiation in the endometrium.

  5. VEGF-A immunohistochemical and mRNA expression in tissues and its serum levels in potentially malignant oral lesions and oral squamous cell carcinomas.

    PubMed

    Nayak, Seema; Goel, Madhu Mati; Chandra, Saumya; Bhatia, Vikram; Mehrotra, Divya; Kumar, Sandeep; Makker, Annu; Rath, S K; Agarwal, S P

    2012-03-01

    The aim of the study was to investigate whether the estimation of circulating Vascular endothelial growth factor-A (VEGF-A) levels by ELISA could be used as surrogate of VEGF-A expression in tissues of pre-malignant oral lesions (PMOLs) and oral squamous cell carcinoma (OSCC) as compared to that in healthy controls. The study samples comprised of tissue and blood samples from 60 PMOLs, 60 OSCC, and 20 healthy controls. Serum VEGF-A levels were determined by an ELISA based assay (Quantikine human VEGF; R & D System, Minneapolis USA). Tissue VEGF-A expression and microvessel density (MVD) were assessed by immunohistochemistry (IHC) using antibodies against VEGF-A and CD-34 on formalin fixed paraffin embedded (FFPE) tissue sections. VEGF-A mRNA expression was analyzed by real-time PCR in snap frozen tissues. Serum VEGF-A levels and immunohistochemical VEGF-A expression were significantly high in PMOLs and OSCC in comparison with controls. VEGF mRNA gene expression showed more than 50-fold increase in PMOLs and OSCC. VEGF-A levels in serum correlated in a linear fashion with the tissue expression in oral pre-malignant and malignant lesions, suggesting that the serum levels may serve as surrogate material for tissue expression of VEGF-A.

  6. cAMP-induced actin cytoskeleton remodelling inhibits MKL1-dependent expression of the chemotactic and pro-proliferative factor, CCN1

    PubMed Central

    Duggirala, Aparna; Kimura, Tomomi E.; Sala-Newby, Graciela B.; Johnson, Jason L.; Wu, Yih-Jer; Newby, Andrew C.; Bond, Mark

    2015-01-01

    Elevation of intracellular cAMP concentration has numerous vascular protective effects that are in part mediated via actin cytoskeleton-remodelling and subsequent regulation of gene expression. However, the mechanisms are incompletely understood. Here we investigated whether cAMP-induced actin-cytoskeleton remodelling modulates VSMC behaviour by inhibiting expression of CCN1. In cultured rat VSMC, CCN1-silencing significantly inhibited BrdU incorporation and migration in a wound healing assay. Recombinant CCN1 enhanced chemotaxis in a Boyden chamber. Adding db-cAMP, or elevating cAMP using forskolin, significantly inhibited CCN1 mRNA and protein expression in vitro; transcriptional regulation was demonstrated by measuring pre-spliced CCN1 mRNA and CCN1-promoter activity. Forskolin also inhibited CCN1 expression in balloon injured rat carotid arteries in vivo. Inhibiting RhoA activity, which regulates actin-polymerisation, by cAMP-elevation or pharmacologically with C3-transferase, or inhibiting its downstream kinase, ROCK, with Y27632, significantly inhibited CCN1 expression. Conversely, expression of constitutively active RhoA reversed the inhibitory effects of forskolin on CCN1 mRNA. Furthermore, CCN1 mRNA levels were significantly decreased by inhibiting actin-polymerisation with latrunculin B or increased by stimulating actin-polymerisation with Jasplakinolide. We next tested the role of the actin-dependent SRF co-factor, MKL1, in CCN1 expression. Forskolin inhibited nuclear translocation of MKL1 and binding of MKL1 to the CCN1 promoter. Constitutively-active MKL1 enhanced basal promoter activity of wild-type but not SRE-mutated CCN1; and prevented forskolin inhibition. Furthermore, pharmacological MKL-inhibition with CCG-1423 significantly inhibited CCN1 promoter activity as well as mRNA and protein expression. Our data demonstrates that cAMP-induced actin-cytoskeleton remodelling regulates expression of CCN1 through MKL1: it highlights a novel c

  7. cAMP-induced actin cytoskeleton remodelling inhibits MKL1-dependent expression of the chemotactic and pro-proliferative factor, CCN1.

    PubMed

    Duggirala, Aparna; Kimura, Tomomi E; Sala-Newby, Graciela B; Johnson, Jason L; Wu, Yih-Jer; Newby, Andrew C; Bond, Mark

    2015-02-01

    Elevation of intracellular cAMP concentration has numerous vascular protective effects that are in part mediated via actin cytoskeleton-remodelling and subsequent regulation of gene expression. However, the mechanisms are incompletely understood. Here we investigated whether cAMP-induced actin-cytoskeleton remodelling modulates VSMC behaviour by inhibiting expression of CCN1. In cultured rat VSMC, CCN1-silencing significantly inhibited BrdU incorporation and migration in a wound healing assay. Recombinant CCN1 enhanced chemotaxis in a Boyden chamber. Adding db-cAMP, or elevating cAMP using forskolin, significantly inhibited CCN1 mRNA and protein expression in vitro; transcriptional regulation was demonstrated by measuring pre-spliced CCN1 mRNA and CCN1-promoter activity. Forskolin also inhibited CCN1 expression in balloon injured rat carotid arteries in vivo. Inhibiting RhoA activity, which regulates actin-polymerisation, by cAMP-elevation or pharmacologically with C3-transferase, or inhibiting its downstream kinase, ROCK, with Y27632, significantly inhibited CCN1 expression. Conversely, expression of constitutively active RhoA reversed the inhibitory effects of forskolin on CCN1 mRNA. Furthermore, CCN1 mRNA levels were significantly decreased by inhibiting actin-polymerisation with latrunculin B or increased by stimulating actin-polymerisation with Jasplakinolide. We next tested the role of the actin-dependent SRF co-factor, MKL1, in CCN1 expression. Forskolin inhibited nuclear translocation of MKL1 and binding of MKL1 to the CCN1 promoter. Constitutively-active MKL1 enhanced basal promoter activity of wild-type but not SRE-mutated CCN1; and prevented forskolin inhibition. Furthermore, pharmacological MKL-inhibition with CCG-1423 significantly inhibited CCN1 promoter activity as well as mRNA and protein expression. Our data demonstrates that cAMP-induced actin-cytoskeleton remodelling regulates expression of CCN1 through MKL1: it highlights a novel c

  8. Altered sex hormone concentrations and gonadal mRNA expression levels of activin signaling factors in hatchling alligators from a contaminated Florida lake.

    PubMed

    Moore, Brandon C; Kohno, Satomi; Cook, Robert W; Alvers, Ashley L; Hamlin, Heather J; Woodruff, Teresa K; Guillette, Louis J

    2010-04-01

    Activins and estrogens participate in regulating the breakdown of ovarian germ cell nests and follicle assembly in mammals. In 1994, our group reported elevated frequencies of abnormal, multioocytic ovarian follicles in 6 month old, environmental contaminant-exposed female alligators after gonadotropin challenge. Here, we investigated if maternal contribution of endocrine disrupting contaminants to the egg subsequently alters estrogen/inhibin/activin signaling in hatchling female offspring, putatively predisposing an increased frequency of multioocytic follicle formation. We quantified basal and exogenous gonadotropin-stimulated concentrations of circulating plasma steroid hormones and ovarian activin signaling factor mRNA abundance in hatchling alligators from the same contaminated (Lake Apopka) and reference (Lake Woodruff) Florida lakes, as examined in 1994. Basal circulating plasma estradiol and testosterone concentrations were greater in alligators from the contaminated environment, whereas activin/inhibin betaA subunit and follistatin mRNA abundances were lower than values measured in ovaries from reference lake animals. Challenged, contaminant-exposed animals showed a more robust increase in plasma estradiol concentration following an acute follicle stimulating hormone (FSH) challenge compared with reference site alligators. Aromatase and follistatin mRNA levels increased in response to an extended FSH challenge in the reference site animals, but not in the contaminant-exposed animals. In hatchling alligators, ovarian follicles have not yet formed; therefore, these endocrine differences are likely to affect subsequent ovarian development, including ovarian follicle assembly.

  9. Quantitation of mRNA levels of steroid 5alpha-reductase isozymes: a novel method that combines quantitative RT-PCR and capillary electrophoresis.

    PubMed

    Torres, Jesús M; Ortega, Esperanza

    2004-01-01

    A novel, accurate, rapid and modestly labor-intensive method has been developed to quantitate specific mRNA species by reverse transcription-polymerase chain reaction (RT-PCR). This strategy combines the high degree of specificity of competitive PCR with the sensitivity of laser-induced fluorescence capillary electrophoresis (LIF-CE). The specific target mRNA and a mimic DNA fragment, used as an internal standard (IS), were co-amplified in a single reaction in which the same primers are used. The amount of mRNA was then quantitated by extrapolation from the standard curve generated with the internal standard. PCR primers were designed to amplify both a 185 bp fragment of the target cDNA for steroid 5alpha-reductase 1 (5alpha-R1) and a 192 bp fragment of the target cDNA for steroid 5alpha-reductase type 2 (5alpha-R2). The 5' forward primers were end-labeled with 6-carboxy-fluorescein (6-FAM). Two synthetic internal standard DNAs of 300 bp were synthesized from the sequence of plasmid pEGFP-C1. The ratio of fluorescence intensity between amplified products of the target cDNA (185 or 192 bp fragments) and the competitive DNA (300 bp fragment) was determined quantitatively after separation by capillary electrophoresis and fluorescence analysis. The accurate quantitation of low-abundance mRNAs by the present method allows low-level gene expression to be characterized.

  10. High throughput detection of miRNAs and gene-specific mRNA at the single-cell level by flow cytometry

    PubMed Central

    Porichis, Filippos; Hart, Meghan G.; Griesbeck, Morgane; Everett, Holly L.; Hassan, Muska; Baxter, Amy E.; Lindqvist, Madelene; Miller, Sara M.; Soghoian, Damien Z.; Kavanagh, Daniel G.; Reynolds, Susan; Norris, Brett; Mordecai, Scott K.; Nguyen, Quan; Lai, Chunfai; Kaufmann, Daniel E.

    2014-01-01

    Fluorescent in situ hybridization (FISH) is a method that uses fluorescent probes to detect specific nucleic acid sequences at the single cell level. Here we describe optimized protocols that exploit a highly sensitive FISH method based on branched DNA technology to detect mRNA and miRNA in human leukocytes. This technique can be multiplexed and combined with fluorescent antibody protein staining to addressa variety of questions in heterogeneous cell populations. We demonstrate antigen-specific upregulation of IFNγ and IL-2 mRNAs in HIV- and CMV-specific T cells. We show simultaneous detection of cytokine mRNA and corresponding protein in single cells. We apply this method to detect mRNAs for which flow antibodies against the corresponding proteins are poor or are not available. We use this technique to show modulation of a microRNA critical for T cell function, miR-155. We adapt this assay for simultaneous detection of mRNA and proteins by Image Stream technology. PMID:25472703

  11. Alpha1-chimaerin, a Rac1 GTPase-activating protein, is expressed at reduced mRNA levels in the brain of Alzheimer's disease patients

    PubMed Central

    Kato, Tomoko; Konishi, Yoshihiro; Shimohama, Shun; Beach, Thomas G.; Akatsu, Hiroyasu; Tooyama, Ikuo

    2015-01-01

    Alpha1-chimaerin is a GTPase-activating protein (GAP) for Rac1, a member of the Rho small GTPase family, whose action leads to the inactivation of Rac1. Rac1 activity is upregulated in Alzheimer's disease, but little is known about the role of α1-chimaerin. In this study, we investigated the expression and localization of α1-chimaerin mRNA in postmortem human brains from patients with Alzheimer's disease and control subjects. In situ hybridization studies demonstrated that α1-chimaerin was expressed by neurons in the neo-cortex of the temporal lobe and the hippocampus of both controls and Alzheimer's disease cases, with the signal intensity dramatically decreased in patients with Alzheimer's disease. Real-time PCR analysis confirmed a significant reduction of α1-chimaerin mRNA expression in the temporal cortex of Alzheimer's disease cases. In contrast, α2-chimaerin mRNA levels showed no significant difference between the groups. The present study showed reduced α1-chimaerin expression in the brain of Alzheimer's disease cases, suggesting a role in the upregulation of Rac1 activity during the disease process. PMID:25676811

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

    PubMed

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

    2006-10-01

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

  13. Changes in serum hyaluronic acid levels and expression of CD44 and CD44 mRNA in hepatic sinusoidal endothelial cells after major hepatectomy in cirrhotic rats.

    PubMed

    Saegusa, Shotaro; Isaji, Shuji; Kawarada, Yoshifumi

    2002-06-01

    Serum hyaluronic acid (HA) is widely distributed in connective tissues, and the majority of circulating HA is degraded by hepatic sinusoidal endothelial cells (SECs) via a receptor recycling pathway. Our previous clinical study revealed that monitoring serum HA levels after hepatectomy is useful in predicting the development of liver failure. In the present study, to determine the mechanism of the high HA levels after hepatectomy, especially in patients with liver cirrhosis, expression of the major HA receptor, CD44, and its mRNA was investigated in SECs isolated from rats with thioacetamide-induced liver cirrhosis subjected to 70% hepatectomy (group I) and from rats with a normal liver that were subjected to 70% hepatectomy (group II). The 48-hour postoperative survival rate in group I (13.3%) was significantly lower than in group II (100%). In group II, the expression of CD44 mRNA had increased significantly at 6 hours after hepatectomy, and this was followed by progressive increases in expression of CD44, indicating activation of SEC function. The increased serum HA levels after hepatectomy in group II became normal as CD44 expression increased. By contrast, the expression of CD44 and CD44 mRNA in group I was markedly attenuated after hepatectomy. The very low CD44 expression was followed by a significant and sustained increase in serum HA levels, indicating functional failure of the SECs. These results suggest that the significantly impaired functional reserve of SECs in liver cirrhosis is associated with increased mortality after 70% hepatectomy.

  14. Tudor-SN, a component of stress granules, regulates growth under salt stress by modulating GA20ox3 mRNA levels in Arabidopsis.

    PubMed

    Yan, Chunxia; Yan, Zongyun; Wang, Yizheng; Yan, Xiaoyuan; Han, Yuzhen

    2014-11-01

    The Tudor-SN protein (TSN) is universally expressed and highly conserved in eukaryotes. In Arabidopsis, TSN is reportedly involved in stress adaptation, but the mechanism involved in this adaptation is not understood. Here, we provide evidence that TSN regulates the mRNA levels of GA20ox3, a key enzyme for gibberellin (GA) biosynthesis. The levels of GA20ox3 transcripts decreased in TSN1/TSN2 RNA interference (RNAi) transgenic lines and increased in TSN1 over-expression (OE) transgenic lines. The TSN1 OE lines displayed phenotypes that may be attributed to the overproduction of GA. No obvious defects were observed in the RNAi transgenic lines under normal conditions, but under salt stress conditions these lines displayed slower growth than wild-type (WT) plants. Two mutants of GA20ox3, ga20ox3-1 and -2, also showed slower growth under stress than WT plants. Moreover, a higher accumulation of GA20ox3 transcripts was observed under salt stress. The results of a western blot analysis indicated that higher levels of TSN1 accumulated after salt treatment than under normal conditions. Subcellular localization studies showed that TSN1 was uniformly distributed in the cytoplasm under normal conditions but accumulated in small granules and co-localized with RBP47, a marker protein for stress granules (SGs), in response to salt stress. The results of RNA immunoprecipitation experiments indicated that TSN1 bound GA20ox3 mRNA in vivo. On the basis of these findings, we conclude that TSN is a novel component of plant SGs that regulates growth under salt stress by modulating levels of GA20ox3 mRNA.

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

    PubMed

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

    2015-08-10

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

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

    PubMed

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

    2014-01-01

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

  17. Absolute Measurements of Macrophage Migration Inhibitory Factor and Interleukin-1-β mRNA Levels Accurately Predict Treatment Response in Depressed Patients

    PubMed Central

    Ferrari, Clarissa; Uher, Rudolf; Bocchio-Chiavetto, Luisella; Riva, Marco Andrea; Pariante, Carmine M.

    2016-01-01

    Background: Increased levels of inflammation have been associated with a poorer response to antidepressants in several clinical samples, but these findings have had been limited by low reproducibility of biomarker assays across laboratories, difficulty in predicting response probability on an individual basis, and unclear molecular mechanisms. Methods: Here we measured absolute mRNA values (a reliable quantitation of number of molecules) of Macrophage Migration Inhibitory Factor and interleukin-1β in a previously published sample from a randomized controlled trial comparing escitalopram vs nortriptyline (GENDEP) as well as in an independent, naturalistic replication sample. We then used linear discriminant analysis to calculate mRNA values cutoffs that best discriminated between responders and nonresponders after 12 weeks of antidepressants. As Macrophage Migration Inhibitory Factor and interleukin-1β might be involved in different pathways, we constructed a protein-protein interaction network by the Search Tool for the Retrieval of Interacting Genes/Proteins. Results: We identified cutoff values for the absolute mRNA measures that accurately predicted response probability on an individual basis, with positive predictive values and specificity for nonresponders of 100% in both samples (negative predictive value=82% to 85%, sensitivity=52% to 61%). Using network analysis, we identified different clusters of targets for these 2 cytokines, with Macrophage Migration Inhibitory Factor interacting predominantly with pathways involved in neurogenesis, neuroplasticity, and cell proliferation, and interleukin-1β interacting predominantly with pathways involved in the inflammasome complex, oxidative stress, and neurodegeneration. Conclusion: We believe that these data provide a clinically suitable approach to the personalization of antidepressant therapy: patients who have absolute mRNA values above the suggested cutoffs could be directed toward earlier access to more

  18. Facioscapulohumeral muscular dystrophy region gene 1 is a dynamic RNA-associated and actin-bundling protein.

    PubMed

    Sun, Chia-Yun Jessica; van Koningsbruggen, Silvana; Long, Steven W; Straasheijm, Kirsten; Klooster, Rinse; Jones, Takako I; Bellini, Michel; Levesque, Lyne; Brieher, William M; van der Maarel, Silvère M; Jones, Peter L

    2011-08-12

    FSHD region gene 1 (FRG1) is a dynamic nuclear and cytoplasmic protein that, in skeletal muscle, shows additional localization to the sarcomere. Maintaining appropriate levels of FRG1 protein is critical for muscular and vascular development in vertebrates; however, its precise molecular function is unknown. This study investigates the molecular functions of human FRG1, along with mouse FRG1 and Xenopus frg1, using molecular, biochemical, and cellular-biological approaches, to provide further insight into its roles in vertebrate development. The nuclear fraction of the endogenous FRG1 is localized in nucleoli, Cajal bodies, and actively transcribed chromatin; however, contrary to overexpressed FRG1, the endogenous FRG1 is not associated with nuclear speckles. We characterize the nuclear and nucleolar import of FRG1, the potential effect of phosphorylation, and its interaction with the importin karyopherin α2. Consistent with a role in RNA biogenesis, human FRG1 is associated with mRNA in vivo and invitro, interacts directly with TAP (Tip-associated protein; the major mRNA export receptor), and is a dynamic nuclear-cytoplasmic shuttling protein supporting a function for FRG1 in mRNA transport. Biochemically, we characterize FRG1 actin binding activity and show that the cytoplasmic pool of FRG1 is dependent on an intact actin cytoskeleton for its localization. These data provide the first biochemical activities (actin binding and RNA binding) for human FRG1 and the characterization of the endogenous human FRG1, together indicating that FRG1 is involved in multiple aspects of RNA biogenesis, including mRNA transport and, potentially, cytoplasmic mRNA localization.

  19. IMP1, an mRNA binding protein that reduces the metastatic potential of breast cancer in a mouse model

    PubMed Central

    Nwokafor, Chiso U.; Sellers, Rani S.; Singer, Robert H.

    2016-01-01

    Cells that are able to localize β-actin mRNA efficiently have decreased metastatic potential. Invasive carcinoma cells derived from primary mammary tumors have reduced levels of an RNA binding protein IMP1/ZBP1/IGF2BP1, required for β-actin mRNA localization. We showed previously that in human breast carcinoma cells in vitro, this protein suppresses invasion. In this work we examined whether its re-expression can suppress breast cancer metastasis in a breast cancer mouse model. We developed a mouse conditionally expressing IMP1-GFP (hereinafter referred to as the IMP1 transgene) specifically in the mammary gland of a PYMT breast cancer mouse. We found that mice conditionally expressing the IMP1 transgene showed little or no metastases to the lungs from the primary tumor in contrast to PYMT mice not expressing IMP1, which uniformly develop metastases at an early stage. PMID:27655671

  20. Anguillicola crassus Infection Significantly Affects the Silvering Related Modifications in Steady State mRNA Levels in Gas Gland Tissue of the European Eel

    PubMed Central

    Pelster, Bernd; Schneebauer, Gabriel; Dirks, Ron P.

    2016-01-01

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

  1. Anguillicola crassus Infection Significantly Affects the Silvering Related Modifications in Steady State mRNA Levels in Gas Gland Tissue of the European Eel.

    PubMed

    Pelster, Bernd; Schneebauer, Gabriel; Dirks, Ron P

    2016-01-01

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

  2. Correlative nanoscale imaging of actin filaments and their complexes

    NASA Astrophysics Data System (ADS)

    Sharma, Shivani; Zhu, Huanqi; Grintsevich, Elena E.; Reisler, Emil; Gimzewski, James K.

    2013-06-01

    Actin remodeling is an area of interest in biology in which correlative microscopy can bring a new way to analyze protein complexes at the nanoscale. Advances in EM, X-ray diffraction, fluorescence, and single molecule techniques have provided a wealth of information about the modulation of the F-actin structure and its regulation by actin binding proteins (ABPs). Yet, there are technological limitations of these approaches to achieving quantitative molecular level information on the structural and biophysical changes resulting from ABPs interaction with F-actin. Fundamental questions about the actin structure and dynamics and how these determine the function of ABPs remain unanswered. Specifically, how local and long-range structural and conformational changes result in ABPs induced remodeling of F-actin needs to be addressed at the single filament level. Advanced, sensitive and accurate experimental tools for detailed understanding of ABP-actin interactions are much needed. This article discusses the current understanding of nanoscale structural and mechanical modulation of F-actin by ABPs at the single filament level using several correlative microscopic techniques, focusing mainly on results obtained by Atomic Force Microscopy (AFM) analysis of ABP-actin complexes.

  3. All histological types of primary human rhabdomyosarcoma express alpha-cardiac and not alpha-skeletal actin messenger RNA.

    PubMed Central

    Schürch, W.; Bochaton-Piallat, M. L.; Geinoz, A.; d'Amore, E.; Laurini, R. N.; Cintorino, M.; Bégin, L. R.; Boivin, Y.; Gabbiani, G.

    1994-01-01

    Eleven human primary rhabdomyosarcomas (RMSs), including all histological variants, were analyzed morphologically, immunohistochemically for intermediate filament proteins and actin isoforms, and by means of Northern blots with probes specific for total actin, alpha-skeletal (SK), alpha-cardiac (CARD), and alpha-smooth muscle actin messenger (m)RNAs. All tumors disclosed ultrastructural evidence of skeletal muscle features with terminal differentiation in three cases. The RMSs contained immunohistochemically the intermediate filament proteins vimentin and desmin and reacted positively with the alpha-sarcomeric actin antibody, which recognizes alpha-SK and alpha-CARD actin isoforms. All RMSs reacted with the total actin probe, recognizing at 2.1 kb cytoplasmic actin mRNAs and at 1.7 kb alpha-actin mRNAs. With the specific probes, all RMSs expressed alpha-CARD actin mRNA, four neoplasms expressed also alpha-smooth muscle actin mRNA, whereas the probe for alpha-SK actin mRNA never produced a signal except in one case, in which the tumor masses were intermingled with non-neoplastic preexistent striated muscle fibers. Because alpha-CARD and alpha-smooth muscle actins are transiently expressed during normal skeletal muscle development, RMSs seem to follow normal skeletal myogenesis without completing the final step, consisting of alpha-SK actin mRNA expression. The use of Northern blots for alpha-CARD actin as an adjunct to conventional techniques may be helpful for the precise identification of primary RMSs compared to other soft tissue neoplasms. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8160781

  4. HER2 and ESR1 mRNA expression levels and response to neoadjuvant trastuzumab plus chemotherapy in patients with primary breast cancer

    PubMed Central

    2013-01-01

    Introduction Recent data suggest that benefit from trastuzumab and chemotherapy might be related to expression of HER2 and estrogen receptor (ESR1). Therefore, we investigated HER2 and ESR1 mRNA levels in core biopsies of HER2-positive breast carcinomas from patients treated within the neoadjuvant GeparQuattro trial. Methods HER2 levels were centrally analyzed by immunohistochemistry (IHC), silver in situ hybridization (SISH) and qRT-PCR in 217 pretherapeutic formalin-fixed, paraffin-embedded (FFPE) core biopsies. All tumors had been HER2-positive by local pathology and had been treated with neoadjuvant trastuzumab/ chemotherapy in GeparQuattro. Results Only 73% of the tumors (158 of 217) were centrally HER2-positive (cHER2-positive) by IHC/SISH, with cHER2-positive tumors showing a significantly higher pCR rate (46.8% vs. 20.3%, P <0.0005). HER2 status by qRT-PCR showed a concordance of 88.5% with the central IHC/SISH status, with a low pCR rate in those tumors that were HER2-negative by mRNA analysis (21.1% vs. 49.6%, P <0.0005). The level of HER2 mRNA expression was linked to response rate in ESR1-positive tumors, but not in ESR1-negative tumors. HER2 mRNA expression was significantly associated with pCR in the HER2-positive/ESR1-positive tumors (P = 0.004), but not in HER2-positive/ESR1-negative tumors. Conclusions Only patients with cHER2-positive tumors - irrespective of the method used - have an increased pCR rate with trastuzumab plus chemotherapy. In patients with cHER2-negative tumors the pCR rate is comparable to the pCR rate in the non-trastuzumab treated HER-negative population. Response to trastuzumab is correlated to HER2 mRNA levels only in ESR1-positive tumors. This study adds further evidence to the different biology of both subsets within the HER2-positive group. Introduction The human epidermal growth factor receptor 2 (HER2) is the prototype of a predictive biomarker for targeted treatment [1-8]. International initiatives have established the

  5. Actin and myosin isoforms in aneural and malformed chick hearts.

    PubMed

    Kirby, M L; Shimizu, N; Gagnon, J; Toyofuku, T; Kennedy, J; Conrad, D C; Zak, R

    1990-09-01

    Although it is generally accepted that actin and myosin isoforms adapt to their functional requirements, the sequence of expression of these proteins in hearts developing abnormally is unknown. In the chick embryo it is possible to change various aspects of heart development without direct manipulation of the cardiovascular system, by removing various regions of the neural crest from early embryos. The neural crest provides both neural (sympathetic and parasympathetic) and ectomesenchymal components to the heart, and selective removal of various areas results in embryos with sympathetically aneural hearts, or persistent truncus arteriosus with or without parasympathetic denervation. Myosin isoform expression was studied in each of these types of hearts using an array of myosin antibodies specific for atrium, ventricle or the conduction system. Myosin expression in experimental hearts was found to follow the normal pattern of development using these antibodies. Actin expression was studied using cDNA probes for the 3' untranslated region of actin mRNA of the alpha-skeletal, alpha-cardiac and beta-actin isoforms. Using slot-blot hybridization analysis, the pattern of actin expression in atrium and ventricle was followed throughout the period of incubation in normal hearts. The pattern of actin expression was found to be abnormal in hearts which were sympathetically aneural and those which had persistent truncus arteriosus combined with parasympathetic denervation. ATPase activity was increased only in atria of hearts with persistent truncus arteriosus. It appears from these experiments that actin isoform expression is influenced in the chick heart by autonomic innervation.

  6. Increased duodenal DMT-1 expression and unchanged HFE mRNA levels in HFE-associated hereditary hemochromatosis and iron deficiency.

    PubMed

    Byrnes, V; Barrett, S; Ryan, E; Kelleher, T; O'Keane, C; Coughlan, B; Crowe, J

    2002-01-01

    HFE-associated hereditary hemochromatosis is characterized by imbalances of iron homeostasis and alterations in intestinal iron absorption. The identification of the HFE gene and the apical iron transporter divalent metal transporter-1, DMT-1, provide a direct method to address the mechanisms of iron overload in this disease. The aim of this study was to evaluate the regulation of duodenal HFE and DMT-1 gene expression in HFE-associated hereditary hemochromatosis. Small bowel biopsies and serum iron indices were obtained from a total of 33 patients. The study population comprised 13 patients with hereditary hemochromatosis (C282Y homozygous), 10 patients with iron deficiency anemia, and 10 apparently healthy controls, all of whom were genotyped for the two common mutations in the HFE gene (C282Y and H63D). Total RNA was isolated from tissue and amplified via RT-PCR for HFE, DMT-1, and the internal control GAPDH. DMT-1 protein expression was additionally assessed by immunohistochemistry. Levels of HFE mRNA did not differ significantly between patient groups (P = 0.09), specifically between C282Y homozygotes and iron deficiency anemic patients, when compared to controls (P = 0.09, P = 0.9, respectively). In contrast, DMT-1 mRNA levels were at least twofold greater in patients with hereditary hemochromatosis and iron deficiency anemia when compared to controls (P = 0.02, P = 0.01, respectively). Heightened DMT-1 protein expression correlated with mRNA levels in all patients. Loss of HFE function in hereditary hemochromatosis is not derived from inhibition of its gene expression. DMT-1 expression in C282Y homozygote subjects is consistent with the hypothesis of a "paradoxical" duodenal iron deficiency in hereditary hemochromatosis. The observed twofold upregulation of the DMT-1 is consistent with the slow but steady increase in body iron stores observed in those presenting with clinical features of hereditary hemochromatosis.

  7. Effects of 11-ketotestosterone and temperature on inhibin subunit mRNA levels in the ovary of the shortfinned eel, Anguilla australis.

    PubMed

    Zadmajid, Vahid; Falahatimarvast, Ali; Damsteegt, Erin L; Setiawan, Alvin N; Ozaki, Yuichi; Shoae, Alireza; Lokman, P Mark

    2015-09-01

    Members of the transforming growth factor-b (TGFb) superfamily are important during early oogenesis in mammals. In this study, we tested whether documented effects of 11-ketotestosterone (11KT) on previtellogenic eel ovaries are mediated through affecting the expression of key ovarian TGFb genes. Furthermore, we investigated whether 11KT effects interacted with temperature. Accordingly, three thermal regimes were compared and their interaction with 11KT-mediated actions on expression of TGFb superfamily genes (chiefly inhibin subunits) evaluated in the eel (Anguilla australis). Inhibin subunit mRNA levels were also measured in ovarian explants cultured in vitro with 11KT and in ovaries from eels collected from the wild. In wild eels, inhibin-bA mRNA levels were higher in early than in previtellogenic eels; inhibin-a expression did not differ between stages, whereas that of inhibin-bB first decreased, then recovered with advanced developmental stage. Temperature was ineffective in modulating any of the end points, at least as long as a Q10 adjustment was made to correct for 'metabolic dose'. However, 11KT affected the expression of inhibin-a compared to control fish, while those of inhibin-b subunit genes remained unaffected. In contrast, 11KT dramatically reduced mRNA levels of inhibin-b subunits in vitro, but had inconsistent effects on inhibin-a transcript abundance. We conclude that 11KT affects ovarian inhibin subunit gene expression, but effects are not in keeping with the changes seen during early oogenesis in eels from the wild. We further contend that in vivo temperature experiments are easily biased and that Q10 corrections may be required to identify 'true' temperature effects.

  8. Effects of Molybdenum or/and Cadmium on mRNA Expression Levels of Inflammatory Cytokines and HSPs in Duck Spleens.

    PubMed

    Cao, Huabin; Zhang, Mengmeng; Xia, Bing; Xiong, Jin; Zong, Yibo; Hu, Guoliang; Zhang, Caiying

    2016-03-01

    Cadmium (Cd) and high dietary intake of molybdenum (Mo) can cause multiple-organ injury in animals, but the co-induced toxicity of Mo and Cd to spleen in ducks is not well understood. The aim of this study was to investigate the co-induced effects of Mo and Cd on the mRNA expression levels of inflammatory cytokines and heat shock proteins (HSPs) in duck spleens. Two hundred forty healthy 11-day-old ducks were randomly divided into six groups and treated with a commercial diet containing Mo or/and Cd. After being treated with Mo or/and Cd for 30, 60, 90, and 120 days, the mRNA expression levels of nuclear factor-kappa B (NF-κB), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), HSP60, HSP70, and HSP90 were examined in duck spleens. Histopathology was examined in duck spleens at 120 days. The results indicated that the mRNA expression levels of HSPs were significantly upregulated in the co-induced groups (P < 0.01), while these decreased in the high dietary intake of Mo combined with Cd group at 120 days. Exposure to Mo or/and Cd upregulated the mRNA expression levels of NF-κB, COX-2, and TNF-α in the combination groups (P < 0.01). Furthermore, severe congestion, bleeding, splenic corpuscle structure fuzzy, wall thickness of sheath artery thickening, and oxyhematin were observed in the spleens of combination groups. Meanwhile, the organizational structure damage of the combined groups was more severe than that of the other groups. These results suggested that exposure to Mo or/and Cd might lead to tissue damage, and high expression of HSPs and inflammatory cytokines may play a role in the resistance of spleen toxicity induced by Mo or/and Cd. Interaction of Mo and Cd may have a synergistic effect on spleen toxicity.

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

    PubMed

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

    2014-08-01

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

  10. Effects of cysteamine on mRNA levels of growth hormone and its receptors and growth in orange-spotted grouper (Epinephelus coioides).

    PubMed

    Li, Yun; Liu, Xiaochun; Zhang, Yong; Ma, Xilan; Lin, Haoran

    2013-06-01

    Effects of cysteamine (CS) on growth hormone (GH) mRNA, two types of growth hormone receptor (GHR) mRNAs and growth rate in orange-spotted grouper (Epinephelus coioides) were investigated. CS could cause a modification in the structure of somatostatin, which is the most important neuroendocrine inhibitor of basal and stimulated growth hormone synthesis and release, and renders it nonimmunoreactive probably through interaction with the disulfide bonds. In the present study, cysteamine hydrochloride (CSH) enhanced the level of pituitary GH mRNA in a dose-dependent manner through attenuating or deleting the inhibiting action of somatostatin on GH mRNA expression. CSH at relatively low doses (from 1 to 3 mg/g diet) enhanced the levels of two types of GHR mRNAs in dose-dependent manner, whereas the stimulation induced by CSH declined from the peak at higher dose of CSH (4 mg/g diet). It might be attributed to the variation in GH-induced up-regulation of GHRs at different doses of GH. Feeding of CSH could induce remarkable enhancement of growth rate in orange-spotted grouper. In addition, the stimulatory effect of CSH could be potentiated by the additive effect of luteinizing hormone-releasing hormone analog (LHRH-A). Compared with individual treatments, combined feeding of CSH and LHRH-A caused more efficient elevation of growth rate after 8 weeks of feeding. CSH and LHRH-A individually and in combination remarkably increased the levels of GH and GHR mRNAs compared with the control. The combined administration of CSH and LHRH-A in diet was most effective to enhance the level of GH and GHR1 mRNA. The morphological characteristics of the experimental fish were evaluated. Compared with control, the ratios of muscle RNA/DNA, condition factors (CF) and feed conversion efficiency (FCE) were significantly enhanced in the treated groups, while the highest values were observed in the combined treatment. All the results suggested that CSH (1-3 mg/g diet) is an effective

  11. Chromium improves glucose uptake and metabolism through upregulating the mRNA levels of IR, GLUT4, GS, and UCP3 in skeletal muscle cells.

    PubMed

    Qiao, Wei; Peng, Zhongli; Wang, Zhisheng; Wei, Jing; Zhou, Anguo

    2009-11-01

    The aim of this study was to evaluate the impact of three different chromium forms as chromic chloride (CrCl), chromium picolinate (CrPic), and a newly synthesized complex of chromium chelated with small peptides (CrSP) on glucose uptake and metabolism in vitro. In cultured skeletal muscle cells, chromium augmented insulin-stimulated glucose uptake and metabolism as assessed by a reduced glucose concentration of culture medium. At the molecular level, insulin significantly increased the mRNA levels of insulin receptor (IR), glucose transporter 4 (GLUT4), glycogen synthase (GS), and uncoupling protein-3 (UCP3), and these impacts can be enhanced by the addition of chromium, especially in the form of CrSP. Collectively, results of this study demonstrate that chromium improves glucose uptake and metabolism through upregulating the mRNA levels of IR, GLUT4, GS, and UCP3 in skeletal muscle cells, and CrSP has higher efficacy on glucose uptake and metabolism compared to the forms of CrCl and CrPic.

  12. Directed actin assembly and motility.

    PubMed

    Boujemaa-Paterski, Rajaa; Galland, Rémi; Suarez, Cristian; Guérin, Christophe; Théry, Manuel; Blanchoin, Laurent

    2014-01-01

    The actin cytoskeleton is a key component of the cellular architecture. However, understanding actin organization and dynamics in vivo is a complex challenge. Reconstitution of actin structures in vitro, in simplified media, allows one to pinpoint the cellular biochemical components and their molecular interactions underlying the architecture and dynamics of the actin network. Previously, little was known about the extent to which geometrical constraints influence the dynamic ultrastructure of these networks. Therefore, in order to study the balance between biochemical and geometrical control of complex actin organization, we used the innovative methodologies of UV and laser patterning to design a wide repertoire of nucleation geometries from which we assembled branched actin networks. Using these methods, we were able to reconstitute complex actin network organizations, closely related to cellular architecture, to precisely direct and control their 3D connections. This methodology mimics the actin networks encountered in cells and can serve in the fabrication of innovative bioinspired systems.

  13. Rab11 and Actin Cytoskeleton Participate in Giardia lamblia Encystation, Guiding the Specific Vesicles to the Cyst Wall

    PubMed Central

    Castillo-Romero, Araceli; Leon-Avila, Gloria; Wang, Ching C.; Perez Rangel, Armando; Camacho Nuez, Minerva; Garcia Tovar, Carlos; Ayala-Sumuano, Jorge Tonatiuh; Luna-Arias, Juan Pedro; Hernandez, Jose Manuel

    2010-01-01

    Background Giardia passes through two stages during its life cycle, the trophozoite and the cyst. Cyst formation involves the synthesis of cyst wall proteins (CWPs) and the transport of CWPs into encystation-specific vesicles (ESVs). Active vesicular trafficking is essential for encystation, but the molecular machinery driving vesicular trafficking remains unknown. The Rab proteins are involved in the targeting of vesicles to several intracellular compartments through their association with cytoskeletal motor proteins. Methodology and Principal Findings In this study, we found a relationship between Rab11 and the actin cytoskeleton in CWP1 transport. Confocal microscopy showed Rab11 was distributed throughout the entire trophozoite, while in cysts it was translocated to the periphery of the cell, where it colocalized with ESVs and microfilaments. Encystation was also accompanied by changes in rab11 mRNA expression. To evaluate the role of microfilaments in encystation, the cells were treated with latrunculin A. Scanning electron microscopy showed this treatment resulted in morphological damages to encysted parasites. The intensity of fluorescence-labeled Rab11 and CWP1 in ESVs and cyst walls was reduced, and rab11 and cwp1 mRNA levels were down-regulated. Furthermore, knocking down Rab11 with a hammerhead ribozyme resulted in an up to 80% down-regulation of rab11 mRNA. Although this knockdown did not appear lethal for trophozoites and did not affect cwp1 expression during the encystation, confocal images showed CWP1 was redistributed throughout the cytosol. Conclusions and Significance Our results indicate that Rab11 participates in the early and late encystation stages by regulating CWP1 localization and the actin-mediated transport of ESVs towards the periphery. In addition, alterations in the dynamics of actin affected rab11 and cwp1 expression. Our results provide new information about the molecules involved in Giardia encystation and suggest that Rab11 and

  14. Acute hypoxia up-regulates HIF-1α and VEGF mRNA levels in Amazon hypoxia-tolerant Oscar (Astronotus ocellatus).

    PubMed

    Baptista, R B; Souza-Castro, N; Almeida-Val, V M F

    2016-10-01

    Amazon fish maintain oxygen uptake through a variety of strategies considered evolutionary and adaptive responses to the low water oxygen saturation, commonly found in Amazon waters. Oscar (Astronotus ocellatus) is among the most hypoxia-tolerant fish in Amazon, considering its intriguing anaerobic capacity and ability to depress oxidative metabolism. Previous studies in hypoxia-tolerant and non-tolerant fish have shown that hypoxia-inducible factor-1α (HIF-1α) gene expression is positively regulated during low oxygen exposure, affecting vascular endothelial growth factor (VEGF) transcription and fish development or tolerance in different manners. However, whether similar isoforms exists in tolerant Amazon fish and whether they are affected similarly to others physiological responses to improve hypoxia tolerance remain unknown. Here we evaluate the hepatic HIF-1α and VEGF mRNA levels after 3 h of acute hypoxia exposure (0.5 mgO2/l) and 3 h of post-hypoxia recovery. Additionally, hematological parameters and oxidative enzyme activities of citrate synthase (CS) and malate dehydrogenase (MDH) were analyzed in muscle and liver tissues. Overall, three sets of responses were detected: (1) as expected, hematocrit, hemoglobin concentration, red blood cells, and blood glucose increased, improving oxygen carrying capacity and glycolysis potential; (2) oxidative enzymes from liver decreased, corroborating the tendency to a widespread metabolic suppression; and (3) HIF-1α and VEGF increased mRNA levels in liver, revealing their role in the oxygen homeostasis through, respectively, activation of target genes and vascularization. This is the first study to investigate a hypoxia-related transcription factor in a representative Amazon hypoxia-tolerant fish and suggests that HIF-1α and VEGF mRNA regulation have an important role in enhancing hypoxia tolerance in extreme tolerant species.

  15. Visualization of dynamics of single endogenous mRNA labeled in live mouse.

    PubMed

    Park, Hye Yoon; Lim, Hyungsik; Yoon, Young J; Follenzi, Antonia; Nwokafor, Chiso; Lopez-Jones, Melissa; Meng, Xiuhua; Singer, Robert H

    2014-01-24

    The transcription and transport of messenger RNA (mRNA) are critical steps in regulating the spatial and temporal components of gene expression, but it has not been possible to observe the dynamics of endogenous mRNA in primary mammalian tissues. We have developed a transgenic mouse in which all β-actin mRNA is fluorescently labeled. We found that β-actin mRNA in primary fibroblasts localizes predominantly by diffusion and trapping as single mRNAs. In cultured neurons and acute brain slices, we found that multiple β-actin mRNAs can assemble together, travel by active transport, and disassemble upon depolarization by potassium chloride. Imaging of brain slices revealed immediate early induction of β-actin transcription after depolarization. Studying endogenous mRNA in live mouse tissues provides insight into its dynamic regulation within the context of the cellular and tissue microenvironment.

  16. Actin organization and gene expression in Beta vulgaris seedlings under clinorotation

    NASA Astrophysics Data System (ADS)

    Kozeko, L. Y.; Shevchenko, G. V.; Artemenko, O. A.; Martyn, G. G.; Kordyum, E. L.

    2005-08-01

    Actin microfilaments (MFs) as highly dynamic structure respond by rapid reorganization to different external influences, including gravity. The object of our experiments was to examine both the actin organization and actin gene expression during growth and differentiation of root cells under clinorotation. It was shown that MFs acted as the indicator of changes caused by altered gravity in distal elongation zone (DEZ) cells, particularly actin cytoskeleton is enhanced in cortex cells. The data testify stable actin expression under altered gravity. The F-actin MFs enhancement in cortex cells of the DEZ occurred under clinorotation at the same level of the total actin content as in the stationary conditions is suggested to be caused by transformation of G-actin into F-actin.

  17. Coactosin-like protein, a human F-actin-binding protein: critical role of lysine-75.

    PubMed Central

    Provost, P; Doucet, J; Stock, A; Gerisch, G; Samuelsson, B; Rådmark, O

    2001-01-01

    Coactosin-like protein (CLP) was recently identified in a yeast two-hybrid screen using 5-lipoxygenase as bait. In the present study, we report the functional characterization of CLP as a human filamentous actin (F-actin)-binding protein. CLP mRNA shows a wide tissue distribution and is predominantly expressed in placenta, lung, kidney and peripheral-blood leucocytes. Endogenous CLP is localized in the cytosol of myeloid cells. Using a two-hybrid approach, actin was identified as a CLP-interacting protein. Binding experiments indicated that CLP associates with F-actin, but does not form a stable complex with globular actin. In transfected mammalian cells, CLP co-localized with actin stress fibres. CLP bound to actin filaments with a stoichiometry of 1:2 (CLP: actin subunits), but could be cross-linked to only one subunit of actin. Site-directed mutagenesis revealed the involvement of Lys(75) of CLP in actin binding, a residue highly conserved in related proteins and supposed to be exposed on the surface of the CLP protein. Our results identify CLP as a new human protein that binds F-actin in vitro and in vivo, and indicate that Lys(75) is essential for this interaction. PMID:11583571

  18. Swimming training down-regulates plasma leptin levels, but not adipose tissue ob mRNA expression.

    PubMed

    Benatti, F B; Polacow, V O; Ribeiro, S M L; Gualano, B; Coelho, D F; Rogeri, P S; Costa, A S; Lancha Junior, A H

    2008-10-01

    The aim of the present study was to assess the effects of endurance training on leptin levels and adipose tissue gene expression and their association with insulin, body composition and energy intake. Male Wistar rats were randomly divided into two groups: trained (N = 18) and sedentary controls (N = 20). The trained group underwent swimming training for 9 weeks. Leptin and insulin levels, adiposity and leptin gene expression in epididymal and inguinal adipose tissue were determined after training. There were no differences in energy intake between groups. Trained rats had a decreased final body weight (-10%), relative and total body fat (-36 and -55%, respectively) and insulin levels (-55%) compared with controls (P < 0.05). Although trained animals showed 56% lower leptin levels (2.58 +/- 1.05 vs 5.89 +/- 2.89 ng/mL in control; P < 0.05), no difference in leptin gene expression in either fat depot was demonstrable between groups. Stepwise multiple regression analysis showed that lower leptin levels in trained rats were due primarily to their lower body fat mass. After adjustment for total body fat, leptin levels were still 20% (P < 0.05) lower in exercised rats. In conclusion, nine weeks of swimming training did not affect leptin gene expression, but did lead to a decrease in leptin levels that was independent of changes in body fat.

  19. Amplification of actin polymerization forces

    PubMed Central

    Dmitrieff, Serge; Nédélec, François

    2016-01-01

    The actin cytoskeleton drives many essential processes in vivo, using molecular motors and actin assembly as force generators. We discuss here the propagation of forces caused by actin polymerization, highlighting simple configurations where the force developed by the network can exceed the sum of the polymerization forces from all filaments. PMID:27002174

  20. Amplification of actin polymerization forces.

    PubMed

    Dmitrieff, Serge; Nédélec, François

    2016-03-28

    The actin cytoskeleton drives many essential processes in vivo, using molecular motors and actin assembly as force generators. We discuss here the propagation of forces caused by actin polymerization, highlighting simple configurations where the force developed by the network can exceed the sum of the polymerization forces from all filaments.

  1. The effect of mechanical force on mRNA levels of collagenase, collagen type I, and tissue inhibitors of metalloproteinases in gingivae of dogs.

    PubMed

    Redlich, M; Reichenberg, E; Harari, D; Zaks, B; Shoshan, S; Palmon, A

    2001-12-01

    Orthodontic force causes an injury to and subsequent degradation of the attachment apparatus, thus leading to the transposition of the tooth. The gingiva, however, is compressed and sometimes becomes hypertrophic with tooth movement and often shrinks after treatment. To study the effect of force on the gingiva, we applied orthodontic force in dogs and analyzed gingival tissues 1, 2, 3, 7, 14, and 28 days later as well as after removing the force. The effect of force on mRNA levels of collagen type I (col-I), matrix metalloproteinase-1 (MMP- 1), and tissue inhibitors 1 and 2 (TIMPs) genes was analyzed by RT-PCR, and MMP-1 activity was determined by zymography. The results showed that force significantly increased both the mRNA levels of MMP-1 and its interstitial activity. After the removal of force, MMP-1 gene expression was significantly decreased. The results could partly explain the clinically observed shrinkage and adaptation of the gingiva during tooth movement.

  2. Cinnamaldehyde up-regulates the mRNA expression level of TRPV1 receptor potential ion channel protein and its function in primary rat DRG neurons in vitro.

    PubMed

    Sui, Feng; Lin, Na; Guo, Jian-You; Zhang, Chang-Bin; Du, Xin-Liang; Zhao, Bao-Sheng; Liu, Hong-Bin; Yang, Na; Li, Lan-Fang; Guo, Shu-Ying; Huo, Hai-Ru; Jiang, Ting-Liang

    2010-01-01

    Cinnamaldehyde (1) is a pharmacologically active ingredient isolated from cassia twig (Ramulus Cinnamomi), which is commonly used in herbal remedies to treat fever-related diseases. Both TRPV1 and TRPM8 ion channel proteins are abundantly expressed in sensory neurons, and are assumed to act as a thermosensor, with the former mediating the feeling of warmth and the latter the feeling of cold in the body. Both of them have recently been reported to be involved in thermoregulation. The purpose of this paper is to further uncover the antipyretic mechanisms of 1 by investigating its effects on the mRNA expression levels and functions of both TRPV1 and TRPM8. The results showed that 1 could up-regulate the mRNA expression levels of TRPV1 at both 37 and 39 degrees C, and its calcium-mediating function was significantly increased at 39 degrees C, all of which could not be blocked by pretreatment of the neuronal cells with ruthenium red, a general transient receptor potential (TRP) blocker, indicating that the action of 1 was achieved through a non-TRPA1 channel pathway. In conclusion, the findings in our in vitro studies might account for part of the peripheral molecular mechanisms for the antipyretic action of 1.

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

    NASA Astrophysics Data System (ADS)

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

    1988-12-01

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

  4. DIFFERENTIAL REGULATION OF POLYSOME mRNA LEVELS IN MOUSE HEPA-1C1C7 CELLS EXPOSED TO DIOXIN

    PubMed Central

    Thornley, Jessica A.; Trask, Heidi W.; Ridley, Christian J. A.; Korc, Murray; Gui, Jiang; Ringelberg, Carol S.; Wang, Sinny; Tomlinson, Craig R.

    2011-01-01

    The environmental agent 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) causes a multitude of human illnesses. In order to more fully understand the underlying biology of TCDD toxicity, we tested the hypothesis that new candidate genes could be identified using polysome RNA from TCDD-treated mouse Hepa-1c1c7 cells. We found that (i) differentially expressed whole cell and cytoplasm RNA levels are both poor predictors of polysome RNA levels; (ii) for a majority of RNAs, differential RNA levels are regulated independently in the nucleus, cytoplasm, and polysomes; (iii) for the remaining polysome RNAs, levels are regulated via several different mechanisms, including a “tagging” of mRNAs in the nucleus for immediate polysome entry; and (iv) most importantly, a gene list derived from differentially expressed polysome RNA generated new genes and cell pathways potentially related to TCDD biology. PMID:21570461

  5. Actinic keratosis. Current treatment options.

    PubMed

    Jeffes, E W; Tang, E H

    2000-01-01

    Actinic keratoses are hyperkeratotic skin lesions that represent focal abnormal proliferation of epidermal keratinocytes. Some actinic keratoses evolve into squamous cell carcinoma of the skin, while others resolve spontaneously. The conversion rate of actinic keratosis to squamous cell carcinoma is not accurately known, but appears to be in the range of 0.25 to 1% per year. Although there is a low rate of conversion of actinic keratoses to squamous cell carcinoma, 60% of squamous cell carcinomas of the skin probably arise from actinic keratoses. The main cause of actinic keratoses in otherwise healthy Caucasians appears to be the sun. Therapy for actinic keratoses begins with prevention which starts with sun avoidance and physical protection. Sunprotection with sunscreens actually slows the return of actinic keratoses in patients already getting actinic keratoses. Interestingly, a few studies are available that demonstrate that a high fat diet is associated with the production of more actinic keratoses than is a low fat diet. One of the mainstays of therapy has been local destruction of the actinic keratoses with cryotherapy, and curettage and electrodesiccation. A new addition to this group of therapies to treat individual actinic keratoses is photodynamic therapy with topical aminolevulinic acid and light. In patients who have numerous actinic keratoses in an area of severely sun damaged skin, therapies which are applied to the whole actinic keratosis area are used. The goal of treating such an area of skin is to treat all of the early as well as the numerous clinically evident actinic keratoses at the same time. The classical approaches for treating areas of photodamaged skin without treating actinic keratoses individually include: the use of topically applied fluorouracil cream, dermabrasion, and cutaneous peels with various agents like trichloroacetic acid. Both topically as well as orally administered retinoids have been used to treat actinic keratoses but

  6. Cyp1B1 mRNA expression in correlation to cotinine levels with respect to the Cyp1B1 L432V gene polymorphism.

    PubMed

    Helmig, Simone; Seelinger, Jens Udo; Philipp-Gehlhaar, Monika; Döhrel, Juliane; Schneider, Joachim

    2010-12-01

    Cytochrome P450 1B1 (CYP1B1) is involved in the activation of a broad spectrum of procarcinogens. An association of the Cyp1B1 Leu432Val polymorphism with cancer as well as an impact on the enzyme activity has been described. To study gene-environmental interactions we investigated the quantitative Cyp1B1 mRNA expression in smokers (N = 102) and non-smokers (N = 192) with regards to the Cyp1B1 L432V gene polymorphism. Tobacco smoke exposure was assessed by serum cotinine levels. Genotypes were analysed by melting curve analysis and quantification of Cyp1B1 mRNA by real-time PCR. In comparing Cyp1B1 expression, significant differences between the two homozygote genotypes *1/*1 and *3/*3 (0.105 ± 0.019; n = 26 vs. 0.051 ± 0.017; n = 14; P = 0.039) and between the heterozygote genotype *1/*3 and *3/*3 (0.121 ± 0.029; n = 55 vs. 0.051 ± 0.017; n = 14; P = 0.039) of smokers were revealed. According to the serum cotinine levels, three subgroups (low; medium; high) were build. The group "high" (0.248 ± 0.089; n = 32) showed proportionally high Cyp1B1 mRNA expression compared to "medium" (0.101 ± 0.024; n = 33), "low" (0.086 ± 0.015; n = 32) and non-smokers (0.084 ± 0.007; n = 176). This result was reflected in the homozygote *1/*1 and the heterozygote *1/*3 genotypes. In contrast the homozygote *3/*3 genotype was missing the high Cyp1B1 mRNA expression in the cotinine subgroup "high". Our results suggest that genotypes carrying the C-allele (*1/*1 and *1/*3) at Cyp1B1 Leu432Val polymorphism show a higher response to environmental factors, such as tobacco smoke than homozygote *3/*3 genotypes.

  7. A HuD-ZBP1 ribonucleoprotein complex localizes GAP-43 mRNA into axons through its 3' untranslated region AU-rich regulatory element.

    PubMed

    Yoo, Soonmoon; Kim, Hak H; Kim, Paul; Donnelly, Christopher J; Kalinski, Ashley L; Vuppalanchi, Deepika; Park, Michael; Lee, Seung J; Merianda, Tanuja T; Perrone-Bizzozero, Nora I; Twiss, Jeffery L

    2013-09-01

    Localized translation of axonal mRNAs contributes to developmental and regenerative axon growth. Although untranslated regions (UTRs) of many different axonal mRNAs appear to drive their localization, there has been no consensus RNA structure responsible for this localization. We recently showed that limited expression of ZBP1 protein restricts axonal localization of both β-actin and GAP-43 mRNAs. β-actin 3'UTR has a defined element for interaction with ZBP1, but GAP-43 mRNA shows no homology to this RNA sequence. Here, we show that an AU-rich regulatory element (ARE) in GAP-43's 3'UTR is necessary and sufficient for its axonal localization. Axonal GAP-43 mRNA levels increase after in vivo injury, and GAP-43 mRNA shows an increased half-life in regenerating axons. GAP-43 mRNA interacts with both HuD and ZBP1, and HuD and ZBP1 co-immunoprecipitate in an RNA-dependent fashion. Reporter mRNA with the GAP-43 ARE competes with endogenous β-actin mRNA for axonal localization and decreases axon length and branching similar to the β-actin 3'UTR competing with endogenous GAP-43 mRNA. Conversely, over-expressing GAP-43 coding sequence with its 3'UTR ARE increases axonal elongation and this effect is lost when just the ARE is deleted from GAP-43's 3'UTR. We have recently found that over-expression of GAP-43 using an axonally targeted construct with the 3'UTRs of GAP-43 promoted elongating growth of axons, while restricting the mRNA to the cell body with the 3'UTR of γ-actin had minimal effect on axon length. In this study, we show that the ARE in GAP-43's 3'UTR is responsible for localization of GAP-43 mRNA into axons and is sufficient for GAP-43 protein's role in elongating axonal growth.

  8. Low Incidence along with Low mRNA Levels of EGFRvIII in Prostate and Colorectal Cancers Compared to Glioblastoma

    PubMed Central

    Peciak, Joanna; Stec, Wojciech J; Treda, Cezary; Ksiazkiewicz, Magdalena; Janik, Karolina; Popeda, Marta; Smolarz, Maciej; Rosiak, Kamila; Hulas-Bigoszewska, Krystyna; Och, Waldemar; Rieske, Piotr; Stoczynska-Fidelus, Ewelina

    2017-01-01

    Background: The presence as well as the potential role of EGFRvIII in tumors other than glioblastoma still remains a controversial subject with many contradictory data published. Previous analyses, however, did not consider the level of EGFRvIII mRNA expression in different tumor types. Methods: Appropriately designed protocol for Real-time quantitative reverse-transcription PCR (Real-time qRT-PCR) was applied to analyze EGFRvIII and EGFRWT mRNA expression in 155 tumor specimens. Additionally, Western Blot (WB) analysis was performed for selected samples. Stable cell lines showing EGFRvIII expression (CAS-1 and DK-MG) were analyzed by means of WB, immunocytochemistry (ICC) and fluorescence in situ hybridization (FISH). Results: Our analyses revealed EGFRvIII expression in 27.59% of glioblastomas (8/29), 8.11% of colorectal cancers (3/37), 6.52% of prostate cancers (3/46) and none of breast cancers (0/43). Despite the average relative expression of EGFRvIII varying greatly among tumors of different tissues (approximately 800-fold) or even within the same tissue group (up to 8000-fold for GB), even the marginal expression of EGFRvIII mRNA can be detrimental to cancer progression, as determined by the analysis of stable cell lines endogenously expressing the oncogene. Conclusion: EGFRvIII plays an unquestionable role in glioblastomas with high expression of this oncogene. Our data suggests that EGFRvIII importance should not be underestimated even in tumors with relatively low expression of this oncogene. PMID:28123609

  9. Low Incidence along with Low mRNA Levels of EGFR(vIII) in Prostate and Colorectal Cancers Compared to Glioblastoma.

    PubMed

    Peciak, Joanna; Stec, Wojciech J; Treda, Cezary; Ksiazkiewicz, Magdalena; Janik, Karolina; Popeda, Marta; Smolarz, Maciej; Rosiak, Kamila; Hulas-Bigoszewska, Krystyna; Och, Waldemar; Rieske, Piotr; Stoczynska-Fidelus, Ewelina

    2017-01-01

    Background: The presence as well as the potential role of EGFR(vIII) in tumors other than glioblastoma still remains a controversial subject with many contradictory data published. Previous analyses, however, did not consider the level of EGFR(vIII) mRNA expression in different tumor types. Methods: Appropriately designed protocol for Real-time quantitative reverse-transcription PCR (Real-time qRT-PCR) was applied to analyze EGFR(vIII) and EGFR(WT) mRNA expression in 155 tumor specimens. Additionally, Western Blot (WB) analysis was performed for selected samples. Stable cell lines showing EGFR(vIII) expression (CAS-1 and DK-MG) were analyzed by means of WB, immunocytochemistry (ICC) and fluorescence in situ hybridization (FISH). Results: Our analyses revealed EGFR(vIII) expression in 27.59% of glioblastomas (8/29), 8.11% of colorectal cancers (3/37), 6.52% of prostate cancers (3/46) and none of breast cancers (0/43). Despite the average relative expression of EGFR(vIII) varying greatly among tumors of different tissues (approximately 800-fold) or even within the same tissue group (up to 8000-fold for GB), even the marginal expression of EGFR(vIII) mRNA can be detrimental to cancer progression, as determined by the analysis of stable cell lines endogenously expressing the oncogene. Conclusion: EGFR(vIII) plays an unquestionable role in glioblastomas with high expression of this oncogene. Our data suggests that EGFR(vIII) importance should not be underestimated even in tumors with relatively low expression of this oncogene.

  10. Influence of functional polymorphisms in TNF-α, IL-8, and IL-10 cytokine genes on mRNA expression levels and risk of gastric cancer.

    PubMed

    de Oliveira, Juliana Garcia; Rossi, Ana Flávia Teixeira; Nizato, Daniela Manchini; Cadamuro, Aline Cristina Targa; Jorge, Yvana Cristina; Valsechi, Marina Curado; Venâncio, Larissa Paola Rodrigues; Rahal, Paula; Pavarino, Érika Cristina; Goloni-Bertollo, Eny Maria; Silva, Ana Elizabete

    2015-12-01

    Functional polymorphisms in promoter regions can produce changes in the affinity of transcription factors, thus altering the messenger ribonucleic acid (mRNA) expression levels of inflammatory cytokines associated with the risk of cancer development. The goal of this study was to evaluate the influence that polymorphisms in the cytokine genes known as TNF-α-308 G/A (rs1800629), TNF-α-857 C/T (rs1799724), IL-8-251 T/A (rs4073), IL-8-845 T/C (rs2227532), and IL-10-592 C/A (rs1800872) have on changes to mRNA expression levels and on the risks of chronic gastritis (CG) and gastric cancer (GC). A sample of 723 individuals was genotyped using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Relative mRNA expression levels were measured using quantitative real-time PCR (qPCR). Polymorphisms TNF-α-308 G/A and IL-8-251 A/T were not associated with risks of these gastric lesions. However, TNF-α-857 C/T, IL-8-845 T/C, and IL-10-592 C/A were found to be associated with a higher risk of GC, and IL-10-592 C/A was found to be associated with a higher risk of CG. The relative mRNA expression levels (RQ) of TNF-α, IL-8, and IL-10 were markedly downregulated in the CG group (median RQs = 0.128, 0.247, and 0.614, respectively), while the RQ levels of TNF-α in the GC group were upregulated (RQ = 2.749), but were basal for IL-8 (RQ = 1.053) and downregulated for IL-10 (RQ = 0.179). When the groups were stratified according to wild-type and polymorphic alleles, only for IL-8-845 T/C the polymorphic allele was found to influence the expression levels of this cytokine. IL-8-845 C allele carriers were significantly upregulated in both groups (GC and CG; RQ = 3.138 and 2.181, respectively) when compared to TT homozygotes (RQ = -0.407 and 0.165, respectively). In silico analysis in the IL-8 promoter region revealed that the presence of the variant C allele in position -845 is responsible for the presence of the binding

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

    PubMed

    Ma, Jiangwei; Qiao, Zengyong; Xu, Biao

    2013-10-01

    The aim of this study was to characterise the effects of ischemic preconditioning (IP) on heart function parameters (ΔST and ΔT), activities of serum creatine kinase (CK), lactate dehydrogenase (LDH), and levels of serum nitric oxide (NO), malondialdehyde (MDA), and myocardium Caspase-3 mRNA, SOCS-1, SOCS-3, tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) expression levels and Apoptosis index in myocardium IR rats. Results showed that ΔST and ΔST values in IP group were markedly lower than those in IR group. Compared with IR group, IP significantly (p < 0.01) decreased serum CK (0.83 ± 0.09 vs 1.36 ± 0.15), LDH (5613 ± 462 vs 7106 ± 492) activities and MDA (11.32 ± 1.05 vs 15.49 ± 1.26) level, increased the serum NO (86.39 ± 7.03 vs 53.77 ± 4.27) level in IR group. The IP induced a significant decreased in myocardium Caspase-3 mRNA (0.303 ± 0.021 vs 0.515 ± 0.022) gene expression (p < 0.01) compared to IR model group. The IP induced a significant decreased in myocardium SOCS-1 (0.241 ± 0.031 vs 0.596 ± 0.036), SOCS-3 (0.258 ± 0.031 vs 0.713 ± 0.057), TNF-α (0.137 ± 0.011 vs 0.427 ± 0.035) and IL-6 (0.314 ± 0.021 vs 0.719 ± 0.064) mRNA gene expression (p < 0.01) compared to IR model group. We conclude that IP is effective in the therapy of heart disease. These findings may have implications for the clinical development of preconditioning-based therapies for ischemic heart disease.

  12. Effects of Dietary Selenium Against Lead Toxicity on mRNA Levels of 25 Selenoprotein Genes in the Cartilage Tissue of Broiler Chicken.

    PubMed

    Gao, H; Liu, C P; Song, S Q; Fu, J

    2016-07-01

    The interactions between the essential element selenium (Se) and the toxic element lead (Pb) have been reported extensively; however, little is known about the effect of Se on Pb toxicity and the expression pattern of selenoproteins in the cartilage of chicken. To investigate the effects of Se on Pb toxicity and the messenger RNA (mRNA) expressions of selenoproteins in cartilage tissue, an in vitro study was performed on 1-day-old broiler chickens (randomly allocated into four groups) with diet of different concentration of Se and Pb. After 90 days, the meniscus cartilage and sword cartilage tissue were examined for the mRNA levels of 25 selenoprotein genes. The results showed that Se and Pb influenced the expression of selenoprotein genes in the chicken cartilage tissue. In detail, Se could alleviate the downtrend of the expression of Gpx1, Gpx2, Gpx4, Txnrd2, Txnrd3, Dio1, Dio2, Seli, Selu, Sepx1, Selk, Selw, Selo, Selm, Sep15, Sepnn1, Sels, and Selt induced by Pb exposure in the meniscus cartilage. In the sword cartilage, Se alleviated the downtrend of the expression of Gpx2, Gpx3, Gpx4, Txnrd1, Txnrd2, Dio2, Dio3, Seli, Selh, SPS2, Sepx1, Selk, Selw, Selo, Selm, Sep15, Selpb, Sepn1, and Selt induced by Pb exposure. The present study provided some compensated data about the roles of Se against Pb toxicity in the regulation of selenoprotein expression.

  13. Actin stress in cell reprogramming

    PubMed Central

    Guo, Jun; Wang, Yuexiu; Sachs, Frederick; Meng, Fanjie

    2014-01-01

    Cell mechanics plays a role in stem cell reprogramming and differentiation. To understand this process better, we created a genetically encoded optical probe, named actin–cpstFRET–actin (AcpA), to report forces in actin in living cells in real time. We showed that stemness was associated with increased force in actin. We reprogrammed HEK-293 cells into stem-like cells using no transcription factors but simply by softening the substrate. However, Madin-Darby canine kidney (MDCK) cell reprogramming required, in addition to a soft substrate, Harvey rat sarcoma viral oncogene homolog expression. Replating the stem-like cells on glass led to redifferentiation and reduced force in actin. The actin force probe was a FRET sensor, called cpstFRET (circularly permuted stretch sensitive FRET), flanked by g-actin subunits. The labeled actin expressed efficiently in HEK, MDCK, 3T3, and bovine aortic endothelial cells and in multiple stable cell lines created from those cells. The viability of the cell lines demonstrated that labeled actin did not significantly affect cell physiology. The labeled actin distribution was similar to that observed with GFP-tagged actin. We also examined the stress in the actin cross-linker actinin. Actinin force was not always correlated with actin force, emphasizing the need for addressing protein specificity when discussing forces. Because actin is a primary structural protein in animal cells, understanding its force distribution is central to understanding animal cell physiology and the many linked reactions such as stress-induced gene expression. This new probe permits measuring actin forces in a wide range of experiments on preparations ranging from isolated proteins to transgenic animals. PMID:25422450

  14. Resistance to antidepressant treatment is associated with polymorphisms in the leptin gene, decreased leptin mRNA expression, and decreased leptin serum levels.

    PubMed

    Kloiber, Stefan; Ripke, Stephan; Kohli, Martin A; Reppermund, Simone; Salyakina, Daria; Uher, Rudolf; McGuffin, Peter; Perlis, Roy H; Hamilton, Steven P; Pütz, Benno; Hennings, Johannes; Brückl, Tanja; Klengel, Torsten; Bettecken, Thomas; Ising, Marcus; Uhr, Manfred; Dose, Tatjana; Unschuld, Paul G; Zihl, Josef; Binder, Elisabeth; Müller-Myhsok, Bertram; Holsboer, Florian; Lucae, Susanne

    2013-07-01

    Leptin, a peptide hormone from adipose tissue and key player in weight regulation, has been suggested to be involved in sleep and cognition and to exert antidepressant-like effects, presumably via its action on the HPA-axis and hippocampal function. This led us to investigate whether genetic variants in the leptin gene, the level of leptin mRNA-expression and leptin serum concentrations are associated with response to antidepressant treatment. Our sample consisted of inpatients from the Munich Antidepressant Response Signature (MARS) project with weekly Hamilton Depression ratings, divided into two subsamples. In the exploratory sample (n=251) 17 single nucleotide polymorphisms (SNPs) covering the leptin gene region were genotyped. We found significant associations of several SNPs with impaired antidepressant treatment outcome and impaired cognitive performance after correction for multiple testing. The SNP (rs10487506) showing the highest association with treatment response (p=3.9×10(-5)) was analyzed in the replication sample (n=358) and the association could be verified (p=0.021) with response to tricyclic antidepressants. In an additional meta-analysis combining results from the MARS study with data from the Genome-based Therapeutic Drugs for Depression (GENDEP) and the Sequenced Treatment Alternatives to Relieve Depression (STAR(⁎)D) studies, nominal associations of several polymorphisms in the upstream vicinity of rs10487506 with treatment outcome were detected (p=0.001). In addition, we determined leptin mRNA expression in lymphocytes and leptin serum levels in subsamples of the MARS study. Unfavorable treatment outcome was accompanied with decreased leptin mRNA and leptin serum levels. Our results suggest an involvement of leptin in antidepressant action and cognitive function in depression with genetic polymorphisms in the leptin gene, decreased leptin gene expression and leptin deficiency in serum being risk factors for resistance to antidepressant

  15. Effects of acute diuresis stress on egr-1 (zif268) mRNA levels in brain regions associated with motivated behavior.

    PubMed

    Aher, Chetan V; Duwaerts, Caroline C; Akama, Keith T; Lucas, Louis R

    2010-01-15

    Stressors evoke a well-studied physiological stress-response, namely, an immediate systemic release of catecholamines from the adrenals followed shortly afterwards by the release of adrenal steroids. The intensity of that response can often be inferred by the amount of adrenal steroids released into the circulatory system. It is still unclear however how the intensity and duration of the stressor affect a number of brain regions, including those in the motivational system. The present study sought to determine whether a brief stressor, such as an isotonic saline injection, activated the brain's motivational system in mesolimbic regions compared with a more intense stressor exemplified by pharmacological challenges caused by the administration of a diuretic. Adult male Sprague-Dawley rats were either injected (s.c.) with isotonic saline or 5mg of the diuretic, furosemide. Controls did not receive any injections. Animals were sacrificed at 30, 60, 120, and 240 min after injection and trunk blood and brains were collected. Serum corticosterone and aldosterone levels were assessed through radioimmunoassay and mesolimbic brain activity was determined through in situ hybridization of mRNA expression of the immediate-early gene egr-1 in the caudate-putamen and nucleus accumbens. While both adrenal steroids demonstrated an initial peak in both stress groups, levels were higher and longer lasting in rats treated with furosemide. Interestingly, egr-1 mRNA levels were significantly higher only in the furosemide-treated group compared with controls. These findings suggest that a selective activation of motivational circuits occurs under thirst and salt-appetite-induced conditions such as those caused by diuresis.

  16. Identification of condition-specific regulatory modules through multi-level motif and mRNA expression analysis

    PubMed Central

    Chen, Li; Wang, Yue; Hoffman, Eric P.; Riggins, Rebecca B.; Clarke, Robert

    2013-01-01

    Many computational methods for identification of transcription regulatory modules often result in many false positives in practice due to noise sources of binding information and gene expression profiling data. In this paper, we propose a multi-level strategy for condition-specific gene regulatory module identification by integrating motif binding information and gene expression data through support vector regression and significant analysis. We have demonstrated the feasibility of the proposed method on a yeast cell cycle data set. The study on a breast cancer microarray data set shows that it can successfully identify the significant and reliable regulatory modules associated with breast cancer. PMID:20054984

  17. Targeting Fyn in Ras-transformed cells induces F-actin to promote adherens junction-mediated cell-cell adhesion.

    PubMed

    Fenton, Sarah E; Hutchens, Kelli A; Denning, Mitchell F

    2015-10-01

    Fyn, a member of the Src family kinases (SFK), is an oncogene in murine epidermis and is associated with cell-cell adhesion turnover and induction of cell migration. Additionally, Fyn upregulation has been reported in multiple tumor types, including cutaneous squamous cell carcinoma (cSCC). Introduction of active H-Ras(G12V) into the HaCaT human keratinocyte cell line resulted in upregulation of Fyn mRNA (200-fold) and protein, while expression of other SFKs remained unaltered. Transduction of active Ras or Fyn was sufficient to induce an epithelial-to-mesenchymal transition in HaCaT cells. Inhibition of Fyn activity, using siRNA or the clinical SFK inhibitor Dasatinib, increased cell-cell adhesion and rapidly (5-60 min) increased levels of cortical F-actin. Fyn inhibition with siRNA or Dasatinib also induced F-actin in MDA-MB-231 breast cancer cells, which have elevated Fyn. F-actin co-localized with adherens junction proteins, and Dasatinib-induced cell-cell adhesion could be blocked by Cytochalasin D, indicating that F-actin polymerization was a key initiator of cell-cell adhesion through the adherens junction. Conversely, inhibiting cell-cell adhesion with low Ca(2+) media did not block Dasatinib-induced F-actin polymerization. Inhibition of the Rho effector kinase ROCK blocked Dasatinib-induced F-actin and cell-cell adhesion, implicating relief of Rho GTPase inhibition as a mechanism of Dasatinib-induced cell-cell adhesion. Finally, topical Dasatinib treatment significantly reduced total tumor burden in the SKH1 mouse model of UV-induced skin carcinogenesis. Together these results identify the promotion of actin-based cell-cell adhesion as a newly described mechanism of action for Dasatinib and suggest that Fyn inhibition may be an effective therapeutic approach in treating cSCC.

  18. Actin-filament disassembly: it takes two to shrink them fast.

    PubMed

    Winterhoff, Moritz; Faix, Jan

    2015-06-01

    Actin-filament disassembly is indispensable for replenishing the pool of polymerizable actin and allows continuous dynamic remodelling of the actin cytoskeleton. A new study now reveals that ADF/cofilin preferentially dismantles branched networks and provides new insights into the collaborative work of ADF/cofilin and Aip1 on filament disassembly at the molecular level.

  19. Force of an actin spring

    NASA Astrophysics Data System (ADS)

    Shin, Jennifer; Mahadevan, L.; Matsudaira, Paul

    2003-03-01

    The acrosomal process of the horseshoe crab sperm is a novel mechanochemical molecular spring that converts its elastic stain energy to mechanical work upon the chemical activation by Ca2+. Twisted and bent, the initial state of the acrosomal bundle features a high degree of complexity in its structure and the energy is believed to be stored in the highly strained actin filaments as an elastic potential energy. When activated, the bundle relaxes from the coil of the highly twisted and bent filaments to its straight conformation at a mean velocity of 15um/s. The mean extension velocity increases dramatically from 3um/s to 27um/s when temperature of the medium is changed from 9.6C to 32C (respective viscosities of 1.25-0.75cp), yet it exhibits a very weak dependence on changes in the medium viscosity (1cp-33cp). These experiments suggest that the uncoiling of the actin spring should be limited not by the viscosity of the medium but by the unlatching events of involved proteins at a molecular level. Unlike the viscosity-limited processes, where force is directly related to the rate of the reaction, a direct measurement is required to obtain the spring force of the acrosomal process. The extending acrosomal bundle is forced to push against a barrier and its elastic buckling response is analyzed to measure the force generated during the uncoiling.

  20. 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.

  1. Relationship of high CH50 level and interruption of cascade reaction of complement mRNA expression in acute venous thromboembolism patients

    PubMed Central

    Wen, Siwan; Yang, Fan; Wang, Lemin; Duan, Qianglin; Gong, Zhu; Lv, Wei

    2014-01-01

    In patients with pulmonary embolism (PE), forepart components of complements were activated. However there are interruption/decrease of cascade reaction and cytolytic effects in complement system. This study detected CRP, CH50, C3 and C4 levels in patients with venous thromboembolism (VTE) and compare with the imbalance of complement associated gene mRNA expression in PE patients. There was significant increase of CH50 in acute VTE patients. Even though CH50 increased significantly in acute VTE patients and had a relatively high sensitivity, cytolytic effects of complements might decrease, based on the genomics results of complement cascade reactions imbalance/interruption and increased total complements in VTE patients. PMID:25232435

  2. Manganese peroxidase mRNA and enzyme activity levels during bioremediation of polycyclic aromatic hydrocarbon-contaminated soil with Phanerochaete chrysosporium.

    PubMed Central

    Bogan, B W; Schoenike, B; Lamar, R T; Cullen, D

    1996-01-01

    mRNA extraction from soil and quantitation by competitive reverse transcription-PCR were combined to study the expression of three manganese peroxidase (MnP) genes during removal of polycyclic aromatic hydrocarbons from cultures of Phanerochaete chrysosporium grown in presterilized soil. Periods of high mnp transcript levels and extractable MnP enzyme activity were temporally correlated, although separated by a short (1- to 2-day) lag period. This time frame also coincided with maximal rates of fluorene oxidation and chrysene disappearance in soil cultures, supporting the hypothesis that high ionization potential polycyclic aromatic hydrocarbons are oxidized in soil via MnP-dependent mechanisms. The patterns of transcript abundance over time in soil-grown P. chrysosporium were similar for all three of the mnp mRNAs studied, indicating that transcription of this gene family may be coordinately regulated under these growth conditions. PMID:8779576

  3. Changes of Antioxidant Function and the mRNA Expression Levels of Apoptosis Genes in Duck Ovaries Caused by Molybdenum or/and Cadmium.

    PubMed

    Cao, Huabin; Xia, Bing; Zhang, Mengmeng; Liao, Yilin; Yang, Zhi; Hu, Guoliang; Zhang, Caiying

    2016-06-01

    To investigate the effects of molybdenum (Mo) combined with cadmium (Cd) on the antioxidant function and the mRNA expression levels of apoptosis-related genes in duck ovaries, 60 healthy 11-old-day female ducks were treated with hexaammonium molybdate ([(NH4)6Mo7O24·4H2O]) or/and cadmium sulfate (3CdSO4·8H2O) at different doses on a daily basis for 120 days. On the 120th day, ten female birds in each group were euthanized, and the ovaries and blood were collected to determine the antioxidant indexes and the mRNA expression levels of Bak-1, Bcl-2, and caspase-3 in ovaries. In addition, ovary tissues were subjected to histopathological analysis with optical microscope. The total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activity decreased significantly (P < 0.01) in treated groups comparing with control while the nitric oxide synthase (NOS) activity increased (P < 0.01) both in ovary tissue and serum. The Bak-1 and caspase-3 expressions were upregulated while the Bcl-2 was downgraded by Mo or/and Cd. Biomolecules were affected in all metal-treated groups, whereas combined-treated animals showed greater effects. What is more, pathological damage in Mo and Cd combination treated groups was more severe. The results from the present study indicated that Mo or/and Cd caused oxidative stress and apoptosis in duck ovaries. Combination of Mo and Cd showed additive or synergistic effect leading to apoptosis and oxidative stress, and the pathway might be the mitochondrial pathway.

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

    PubMed Central

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

    2016-01-01

    Erythrina mulungu Benth. (Fabaceae), Cordia verbenacea A. DC. (Boraginaceae), Solanum paniculatum L. (Solanaceae) and Lippia sidoides Cham. (Verbenaceae) are medicinal plant species native to Brazil shortlisted by the Brazilian National Health System for future clinical use. However, nothing is known about their effects in metabolic and transporter proteins, which could potentially lead to herb-drug interactions (HDI). In this work, we assess non-toxic concentrations (100 μg/mL) of the plant infusions for their in vitro ability to modulate CYP3A4 mRNA gene expression and intracellular glutathione levels in HepG2 cells, as well as P-glycoprotein (P-gp) activity in vincristine-resistant Caco-2 cells (Caco-2 VCR). Their mechanisms of action were further studied by measuring the activation of human pregnane X receptor (hPXR) in transiently co-transfected HeLa cells and the inhibition of γ-glutamyl transferase (GGT) in HepG2 cells. Our results show that P-gp activity was not affected in any case and that only Solanum paniculatum was able to significantly change CYP3A4 mRNA gene expression (twofold decrease, p < 0.05), this being correlated with an antagonist effect upon hPXR (EC50 = 0.38 mg/mL). Total intracellular glutathione levels were significantly depleted by exposure to Solanum paniculatum (-44%, p < 0.001), Lippia sidoides (-12%, p < 0.05) and Cordia verbenacea (-47%, p < 0.001). The latter plant extract was able to decrease GGT activity (-48%, p < 0.01). In conclusion, this preclinical study shows that the administration of some of these herbal medicines may be able to cause disturbances to metabolic mechanisms in vitro. Although Erythrina mulungu appears safe in our tests, active pharmacovigilance is recommended for the other three species, especially in the case of Solanum paniculatum. PMID:27594838

  5. Chronic central infusion of ghrelin increases hypothalamic neuropeptide Y and Agouti-related protein mRNA levels and body weight in rats.

    PubMed

    Kamegai, J; Tamura, H; Shimizu, T; Ishii, S; Sugihara, H; Wakabayashi, I

    2001-11-01

    Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), was originally purified from the rat stomach. Like the synthetic growth hormone secretagogues (GHSs), ghrelin specifically releases growth hormone (GH) after intravenous administration. Also consistent with the central actions of GHSs, ghrelin-immunoreactive cells were shown to be located in the hypothalamic arcuate nucleus as well as the stomach. Recently, we showed that a single central administration of ghrelin increased food intake and hypothalamic agouti-related protein (AGRP) gene expression in rodents, and the orexigenic effect of this peptide seems to be independent of its GH-releasing activity. However, the effect of chronic infusion of ghrelin on food consumption and body weight and their possible mechanisms have not been elucidated. In this study, we determined the effects of chronic intracerebroventricular treatment with ghrelin on metabolic factors and on neuropeptide genes that are expressed in hypothalamic neurons that have been previously shown to express the GHS-R and to regulate food consumption. Chronic central administration of rat ghrelin (1 microg/rat every 12 h for 72 h) significantly increased food intake and body weight. However, it did not affect plasma insulin, glucose, leptin, or GH concentrations. We also found that chronic central administration of ghrelin increased both neuropeptide Y (NPY) mRNA levels (151.0 +/- 10.1% of saline-treated controls; P < 0.05) and AGRP mRNA levels (160.0 +/- 22.5% of saline-treated controls; P < 0.05) in the arcuate nucleus. Thus, the primary hypothalamic targets of ghrelin are NPY/AGRP-containing neurons, and ghrelin is a newly discovered orexigenic peptide in the brain and stomach.

  6. Targeting the actin cytoskeleton: selective antitumor action via trapping PKCɛ

    PubMed Central

    Foerster, F; Braig, S; Moser, C; Kubisch, R; Busse, J; Wagner, E; Schmoeckel, E; Mayr, D; Schmitt, S; Huettel, S; Zischka, H; Mueller, R; Vollmar, A M

    2014-01-01

    Targeting the actin cytoskeleton (CSK) of cancer cells offers a valuable strategy in cancer therapy. There are a number of natural compounds that interfere with the actin CSK, but the mode of their cytotoxic action and, moreover, their tumor-specific mechanisms are quite elusive. We used the myxobacterial compound Chondramide as a tool to first elucidate the mechanisms of cytotoxicity of actin targeting in breast cancer cells (MCF7, MDA-MB-231). Chondramide inhibits cellular actin filament dynamics shown by a fluorescence-based analysis (fluorescence recovery after photobleaching (FRAP)) and leads to apoptosis characterized by phosphatidylserine exposure, release of cytochrome C from mitochondria and finally activation of caspases. Chondramide enhances the occurrence of mitochondrial permeability transition (MPT) by affecting known MPT modulators: Hexokinase II bound to the voltage-dependent anion channel (VDAC) translocated from the outer mitochondrial membrane to the cytosol and the proapoptotic protein Bad were recruited to the mitochondria. Importantly, protein kinase C-ɛ (PKCɛ), a prosurvival kinase possessing an actin-binding site and known to regulate the hexokinase/VDAC interaction as well as Bad phosphorylation was identified as the link between actin CSK and apoptosis induction. PKCɛ, which was found overexpressed in breast cancer cells, accumulated in actin bundles induced by Chondramide and lost its activity. Our second goal was to characterize the potential tumor-specific action of actin-binding agents. As the nontumor breast epithelial cell line MCF-10A in fact shows resistance to Chondramide-induced apoptosis and notably express low level of PKCɛ, we suggest that trapping PKCɛ via Chondramide-induced actin hyperpolymerization displays tumor cell specificity. Our work provides a link between targeting the ubiquitously occurring actin CSK and selective inhibition of pro-tumorigenic PKCɛ, thus setting the stage for actin-stabilizing agents as

  7. Efficacy of CDK4 inhibition against sarcomas depends on their levels of CDK4 and p16ink4 mRNA

    PubMed Central

    Perez, Marco; Muñoz-Galván, Sandra; Jiménez-García, Manuel P.; Marín, Juan J.; Carnero, Amancio

    2015-01-01

    Sarcomas are malignant tumors accounting for a high percentage of cancer morbidity and mortality in children and young adults. Surgery and radiation therapy are the accepted treatments for most sarcomas; however, patients with metastatic disease are treated with systemic chemotherapy. Many tumors display marginal levels of chemoresponsiveness and new treatment approaches are needed. Deregulation of the G1 checkpoint is crucial for various oncogenic transformation processes, suggesting that many cancer cell types depend on CDK4/6 activity. Thus, CDK4/6 activity appears to represent a promising therapeutic target for cancer treatment. In the present work, we explore the efficacy of CDK4 inhibition using palbociclib (PD0332991), a highly selective inhibitor of CDK4/6, in a panel of sarcoma cell lines and sarcoma tumor xenografts (PDXs). Palbociclib induces senescence in these cell lines and the responsiveness of these cell lines correlated with their levels of CDK4 mRNA. Palbociclib is also active in vivo against sarcomas displaying high levels of CDK4 but not against sarcomas displaying low levels of CDK4 and high levels of p16ink4a. The analysis of tumors growing after palbociclib showed a clear decrease in the CDK4 levels, indicating that clonal selection occurred in these treated tumors. In summary, our data support the efficacy of CDK4 inhibitors against sarcomas displaying increased CDK4 levels, particularly fibrosarcomas and MPNST. Our results also suggest that high levels of p16ink4a may indicate poor efficacy of CDK4 inhibitors. PMID:26528855

  8. Low-level laser irradiation alters mRNA expression from genes involved in DNA repair and genomic stabilization in myoblasts

    NASA Astrophysics Data System (ADS)

    Trajano, L. A. S. N.; Sergio, L. P. S.; Silva, C. L.; Carvalho, L.; Mencalha, A. L.; Stumbo, A. C.; Fonseca, A. S.

    2016-07-01

    Low-level lasers are used for the treatment of diseases in soft and bone tissues, but few data are available regarding their effects on genomic stability. In this study, we investigated mRNA expression from genes involved in DNA repair and genomic stabilization in myoblasts exposed to low-level infrared laser. C2C12 myoblast cultures in different fetal bovine serum concentrations were exposed to low-level infrared laser (10, 35 and 70 J cm-2), and collected for the evaluation of DNA repair gene expression. Laser exposure increased gene expression related to base excision repair (8-oxoguanine DNA glycosylase and apurinic/apyrimidinic endonuclease 1), nucleotide excision repair (excision repair cross-complementation group 1 and xeroderma pigmentosum C protein) and genomic stabilization (ATM serine/threonine kinase and tumor protein p53) in normal and low fetal bovine serum concentrations. Results suggest that genomic stability could be part of a biostimulation effect of low-level laser therapy in injured muscles.

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

    PubMed Central

    2013-01-01

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

  10. G-actin guides p53 nuclear transport: potential contribution of monomeric actin in altered localization of mutant p53

    PubMed Central

    Saha, Taniya; Guha, Deblina; Manna, Argha; Panda, Abir Kumar; Bhat, Jyotsna; Chatterjee, Subhrangsu; Sa, Gaurisankar

    2016-01-01

    p53 preserves genomic integrity by restricting anomaly at the gene level. Till date, limited information is available for cytosol to nuclear shuttling of p53; except microtubule-based trafficking route, which utilizes minus-end directed motor dynein. The present study suggests that monomeric actin (G-actin) guides p53 traffic towards the nucleus. Histidine-tag pull-down assay using purified p53(1–393)-His and G-actin confirms direct physical association between p53 and monomeric G-actin. Co-immunoprecipitation data supports the same. Confocal imaging explores intense perinuclear colocalization between p53 and G-actin. To address atomistic details of the complex, constraint-based docked model of p53:G-actin complex was generated based on crystal structures. MD simulation reveals that p53 DNA-binding domain arrests very well the G-actin protein. Docking benchmark studies have been carried out for a known crystal structure, 1YCS (complex between p53DBD and BP2), which validates the docking protocol we adopted. Co-immunoprecipitation study using “hot-spot” p53 mutants suggested reduced G-actin association with cancer-associated p53 conformational mutants (R175H and R249S). Considering these findings, we hypothesized that point mutation in p53 structure, which diminishes p53:G-actin complexation results in mutant p53 altered subcellular localization. Our model suggests p53Arg249 form polar-contact with Arg357 of G-actin, which upon mutation, destabilizes p53:G-actin interaction and results in cytoplasmic retention of p53R249S. PMID:27601274

  11. The effects of recombinant bovine somatotropin (rbST) on tissue IGF-I, IGF-I receptor, and GH mRNA levels in rainbow trout, Oncorhynchus mykiss.

    PubMed

    Biga, Peggy R; Schelling, Gerald T; Hardy, Ronald W; Cain, Kenneth D; Overturf, Kenneth; Ott, Troy L

    2004-02-01

    Numerous studies demonstrated that rbST increased growth rates in several fish species, and several species exhibit GH production in tissues other than the pituitary. The role of tissue GH and IGF-I in regulating fish growth is poorly understood. Therefore an experiment was conducted to examine the effects of rbST treatment on tissue GH, IGF-I, and IGF-I receptor-A (rA) expression in rainbow trout. Rainbow trout (550 +/- 10 g) received either intra-peritoneal injections of rbST (120 microg/g body weight) or vehicle on days 0 and 21, and tissue samples were collected on days 0, 0.5, 1, 3, 7, and 28 (n = 6/day/trt). Total RNA was isolated and assayed for steady-state levels of IGF-I, IGF-IrA, and GH mRNA using quantitative RT-PCR. Insulin-like growth factor-I mRNA levels increased in liver, gill, gonad, muscle, brain, and intestine in response to rbST treatment (P < 0.10). Liver IGF-I mRNA increased (P < 0.01) 0.5 day after treatment and remained elevated throughout the trial. Intestine IGF-I mRNA increased (P < 0.05) in treated fish from day 1 to day 3, then decreased to day 7 and increased again at day 28, and remained elevated above control levels throughout the trial. Gill IGF-I mRNA levels increased (P < 0.05) 1 day after treatment and remained elevated throughout the trial. Heart IGF-IrA mRNA levels decreased (P < 0.05) while gonad GH mRNA levels increased (P < 0.10) following rbST treatment. These results demonstrate that rbST treatment increased IGF-I mRNA levels in extra-hepatic tissues, and decreased heart IGF-IrA and increased gonad GH mRNA levels. Because the primary source for endocrine IGF-I is liver, the increased IGF-I mRNA reported in extra-hepatic tissues may indicate local paracrine/autocrine actions for IGF-I for local physiological functions.

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

    PubMed

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

    2014-07-01

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

  13. Effect of low levels of dietary available phosphorus on phosphorus utilization, bone mineralization, phosphorus transporter mRNA expression and performance in growing pigs.

    PubMed

    Pokharel, Bishwo B; Regassa, Alemu; Nyachoti, Charles M; Kim, Woo K

    2017-03-03

    A study was conducted to examine the effects of different dietary levels of available phosphorus (aP) on P excretion, bone mineralization, performance and the mRNA expression of sodium-dependent P transporters in growing pigs. Sixty-day old growing pigs (n = 54) with an average initial BW of 19.50 ± 1.11 kg were randomly allocated to a control diet (C) containing 0.23% available phosphorus (aP), T1 containing 0.17% aP and T2 containing 0.11% aP. There were 6 pens per treatment with 3 pigs per pen. Body weight and feed intake were measured weekly. At the end of each week, one pig from each pen was housed in a metabolic crate for 24 h to collect fecal and urine samples and then sacrificed to obtain third metacarpal (MC3) bones and jejunal and kidney samples. Bones were scanned by Dual Energy X-ray Absorptiometry (DEXA). Fecal and urine samples were sub-sampled and analyzed for P content. The expression of P transporter mRNA in jejunum and kidney samples was measured using quantitative real-time polymerase chain reaction (qRT-PCR). Data were analyzed using GLM procedure of the Statistical Analysis System (SAS Institute version 9.2). Pigs fed the T2 diet had reduced (P < 0.05) average daily gain (ADG) and gain to feed (G:F) compared to those fed the C diet during week 2. Overall, ADG and G:F were also reduced (P < 0.05) in pigs fed the T2 diet compared to those fed the C and T1 diets. Bone mineral density (BMD) and bone mineral content (BMC) were reduced (P < 0.05) in pigs fed the T2 diet compared to those fed the C diet throughout the experiment. At week 1, jejunal mRNA expression of Na (+)-dependent phosphate transporter 2 (SLC34A2) was increased (P < 0.01) in pigs fed the T2 diet compared to C diet. Renal mRNA expression of Na(+)-dependent phosphate transporter 1 (SLC34A1) and SLC34A3 were increased (P < 0.05) in pigs fed the T2 diet compared to those fed the C diet at week 2 and was accompanied by lower (P < 0.05) urinary P in pigs fed the T2 diet during week 2

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

    PubMed Central

    Soyal, Selma M.; Felder, Thomas; Auer, Simon; Oberkofler, Hannes; Iglseder, Bernhard; Paulweber, Bernhard; Dossena, Silvia; Nofziger, Charity; Paulmichl, Markus; Esterbauer, Harald; Krempler, Franz; Patsch, Wolfgang

    2015-01-01

    The genomic region ~500 kb upstream of IRS1 has been implicated in insulin resistance, type 2 diabetes, adverse lipid profile, and cardiovascular risk. To gain further insight into this chromosomal region, we typed four SNPs in a cross-sectional cohort and subjects with type 2 diabetes recruited from the same geographic region. From 16 possible haplotypes, 6 haplotypes with frequencies >0.01 were observed. We identified one haplotype that was protective against insulin resistance (determined by HOMA-IR and fasting plasma insulin levels), type 2 diabetes, an adverse lipid profile, increased C-reactive protein, and asymptomatic atherosclerotic disease (assessed by intima media thickness of the common carotid arteries). BMI and total adipose tissue mass as well as visceral and subcutaneous adipose tissue mass did not differ between the reference and protective haplotypes. In 92 subjects, we observed an association of the protective haplotype with higher skeletal muscle mRNA levels of LOC646736, which is located in the same haplotype block as the informative SNPs and is mainly expressed in skeletal muscle, but only at very low levels in liver or adipose tissues. These data suggest a role for LOC646736 in human insulin resistance and warrant further studies on the functional effects of this locus. PMID:26090471

  15. Transplantable rat glucagonomas cause acute onset of severe anorexia and adipsia despite highly elevated NPY mRNA levels in the hypothalamic arcuate nucleus.

    PubMed Central

    Jensen, P B; Blume, N; Mikkelsen, J D; Larsen, P J; Jensen, H I; Holst, J J; Madsen, O D

    1998-01-01

    We have isolated a stable, transplantable, and small glucagonoma (MSL-G-AN) associated with abrupt onset of severe anorexia occurring 2-3 wk after subcutaneous transplantation. Before onset of anorexia, food consumption is comparable to untreated controls. Anorexia is followed by adipsia and weight loss, and progresses rapidly in severity, eventually resulting in reduction of food and water intake of 100 and 80%, respectively. During the anorectic phase, the rats eventually become hypoglycemic and hypothermic. The tumor-associated anorexia shows no sex difference, and is not affected by bilateral abdominal vagotomy, indicating a direct central effect. The adipose satiety factor leptin, known to suppress food intake by reducing hypothalamic neuropeptide Y (NPY) levels, was not found to be expressed by the tumor, and circulating leptin levels were reduced twofold in the anorectic phase. A highly significant increase in hypothalamic (arcuate nucleus) NPY mRNA levels was found in anorectic rats compared with control animals. Since elevated hypothalamic NPY is among the most potent stimulators of feeding and a characteristic of most animal models of hyperphagia, we conclude that the MSL-G-AN glucagonoma releases circulating factor(s) that overrides the hypothalamic NPY-ergic system, thereby eliminating the orexigenic effect of NPY. We hypothesize a possible central role of proglucagon-derived peptides in the observed anorexia. PMID:9435324

  16. The Drosophila tricellular junction protein Gliotactin regulates its own mRNA levels through BMP-mediated induction of miR-184

    PubMed Central

    Sharifkhodaei, Zohreh; Padash-Barmchi, Mojgan; Gilbert, Mary M.; Samarasekera, Gayathri; Fulga, Tudor A.; Van Vactor, David; Auld, Vanessa J.

    2016-01-01

    ABSTRACT Epithelial bicellular and tricellular junctions are essential for establishing and maintaining permeability barriers. Tricellular junctions are formed by the convergence of three bicellular junctions at the corners of neighbouring epithelia. Gliotactin, a member of the Neuroligin family, is located at the Drosophila tricellular junction, and is crucial for the formation of tricellular and septate junctions, as well as permeability barrier function. Gliotactin protein levels are tightly controlled by phosphorylation at tyrosine residues and endocytosis. Blocking endocytosis or overexpressing Gliotactin results in the spread of Gliotactin from the tricellular junction, resulting in apoptosis, delamination and migration of epithelial cells. We show that Gliotactin levels are also regulated at the mRNA level by micro (mi)RNA-mediated degradation and that miRNAs are targeted to a short region in the 3′UTR that includes a conserved miR-184 target site. miR-184 also targets a suite of septate junction proteins, including NrxIV, coracle and Mcr. miR-184 expression is triggered when Gliotactin is overexpressed, leading to activation of the BMP signalling pathway. Gliotactin specifically interferes with Dad, an inhibitory SMAD, leading to activation of the Tkv type-I receptor and activation of Mad to elevate the biogenesis and expression of miR-184. PMID:26906422

  17. Exposure to bis(maltolato)oxovanadium(IV) increases levels of hepcidin mRNA and impairs the homeostasis of iron but not that of manganese.

    PubMed

    Sánchez-González, Cristina; Rivas-García, Lorenzo; López-Chaves, Carlos; Rodríguez-Nogales, Alba; Algieri, Francesca; Gálvez, Julio; Gómez-Aracena, Jorge; Vera-Ramírez, Laura; Montes-Bayon, Maria; Sanz-Medel, Alfredo; Llopis, Juan

    2014-11-01

    The aim of this study was to examine whether alterations in iron homeostasis, caused by exposure to vanadium, are related to changes in the gene expression of hepatic hepcidin. Two groups of rats were examined: control and vanadium-exposed. Vanadium, as bis(maltolato)oxovanadium(IV) was supplied in the drinking water. The experiment had a duration of five weeks. Iron and manganese were measured in excreta, serum and tissues. Leptin, ferritin, IL-1β, IL-6, TNF-α, red blood cells, haemoglobin and haematocrit were determined. Protein carbonyl group levels and hepcidin gene expression were determined in the liver. In the vanadium-exposed rats, iron absorption, serum iron and leptin and all haematological parameters decreased. Levels of IL-6, TNF-α and ferritin in serum and of iron in the liver, spleen and heart increased. In the liver, levels of protein carbonyl groups and hepcidin mRNA were also higher in the vanadium-exposed group. Exposure to vanadium did not modify manganese homeostasis. The results obtained from this study provide the first evidence that bis(maltolato)oxovanadium(IV) produces an increase in the gene expression of the hepcidin, possibly caused by an inflammatory process. Both factors could be the cause of alterations in Fe homeostasis and the appearance of anaemia. However, Mn homeostasis was not affected.

  18. Enhanced surfactant protein and defensin mRNA levels and reduced viral replication during parainfluenza virus type 3 pneumonia in neonatal lambs.

    PubMed

    Grubor, Branka; Gallup, Jack M; Meyerholz, David K; Crouch, Erika C; Evans, Richard B; Brogden, Kim A; Lehmkuhl, Howard D; Ackermann, Mark R

    2004-05-01

    Defensins and surfactant protein A (SP-A) and SP-D are antimicrobial components of the pulmonary innate immune system. The purpose of this study was to determine the extent to which parainfluenza type 3 virus infection in neonatal lambs alters expression of sheep beta-defensin 1 (SBD-1), SP-A, and SP-D, all of which are constitutively transcribed by respiratory epithelia. Parainfluenza type 3 viral antigen was detected by immunohistochemistry (IHC) in the bronchioles of all infected lambs 3 days postinoculation and at diminished levels 6 days postinoculation, but it was absent 17 days postinoculation. At all times postinoculation, lung homogenates from parainfluenza type 3 virus-inoculated animals had increased SBD-1, SP-A, and SP-D mRNA levels as detected by fluorogenic real-time reverse transcriptase PCR. Protein levels of SP-A in lung homogenates detected by quantitative-competitive enzyme-linked immunosorbent assay and protein antigen of SP-A detected by IHC were not altered. These studies demonstrate that parainfluenza type 3 virus infection results in enhanced expression of constitutively transcribed innate immune factors expressed by respiratory epithelia and that this increased expression occurs concurrently with decreased viral replication.

  19. Expression of insulin-like growth factor I receptors at mRNA and protein levels during metamorphosis of Japanese flounder (Paralichthys olivaceus).

    PubMed

    Zhang, Junling; Shi, Zhiyi; Cheng, Qi; Chen, Xiaowu

    2011-08-01

    Insulin-like growth factor I (IGF-I) is an important regulator of fish growth and development, and its biological actions are initiated by binding to IGF-I receptor (IGF-IR). Our previous study has revealed that IGF-I could play an important role during metamorphosis of Japanese flounder, Paralichthys olivaceus. The analysis of IGF-IR expression thus helps further elucidate the IGF-I regulation of metamorphic processes. In this study, the spatial-temporal expression of two distinct IGF-IR mRNAs was investigated by real-time RT-PCR. The spatial distribution of two IGF-IR mRNAs in adult tissues is largely overlapped, but they exhibit distinct temporal expression patterns during larval development. A remarkable decrease in IGF-IR-2 mRNA was detected during metamorphosis. In contrast, a significant increase in IGF-IR-1 mRNA was determined from pre-metamorphosis to metamorphic completion. These indicate that they may play different function roles during the flounder metamorphosis. The levels and localization of IGF-IR proteins during larval development were further studied by Western blotting and immunohistochemistry. Immunoreactive IGF-IRs were detected throughout larval development, and the IGF-IR proteins displayed a relatively abundant expression during metamorphosis. Moreover, the IGF-IR proteins appeared in key tissues, such as thickened skin beneath the migrating eye, developing intestine, gills and kidney during metamorphosis. These results further suggest that the IGF-I system may be involved in metamorphic development of Japanese flounder.

  20. Nerve growth factor affects Ca2+ currents via the p75 receptor to enhance prolactin mRNA levels in GH3 rat pituitary cells

    PubMed Central

    López-Domínguez, Adriana M; Espinosa, Juan Luis; Navarrete, Araceli; Avila, Guillermo; Cota, Gabriel

    2006-01-01

    In clonal pituitary GH3 cells, spontaneous action potentials drive the opening of Cav1 (L-type) channels, leading to Ca2+ transients that are coupled to prolactin gene transcription. Nerve growth factor (NGF) has been shown to stimulate prolactin synthesis by GH3 cells, but the underlying mechanisms are unknown. Here we studied whether NGF influences prolactin gene expression and Ca2+ currents. By using RT-PCR, NGF (50 ng ml−1) was found to augment prolactin mRNA levels by ∼80% when applied to GH3 cells for 3 days. A parallel change in the prolactin content was detected by Western blotting. Both NGF-induced responses were mimicked by an agonist (Bay K 8644) and prevented by a blocker (nimodipine) of L-type channels. In whole-cell patch-clamp experiments, NGF enhanced the L-type Ca2+ current by ∼2-fold within 60 min. This effect reversed quickly upon growth factor withdrawal, but was maintained for days in the continued presence of NGF. In addition, chronic treatment (≥ 24 h) with NGF amplified the T-type current, which flows through Cav3 channels and is thought to support pacemaking activity. Thus, NGF probably increases the amount of Ca2+ that enters per action potential and may also induce a late increase in spike frequency. MC192, a specific antibody for the p75 neurotrophin receptor, but not tyrosine kinase inhibitors (K252a and lavendustin A), blocked the effects of NGF on Ca2+ currents. Overall, the results indicate that NGF activates the p75 receptor to cause a prolonged increase in Ca2+ influx through L-type channels, which in turn up-regulates the prolactin mRNA. PMID:16690703

  1. Aestivation Induces Changes in the mRNA Expression Levels and Protein Abundance of Two Isoforms of Urea Transporters in the Gills of the African Lungfish, Protopterus annectens.

    PubMed

    Chng, You R; Ong, Jasmine L Y; Ching, Biyun; Chen, Xiu L; Hiong, Kum C; Wong, Wai P; Chew, Shit F; Lam, Siew H; Ip, Yuen K

    2017-01-01

    The African lungfish, Protopterus annectens, is ammonotelic in water despite being ureogenic. When it aestivates in mucus cocoon on land, ammonia is detoxified to urea. During the maintenance phase of aestivation, urea accumulates in the body, which is subsequently excreted upon arousal. Urea excretion involves urea transporters (UT/Ut). This study aimed to clone and sequence the ut isoforms from the gills of P. annectens, and to test the hypothesis that the mRNA and/or protein expression levels of ut/Ut isoforms could vary in the gills of P. annectens during the induction, maintenance, and arousal phases of aestivation. Two isoforms of ut, ut-a2a and ut-a2b, were obtained from the gills of P. annectens. ut-a2a consisted of 1227 bp and coded for 408 amino acids with an estimated molecular mass of 44.7 kDa, while ut-a2b consisted of 1392 bp and coded for 464 amino acids with an estimated molecular mass of 51.2 kDa. Ut-a2a and Ut-a2b of P. annectens had a closer phylogenetic relationship with Ut/UT of tetrapods than Ut of fishes. While the mRNA expression pattern of ut-a2a and ut-a2b across various tissues of P. annectens differed, the transcript levels of ut-a2a and ut-a2b in the gills were comparable, indicating that they might be equally important for branchial urea excretion during the initial arousal phase of aestivation. During the maintenance phase of aestivation, the transcript level of ut-a2a increased significantly, but the protein abundance of Ut-a2a remained unchanged in the gills of P. annectens. This could be an adaptive feature to prepare for an increase in the production of Ut-a2a upon arousal. Indeed, arousal led to a significant increase in the branchial Ut-a2a protein abundance. Although the transcript level of ut-a2b remained unchanged, there were significant increases in the protein abundance of Ut-a2b in the gills of P. annectens throughout the three phases of aestivation. The increase in the protein abundance of Ut-a2b during the maintenance

  2. Aestivation Induces Changes in the mRNA Expression Levels and Protein Abundance of Two Isoforms of Urea Transporters in the Gills of the African Lungfish, Protopterus annectens

    PubMed Central

    Chng, You R.; Ong, Jasmine L. Y.; Ching, Biyun; Chen, Xiu L.; Hiong, Kum C.; Wong, Wai P.; Chew, Shit F.; Lam, Siew H.; Ip, Yuen K.

    2017-01-01

    The African lungfish, Protopterus annectens, is ammonotelic in water despite being ureogenic. When it aestivates in mucus cocoon on land, ammonia is detoxified to urea. During the maintenance phase of aestivation, urea accumulates in the body, which is subsequently excreted upon arousal. Urea excretion involves urea transporters (UT/Ut). This study aimed to clone and sequence the ut isoforms from the gills of P. annectens, and to test the hypothesis that the mRNA and/or protein expression levels of ut/Ut isoforms could vary in the gills of P. annectens during the induction, maintenance, and arousal phases of aestivation. Two isoforms of ut, ut-a2a and ut-a2b, were obtained from the gills of P. annectens. ut-a2a consisted of 1227 bp and coded for 408 amino acids with an estimated molecular mass of 44.7 kDa, while ut-a2b consisted of 1392 bp and coded for 464 amino acids with an estimated molecular mass of 51.2 kDa. Ut-a2a and Ut-a2b of P. annectens had a closer phylogenetic relationship with Ut/UT of tetrapods than Ut of fishes. While the mRNA expression pattern of ut-a2a and ut-a2b across various tissues of P. annectens differed, the transcript levels of ut-a2a and ut-a2b in the gills were comparable, indicating that they might be equally important for branchial urea excretion during the initial arousal phase of aestivation. During the maintenance phase of aestivation, the transcript level of ut-a2a increased significantly, but the protein abundance of Ut-a2a remained unchanged in the gills of P. annectens. This could be an adaptive feature to prepare for an increase in the production of Ut-a2a upon arousal. Indeed, arousal led to a significant increase in the branchial Ut-a2a protein abundance. Although the transcript level of ut-a2b remained unchanged, there were significant increases in the protein abundance of Ut-a2b in the gills of P. annectens throughout the three phases of aestivation. The increase in the protein abundance of Ut-a2b during the maintenance

  3. Force Generation, Polymerization Dynamics and Nucleation of Actin Filaments

    NASA Astrophysics Data System (ADS)

    Wang, Ruizhe

    We study force generation and actin filament dynamics using stochastic and deterministic methods. First, we treat force generation of bundled actin filaments by polymerization via molecular-level stochastic simulations. In the widely-used Brownian Ratchet model, actin filaments grow freely whenever the tip-obstacle gap created by thermal fluctuation exceeds the monomer size. We name this model the Perfect Brownian Ratchet (PBR) model. In the PBR model, actin monomer diffusion is treated implicitly. We perform a series of simulations based on the PBR, in which obstacle motion is treated explicitly; in most previous studies, obstacle motion has been treated implicitly. We find that the cooperativity of filaments is generally weak in the PBR model, meaning that more filaments would grow more slowly given the same force per filament. Closed-form formulas are also developed, which match the simulation results. These portable and accurate formulas provide guidance for experiments and upper and lower bounds for theoretical analyses. We also studied a variation of the PBR, called the Diffusing Brownian Ratchet (DBR) model, in which both actin monomer and obstacle diffusion are treated explicitly. We find that the growth rate of multiple filaments is even lower, compared with that in PBR. This finding challenges the widely-accepted PBR assumption and suggests that pushing the study of actin dynamics down to the sub-nanometer level yields new insights. We subsequently used a rate equation approach to model the effect of local depletion of actin monomers on the nucleation of actin filaments on biomimetic beads, and how the effect is regulated by capping protein (CP). We find that near the bead surface, a higher CP concentration increases local actin concentration, which leads to an enhanced activities of actin filaments' nucleation. Our model analysis matches the experimental results and lends support to an important but undervalued hypothesis proposed by Carlier and

  4. Altered mRNA Levels of Glucocorticoid Receptor, Mineralocorticoid Receptor, and Co-Chaperones (FKBP5 and PTGES3) in the Middle Frontal Gyrus of Autism Spectrum Disorder Subjects.

    PubMed

    Patel, Neil; Crider, Amanda; Pandya, Chirayu D; Ahmed, Anthony O; Pillai, Anilkumar

    2016-05-01

    Although stress has been implicated in the pathophysiology of autistic spectrum disorder (ASD), it is not known whether glucocorticoid receptor (GR) levels are altered in the brain of subjects with ASD. The messenger RNA (mRNA) levels of GR isoforms (GRα, GRβ, GRγ, and GRP), mineralocorticoid receptor (MR), GR co-chaperones (FKBP5, PTGES3, and BAG1), and inflammatory cytokines (IL-6, IL-1β, and IFN-γ) were examined in the postmortem middle frontal gyrus tissues of 13 ASD and 13 age-matched controls by qRT-PCR. The protein levels were examined by Western blotting. We found significant decreases in GRα (64%), GRγ (48%), GRP (20%) and MR (46%) mRNA levels in ASD subjects as compared to controls. However, significant increases in FKBP5 (42%) and PTGES3 (35%) mRNA levels were observed in ASD subjects. There were no differences in the mRNA levels of GRβ and BAG1 in ASD subjects as compared to controls. MR mRNA was found to be negatively correlated with the diagnostic score for abnormality of development. On the protein level, significant reductions in GR and MR, but no change in FKBP5 and PTGES3 were found in ASD subjects as compared to controls. Moreover, we observed significant increases in IL-1β and IFN-γ mRNA levels in ASD subjects, and these cytokines were negatively associated with GR levels. Our data, for the first time, reports dysregulation of GR, MR, FKBP5, and PTGES3 in ASD and suggest a possible role of inflammation in altered GR function in ASD.

  5. Expression of retinoblastoma gene product (pRb) in mantle cell lymphomas. Correlation with cyclin D1 (PRAD1/CCND1) mRNA levels and proliferative activity.

    PubMed Central

    Jares, P.; Campo, E.; Pinyol, M.; Bosch, F.; Miquel, R.; Fernandez, P. L.; Sanchez-Beato, M.; Soler, F.; Perez-Losada, A.; Nayach, I.; Mallofré, C.; Piris, M. A.; Montserrat, E.; Cardesa, A.

    1996-01-01

    Mantle cell lymphomas (MCLs) are molecularly characterized by bcl-1 rearrangement and constant cyclin D1 (PRAD-1/CCND1) gene overexpression. Cyclin D1 is a G1 cyclin that participates in the control of the cell cycle progression by interacting with the retinoblastoma gene product (pRb). Inactivation of the Rb tumor suppressor gene has been implicated in the development of different types of human tumors including some high grade non-Hodgkin's lymphomas. To determine the role of the retinoblastoma gene in the pathogenesis of MCLs and its possible interaction with cyclin D1, pRb expression was examined in 23 MCLs including 17 typical and 6 blastic variants by immunohistochemistry and Western blot. Rb gene structure was studied in 13 cases by Southern blot. Cytogenetic analysis was performed in 5 cases. The results were compared with the cyclin D1 mRNA levels examined by Northern analysis, and the proliferative activity of the tumors was measured by Ki-67 growth fraction and flow cytometry. pRb was expressed in all MCLs. The expression varied from case to case (mean, 14.1% of positive cells; range, 1.3 to 42%) with a significant correlation with the proliferative activity of the tumors (mitotic index r = 0.85; Ki-67 r = 0.7; S phase = 0.73). Blastic variants showed higher numbers of pRb-positive cells (mean, 29%) than the typical cases (10%; P < 0.005) by immunohistochemistry and, concordantly, higher levels of expression by Western blot. In addition, the blastic cases also had an increased expression of the phosphorylated protein. No alterations in Rb gene structure were observed by Southern blot analysis. Cyclin D1 mRNA levels were independent of pRb expression and the proliferative activity of the tumors. These findings suggest that pRb in MCLs is normally regulated in relation to the proliferative activity of the tumors. Cyclin D1 overexpression may play a role in the maintenance of cell proliferation by overcoming the suppressive growth control of pRb. Images

  6. Regulation of c-myc and c-fos mRNA levels by polyomavirus: distinct roles for the capsid protein VP/sub 1/ and the viral early proteins

    SciTech Connect

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

    1987-03-01

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

  7. mRNA levels of kisspeptins, kisspeptin receptors, and GnRH1 in the brain of chub mackerel during puberty.

    PubMed

    Ohga, Hirofumi; Adachi, Hayato; Matsumori, Kojiro; Kodama, Ryoko; Nyuji, Mitsuo; Selvaraj, Sethu; Kato, Keitaro; Yamamoto, Shinji; Yamaguchi, Akihiko; Matsuyama, Michiya

    2015-01-01

    Kisspeptin (Kiss) and its cognate receptor (Kiss1R), implicated in the neuroendocrine control of GnRH secretion in mammals, have been proposed to be the key factors in regulating puberty. However, the mechanisms underlying the initiation of puberty in fish are poorly understood. The chub mackerel Scomber japonicus expresses two forms of Kiss (kiss1 and kiss2) and two Kiss receptor (kissr1 and kissr2) genes in the brain, which exhibit sexually dimorphic changes during the seasonal reproductive cycle. This indicates that the kisspeptin system plays an important role in gonadal recrudescence of chub mackerel; however, the involvement of the kisspeptin system in the pubertal process has not been identified. In the present study, we examined the mRNA expression of kiss1, kiss2, kissr1, kissr2, and gnrh1 (hypophysiotropic form) in the brain of a chub mackerel during puberty. In male fish, kiss2, kissr1 and kissr2 levels increased significantly at 14weeks post-hatch (wph), synchronously with an increase in type A spermatogonial populations in the testis; kiss2 and gnrh1 levels significantly increased at 22wph, just before the onset of meiosis in the testes. In female fish, kiss2 increased significantly at 14wph, synchronously with an increase in the number of perinucleolar oocytes in the ovary; kiss1 and kiss2 levels significantly increased concomitantly with an increase in the kissr1, kissr2, and gnrh1 levels at 24wph, just before the onset of vitellogenesis in oocytes. The present results suggest positive involvement of the kisspeptin-GnRH system in the pubertal process in the captive reared chub mackerel.

  8. Cells Lacking β-Actin are Genetically Reprogrammed and Maintain Conditional Migratory Capacity*

    PubMed Central

    Tondeleir, Davina; Lambrechts, Anja; Müller, Matthias; Jonckheere, Veronique; Doll, Thierry; Vandamme, Drieke; Bakkali, Karima; Waterschoot, Davy; Lemaistre, Marianne; Debeir, Olivier; Decaestecker, Christine; Hinz, Boris; Staes, An; Timmerman, Evy; Colaert, Niklaas; Gevaert, Kris; Vandekerckhove, Joël; Ampe, Christophe

    2012-01-01

    Vertebrate nonmuscle cells express two actin isoforms: cytoplasmic β- and γ-actin. Because of the presence and localized translation of β-actin at the leading edge, this isoform is generally accepted to specifically generate protrusive forces for cell migration. Recent evidence also implicates β-actin in gene regulation. Cell migration without β-actin has remained unstudied until recently and it is unclear whether other actin isoforms can compensate for this cytoplasmic function and/or for its nuclear role. Primary mouse embryonic fibroblasts lacking β-actin display compensatory expression of other actin isoforms. Consistent with this preservation of polymerization capacity, β-actin knockout cells have unchanged lamellipodial protrusion rates despite a severe migration defect. To solve this paradox we applied quantitative proteomics revealing a broad genetic reprogramming of β-actin knockout cells. This also explains why reintroducing β-actin in knockout cells does not restore the affected cell migration. Pathway analysis suggested increased Rho-ROCK signaling, consistent with observed phenotypic changes. We therefore developed and tested a model explaining the phenotypes in β-actin knockout cells based on increased Rho-ROCK signaling and increased TGFβ production resulting in increased adhesion and contractility in the knockout cells. Inhibiting ROCK or myosin restores migration of β-actin knockout cells indicating that other actins compensate for β-actin in this process. Consequently, isoactins act redundantly in providing propulsive forces for cell migration, but β-actin has a unique nuclear function, regulating expression on transcriptional and post-translational levels, thereby preventing myogenic differentiation. PMID:22448045

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

    PubMed Central

    Kametani, Yoshie; Ohshima, Shino; Miyamoto, Asuka; Shigenari, Atsuko; Takasu, Masaki; Imaeda, Noriaki; Matsubara, Tatsuya; Tanaka, Masafumi; Shiina, Takashi; Kamiguchi, Hiroshi; Suzuki, Ryuji; Kitagawa, Hitoshi; Kulski, Jerzy K.; Hirayama, Noriaki; Inoko, Hidetoshi; Ando, Asako

    2016-01-01

    The class I major histocompatibility complex (MHC) presents self-developed peptides to specific T cells to induce cytotoxity against infection. The MHC proteins are encoded by multiple loci that express numerous alleles to preserve the variability of the antigen-presenting ability in each species. The mechanism regulating MHC mRNA and protein expression at each locus is difficult to analyze because of the structural and sequence similarities between alleles. In this study, we examined the correlation between the mRNA and surface protein expression of swine leukocyte antigen (SLA)-1*0401 after the stimulation of peripheral blood mononuclear cells (PBMCs) by Staphylococcus aureus superantigen toxic shock syndrome toxin-1 (TSST-1). We prepared a monoclonal antibody (mAb) against a domain composed of Y102, L103 and L109 in the α2 domain. The Hp-16.0 haplotype swine possess only SLA-1*0401, which has the mAb epitope, while other haplotypes possess 0 to 3 SLA classical class I loci with the mAb epitopes. When PBMCs from SLA-1*0401 homozygous pigs were stimulated, the SLA-1*0401 mRNA expression level increased until 24 hrs and decreased at 48 hrs. The kinetics of the interferon regulatory transcription factor-1 (IRF-1) mRNA level were similar to those of the SLA-1*0401 mRNA. However, the surface protein expression level continued to increase until 72 hrs. Similar results were observed in the Hp-10.0 pigs with three mAb epitopes. These results suggest that TSST-1 stimulation induced both mRNA and surface protein expression of class I SLA in the swine PBMCs differentially and that the surface protein level was sustained independently of mRNA regulation. PMID:27760184

  10. Effect of dark exposure in the middle of the day on Period1, Period2, and arylalkylamine N-acetyltransferase mRNA levels in the rat suprachiasmatic nucleus and pineal gland.

    PubMed

    Fukuhara, Chiaki

    2004-11-04

    The suprachiasmatic nucleus (SCN) of the mammalian hypothalamus contains a central circadian pacemaker, which adjusts circadian rhythms within the body to environmental light-dark cycles. It has been shown that dark exposure in the day causes phase shifts in circadian rhythms, but it does not induce changes in the melatonin levels in the pineal gland. In this study, we examined the effect of dark exposure on two "circadian clock" genes Period1 and Period2 mRNA levels in the rat SCN, and on Period1, Period2, and arylalkylamine N-acetyltransferase (Aa-Nat, the rate-limiting enzyme in melatonin synthesis) gene expression in the pineal gland. Period1 and Period2 mRNA levels were significantly decreased in the SCN after 0.5 and 2 h, respectively, therefore suggesting that changes in those mRNA levels may be the part of the mechanisms of dark-induced phase shifts. Period1 and Aa-Nat mRNA levels in the pineal gland were not affected by darkness, but Period2 was moderately affected. Since Period1 and Aa-Nat mRNA levels in the pineal gland did not respond to dark stimulation, we further examined whether the pineal gland itself is capable of responding to adrenergic stimulation at this time of the day. Isoproterenol significantly induced Period1 and Aa-Nat mRNA levels; however, it did not affect Period2. Although previous studies have reported that during the day the SCN "gates" the dark information reaching the pineal, our data demonstrate that dark information may reach the pineal during the daytime.

  11. β- and γ-Actins in the nucleus of human melanoma A375 cells.

    PubMed

    Migocka-Patrzałek, Marta; Makowiecka, Aleksandra; Nowak, Dorota; Mazur, Antonina J; Hofmann, Wilma A; Malicka-Błaszkiewicz, Maria

    2015-11-01

    Actin is a highly conserved protein that is expressed in all eukaryotic cells and has essential functions in the cytoplasm and the nucleus. Nuclear actin is involved in transcription by all three RNA polymerases, chromatin remodelling, RNA processing, intranuclear transport, nuclear export and in maintenance of the nuclear architecture. The nuclear actin level and polymerization state are important factors regulating nuclear processes such as transcription. Our study shows that, in contrast to the cytoplasm, the majority of endogenous nuclear actin is unpolymerized in human melanoma A375 cells. Most mammalian cells express the two non-muscle β- and γ-actin isoforms that differ in only four amino acids. Despite their sequence similarity, studies analysing the cytoplasmic functions of these isoforms demonstrated that β- and γ-actins show differences in localization and function. However, little is known about the involvement of the individual actin isoforms in nuclear processes. Here, we used the human melanoma A375 cell line to analyse actin isoforms in regard to their nuclear localization. We show that both β- and γ-non-muscle actin isoforms are present in nuclei of these cells. Immunolocalization studies demonstrate that both isoforms co-localize with RNA polymerase II and hnRNP U. However, we observe differences in the ratio of cytoplasmic to nuclear actin distribution between the isoforms. We show that β-actin has a significantly higher nucleus-to-cytoplasm ratio than γ-actin.

  12. Evolutionarily divergent, unstable filamentous actin is essential for gliding motility in apicomplexan parasites.

    PubMed

    Skillman, Kristen M; Diraviyam, Karthikeyan; Khan, Asis; Tang, Keliang; Sept, David; Sibley, L David

    2011-10-01

    Apicomplexan parasites rely on a novel form of actin-based motility called gliding, which depends on parasite actin polymerization, to migrate through their hosts and invade cells. However, parasite actins are divergent both in sequence and function and only form short, unstable filaments in contrast to the stability of conventional actin filaments. The molecular basis for parasite actin filament instability and its relationship to gliding motility remain unresolved. We demonstrate that recombinant Toxoplasma (TgACTI) and Plasmodium (PfACTI and PfACTII) actins polymerized into very short filaments in vitro but were induced to form long, stable filaments by addition of equimolar levels of phalloidin. Parasite actins contain a conserved phalloidin-binding site as determined by molecular modeling and computational docking, yet vary in several residues that are predicted to impact filament stability. In particular, two residues were identified that form intermolecular contacts between different protomers in conventional actin filaments and these residues showed non-conservative differences in apicomplexan parasites. Substitution of divergent residues found in TgACTI with those from mammalian actin resulted in formation of longer, more stable filaments in vitro. Expression of these stabilized actins in T. gondii increased sensitivity to the actin-stabilizing compound jasplakinolide and disrupted normal gliding motility in the absence of treatment. These results identify the molecular basis for short, dynamic filaments in apicomplexan parasites and demonstrate that inherent instability of parasite actin filaments is a critical adaptation for gliding motility.

  13. Evolutionarily Divergent, Unstable Filamentous Actin Is Essential for Gliding Motility in Apicomplexan Parasites

    PubMed Central

    Skillman, Kristen M.; Diraviyam, Karthikeyan; Khan, Asis; Tang, Keliang; Sept, David; Sibley, L. David

    2011-01-01

    Apicomplexan parasites rely on a novel form of actin-based motility called gliding, which depends on parasite actin polymerization, to migrate through their hosts and invade cells. However, parasite actins are divergent both in sequence and function and only form short, unstable filaments in contrast to the stability of conventional actin filaments. The molecular basis for parasite actin filament instability and its relationship to gliding motility remain unresolved. We demonstrate that recombinant Toxoplasma (TgACTI) and Plasmodium (PfACTI and PfACTII) actins polymerized into very short filaments in vitro but were induced to form long, stable filaments by addition of equimolar levels of phalloidin. Parasite actins contain a conserved phalloidin-binding site as determined by molecular modeling and computational docking, yet vary in several residues that are predicted to impact filament stability. In particular, two residues were identified that form intermolecular contacts between different protomers in conventional actin filaments and these residues showed non-conservative differences in apicomplexan parasites. Substitution of divergent residues found in TgACTI with those from mammalian actin resulted in formation of longer, more stable filaments in vitro. Expression of these stabilized actins in T. gondii increased sensitivity to the actin-stabilizing compound jasplakinolide and disrupted normal gliding motility in the absence of treatment. These results identify the molecular basis for short, dynamic filaments in apicomplexan parasites and demonstrate that inherent instability of parasite actin filaments is a critical adaptation for gliding motility. PMID:21998582

  14. Ring closure in actin polymers

    NASA Astrophysics Data System (ADS)

    Sinha, Supurna; Chattopadhyay, Sebanti

    2017-03-01

    We present an analysis for the ring closure probability of semiflexible polymers within the pure bend Worm Like Chain (WLC) model. The ring closure probability predicted from our analysis can be tested against fluorescent actin cyclization experiments. We also discuss the effect of ring closure on bend angle fluctuations in actin polymers.

  15. Simulated microgravity reduces mRNA levels of multidrug resistance genes 4 and 5 in non-metastatic human melanoma cells

    NASA Astrophysics Data System (ADS)

    Eiermann, Peter; Tsiockas, Wasiliki; Hauslage, Jens; Hemmersbach, Ruth; Gerzer, Rupert; Ivanova, Krassimira

    mRNA levels of sGC α and β were down-regulated by about 31% and 22%, respectively. Thus, the reduced expression of MRP4/5 could be related to the decrease in mRNA levels for the sGC subunits. In addition, the long-term exposure to simulated microgravity did not alter cellular morphology. Taken together, the results of our studies indicate that the expression of MRP4/5 in non-metastatic melanoma cells is inversely regulated by hypergravity and simulated microgravity. Finally, a reduced expression of MRP4 and MRP5 may increase the availability of drugs in cells and influence astronaut medication.

  16. Participation of Actin on Giardia lamblia Growth and Encystation

    PubMed Central

    Castillo-Romero, Araceli; Leon-Avila, Gloria; Perez Rangel, Armando; Cortes Zarate, Rafael; Garcia Tovar, Carlos; Hernandez, Jose Manuel

    2009-01-01

    Background Microfilaments play a determinant role in different cell processes such as: motility, cell division, phagocytosis and intracellular transport; however, these structures are poorly understood in the parasite Giardia lamblia. Methodology and Principal Findings By confocal microscopy using TRITC-phalloidin, we found structured actin distributed in the entire trophozoite, the label stand out at the ventral disc, median body, flagella and around the nuclei. During Giardia encystation, a sequence of morphological changes concurrent to modifications on the distribution of structured actin and in the expression of actin mRNA were observed. To elucidate whether actin participates actively on growth and encystation, cells were treated with Cytochalasin D, Latrunculin A and Jasplakinolide and analyzed by confocal and scanning electron microscopy. All drugs caused a growth reduction (27 to 45%) and changes on the distribution of actin. Besides, 60 to 80% of trophozoites treated with the drugs, exhibited damage at the caudal region, alterations in the flagella and wrinkles-like on the plasma membrane. The drugs also altered the cyst-yield and the morphology, scanning electron microscopy revealed diminished cytokinesis, cysts with damages in the wall and alterations in the size and on the intermembranal space. Furthermore, the drugs caused a significant reduction of the intensity of flourescence-labeled CWP1 on ESV and on cyst wall, this was coincident with a reduction of CWP1 gene expression (34%). Conclusions and Significance All our results, indicated an important role of actin in the morphology, growth and encystation and indirectly suggested an actin role in gene expression. PMID:19774081

  17. Viral Replication Protein Inhibits Cellular Cofilin Actin Depolymerization Factor to Regulate the Actin Network and Promote Viral Replicase Assembly

    PubMed Central

    Kovalev, Nikolay; de Castro Martín, Isabel Fernández; Barajas, Daniel; Risco, Cristina; Nagy, Peter D.

    2016-01-01

    RNA viruses exploit host cells by co-opting host factors and lipids and escaping host antiviral responses. Previous genome-wide screens with Tomato bushy stunt virus (TBSV) in the model host yeast have identified 18 cellular genes that are part of the actin network. In this paper, we show that the p33 viral replication factor interacts with the cellular cofilin (Cof1p), which is an actin depolymerization factor. Using temperature-sensitive (ts) Cof1p or actin (Act1p) mutants at a semi-permissive temperature, we find an increased level of TBSV RNA accumulation in yeast cells and elevated in vitro activity of the tombusvirus replicase. We show that the large p33 containing replication organelle-like structures are located in the close vicinity of actin patches in yeast cells or around actin cable hubs in infected plant cells. Therefore, the actin filaments could be involved in VRC assembly and the formation of large viral replication compartments containing many individual VRCs. Moreover, we show that the actin network affects the recruitment of viral and cellular components, including oxysterol binding proteins and VAP proteins to form membrane contact sites for efficient transfer of sterols to the sites of replication. Altogether, the emerging picture is that TBSV, via direct interaction between the p33 replication protein and Cof1p, controls cofilin activities to obstruct the dynamic actin network that leads to efficient subversion of cellular factors for pro-viral functions. In summary, the discovery that TBSV interacts with cellular cofilin and blocks the severing of existing filaments and the formation of new actin filaments in infected cells opens a new window to unravel the way by which viruses could subvert/co-opt cellular proteins and lipids. By regulating the functions of cofilin and the actin network, which are central nodes in cellular pathways, viruses could gain supremacy in subversion of cellular factors for pro-viral functions. PMID:26863541

  18. Effects of different dietary intake on mRNA levels of MSTN, IGF-I, and IGF-II in the skeletal muscle of Dorper and Hu sheep hybrid F1 rams.

    PubMed

    Xing, H J; Wang, Z Y; Zhong, B S; Ying, S J; Nie, H T; Zhou, Z R; Fan, Y X; Wang, F

    2014-07-24

    MSTN, IGF-І(insulin-like growth factor-І) and IGF-II (insulin-like growth factor-II) regulate skeletal muscle growth. This study investigated the effects of different dietary intake levels on skeletal muscles. Sheep was randomly assigned to 3 feeding groups: 1) the maintenance diet (M), 2) 1.4 x the maintenance diet (1.4M), and 3) 2.15 x the maintenance diet (2.15M). Before slaughtering the animals, blood samples were collected to measure plasma urea, growth hormone, and insulin concentrations. After slaughtering, the longissimus dorsi, semitendinosus, semimembranosus, gastrocnemius, soleus, and chest muscle were removed to record various parameters, including the mRNA expression levels of MSTN and IGFs, in addition to skeletal muscle fiber diameter and cross-sectional area. The result showed that as dietary intake improved, the mRNA expression levels of MSTN and IGF-II decreased, whereas IGF-Іexpression increased. The mRNA expression levels of MSTN and IGFs were significantly different in the same skeletal muscle under different dietary intake. The skeletal muscle fiber diameter and cross-sectional area increased with greater dietary intake, as observed for the mRNA expression of IGF-І; however, it contrasted to that observed for the mRNA expression of MSTN and IGF-II. In conclusion, dietary intake levels have a certain influence on MSTN and IGFs mRNA expression levels, in addition to skeletal muscle fiber diameter and cross-sectional area. This study contributes valuable information for enhancing the molecular-based breeding of sheep.

  19. Immunolabelling, histochemistry and in situ hybridisation in human skeletal muscle fibres to detect myosin heavy chain expression at the protein and mRNA level

    PubMed Central

    SERRANO, A. L.; PÉREZ, MARGARITA; LUCÍA, A.; CHICHARRO, J. L.; QUIROZ-ROTHE, E.; RIVERO, J. L. L.

    2001-01-01

    The distribution of muscle fibres classified on the basis of their content of different myosin heavy chain (MHC) isoforms was analysed in vastus lateralis muscle biopsies of 15 young men (with an average age of 22 y) by correlating immunohistochemistry with specific anti-MHC monoclonal antibodies, myofibrillar ATPase (mATPase) histochemistry and in situ hybridisation with probes specific for MHC β-slow, MHC-IIA and MHC-IIX. The characterisation of a large number of individual fibres was compared and correlated on a fibre-to-fibre basis. The panel of monoclonal antibodies used in the study allowed classification of human skeletal muscle fibres into 5 categories according to the MHC isoform they express at the protein level, types I, I+IIA, IIA, IIAX and IIX. Hybrid fibres coexpressing two isoforms represented a considerable proportion of the fibre composition (about 14%) and were clearly underestimated by mATPase histochemistry. For a very high percentage of fibres there was a precise correspondence between the MHC protein isoforms and mRNA transcripts. The integrated methods used demonstrate a high degree of precision of the immunohistochemical procedure used for the identification and quantification of human skeletal muscle fibre types. The monoclonal antibody S5-8H2 is particularly useful for identifying hybrid IIAX fibres. This protocol offers new prospects for muscle fibre classification in human experimental studies. PMID:11554510

  20. Effects of nefiracetam on the levels of brain-derived neurotrophic factor and synapsin I mRNA and protein in the hippocampus of microsphere-embolized rats.

    PubMed

    Ando, Tsuyoshi; Takagi, Norio; Takagi, Keiko; Kago, Tomoyuki; Takeo, Satoshi

    2005-01-10

    Our recent study demonstrated that nefiracetam, N-(2,6-dimethylphenyl)-2-(2-oxo-1-pyrrolidinyl) acetamide, prevented impairment of the cyclic AMP (cAMP)/cAMP-responsive element binding (CREB) protein signaling pathway in sustained cerebral ischemia. The purpose of the present study was to determine whether nefiracetam has an effect on the expression of brain-derived neurotrophic factor (BDNF) and synapsin I mRNAs that are believed to be produced via CREB, and the alteration in their protein contents in the hippocampus after cerebral ischemia. Sustained cerebral ischemia was induced by injection of 700 microspheres into the right hemisphere of each rat. The rats were treated once daily with 10 mg/kg nefiracetam, p.o., from 15 h after the operation. Treatment with nefiracetam reduced the prolongation of the escape latency in the water maze test on days 7-9 after microsphere embolism-induced sustained cerebral ischemia, suggesting an improvement in the spatial learning function. Microsphere-embolized rats on day 5 showed decreases in BDNF and synapsin I mRNA levels and their protein contents in the ipsilateral hippocampus. Treatment with nefiracetam partially attenuated the decreases. These results suggest that enhancement of BDNF and synapsin I expression by nefiracetam treatment may be, at least in part, due to the improvement in the CREB binding activity, contributing to the prevention of learning and memory dysfunction after sustained cerebral ischemia.

  1. Effect of α-Hederin on IL-2 and IL-17 mRNA and miRNA-133a Levels in Lungs of Ovalbumin-Sensitized Male Rats.

    PubMed

    Ebrahimi, Hadi; Fallahi, Maryam; Khamaneh, Amir Mahdi; Ebrahimi Saadatlou, Mohammad Ali; Saadat, Saeideh; Keyhanmanesh, Rana

    2016-03-01

    α-hederin, a saponin that is a major constituent of English Ivy (Hedera helix) is effective in the treatment of asthma. In the present study, the effect of α-hederin on lung tissue pathology and the levels of the inflammatory mediators; IL-2 mRNA, IL-17 mRNA, and MicroRNAs (miRNA)-133a was evaluated in a rat ovalbumin (OVA)-sensitized model of asthma. Rats were divided randomly into control (C), OVA-sensitized (S), OVA-sensitized pretreated with the antioxidant, thymoquinone (3 mg/kg, S + TQ) or OVA-sensitized pretreated with α-hederin (0.02 mg/kg, S + AH) groups. Levels of IL-2 and IL-17 mRNA were higher in the OVA-sensitized group than controls while the level of miRNA-133a gene expression was lower. IL-2 mRNA and miRNA-133a gene expression in the S + TQ group was higher than in the control and OVA-sensitized groups while the level of IL-17 mRNA in the S + TQ group was lower than in the OVA-sensitized group. Pretreatment with α-hederin decreased IL-17 mRNA levels and increased miRNA-133a gene expression compared with OVA-sensitized animals. All pathological changes in pretreated groups were lower than the OVA-sensitized group. These results showed a beneficial effect of α-hederin in OVA-sensitized rats, suggesting that α-hederin affects the IL-2 and IL-17 secretion pathways, altering miRNA-133a expression.

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

    PubMed

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

    2017-05-01

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

  3. Novel actin depolymerizing macrolide aplyronine A.

    PubMed

    Saito, S; Watabe, S; Ozaki, H; Kigoshi, H; Yamada, K; Fusetani, N; Karaki, H

    1996-09-01

    Aplyronine A is a macrolide isolated from Aplysia kurodai. By monitoring fluorescent intensity of pyrenyl-actin, it was found that aplyronine A inhibited both the velocity and the degree of actin polymerization. Aplyronine A also quickly depolymerized F-actin. The kinetics of depolymerization suggest that aplyronine A severs F-actin. The relationship between the concentration of total actin and F-actin at different concentrations of aplyronine A suggests that aplyronine A forms a 1:1 complex with G-actin. From these results, it is concluded that aplyronine A inhibits actin polymerization and depolymerizes F-actin by nibbling. Comparison of the chemical structure of aplyronine A and another actin-depolymerizing macrolide, mycalolide B, suggests that the side-chain but not the macrolide ring of aplyronine A may account for its actin binding and severing activity.

  4. Bacterial Actins and Their Interactors.

    PubMed

    Gayathri, Pananghat

    2017-01-01

    Bacterial actins polymerize in the presence of nucleotide (preferably ATP), form a common arrangement of monomeric interfaces within a protofilament, and undergo ATP hydrolysis-dependent change in stability of the filament-all of which contribute to performing their respective functions. The relative stability of the filament in the ADP-bound form compared to that of ATP and the rate of addition of monomers at the two ends decide the filament dynamics. One of the major differences between eukaryotic actin and bacterial actins is the variety in protofilament arrangements and dynamics exhibited by the latter. The filament structure and the polymerization dynamics enable them to perform various functions such as shape determination in rod-shaped bacteria (MreB), cell division (FtsA), plasmid segregation (ParM family of actin-like proteins), and organelle positioning (MamK). Though the architecture and dynamics of a few representative filaments have been studied, information on the effect of interacting partners on bacterial actin filament dynamics is not very well known. The chapter reviews some of the structural and functional aspects of bacterial actins, with special focus on the effect that interacting partners exert on the dynamics of bacterial actins, and how these assist them to carry out the functions within the bacterial cell.

  5. Requirements for intercistronic distance and level of eukaryotic initiation factor 2 activity in reinitiation on GCN4 mRNA vary with the downstream cistron.

    PubMed Central

    Grant, C M; Miller, P F; Hinnebusch, A G

    1994-01-01

    Translational control of the GCN4 gene in response to amino acid availability is mediated by four short open reading frames in the GCN4 mRNA leader (uORFs) and by phosphorylation of eukaryotic initiation factor 2 (eIF-2). We have proposed that reducing eIF-2 activity by phosphorylation of its alpha subunit or by a mutation in the eIF-2 recycling factor eIF-2B allows ribosomes which have translated the 5'-proximal uORF1 to bypass uORF2 to uORF4 and reinitiate at GCN4 instead. In this report, we present two lines of evidence that all ribosomes which synthesize GCN4 have previously translated uORF1, resumed scanning, and reinitiated at the GCN4 start site. First, GCN4 expression was abolished when uORF1 was elongated to make it overlap the beginning of the GCN4 coding region. Second, GCN4 expression was reduced as uORF1 was moved progressively closer to GCN4, decreasing to only 5% of the level seen in the absence of all uORFs when only 32 nucleotides separated uORF1 from GCN4. We additionally found that inserting small synthetic uORFs between uORF4 and GCN4 inhibited GCN4 expression under derepressing conditions, confirming the idea that reinitiation at GCN4 under conditions of diminished eIF-2 activity is proportional to the distance of the reinitiation site downstream from uORF1. While uORF4 and GCN4 appear to be equally effective at capturing ribosomes scanning downstream from the 5' cap of mRNA, these two ORFs differ greatly in their ability to capture reinitiating ribosomes scanning from uORF1. When the active form of eIF-2 is present at high levels, reinitiation appears to be much more efficient at uORF4 than at GCN4 when each is located very close to uORF1. Under conditions of reduced recycling of eIF-2, reinitiation at uORF4 is substantially suppressed, which allows ribosomes to reach the GCN4 start site; in contrast, reinitiation at GCN4 in constructs lacking uORF4 is unaffected by decreasing the level of eIF-2 activity. This last finding raises the

  6. Peroxisome proliferator-activated receptor alpha (PPARalpha) activators, bezafibrate and Wy-14,643, increase uncoupling protein-3 mRNA levels without modifying the mitochondrial membrane potential in primary culture of rat preadipocytes.

    PubMed

    Cabrero, A; Alegret, M; Sánchez, R; Adzet, T; Laguna, J C; Vázquez, M

    2000-08-15

    Uncoupling proteins (UCPs) are inner mitochondrial membrane transporters which act as pores for H(+) ions, dissipating the electrochemical gradient that develops during mitochondrial respiration at the expense of ATP synthesis. We have studied the effects of two fibrates, bezafibrate and Wy-14,643, on UCP-3 and UCP-2 mRNA levels in primary monolayer cultures of rat adipocytes and undifferentiated preadipocytes. Treatment with both PPARalpha activators for 24 h up-regulated UCP-3 mRNA levels. Thus, bezafibrate treatment resulted in an 8-fold induction in UCP-3 mRNA levels in preadipocytes compared with the 3.5-fold induction observed in adipocytes. Differences in the induction of UCP-3 between these cells correlated well with the higher expression of PPARalpha and RXRalpha mRNA values in preadipocytes compared to adipocytes. Wy-14,643 caused similar effects on UCP-3 mRNA expression. In contrast to UCP-3, UCP-2 mRNA levels were only slightly modified by bezafibrate in adipocytes. The induction in UCP-3 expression was not accompanied by changes in the mitochondrial membrane potential of rat primary preadipocytes after bezafibrate or Wy-14,643 treatment. Since it has been proposed that UCP-3 could be involved in the regulation of the use of fatty acids as fuel substrates, the UCP-3 induction achieved after bezafibrate and Wy-14, 643 treatment may indicate a higher oxidation of fatty acids, limiting their availability to be stored as triglycerides. This change may result in a reduced rate of conversion of preadipocytes to adipocytes, which directly affects fat depots.

  7. Changes in liver PPARalpha mRNA expression in response to two levels of high-safflower-oil diets correlate with changes in adiposity and serum leptin in rats and mice.

    PubMed

    Hsu, Shan-Ching; Huang, Ching-jang

    2007-02-01

    The ligand-dependent transcription factor peroxisome proliferator-activated receptor alpha (PPARalpha) is known to be activated by common fatty acids and to regulate the expression of genes of various lipid oxidation pathways and transport. High-fat diets provide more fatty acids, which presumably could enhance lipid catabolism through up-regulation of PPARalpha signaling. However, high intake of fat could also lead to obesity. To examine PPARalpha signaling in high-fat feeding and obesity, this study examined the hepatic mRNA expression of PPARalpha and some of its target genes in Wistar rats and C57BL/6J mice fed two levels (20% or 30% wt/wt) of high-safflower-oil (SFO; oleic-acid-rich) diets until animals showed significantly higher body weight (13 weeks for rats and 22 weeks for mice) than those of control groups fed a 5% SFO diet. At the end of these respective feeding periods, only the rats fed 30% SFO and the mice fed 20% SFO among the two groups fed high-fat diets showed significantly higher body weight, white adipose tissue weight, serum leptin and mRNA expression of PPARalpha (P<.05) compared to the respective control groups. Despite elevated acyl-CoA (a PPARalpha target gene) protein and activity in both groups fed high-fat diets, the mRNA expression level of most PPARalpha target genes examined correlated mainly to PPARalpha mRNA levels and not to fat intake or liver lipid levels. The observation that the liver PPARalpha mRNA expression in groups fed high-fat diets was significantly higher only in obese animals with elevated serum leptin implied that obesity and associated hyperleptinemia might have a stronger impact than dietary SFO intake per se on PPARalpha-regulated mRNA expression in the liver.

  8. Macrophage Migration Inhibitory Factor Promoter Polymorphisms (−794 CATT5–8 and −173 G>C): Relationship with mRNA Expression and Soluble MIF Levels in Young Obese Subjects

    PubMed Central

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

    2015-01-01

    We analyzed the relationship of −794 CATT5–8 and −173 G>C MIF polymorphisms with mRNA and soluble MIF in young obese subjects. A total of 250 young subjects, 150 normal-weight and 100 obese subjects, were recruited in the study. Genotyping of −794 CATT5–8 and −173 G>C MIF polymorphisms was performed by PCR and PCR-RFLP, respectively. MIF mRNA expression was determined by real-time PCR and serum MIF levels were measured using an ELISA kit. For both MIF promoter polymorphisms, no significant differences in the genotype and allele frequencies between groups were observed. MIF mRNA expression was slightly higher in obese subjects than in normal-weight subjects (1.38-fold), while soluble MIF levels did not show differences between groups. In addition, we found an increase in MIF mRNA expression in carriers of the 6,6 and C/C genotypes and the 6G haplotype of the −794 CATT5–8 and −173 G>C MIF polymorphisms, although it was not significant. In conclusion, this study found no relationship between obesity and MIF gene promoter polymorphisms with MIF mRNA expression in young obese subjects. PMID:25972622

  9. Expression at mRNA level of cytokines and A238L gene in porcine blood-derived macrophages infected in vitro with African swine fever virus (ASFV) isolates of different virulence.

    PubMed

    Gil, S; Spagnuolo-Weaver, M; Canals, A; Sepúlveda, N; Oliveira, J; Aleixo, A; Allan, G; Leitão, A; Martins, C L V

    2003-11-01

    Porcine macrophage cultures were infected with two ASFV isolates of variable virulence and mRNA levels of several relevant macrophage-derived cytokines were quantified by real time PCR. At six hours post infection, a clear enhancement of mRNA expression of TNFalpha, IL6, IL12 and IL15 was observed in macrophages infected with the low virulent ASFV/NH/P68 (NHV) when compared to those infected with the highly virulent ASFV/L60 (L60). The sequence of the A238L gene homologue to the cellular IkappaB was found identical in both viral isolates and its expression at mRNA level was higher in macrophages infected with NHV when compared to macrophages infected with L60. Furthermore our results suggest a negative correlation between the mRNA expression of A238L gene and the mRNA expression of the above mentioned cytokines (with the exception of IL10) in L60 infected macrophages in opposition to the positive correlation (with exception of the IL1) suggested in NHV infection. Overall, our data strongly emphasize that virulence of ASFV isolates may depend on their capacity to regulate the expression of macrophage-derived cytokines relevant for the development of host protective responses by yet unknown mechanisms triggered by the virus at early stages of the cellular infection.

  10. Macrophage migration inhibitory factor promoter polymorphisms (-794 CATT 5-8 and -173 G>C): relationship with mRNA expression and soluble MIF levels in young obese subjects.

    PubMed

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

    2015-01-01

    We analyzed the relationship of -794 CATT5-8 and -173 G>C MIF polymorphisms with mRNA and soluble MIF in young obese subjects. A total of 250 young subjects, 150 normal-weight and 100 obese subjects, were recruited in the study. Genotyping of -794 CATT5-8 and -173 G>C MIF polymorphisms was performed by PCR and PCR-RFLP, respectively. MIF mRNA expression was determined by real-time PCR and serum MIF levels were measured using an ELISA kit. For both MIF promoter polymorphisms, no significant differences in the genotype and allele frequencies between groups were observed. MIF mRNA expression was slightly higher in obese subjects than in normal-weight subjects (1.38-fold), while soluble MIF levels did not show differences between groups. In addition, we found an increase in MIF mRNA expression in carriers of the 6,6 and C/C genotypes and the 6G haplotype of the -794 CATT5-8 and -173 G>C MIF polymorphisms, although it was not significant. In conclusion, this study found no relationship between obesity and MIF gene promoter polymorphisms with MIF mRNA expression in young obese subjects.

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

    PubMed

    Sonne, Si B; Hoei-Hansen, Christina E; Nielsen, John E; Herlihy, Amy S; Andersson, Anna-Maria; Almstrup, Kristian; Daugaard, Gedske; Skakkebaek, Niels E; Leffers, Henrik; Rajpert-De Meyts, Ewa

    2006-01-01

    E-cadherin (CDH1) is a transmembrane glycoprotein involved in cellular adhesion. In our recent microarray studies of testicular germ cell tumours (TGCTs) and the common precursor carcinoma in situ (CIS), CDH1 mRNA was highly expressed in CIS and embryonal carcinoma. It has previously been reported that the CDH1 protein is not expressed in CIS. To resolve the discrepancy, we performed a detailed analysis of the expression of CDH1 mRNA and protein in a series of normal and neoplastic testes. High expression of CDH1 mRNA in CIS was confirmed by real-time PCR and in situ hybridisation. At the protein level, however, CDH1 was only detected with one of three tested antibodies, but Western blotting analysis with this antibody showed additional bands, suggesting unspecific staining. The levels of a CDH1 protein fragment in serum samples from 58 patients with TGCTs were analysed by ELISA; we found significantly higher levels in patients with advanced disease (stage II/III) when compared to healthy individuals and patients with stage I TGCT. In conclusion, despite high mRNA levels, the CDH1 protein is not expressed in CIS, suggesting translational suppression of CDH1 protein expression. CDH1 serum levels may be a serological marker for staging of TGCT patients.

  12. Actin cytoskeleton: putting a CAP on actin polymerization.

    PubMed

    Stevenson, V A; Theurkauf, W E

    2000-10-05

    Two recent studies have identified a Drosophila homolog of cyclase-associated protein (CAP) as a developmentally important negative regulator of actin polymerization that may also directly mediate signal transduction.

  13. Formin' actin in the nucleus.

    PubMed

    Baarlink, Christian; Grosse, Robert

    2014-01-01

    Many if not most proteins can, under certain conditions, change cellular compartments, such as, for example, shuttling from the cytoplasm to the nucleus. Thus, many proteins may exert functions in various and very different subcellular locations, depending on the signaling context. A large amount of actin regulatory proteins has been detected in the mammalian cell nucleus, although their potential roles are much debated and are just beginning to emerge. Recently, members of the formin family of actin nucleators were also reported to dynamically localize to the nuclear environment. Here we discuss our findings that specific diaphanous-related formins can promote nuclear actin assembly in a signal-dependent manner.

  14. The BAX gene as a candidate for negative autophagy-related genes regulator on mRNA levels in colorectal cancer.

    PubMed

    Gil, Justyna; Ramsey, David; Szmida, Elzbieta; Leszczynski, Przemyslaw; Pawlowski, Pawel; Bebenek, Marek; Sasiadek, Maria M

    2017-02-01

    Autophagy is a catabolic process, which is involved in the maintenance of intracellular homeostasis by degrading redundant molecules and organelles. Autophagy begins with the formation of a double-membrane phagophore, followed by its enclosure, thus leading to the appearance of an autophagosome which fuses with lysosome. This process is highly conserved, precisely orchestrated and regulated by autophagy-related genes. Recently, autophagy has been widely studied in different types of cancers, including colorectal cancer. As it has been revealed, autophagy plays two opposite roles in tumorigenesis, as a tumor suppressor and a tumor enhancer/activator, and therefore is called a double-edge sword. Recently, interaction between autophagy and apoptosis has been found. Therefore, we aimed to study the mRNA levels of genes engaged in autophagy and apoptosis in colorectal cancer tissues. Colorectal cancer and adjacent healthy tissues were obtained from 73 patients diagnosed with primary colorectal cancer. Real-time PCR analysis employing Universal Probe Library was used to assess the expression of the seven following selected genes: BECN1, UVRAG, ULK1, ATG13, Bif-1, BCL2 and BAX. For all but one of the tested genes, a decrease in expression was observed. An increase in expression was observed for BAX. BAX expression decreases consistently from early to more advanced stages. High expression of BAX was strongly associated with negative UVRAG expression. The high expression of the BAX gene seems to be a negative regulator of autophagy in colorectal cancer cells. The relative downregulation of autophagy-related genes was observed in colorectal cancer samples.

  15. Reconstitution of actin-based motility of Listeria and Shigella using pure proteins

    NASA Astrophysics Data System (ADS)

    Loisel, Thomas P.; Boujemaa, Rajaa; Pantaloni, Dominique; Carlier, Marie-France

    1999-10-01

    Actin polymerization is essential for cell locomotion and is thought to generate the force responsible for cellular protrusions. The Arp2/3 complex is required to stimulate actin assembly at the leading edge in response to signalling. The bacteria Listeria and Shigella bypass the signalling pathway and harness the Arp2/3 complex to induce actin assembly and to propel themselves in living cells. However, the Arp2/3 complex alone is insufficient to promote movement. Here we have used pure components of the actin cytoskeleton to reconstitute sustained movement in Listeria and Shigella in vitro. Actin-based propulsion is driven by the free energy released by ATP hydrolysis linked to actin polymerization, and does not require myosin. In addition to actin and activated Arp2/3 complex, actin depolymerizing factor (ADF, or cofilin) and capping protein are also required for motility as they maintain a high steady-state level of G-actin, which controls the rate of unidirectional growth of actin filaments at the surface of the bacterium. The movement is more effective when profilin, α-actinin and VASP (for Listeria) are also included. These results have implications for our understanding of the mechanism of actin-based motility in cells.

  16. Lack of Drosophila cytoskeletal tropomyosin affects head morphogenesis and the accumulation of oskar mRNA required for germ cell formation.

    PubMed Central

    Tetzlaff, M T; Jäckle, H; Pankratz, M J

    1996-01-01

    Drosophila encodes five muscle and one cytoskeletal isoform of the actin-binding protein tropomyosin. We have identified a lack-of-function mutation in the cytoskeletal isoform (cTmII). Zygotic mutant embryos show a defect in head morphogenesis, while embryos lacking maternal cTmII are defective in germ cell formation but otherwise give rise to viable adults. oskar mRNA, which is required for both germ cell formation and abdominal segmentation, fails to accumulate at the posterior pole in these embryos. nanos mRNA, however, which is required exclusively for abdominal segmentation, is localized at wild-type levels. These results indicate that head morphogenesis and the accumulation of high levels of oskar mRNA necessary for germ cell formation require tropomyosin-dependent cytoskeleton. Images PMID:8635457

  17. Comparative effects of low-level laser therapy pre- and post-injury on mRNA expression of MyoD, myogenin, and IL-6 during the skeletal muscle repair.

    PubMed

    Alves, Agnelo Neves; Ribeiro, Beatriz Guimarães; Fernandes, Kristianne Porta Santos; Souza, Nadhia Helena Costa; Rocha, Lília Alves; Nunes, Fabio Daumas; Bussadori, Sandra Kalil; Mesquita-Ferrari, Raquel Agnelli

    2016-05-01

    This study analyzed the effect of pre-injury and post-injury irradiation with low-level laser therapy (LLLT) on the mRNA expression of myogenic regulatory factors and interleukin 6 (IL-6) during the skeletal muscle repair. Male rats were divided into six groups: control group, sham group, LLLT group, injury group; pre-injury LLLT group, and post-injury LLLT group. LLLT was performed with a diode laser (wavelength 780 nm; output power 40 mW' and total energy 3.2 J). Cryoinjury was induced by two applications of a metal probe cooled in liquid nitrogen directly onto the belly of the tibialis anterior (TA) muscle. After euthanasia, the TA muscle was removed for the isolation of total RNA and analysis of MyoD, myogenin, and IL-6 using real-time quantitative PCR. Significant increases were found in the expression of MyoD mRNA at 3 and 7 days as well as the expression of myogenin mRNA at 14 days in the post-injury LLLT group in comparison to injury group. A significant reduction was found in the expression of IL-6 mRNA at 3 and 7 days in the pre-injury LLLT and post-injury LLLT groups. A significant increase in IL-6 mRNA was found at 14 days in the post-injury LLLT group in comparison to the injury group. LLLT administered following muscle injury modulates the mRNA expression of MyoD and myogenin. Moreover, the both forms of LLLT administration were able to modulate the mRNA expression of IL-6 during the muscle repair process.

  18. A Kampo (traditional Japanese herbal) medicine, Hochuekkito, pretreatment in mice prevented influenza virus replication accompanied with GM-CSF expression and increase in several defensin mRNA levels.

    PubMed

    Dan, Katsuaki; Akiyoshi, Hiroko; Munakata, Kaori; Hasegawa, Hideki; Watanabe, Kenji

    2013-01-01

    A Kampo medicine, Hochuekkito (TJ-41), with an influenza virus-preventing effect had life-extending effectiveness, and immunological responses other than interferon (IFN)-α release were examined. TJ-41 (1 g/kg) was given to C57BL/6 male mice orally once a day for 2 weeks. Mice were then intranasally infected with influenza virus. After infection, virus titers and various parameters, mRNA levels and protein expression, for immunoresponses in the bronchoalveolar lavage fluid or removed lung homogenate, were measured by plaque assay, quantitative RT-PCR and ELISA. IFN-α and -β levels of TJ-41-treated mice were higher than those of the control. Toll-like receptor TLR7 and TLR9 mRNAs were elevated after infection, but retinoic acid-inducible gene (RIG-1) family mRNA levels, RIG-1, melanoma differentiation-associated gene 5 and Leishmania G protein 2 showed no response in either TJ-41 or control groups. Interferon regulatory transcription factor (IRF)-3 mRNA levels to stimulate type I (α/β) IFN were increased, but IRF-7 did not change. Only granulocyte-macrophage colony-stimulating factor (GM-CSF) after Hochuekkito treatment was significantly elevated 2 and 3 days after infection. The mRNA levels of 7 defensins after infection increased compared to preinfection values. The key roles of TJ-41 were not only stimulation of type I IFN release but also GM-CSF-derived anti-inflammation activity. Furthermore, defensin (antimicrobial peptide) mRNA levels increased by infection and were further enhanced by TJ-41 treatment. Defensin might prevent influenza virus replication.

  19. Dual pools of actin at presynaptic terminals.

    PubMed

    Bleckert, Adam; Photowala, Huzefa; Alford, Simon

    2012-06-01

    We investigated actin's function in vesicle recycling and exocytosis at lamprey synapses and show that FM1-43 puncta and phalloidin-labeled filamentous actin (F-actin) structures are colocalized, yet recycling vesicles are not contained within F-actin clusters. Additionally, phalloidin also labels a plasma membrane-associated cortical actin. Injection of fluorescent G-actin revealed activity-independent dynamic actin incorporation into presynaptic synaptic vesicle clusters but not into cortical actin. Latrunculin-A, which sequesters G-actin, dispersed vesicle-associated actin structures and prevented subsequent labeled G-actin and phalloidin accumulation at presynaptic puncta, yet cortical phalloidin labeling persisted. Dispersal of presynaptic F-actin structures by latrunculin-A did not disrupt vesicle clustering or recycling or alter the amplitude or kinetics of excitatory postsynaptic currents (EPSCs). However, it slightly enhanced release during repetitive stimulation. While dispersal of presynaptic actin puncta with latrunculin-A failed to disperse synaptic vesicles or inhibit synaptic transmission, presynaptic phalloidin injection blocked exocytosis and reduced endocytosis measured by action potential-evoked FM1-43 staining. Furthermore, phalloidin stabilization of only cortical actin following pretreatment with latrunculin-A was sufficient to inhibit synaptic transmission. Conversely, treatment of axons with jasplakinolide, which induces F-actin accumulation but disrupts F-actin structures in vivo, resulted in increased synaptic transmission accompanied by a loss of phalloidin labeling of cortical actin but no loss of actin labeling within vesicle clusters. Marked synaptic deficits seen with phalloidin stabilization of cortical F-actin, in contrast to the minimal effects of disruption of a synaptic vesicle-associated F-actin, led us to conclude that two structurally and functionally distinct pools of actin exist at presynaptic sites.

  20. Robust changes in expression of brain-derived neurotrophic factor (BDNF) mRNA and protein across the brain do not translate to detectable changes in BDNF levels in CSF or plasma.

    PubMed

    Lanz, Thomas A; Bove, Susan E; Pilsmaker, Catherine D; Mariga, Abigail; Drummond, Elena M; Cadelina, Gregory W; Adamowicz, Wendy O; Swetter, Brentt J; Carmel, Sharon; Dumin, Jo Ann; Kleiman, Robin J

    2012-09-01

    Adult rats were treated acutely with peripheral kainic acid (KA), and changes in brain-derived neurotrophic factor (BDNF) mRNA and protein were tracked over time across multiple brain regions. Despite robust elevation in both mRNA and protein in multiple brain regions, plasma BDNF was unchanged and cerebrospinal fluid (CSF) BDNF levels remained undetectable. Primary neurons were then treated with KA. BDNF was similarly elevated within neurons, but was undetectable in neuronal media. Thus, while deficits in BDNF signaling have been implicated in a number of diseases, these data suggest that extracellular concentrations of BDNF may not be a facile biomarker for changes in neurons.

  1. [Photodynamic therapy for actinic cheilitis].

    PubMed

    Castaño, E; Comunión, A; Arias, D; Miñano, R; Romero, A; Borbujo, J

    2009-12-01

    Actinic cheilitis is a subtype of actinic keratosis that mainly affects the lower lip and has a higher risk of malignant transformation. Its location on the labial mucosa influences the therapeutic approach. Vermilionectomy requires local or general anesthetic and is associated with a risk of an unsightly scar, and the treatment with 5-fluorouracil or imiquimod lasts for several weeks and the inflammatory reaction can be very intense. A number of authors have used photodynamic therapy as an alternative to the usual treatments. We present 3 patients with histologically confirmed actinic cheilitis treated using photodynamic therapy with methyl aminolevulinic acid as the photosensitizer and red light at 630 nm. The clinical response was good, with no recurrences after 3 to 6 months of follow-up. Our experience supports the use of photodynamic therapy as a good alternative for the treatment of actinic cheilitis.

  2. Analysis of adeno-associated virus (AAV) wild-type and mutant Rep proteins for their abilities to negatively regulate AAV p5 and p19 mRNA levels.

    PubMed Central

    Kyöstiö, S R; Owens, R A; Weitzman, M D; Antoni, B A; Chejanovsky, N; Carter, B J

    1994-01-01

    The rep gene of adeno-associated virus type 2 (AAV) encodes four overlapping Rep proteins that are involved in gene regulation and replication of the virus. We studied here the regulation of mRNA transcribed from the AAV p5 and p19 promoters, using transient expression in human 293 cells followed by Northern (RNA) blot analysis of the mRNA. The p5 transcript encodes the larger Rep proteins, Rep78 and Rep68, while the p19 transcript encodes the smaller proteins, Rep52 and Rep40. A plasmid (pNTC3) containing the entire AAV genome with an amber mutation in the rep gene accumulated higher levels of p5 and p19 mRNA than a plasmid containing the wild-type AAV genome. Addition of increasing amounts of the wild-type rep gene in trans from a heterologous promoter inhibited p5 and p19 mRNA accumulation from pNTC3, indicating that the levels of both transcripts were decreased by the Rep proteins. Cotransfections with plasmids producing individual wild-type Rep proteins in trans showed that p5 and p19 mRNA accumulation was inhibited 5- to 10-fold by Rep78 and Rep68 and 2- to 3-fold by Rep52 and Rep40. Analysis of carboxyl-terminal truncation mutants of Rep78 showed that the ability of Rep78 to decrease p5 and p19 mRNA levels was lost when 159 or more amino acids were deleted. Rep78 and Rep68 mutants deleted for the methionine at residue 225 showed decreased abilities to down-regulate both p5 and p19 transcript levels, while mutants containing a substitution of glycine for the methionine resembled the wild-type Rep78. A Rep78 protein with a mutation in the putative nucleoside triphosphate binding site inhibited expression from p5 but not from p19, suggesting that the regulation of p5 transcript levels by Rep78 and Rep68 differs from that of p19. A deletion analysis of AAV cis sequences revealed that an intact terminal repeat was not required for negative regulation of p5 and p19 transcript levels and that the regulation of p19 mRNA levels by Rep78 did not require the presence

  3. Stearoyl coenzyme A desaturase enzyme activity and mRNA levels are not different in subcutaneous adipose tissue from Angus and American Wagyu steers.

    PubMed

    Cameron, P J; Rogers, M; Oman, J; May, S G; Lunt, D K; Smith, S B

    1994-10-01

    We proposed that greater stearoyl coenzyme A (CoA) desaturase enzyme activity caused the elevated monounsaturated fatty acids observed in American Wagyu adipose tissue. Stearoyl CoA desaturase mRNA concentrations and enzyme activities were measured in subcutaneous adipose samples from Angus (n = 5) and American Wagyu (n = 5), fed to the Japanese market end point. A rat liver stearoyl CoA desaturase cDNA clone was used to measure the relative amounts of stearoyl CoA desaturase mRNA. Enzyme activities and mRNA concentrations, as measured by laser densitometry of slot-blot autoradiograms, were not significantly different between the two breeds at this stage of growth. This investigation has demonstrated that, at this stage of maturity, differences in fatty acid composition between Angus and American Wagyu steers cannot be attributed to differences in stearoyl CoA desaturase enzyme activity.

  4. Growing actin networks regulated by obstacle size and shape

    NASA Astrophysics Data System (ADS)

    Gong, Bo; Lin, Ji; Qian, Jin

    2017-01-01

    Growing actin networks provide the driving force for the motility of cells and intracellular pathogens. Based on the molecular-level processes of actin polymerization, branching, capping, and depolymerization, we have developed a modeling framework to simulate the stochastic and cooperative behaviors of growing actin networks in propelling obstacles, with an emphasis on the size and shape effects on work capacity and filament orientation in the growing process. Our results show that the characteristic size of obstacles changes the protrusion power per unit length, without influencing the orientation distribution of actin filaments in growing networks. In contrast, the geometry of obstacles has a profound effect on filament patterning, which influences the orientation of filaments differently when the drag coefficient of environment is small, intermediate, or large. We also discuss the role of various parameters, such as the aspect ratio of obstacles, branching rate, and capping rate, in affecting the protrusion power of network growth.

  5. Cloning of a long HIV-1 readthrough transcript and detection of an increased level of early growth response protein-1 (Egr-1) mRNA in chronically infected U937 cells.

    PubMed

    Dron, M; Hameau, L; Benboudjema, L; Guymarho, J; Cajean-Feroldi, C; Rizza, P; Godard, C; Jasmin, C; Tovey, M G; Lang, M C

    1999-01-01

    To identify the pathways involved in HIV-1 modification of cellular gene expression, chronically infected U937 cells were screened by mRNA differential display. A chimeric transcript consisting of the 3' end of the LTR of a HIV-1 provirus, followed by 3.7 kb of cellular RNA was identified suggesting that long readthrough transcription might be one of the mechanisms by which gene expression could be modified in individual infected cells. Such a phenomenon may also be the first step towards the potential transduction of cellular sequences. Furthermore, the mRNA encoding for the transcription factor Egr-1 was detected as an over-represented transcript in infected cells. Northern blot analysis confirmed the increase of Egr-1 mRNA content in both HIV-1 infected promonocytic U937 cells and T cell lines such as Jurkat and CEM. Interestingly a similar increase of Egr-1 mRNA has previously been reported to occur in HTLV-1 and HTLV-2 infected T cell lines. Despite the consistent increase in the level of Egr-1 mRNA, the amount of the encoded protein did not appear to be modified in HIV-1 infected cells, suggesting an increased turn over of the protein in chronically infected cells.

  6. Increased levels of cell-free human placental lactogen mRNA at 28-32 gestational weeks in plasma of pregnant women with placenta previa and invasive placenta.

    PubMed

    Kawashima, Akihiro; Sekizawa, Akihiko; Ventura, Walter; Koide, Keiko; Hori, Kyouko; Okai, Takashi; Masashi, Yoshida; Furuya, Kenichi; Mizumoto, Yoshifumi

    2014-02-01

    We compared the levels of cell-free human placental lactogen (hPL) messenger RNA (mRNA) in maternal plasma at 28 to 32 weeks of gestation between women with diagnosis of placenta previa or invasive placenta and women with an uneventful pregnancy. Sensitivity and specificity of hPL mRNA for the prediction of invasive placenta were further explored. Plasma hPL mRNA were quantified by real-time reverse-transcriptase polymerase chain reaction in women with placenta previa (n = 13), invasive placenta (n = 5), and normal pregnancies (n = 92). Median (range) hPL mRNA was significantly higher in women with placenta previa, 782 (10-2301) copies/mL of plasma, and in those with invasive placenta, 615 (522-2102) copies/mL of plasma, when compared to normal pregnancies, 90 (4-4407) copies/mL of plasma, P < .01 and P < .05, respectively. We found a sensitivity of 100% and a specificity of 61.5% for the prediction of invasive placenta among women with placenta previa. In conclusion, expression of hPL mRNA is increased in plasma of women with placenta previa and invasive placenta at 28 to 32 weeks of gestation.

  7. Elevated levels of the polyadenylation factor CstF 64 enhance formation of the 1kB Testis brain RNA-binding protein (TB-RBP) mRNA in male germ cells.

    PubMed

    Chennathukuzhi, V M; Lefrancois, S; Morales, C R; Syed, V; Hecht, N B

    2001-04-01

    The single copy mouse Testis Brain RNA-Binding Protein (TB-RBP) gene encodes three mRNAs of 3.0, 1.7, and 1.0 kb which only differ in their 3' UTRs. The 1 kb TB-RBP mRNA predominates in testis, while somatic cells preferentially express the 3.0 kb TB-RBP mRNA. Here we show that the 1 kb mRNA is translated several-fold more efficiently than the 3 kb TB-RBP in rabbit reticulocyte lysates and cells with elevated levels of the 1 kB TB-RBP mRNA express high levels of TB-RBP. To determine if the cleavage stimulatory factor CstF 64 can modulate the alternative splicing of the TB-RBP pre-mRNA and therefore TB-RBP expression, CstF 64 levels and binding to alternative polyadenylation sites were examined. CstF 64 is abundant in the testis and preferentially binds to a distal site in the TB-RBP pre-mRNA that produces the 3 kb TB-RBP. Moreover, upregulation or overexpression of CstF 64 increases the poly(A) site selection for the 1 kb TB-RBP mRNA. We propose that the level of the polyadenylation factor CstF 64 modulates the level of TB-RBP synthesis in male germ cells by an alternative processing of the TB-RBP pre-mRNA.

  8. Molecular characterization of argininosuccinate synthase and argininosuccinate lyase from the liver of the African lungfish Protopterus annectens, and their mRNA expression levels in the liver, kidney, brain and skeletal muscle during aestivation.

    PubMed

    Chng, You R; Ong, Jasmine L Y; Ching, Biyun; Chen, Xiu L; Wong, Wai P; Chew, Shit F; Ip, Yuen K

    2014-10-01

    Argininosuccinate synthase (Ass) and argininosuccinate lyase (Asl) are involved in arginine synthesis for various purposes. The complete cDNA coding sequences of ass and asl from the liver of Protopterus annectens consisted of 1,296 and 1,398 bp, respectively. Phylogenetic analyses revealed that the deduced Ass and Asl of P. annectens had close relationship with that of the cartilaginous fish Callorhinchus milii. Besides being strongly expressed in the liver, ass and asl expression were detectable in many tissues/organs. In the liver, mRNA expression levels of ass and asl increased significantly during the induction phase of aestivation, probably to increase arginine production to support increased urea synthesis. The increases in ass and asl mRNA expression levels during the prolonged maintenance phase and early arousal phase of aestivation could reflect increased demand on arginine for nitric oxide (NO) production in the liver. In the kidney, there was a significant decrease in ass mRNA expression level after 6 months of aestivation, indicating possible decreases in the synthesis and supply of arginine to other tissues/organs. In the brain, changes in ass and asl mRNA expression levels during the three phases of aestivation could be related to the supply of arginine for NO synthesis in response to conditions that resemble ischaemia and ischaemia-reperfusion during the maintenance and arousal phase of aestivation, respectively. The decrease in ass mRNA expression level, accompanied with decreases in the concentrations of arginine and NO, in the skeletal muscle of aestivating P. annectens might ameliorate the potential of disuse muscle atrophy.

  9. Rho GTPases, phosphoinositides, and actin

    PubMed Central

    Croisé, Pauline; Estay-Ahumada, Catherine; Gasman, Stéphane; Ory, Stéphane

    2014-01-01

    Rho GTPases are well known regulators of the actin cytoskeleton that act by binding and activating actin nucleators. They are therefore involved in many actin-based processes, including cell migration, cell polarity, and membrane trafficking. With the identification of phosphoinositide kinases and phosphatases as potential binding partners or effectors, Rho GTPases also appear to participate in the regulation of phosphoinositide metabolism. Since both actin dynamics and phosphoinositide turnover affect the efficiency and the fidelity of vesicle transport between cell compartments, Rho GTPases have emerged as critical players in membrane trafficking. Rho GTPase activity, actin remodeling, and phosphoinositide metabolism need to be coordinated in both space and time to ensure the progression of vesicles along membrane trafficking pathways. Although most molecular pathways are still unclear, in this review, we will highlight recent advances made in our understanding of how Rho-dependent signaling pathways organize actin dynamics and phosphoinositides and how phosphoinositides potentially provide negative feedback to Rho GTPases during endocytosis, exocytosis and membrane exchange between intracellular compartments. PMID:24914539

  10. High temperature induces cyp26b1 mRNA expression and delays meiotic initiation of germ cells by increasing cortisol levels during gonadal sex differentiation in Japanese flounder.

    PubMed

    Yamaguchi, Toshiya; Kitano, Takeshi

    2012-03-09

    The Japanese flounder (Paralichthys olivaceus) is a teleost fish with an XX/XY sex determination system. XX flounder can be induced to develop into phenotypic females or males, by rearing them at 18°C or 27°C, respectively, during the sex differentiation period. Therefore, the flounder provides an excellent model to study the molecular mechanisms underlying temperature-dependent sex determination. We previously showed that cortisol, the major glucocorticoid produced by the interrenal cells in teleosts, causes female-to-male sex reversal by directly suppressing mRNA expression of ovary-type aromatase (cyp19a1), a steroidogenic enzyme responsible for the conversion of androgens to estrogens in the gonads. Furthermore, an inhibitor of cortisol synthesis prevented masculinization of XX flounder at 27°C, suggesting that masculinization by high temperature is due to the suppression of cyp19a1 mRNA expression by elevated cortisol levels during gonadal sex differentiation in the flounder. In the present study, we found that exposure to high temperature during gonadal sex differentiation upregulates the mRNA expression of retinoid-degrading enzyme (cyp26b1) concomitantly with masculinization of XX gonads and delays meiotic initiation of germ cells. We also found that cortisol induces cyp26b1 mRNA expression and suppresses specific meiotic marker synaptonemal complex protein 3 (sycp3) mRNA expression in gonads during the sexual differentiation. In conclusion, these results suggest that exposure to high temperature induces cyp26b1 mRNA expression and delays meiotic initiation of germ cells by elevating cortisol levels during gonadal sex differentiation in Japanese flounder.

  11. Long-range conformational effects of proteolytic removal of the last three residues of actin.

    PubMed Central

    Strzelecka-Gołaszewska, H; Mossakowska, M; Woźniak, A; Moraczewska, J; Nakayama, H

    1995-01-01

    Truncated derivatives of actin devoid of either the last two (actin-2C) or three residues (actin-3C) were used to study the role of the C-terminal segment in the polymerization of actin. The monomer critical concentration and polymerization rate increased in the order: intact actin < actin-2C < actin-3C. Conversely, the rate of hydrolysis of actin-bound ATP during spontaneous polymerization of Mg-actin decreased in the same order, so that, for actin-3C, the ATP hydrolysis significantly lagged behind the polymer growth. Probing the conformation of the nucleotide site in the monomer form by measuring the rates of the bound nucleotide exchange revealed a similar change upon removal of either the two or three residues from the C-terminus. The C-terminal truncation also resulted in a slight decrease in the rate of subtilisin cleavage of monomeric actin within the DNAse-I binding loop, whereas in F-actin subunits the susceptibility of this and of another site within this loop, specifically cleaved by a proteinase from Escherichia coli A2 strain, gradually increased upon sequential removal of the two and of the third residue from the C-terminus. From these and other observations made in this work it has been concluded that perturbation of the C-terminal structure in monomeric actin is transmitted to the cleft, where nucleotide and bivalent cation are bound, and to the DNAse-I binding loop on the top of subdomain 2. Further changes at these sites, observed on the polymer level, seem to result from elimination of the intersubunit contact between the C-terminal residues and the DNAse-I binding loop. It is suggested that formation of this contact plays an essential role in regulating the hydrolysis of actin-bound ATP associated with the polymerization process. Images Figure 5 Figure 6 Figure 8 PMID:7733893

  12. The role of actin networks in cellular mechanosensing

    NASA Astrophysics Data System (ADS)

    Azatov, Mikheil

    Physical processes play an important role in many biological phenomena, such as wound healing, organ development, and tumor metastasis. During these processes, cells constantly interact with and adapt to their environment by exerting forces to mechanically probe the features of their surroundings and generating appropriate biochemical responses. The mechanisms underlying how cells sense the physical properties of their environment are not well understood. In this thesis, I present my studies to investigate cellular responses to the stiffness and topography of the environment. In order to sense the physical properties of their environment, cells dynamically reorganize the structure of their actin cytoskeleton, a dynamic network of biopolymers, altering the shape and spatial distribution of protein assemblies. Several observations suggest that proteins that crosslink actin filaments may play an important role in cellular mechanosensitivity. Palladin is an actin-crosslinking protein that is found in the lamellar actin network, stress fibers and focal adhesions, cellular structures that are critical for mechanosensing of the physical environment. By virtue of its close interactions with these structures in the cell, palladin may play an important role in cell mechanics. However, the role of actin crosslinkers in general, and palladin in particular, in cellular force generation and mechanosensing is not well known. I have investigated the role of palladin in regulating the plasticity of the actin cytoskeleton and cellular force generation in response to alterations in substrate stiffness. I have shown that the expression levels of palladin modulate the forces exerted by cells and their ability to sense substrate stiffness. Perturbation experiments also suggest that palladin levels in cells altered myosin motor activity. These results suggest that the actin crosslinkers, such as palladin, and myosin motors coordinate for optimal cell function and to prevent aberrant

  13. Differential regulation of igf1 and igf1r mRNA levels in the two hepatic lobes following intrauterine growth restriction and its treatment with intra-amniotic insulin-like growth factor-1 in ovine fetuses.

    PubMed

    Darp, Revati A; de Boo, Hendrina A; Phua, Hui Hui; Oliver, Mark H; Derraik, José G B; Harding, Jane E; Bloomfield, Frank H

    2010-01-01

    Intrauterine growth restriction (IUGR) has life-long health implications, yet there is no effective prenatal treatment. Daily intra-amniotic administration of insulin-like growth factor (IGF)-1 to IUGR fetal sheep improves fetal gut maturation but suppresses hepatic igf1 gene expression. Fetal hepatic blood supply is regulated, in part, by shunting of oxygen- and nutrient-rich umbilical venous blood through the ductus venosus, with the left hepatic lobe predominantly supplied by umbilical venous blood and the right hepatic lobe predominantly supplied by the portal circulation. We hypothesised that: (1) once-weekly intra-amniotic IGF-1 treatment of IUGR would be effective in promoting gut maturation; and (2) IUGR and its treatment with intra-amniotic IGF-1 would differentially affect igf1 and igf1r mRNA expression in the two hepatic lobes. IUGR fetuses received 360 µg IGF-1 or saline intra-amniotically once weekly from 110 until 131 days gestation. Treatment of IUGR fetuses with IGF-1 reversed impaired gut growth. In unembolised, untreated control fetuses, igf1 mRNA levels were 19% lower in the right hepatic lobe than in the left; in IUGR fetuses, igf1 and igf1r mRNA levels were sixfold higher in the right lobe. IGF-1 treatment reduced igf1 and igf1r mRNA levels in both lobes compared with IUGR fetuses. Thus, weekly intra-amniotic IGF-1 treatment, a clinically feasible approach, reverses the impaired gut development seen in IUGR. Furthermore, igf1 and igf1r mRNA levels are differentially expressed in the two hepatic lobes and relative expression in the two lobes is altered by both IUGR and intra-amniotic IGF-1 treatment.

  14. A nonsense mutation causing decreased levels of insulin receptor mRNA: Detection by a simplified technique for direct sequencing of genomic DNA amplified by the polymerase chain reaction

    SciTech Connect

    Kadowaki, T.; Kadowaki, H.; Taylor, S.I. )

    1990-01-01

    Mutations in the insulin receptor gene can render the cell resistant to the biological action of insulin. The authors have studied a patient with leprechaunism (leprechaun/Minn-1), a genetic syndrome associated with intrauterine growth retardation and extreme insulin resistance. Genomic DNA from the patient was amplified by the polymerase chain reaction catalyzed by Thermus aquaticus (Taq) DNA polymerase, and the amplified DNA was directly sequenced. A nonsense mutations was identified at codon 897 in exon 14 in the paternal allele of the patient's insulin receptor gene. Levels of insulin receptor mRNA are decreased to <10% of normal in Epstein-Barr virus-transformed lymphoblasts and cultured skin fibroblasts from this patient. Thus, this nonsense mutation appears to cause a decrease in the levels of insulin receptor mRNA. In addition, they have obtained indirect evidence that the patient's maternal allele of the insulin receptor gene contains a cis-acting dominant mutation that also decreases the level of mRNA, but by a different mechanism. The nucleotide sequence of the entire protein-coding domain and the sequences of the intron-exon boundaries for all 22 exons of the maternal allele were normal. Presumably, the mutation in the maternal allele maps elsewhere in the insulin receptor gene. Thus, they conclude that the patient is a compound heterozygote for two cis-acting dominant mutations in the insulin receptor gene: (i) a nonsense mutation in the paternal allel that reduces the level of insulin receptor mRNA and (ii) an as yet unidentified mutation in the maternal allele that either decreases the rate of transcription or decreases the stability of the mRNA.

  15. Gonadotropin-induced changes in oviducal mRNA expression levels of sex steroid hormone receptors and activin-related signaling factors in the alligator.

    PubMed

    Moore, Brandon C; Forouhar, Sara; Kohno, Satomi; Botteri, Nicole L; Hamlin, Heather J; Guillette, Louis J

    2012-01-15

    Oviducts respond to hormonal cues from ovaries with tissue proliferation and differentiation in preparation of transporting and fostering gametes. These responses produce oviducal microenvironments conducive to reproductive success. Here, we investigated changes in circulating plasma sex steroid hormones concentrations and ovarian and oviducal mRNA expression to an in vivo gonadotropin (FSH) challenge in sexually immature, five-month-old alligators. Further, we investigated differences in these observed responses between alligators hatched from eggs collected at a heavily-polluted (Lake Apopka, FL) and minimally-polluted (Lake Woodruff, FL) site. In oviducts, we measured mRNA expression of estrogen, progesterone, and androgen receptors and also beta A and B subunits which homo- or heterodimerize to produce the transforming growth factor activin. In comparison, minimal inhibin alpha subunit mRNA expression suggests that these oviducts produce a primarily activin-dominated signaling milieu. Ovaries responded to a five-day FSH challenge with increased expression of steroidogenic enzyme mRNA which was concomitant with increased circulating sex steroid hormone concentrations. Oviducts in the FSH-challenged Lake Woodruff alligators increased mRNA expression of progesterone and androgen receptors, proliferating cell nuclear antigen, and the activin signaling antagonist follistatin. In contrast, Lake Apopka alligators displayed a diminished increase in ovarian CYP19A1 aromatase expression and no increase in oviducal AR expression, as compared to those observed in Lake Woodruff alligators. These results demonstrate that five-month-old female alligators display an endocrine-responsive ovarian-oviducal axis and environmental pollution exposure may alter these physiological responses.

  16. Gonadotropin-induced changes in oviducal mRNA expression levels of sex steroid hormone receptors and activin-related signaling factors in the alligator

    PubMed Central

    Moore, Brandon C.; Forouhar, Sara; Kohno, Satomi; Botteri, Nicole L.; Hamlin, Heather J.; Guillette, Louis J.

    2011-01-01

    Oviducts respond to hormonal cues from ovaries with tissue proliferation and differentiation in preparation of transporting and fostering gametes. These responses produce oviducal microenvironments conducive to reproductive success. Here we investigated changes in circulating plasma sex steroid hormones concentrations and ovarian and oviducal mRNA expression to an in vivo gonadotropin (FSH) challenge in sexually immature, five-month-old alligators. Further, we investigated differences in these observed responses between alligators hatched from eggs collected at a heavily-polluted (Lake Apopka, FL) and minimally-polluted (Lake Woodruff, FL) site. In oviducts, we measured mRNA expression of estrogen, progesterone, and androgen receptors and also beta A and B subunits which homo- or heterodimerize to produce the transforming growth factor activin. In comparison, minimal inhibin alpha subunit mRNA expression suggests that these oviducts produce a primarily activin-dominated signaling milieu. Ovaries responded to a five-day FSH challenge with increased expression of steroidogenic enzyme mRNA which was concomitant with increased circulating sex steroid hormone concentrations. Oviducts in the FSH-challenged Lake Woodruff alligators increased mRNA expression of progesterone and androgen receptors, proliferating cell nuclear antigen, and the activin signaling antagonist follistatin. In contrast, Lake Apopka alligators displayed a diminished increase in ovarian CYP19A1 aromatase expression and no increase in oviducal AR expression, as compared to those observed in Lake Woodruff alligators. These results demonstrate that five-month-old female alligators display an endocrine-responsive ovarian-oviducal axis and environmental pollution exposure may alter these physiological responses. PMID:22154572

  17. Nucleus-associated actin in Amoeba proteus.

    PubMed

    Berdieva, Mariia; Bogolyubov, Dmitry; Podlipaeva, Yuliya; Goodkov, Andrew

    2016-10-01

    The presence, spatial distribution and forms of intranuclear and nucleus-associated cytoplasmic actin were studied in Amoeba proteus with immunocytochemical approaches. Labeling with different anti-actin antibodies and staining with TRITC-phalloidin and fluorescent deoxyribonuclease I were used. We showed that actin is abundant within the nucleus as well as in the cytoplasm of A. proteus cells. According to DNase I experiments, the predominant form of intranuclear actin is G-actin which is associated with chromatin strands. Besides, unpolymerized actin was shown to participate in organization of a prominent actin layer adjacent to the outer surface of nuclear envelope. No significant amount of F-actin was found in the nucleus. At the same time, the amoeba nucleus is enclosed in a basket-like structure formed by circumnuclear actin filaments and bundles connected with global cytoplasmic actin cytoskeleton. A supposed architectural function of actin filaments was studied by treatment with actin-depolymerizing agent latrunculin A. It disassembled the circumnuclear actin system, but did not affect the intranuclear chromatin structure. The results obtained for amoeba cells support the modern concept that actin is involved in fundamental nuclear processes that have evolved in the cells of multicellular organisms.

  18. Gastrointestinal stromal tumors - quantitative detection of the Ki-67, TPX2, TOP2A, and hTERT telomerase subunit mRNA levels to determine proliferation activity and a potential for aggressive biological behavior.

    PubMed

    Kalfusova, A; Hilska, I; Krskova, L; Kalinova, M; Linke, Z; Kodet, R

    2016-01-01

    Gastrointestinal stromal tumors (GISTs) have an unpredictable biological potential ranging from benign to malignant. Molecular markers involved in the mechanisms of proliferation and cellular senescence may provide additional information about biological behavior of the tumor. The aim of the present study was to investigate Ki-67, TPX2, TOP2A and hTERT mRNA expression levels in specimens from patients with GISTs to define relationships between proliferation activity and biological potential and progression of the disease. We measured Ki-67, TPX2, TOP2A and hTERT mRNA levels using quantitative real-time reverse transcription PCR (RQ RT PCR). The highest Ki-67, TPX2, TOP2A and hTERT mRNA expression levels were found in the highly proliferative BLs (18 specimens), in comparison with GISTs (137 specimens) and LMSs (9 specimens). Patients with GISTs and adequate information about mitotic activity, tumor size and anatomical site (84 specimens) were divided into two groups - GISTs with benign (29 patients) and with malignant (55 patients) potential. We observed association between higher Ki-67, TPX2 and hTERT mRNA levels and the GISTs with malignant potential. Univariate analysis (57 patients with available follow-up information) of survival (Kaplan Meier curves method) revealed a correlation between higher levels of TPX2, Ki-67 and hTERT markers and shorter event-free survival (EFS) or poorer overall survival (OS). The results demonstrate the importance of quantitative assessment of the proliferation activity in GISTs. Proliferation markers of Ki-67, TPX2, TOP2A and hTERT are suitable markers for detection the proliferation activity and telomerase activity of these tumors. Furthermore, the assessment of TPX2, Ki-67 and hTERT expression levels is appropriate for determination of malignant potential of GISTs.

  19. A Balance of Capping Protein and Profilin Functions Is Required to Regulate Actin Polymerization in Drosophila Bristle

    PubMed Central

    Hopmann, Roberta; Miller, Kathryn G.

    2003-01-01

    Profilin is a well-characterized protein known to be important for regulating actin filament assembly. Relatively few studies have addressed how profilin interacts with other actin-binding proteins in vivo to regulate assembly of complex actin structures. To investigate the function of profilin in the context of a differentiating cell, we have studied an instructive genetic interaction between mutations in profilin (chickadee) and capping protein (cpb). Capping protein is the principal protein in cells that caps actin filament barbed ends. When its function is reduced in the Drosophila bristle, F-actin levels increase and the actin cytoskeleton becomes disorganized, causing abnormal bristle morphology. chickadee mutations suppress the abnormal bristle phenotype and associated abnormalities of the actin cytoskeleton seen in cpb mutants. Furthermore, overexpression of profilin in the bristle mimics many features of the cpb loss-of-function phenotype. The interaction between cpb and chickadee suggests that profilin promotes actin assembly in the bristle and that a balance between capping protein and profilin activities is important for the proper regulation of F-actin levels. Furthermore, this balance of activities affects the association of actin structures with the membrane, suggesting a link between actin filament dynamics and localization of actin structures within the cell. PMID:12529431

  20. Boolean gates on actin filaments

    NASA Astrophysics Data System (ADS)

    Siccardi, Stefano; Tuszynski, Jack A.; Adamatzky, Andrew

    2016-01-01

    Actin is a globular protein which forms long polar filaments in the eukaryotic cytoskeleton. Actin networks play a key role in cell mechanics and cell motility. They have also been implicated in information transmission and processing, memory and learning in neuronal cells. The actin filaments have been shown to support propagation of voltage pulses. Here we apply a coupled nonlinear transmission line model of actin filaments to study interactions between voltage pulses. To represent digital information we assign a logical TRUTH value to the presence of a voltage pulse in a given location of the actin filament, and FALSE to the pulse's absence, so that information flows along the filament with pulse transmission. When two pulses, representing Boolean values of input variables, interact, then they can facilitate or inhibit further propagation of each other. We explore this phenomenon to construct Boolean logical gates and a one-bit half-adder with interacting voltage pulses. We discuss implications of these findings on cellular process and technological applications.

  1. Arabidopsis microtubule-destabilizing protein 25 functions in pollen tube growth by severing actin filaments.

    PubMed

    Qin, Tao; Liu, Xiaomin; Li, Jiejie; Sun, Jingbo; Song, Leina; Mao, Tonglin

    2014-01-01

    The formation of distinct actin filament arrays in the subapical region of pollen tubes is crucial for pollen tube growth. However, the molecular mechanisms underlying the organization and dynamics of the actin filaments in this region remain to be determined. This study shows that Arabidopsis thaliana MICROTUBULE-DESTABILIZING PROTEIN25 (MDP25) has the actin filament-severing activity of an actin binding protein. This protein negatively regulated pollen tube growth by modulating the organization and dynamics of actin filaments in the subapical region of pollen tubes. MDP25 loss of function resulted in enhanced pollen tube elongation and inefficient fertilization. MDP25 bound directly to actin filaments and severed individual actin filaments, in a manner that was dramatically enhanced by Ca(2+), in vitro. Analysis of a mutant that bears a point mutation at the Ca(2+) binding sites demonstrated that the subcellular localization of MDP25 was determined by cytosolic Ca(2+) level in the subapical region of pollen tubes, where MDP25 was disassociated from the plasma membrane and moved into the cytosol. Time-lapse analysis showed that the F-actin-severing frequency significantly decreased and a high density of actin filaments was observed in the subapical region of mdp25-1 pollen tubes. This study reveals a mechanism whereby calcium enhances the actin filament-severing activity of MDP25 in the subapical region of pollen tubes to modulate pollen tube growth.

  2. The Effect of Crosslinking on the Microscale Stress Response and Molecular Deformations in Actin Networks

    NASA Astrophysics Data System (ADS)

    Gurmessa, Bekele; Fitzpatrick, Robert; Valdivia, Jonathon; Anderson, Rae M. R.

    Actin, the most abundant protein in eukaryotic cells, is a semi-flexible biopolymer in the cytoskeleton that plays a crucial structural and mechanical role in cell stability, motion and replication, as well as muscle contraction. Most of these mechanically driven structural changes in cells stem from the complex viscoelastic nature of entangled actin networks and the presence of a myriad of proteins that cross-link actin filaments. Despite their importance, the mechanical response of actin networks is not yet well understood, particularly at the molecular level. Here, we use optical trapping - coupled with fluorescence microscopy - to characterize the microscale stress response and induced filament deformations in entangled and cross-linked actin networks subject to localized mechanical perturbations. In particular, we actively drive a microsphere 10 microns through an entangled or cross- linked actin network at a constant speed and measure the resistive force that the deformed actin filaments exert on the bead during and following strain. We simultaneously visualize and track individual sparsely-labeled actin filaments to directly link force response to molecular deformations, and map the propagation of the initially localized perturbation field throughout the rest of the network (~100 um). By varying the concentration of actin and cross-linkers we directly determine the role of crosslinking and entanglements on the length and time scales of stress propagation, molecular deformation and relaxation mechanisms in actin networks.

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

    PubMed

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

    2005-08-01

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

  4. Formation of long and winding nuclear F-actin bundles by nuclear c-Abl tyrosine kinase

    SciTech Connect

    Aoyama, Kazumasa; Yuki, Ryuzaburo; Horiike, Yasuyoshi; Kubota, Sho; Yamaguchi, Noritaka; Morii, Mariko; Ishibashi, Kenichi; Nakayama, Yuji; Kuga, Takahisa; Hashimoto, Yuuki; Tomonaga, Takeshi; Yamaguchi, Naoto

    2013-12-10

    The non-receptor-type tyrosine kinase c-Abl is involved in actin dynamics in the cytoplasm. Having three nuclear localization signals (NLSs) and one nuclear export signal, c-Abl shuttles between the nucleus and the cytoplasm. Although monomeric actin and filamentous actin (F-actin) are present in the nucleus, little is known about the relationship between c-Abl and nuclear actin dynamics. Here, we show that nuclear-localized c-Abl induces nuclear F-actin formation. Adriamycin-induced DNA damage together with leptomycin B treatment accumulates c-Abl into the nucleus and increases the levels of nuclear F-actin. Treatment of c-Abl-knockdown cells with Adriamycin and leptomycin B barely increases the nuclear F-actin levels. Expression of nuclear-targeted c-Abl (NLS-c-Abl) increases the levels of nuclear F-actin even without Adriamycin, and the increased levels of nuclear F-actin are not inhibited by inactivation of Abl kinase activity. Intriguingly, expression of NLS-c-Abl induces the formation of long and winding bundles of F-actin within the nucleus in a c-Abl kinase activity-dependent manner. Furthermore, NLS-c-AblΔC, which lacks the actin-binding domain but has the full tyrosine kinase activity, is incapable of forming nuclear F-actin and in particular long and winding nuclear F-actin bundles. These results suggest that nuclear c-Abl plays critical roles in actin dynamics within the nucleus. - Highlights: • We show the involvement of c-Abl tyrosine kinase in nuclear actin dynamics. • Nuclear F-actin is formed by nuclear-localized c-Abl and its kinase-dead version. • The c-Abl actin-binding domain is prerequisite for nuclear F-actin formation. • Formation of long nuclear F-actin bundles requires nuclear c-Abl kinase activity. • We discuss a role for nuclear F-actin bundle formation in chromatin regulation.

  5. Tubulin binding protein, CacyBP/SIP, induces actin polymerization and may link actin and tubulin cytoskeletons.

    PubMed

    Schneider, Gabriela; Nieznanski, Krzysztof; Jozwiak, Jolanta; Slomnicki, Lukasz P; Redowicz, Maria J; Filipek, Anna

    2010-11-01

    CacyBP/SIP, originally identified as a S100A6 target, was shown to interact with some other S100 proteins as well as with Siah-1, Skp1, tubulin and ERK1/2 kinases (reviewed in Schneider and Filipek, Amino Acids, 2010). Here, we show that CacyBP/SIP interacts and co-localizes with actin in NB2a cells. Using a zero-length cross-linker we found that both proteins bound directly to each other. Co-sedimentation assays revealed that CacyBP/SIP induced G-actin polymerization and formation of unique circular actin filament bundles. The N-terminal fragment of CacyBP/SIP (residues 1-179) had similar effect on actin polymerization as the entire CacyBP/SIP protein, while the C-terminal one (residues 178-229) had not. To check the influence of CacyBP/SIP on cell morphology as well as on cell adhesion and migration, a stable NIH 3T3 cell line with an increased level of CacyBP/SIP was generated. We found that the adhesion and migration rates of the modified cells were changed in comparison with the control ones. Interestingly, the co-sedimentation and proximity ligation assays indicated that CacyBP/SIP could simultaneously interact with tubulin and actin, suggesting that CacyBP/SIP might link actin and tubulin cytoskeletons.

  6. G-actin regulates rapid induction of actin nucleation by mDia1 to restore cellular actin polymers.

    PubMed

    Higashida, Chiharu; Suetsugu, Shiro; Tsuji, Takahiro; Monypenny, James; Narumiya, Shuh; Watanabe, Naoki

    2008-10-15

    mDia1 belongs to the formin family of proteins that share FH1 and FH2 domains. Although formins play a critical role in the formation of many actin-based cellular structures, the physiological regulation of formin-mediated actin assembly within the cell is still unknown. Here we show that cells possess an acute actin polymer restoration mechanism involving mDia1. By using single-molecule live-cell imaging, we found that several treatments including low-dose G-actin-sequestering drugs and unpolymerizable actin mutants activate mDia1 to initiate fast directional movement. The FH2 region, the core domain for actin nucleation, is sufficient to respond to latrunculin B (LatB) to increase its actin nucleation frequency. Simulation analysis revealed an unexpected paradoxical effect of LatB that leads to a several fold increase in free G-actin along with an increase in total G-actin. These results indicate that in cells, the actin nucleation frequency of mDia1 is enhanced not only by Rho, but also strongly through increased catalytic efficiency of the FH2 domain. Consistently, frequent actin nucleation by mDia1 was found around sites of vigorous actin disassembly. Another major actin nucleator, the Arp2/3 complex, was not affected by the G-actin increase induced by LatB. Taken together, we propose that transient accumulation of G-actin works as a cue to promote mDia1-catalyzed actin nucleation to execute rapid reassembly of actin filaments.

  7. Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

    NASA Technical Reports Server (NTRS)

    Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.

    2000-01-01

    Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

  8. Coordinated oscillations in cortical actin and Ca2+ correlate with cycles of vesicle secretion.

    PubMed

    Wollman, R; Meyer, T

    2012-12-01

    The actin cortex both facilitates and hinders the exocytosis of secretory granules. How cells consolidate these two opposing roles was not well understood. Here we show that antigen activation of mast cells induces oscillations in Ca(2+) and PtdIns(4,5)P(2) lipid levels that in turn drive cyclic recruitment of N-WASP and cortical actin level oscillations. Experimental and computational analysis argues that vesicle fusion correlates with the observed actin and Ca(2+) level oscillations. A vesicle secretion cycle starts with the capture of vesicles by actin when cortical F-actin levels are high, followed by vesicle passage through the cortex when F-actin levels are low, and vesicle fusion with the plasma membrane when Ca(2+) levels subsequently increase. Thus, cells employ oscillating levels of Ca(2+), PtdIns(4,5)P(2) and cortical F-actin to increase secretion efficiency, explaining how the actin cortex can function as a carrier as well as barrier for vesicle secretion.

  9. Cardiac α-actin over-expression therapy in dominant ACTA1 disease.

    PubMed

    Ravenscroft, Gianina; McNamara, Elyshia; Griffiths, Lisa M; Papadimitriou, John M; Hardeman, Edna C; Bakker, Anthony J; Davies, Kay E; Laing, Nigel G; Nowak, Kristen J

    2013-10-01

    More than 200 mutations in the skeletal muscle α-actin gene (ACTA1) cause either dominant or recessive skeletal muscle disease. Currently, there are no specific therapies. Cardiac α-actin is 99% identical to skeletal muscle α-actin and the predominant actin isoform in fetal muscle. We previously showed cardiac α-actin can substitute for skeletal muscle α-actin, preventing the early postnatal death of Acta1 knock-out mice, which model recessive ACTA1 disease. Dominant ACTA1 disease is caused by the presence of 'poison' mutant actin protein. Experimental and anecdotal evidence nevertheless indicates that the severity of dominant ACTA1 disease is modulated by the relative amount of mutant skeletal muscle α-actin protein present. Thus, we investigated whether transgenic over-expression of cardiac α-actin in postnatal skeletal muscle could ameliorate the phenotype of mouse models of severe dominant ACTA1 disease. In one model, lethality of ACTA1(D286G). Acta1(+/-) mice was reduced from ∼59% before 30 days of age to ∼12%. In the other model, Acta1(H40Y), in which ∼80% of male mice die by 5 months of age, the cardiac α-actin transgene did not significantly improve survival. Hence cardiac α-actin over-expression is likely to be therapeutic for at least some dominant ACTA1 mutations. The reason cardiac α-actin was not effective in the Acta1(H40Y) mice is uncertain. We showed that the Acta1(H40Y) mice had endogenously elevated levels of cardiac α-actin in skeletal muscles, a finding not reported in dominant ACTA1 patients.

  10. Bacterial Actins? An Evolutionary Perspective

    NASA Technical Reports Server (NTRS)

    Doolittle, Russell F.; York, Amanda L.

    2003-01-01

    According to the conventional wisdom, the existence of a cytoskeleton in eukaryotes and its absence in prokaryotes constitute a fundamental divide between the two domains of life. An integral part of the dogma is that a cytoskeleton enabled an early eukaryote to feed upon prokaryotes, a consequence of which was the occasional endosymbiosis and the eventual evolution of organelles. Two recent papers present compelling evidence that actin, one of the principal components of a cytoskeleton, has a homolog in Bacteria that behaves in many ways like eukaryotic actin. Sequence comparisons reveml that eukaryotic actin and the bacterial homolog (mreB protein), unlike many other proteins common to eukaryotes and Bacteria, have very different and more highly extended evolutionary histories.

  11. Effect of Cheonggukjang supplementation upon hepatic acyl-CoA synthase, carnitine palmitoyltransferase I, acyl-CoA oxidase and uncoupling protein 2 mRNA levels in C57BL/6J mice fed with high fat diet

    PubMed Central

    Soh, Ju-Ryoun; Shin, Dong-Hwa; Kwon, Dae Young

    2007-01-01

    This study investigated the effect of Cheonggukjang on mRNA levels of hepatic acyl-CoA synthase (ACS), carnitine palmitoyltransferase I (CPT-I), acyl-CoA oxidase (ACO) and uncoupling protein 2 (UCP2), and on serum lipid profiles in C57BL/6J mice. Thirty male C57BL/6J mice were divided into three groups; normal diet (ND), high fat diet (HD) and high fat diet with 40% Cheonggukjang (HDC). Energy intake was significantly higher in the HDC group than in the ND and HD groups. The HDC group normalized in weight gain, epididymal and back fat (g/100 g) accumulation which are increased by high fat diet. Serum concentrations of triglyceride and total cholesterol in the HDC were significantly lower than those in the HD group. These results were confirmed by hepatic mRNA expression of enzymes and protein (ACS, CPT-1, ACO, UCP2) which is related with lipid metabolism by RT-PCR. Hepatic CPT-I, ACO and UCP2 mRNA expression was increased by Cheonggukjang supplementation. We demonstrated that Cheonggukjang supplement leads to increased mRNA expressions of enzymes and protein involved in fatty acid oxidation in liver, reduced accumulation of body fat and improvement of serum lipids in high fat diet fed mice. PMID:18850232

  12. Actin polymerization is stimulated by actin cross-linking protein palladin.

    PubMed

    Gurung, Ritu; Yadav, Rahul; Brungardt, Joseph G; Orlova, Albina; Egelman, Edward H; Beck, Moriah R

    2016-02-15

    The actin scaffold protein palladin regulates both normal cell migration and invasive cell motility, processes that require the co-ordinated regulation of actin dynamics. However, the potential effect of palladin on actin dynamics has remained elusive. In the present study, we show that the actin-binding immunoglobulin-like domain of palladin, which is directly responsible for both actin binding and bundling, also stimulates actin polymerization in vitro. Palladin eliminated the lag phase that is characteristic of the slow nucleation step of actin polymerization. Furthermore, palladin dramatically reduced depolymerization, slightly enhanced the elongation rate, and did not alter the critical concentration. Microscopy and in vitro cross-linking assays reveal differences in actin bundle architecture when palladin is incubated with actin before or after polymerization. These results suggest a model whereby palladin stimulates a polymerization-competent form of globular or monomeric actin (G-actin), akin to metal ions, either through charge neutralization or through conformational changes.

  13. Increased apolipoprotein E and c-fms gene expression without elevated interleukin 1 or 6 mRNA levels indicates selective activation of macrophage functions in advanced human atheroma.

    PubMed Central

    Salomon, R N; Underwood, R; Doyle, M V; Wang, A; Libby, P

    1992-01-01

    Cells found within atherosclerotic lesions can produce in culture protein mediators that may participate in atherogenesis. To test whether human atheromata actually contain transcripts for certain of these genes, we compared levels of mRNAs in carotid or coronary atheromata and in nonatherosclerotic human vessels by polymerase chain reaction (PCR) amplification of cDNAs reverse-transcribed from RNA. We measured PCR products (generated during exponential amplification) by incorporation of 32P-labeled primers. Levels of interleukin 1 alpha, 1 beta, or 6 mRNAs in plaques and controls did not differ. Compared to uninvolved vessels, plaques did contain higher levels of mRNA encoding platelet-derived growth factor A chain (42 +/- 24 vs. 12 +/- 10 fmol of product; mean +/- SD; n = 8 and 8, respectively; P = 0.007) and B chain (41 +/- 36 vs. 4 +/- 3 fmol of product, n = 14 and 6, respectively; P = 0.024). Atherosclerotic lesions consistently had much higher levels of apolipoprotein E (apoE) mRNA than did control vessels (131 +/- 71 vs. 5 +/- 3 fmol of product; n = 12 and 10, respectively; P less than 0.001). Direct RNA blot analyses confirmed elevated levels of apoE mRNA in plaque extracts. To test whether mononuclear phagocytes might be a source of the apoE mRNA, we studied a selective marker for cells of the monocytic lineage, the c-fms protooncogene, which encodes the receptor for macrophage colony-stimulating factor. Plaques also contained elevated levels of c-fms mRNA (30 +/- 17 vs. 5 +/- 3 fmol of product; n = 10 and 7, respectively; P = 0.002). Immunohistochemical colocalization demonstrated apoE protein in association with macrophages in plaques, whereas nonatherosclerotic vessels showed no immunoreactive apoE. ApoE produced locally in atheroma might modulate the functions of lesional T cells or promote "reverse cholesterol transport" by associating with high density lipoprotein particles, thus targeting them for peripheral uptake. Macrophages within the advanced

  14. Mammalian verprolin CR16 acts as a modulator of ITSN scaffold proteins association with actin.

    PubMed

    Kropyvko, Sergii; Gryaznova, Tetyana; Morderer, Dmytro; Rynditch, Alla

    2017-03-18

    Actin cytoskeleton rearrangements are required for normal cell functioning, and their deregulation leads to various pathologies. Members of two mammalian protein families - ITSNs (ITSN1 and ITSN2) and verprolins (WIP, CR16 and WIRE) are involved in Cdc42/N-WASP/Arp2/3 signaling pathway-mediated remodeling of the actin cytoskeleton. Recently we demonstrated that ITSNs interact with the actin-regulating protein WIP. Here, we show that other member of verprolin family, CR16, also forms complexes with ITSN1 and ITSN2 in human cell lines. The actin-binding protein CR16 modulates ITSN/β-actin association. Moreover, overexpressed CR16 promoted co-localization of ITSN1 with F-actin in MCF-7 breast cancer cells. Our data demonstrated that CR16 mRNA is expressed in glioblastoma and breast tumors. These findings provide the basis for further functional investigations of the ITSN/CR16 complex that may play an important role in actin remodeling and cellular invasion.

  15. Expression of constitutive cyclo-oxygenase (COX-1) in rats with streptozotocin-induced diabetes; effects of treatment with evening primrose oil or an aldose reductase inhibitor on COX-1 mRNA levels.

    PubMed

    Fang, C; Jiang, Z; Tomlinson, D R

    1997-02-01

    Altered prostanoid metabolism participates in the pathogenesis of diabetic complications. The rate-limiting enzyme in the control of prostanoid metabolism is constitutive cyclo-oxygenase (COX-1). This study examined the possibility that altered prostanoid metabolism derives from altered COX-1 expression in those tissues from diabetic rats, with characteristic changes in prostanoid production and related haemodynamics. This account also describes a procedure for estimation of minute amounts of COX-1 mRNA by reverse transcription and competitive polymerase chain reaction (RT-cPCR) amplification. In streptozotocin-diabetic rats (STZ-D, 55 mg/kg body weight), compared with age-matched controls, the level of COX-1 mRNA (in attomoles/micrograms tRNA +/- 1SD) was significantly decreased in sciatic nerve (0.50 +/- 0.26 versus 0.89 +/- 0.32 in controls; P < 0.05) and thoracic aorta (3.99 +/- 1.67 versus 8.80 +/- 2.37 in controls; P < 0.05). There were no differences in COX-1 mRNA in diabetic and control rat kidney and retina, though there was a trend towards increased expression with diabetes in the latter. Evening primrose oil (EPO) treatment increased COX-1 mRNA in nerve and retina to levels in diabetic rats that were higher than those of non-diabetic controls (1.21 +/- 0.28 for nerve and 0.065 +/- 0.017 for retina, where control retinae gave 0.031 +/- 0.020-see above for nerve). Treatment of diabetic rats with an aldose reductase inhibitor was without effect on COX-1 mRNA levels in the tissues examined. This study demonstrates that the changes in COX-1 mRNA levels in diabetic rats are organ specific and suggests that altered prostanoid metabolism can, in part, be explained by altered COX-1 expression. Apart from providing arachidonate as substrate for COX, EPO stimulates COX-1 expression in some tissues.

  16. Formation and Destabilization of Actin Filaments with Tetramethylrhodamine-Modified Actin

    PubMed Central

    Kudryashov, Dmitry S.; Phillips, Martin; Reisler, Emil

    2004-01-01

    Actin labeling at Cys374 with tethramethylrhodamine derivatives (TMR-actin) has been widely used for direct observation of the in vitro filaments growth, branching, and treadmilling, as well as for the in vivo visualization of actin cytoskeleton. The advantage of TMR-actin is that it does not lock actin in filaments (as rhodamine-phalloidin does), possibly allowing for its use in investigating the dynamic assembly behavior of actin polymers. Although it is established that TMR-actin alone is polymerization incompetent, the impact of its copolymerization with unlabeled actin on filament structure and dynamics has not been tested yet. In this study, we show that TMR-actin perturbs the filaments structure when copolymerized with unlabeled actin; the resulting filaments are more fragile and shorter than the control filaments. Due to the increased severing of copolymer filaments, TMR-actin accelerates the polymerization of unlabeled actin in solution also at mole ratios lower than those used in most fluorescence microscopy experiments. The destabilizing and severing effect of TMR-actin is countered by filament stabilizing factors, phalloidin, S1, and tropomyosin. These results point to an analogy between the effects of TMR-actin and severing proteins on F-actin, and imply that TMR-actin may be inappropriate for investigations of actin filaments dynamics. PMID:15298916

  17. Optimal treatment of actinic keratoses

    PubMed Central

    Uhlenhake, Elizabeth E

    2013-01-01

    The most compelling reason and primary goal of treating actinic keratoses is to prevent malignant transformation into invasive squamous cell carcinoma, and although there are well established guidelines outlining treatment modalities and regimens for squamous cell carcinoma, the more commonly encountered precancerous actinic lesions have no such standard. Many options are available with variable success and patient compliance rates. Prevention of these lesions is key, with sun protection being a must in treating aging patients with sun damage as it is never too late to begin protecting the skin. PMID:23345970

  18. Cloning and characterization of an abalone (Haliotis discus hannai) actin gene

    NASA Astrophysics Data System (ADS)

    Ma, Hongming; Xu, Wei; Mai, Kangsen; Liufu, Zhiguo; Chen, Hong

    2004-10-01

    An actin encoding gene was cloned by using RT-PCR, 3‧ RACE and 5‧ RACE from abalone Haliotis discus hannai. The full length of the gene is 1532 base pairs, which contains a long 3‧ untranslated region of 307 base pairs and 79 base pairs of 5‧ untranslated sequence. The open reading frame encodes 376 amino acid residues. Sequence comparison with those of human and other mollusks showed high conservation among species at amino acid level. The identities was 96%, 97% and 96% respectively compared with Aplysia californica, Biomphalaria glabrata and Homo sapience β-actin. It is also indicated that this actin is more similar to the human cytoplasmic actin (β-actin) than to human muscle actin.

  19. Regulation of myosin IIA and filamentous actin during insulin-stimulated glucose uptake in 3T3-L1 adipocytes

    SciTech Connect

    Stall, Richard; Ramos, Joseph; Kent Fulcher, F.; Patel, Yashomati M.

    2014-03-10

    Insulin stimulated glucose uptake requires the colocalization of myosin IIA (MyoIIA) and the insulin-responsive glucose transporter 4 (GLUT4) at the plasma membrane for proper GLUT4 fusion. MyoIIA facilitates filamentous actin (F-actin) reorganization in various cell types. In adipocytes F-actin reorganization is required for insulin-stimulated glucose uptake. What is not known is whether MyoIIA interacts with F-actin to regulate insulin-induced GLUT4 fusion at the plasma membrane. To elucidate the relationship between MyoIIA and F-actin, we examined the colocalization of MyoIIA and F-actin at the plasma membrane upon insulin stimulation as well as the regulation of this interaction. Our findings demonstrated that MyoIIA and F-actin colocalized at the site of GLUT4 fusion with the plasma membrane upon insulin stimulation. Furthermore, inhibition of MyoII with blebbistatin impaired F-actin localization at the plasma membrane. Next we examined the regulatory role of calcium in MyoIIA-F-actin colocalization. Reduced calcium or calmodulin levels decreased colocalization of MyoIIA and F-actin at the plasma membrane. While calcium alone can translocate MyoIIA it did not stimulate F-actin accumulation at the plasma membrane. Taken together, we established that while MyoIIA activity is required for F-actin localization at the plasma membrane, it alone is insufficient to localize F-actin to the plasma membrane. - Highlights: • Insulin induces colocalization of MyoIIA and F-actin at the cortex in adipocytes. • MyoIIA is necessary but not sufficient to localize F-actin at the cell cortex. • MyoIIA-F-actin colocalization is regulated by calcium and calmodulin.

  20. Molecular Mechanical Differences between Isoforms of Contractile Actin in the Presence of Isoforms of Smooth Muscle Tropomyosin

    PubMed Central

    Hilbert, Lennart; Bates, Genevieve; Roman, Horia N.; Blumenthal, Jenna L.; Zitouni, Nedjma B.; Sobieszek, Apolinary; Mackey, Michael C.; Lauzon, Anne-Marie

    2013-01-01

    The proteins involved in smooth muscle's molecular contractile mechanism – the anti-parallel motion of actin and myosin filaments driven by myosin heads interacting with actin – are found as different isoforms. While their expression levels are altered in disease states, their relevance to the mechanical interaction of myosin with actin is not sufficiently understood. Here, we analyzed in vitro actin filament propulsion by smooth muscle myosin for -actin (A), -actin-tropomyosin- (A-Tm), -actin-tropomyosin- (A-Tm), -actin (A), -actin-tropomyosin- (A-Tm), and -actin-tropomoysin- (A-Tm). Actin sliding analysis with our specifically developed video analysis software followed by statistical assessment (Bootstrapped Principal Component Analysis) indicated that the in vitro motility of A, A, and A-Tm is not distinguishable. Compared to these three ‘baseline conditions’, statistically significant differences () were: A-Tm – actin sliding velocity increased 1.12-fold, A-Tm – motile fraction decreased to 0.96-fold, stop time elevated 1.6-fold, A-Tm – run time elevated 1.7-fold. We constructed a mathematical model, simulated actin sliding data, and adjusted the kinetic parameters so as to mimic the experimentally observed differences: A-Tm – myosin binding to actin, the main, and the secondary myosin power stroke are accelerated, A-Tm – mechanical coupling between myosins is stronger, A-Tm – the secondary power stroke is decelerated and mechanical coupling between myosins is weaker. In summary, our results explain the different regulatory effects that specific combinations of actin and smooth muscle tropomyosin have on smooth muscle actin-myosin interaction kinetics. PMID:24204225

  1. Evaluation of Alpha 1-Antitrypsin and the Levels of mRNA Expression of Matrix Metalloproteinase 7, Urokinase Type Plasminogen Activator Receptor and COX-2 for the Diagnosis of Colorectal Cancer

    PubMed Central

    Bujanda, Luis; Sarasqueta, Cristina; Cosme, Angel; Hijona, Elizabeth; Enríquez-Navascués, José M.; Placer, Carlos; Villarreal, Eloisa; Herreros-Villanueva, Marta; Giraldez, María D.; Gironella, Meritxell; Balaguer, Francesc; Castells, Antoni

    2013-01-01

    Background Colorectal cancer (CRC) is the second most common cause of death from cancer in both men and women in the majority of developed countries. Molecular tests of blood could potentially provide this ideal screening tool. Aim Our objective was to assess the usefulness of serum markers and mRNA expression levels in the diagnosis of CRC. Methods In a prospective study, we measured mRNA expression levels of 13 markers (carbonic anhydrase, guanylyl cyclase C, plasminogen activator inhibitor, matrix metalloproteinase 7 (MMP7), urokinase-type plasminogen activator receptor (uPAR), urokinase-type plasminogen activator, survivin, tetranectin, vascular endothelial growth factor (VEGF), cytokeratin 20, thymidylate synthase, cyclooxygenase 2 (COX-2), and CD44) and three proteins in serum (alpha 1 antitrypsin, carcinoembryonic antigen (CEA) and activated C3 in 42 patients with CRC and 33 with normal colonoscopy results. Results Alpha 1-antitrypsin was the serum marker that was most useful for CRC diagnosis (1.79±0.25 in the CRC group vs 1.27±0.25 in the control group, P<0.0005). The area under the ROC curve for alpha 1-antitrypsin was 0.88 (0.79–0.96). The mRNA expression levels of five markers were statistically different between CRC cases and controls: those for which the ROC area was over 75% were MMP7 (0.81) and tetranectin (0.80), COX-2 (0.78), uPAR (0.78) and carbonic anhydrase (0.77). The markers which identified early stage CRC (Stages I and II) were alpha 1-antitrypsin, uPAR, COX-2 and MMP7. Conclusions Serum alpha 1-antitrypsin and the levels of mRNA expression of MMP7, COX-2 and uPAR have good diagnostic accuracy for CRC, even in the early stages. PMID:23300952

  2. mRNA and Protein Levels for GABA[subscript A][alpha]4, [alpha]5, [beta]1 and GABA[subscript B]R1 Receptors are Altered in Brains from Subjects with Autism

    ERIC Educational Resources Information Center

    Fatemi, S. Hossein; Reutiman, Teri J.; Folsom, Timothy D.; Rooney, Robert J.; Patel, Diven H.; Thuras, Paul D.

    2010-01-01

    We have shown altered expression of gamma-aminobutyric acid A (GABA[subscript A]) and gamma-aminobutyric acid B (GABA[subscript B]) receptors in the brains of subjects with autism. In the current study, we sought to verify our western blotting data for GABBR1 via qRT-PCR and to expand our previous work to measure mRNA and protein levels of 3…

  3. Structural Analysis of Human Cofilin 2/Filamentous Actin Assemblies: Atomic-Resolution Insights from Magic Angle Spinning NMR Spectroscopy

    PubMed Central

    Yehl, Jenna; Kudryashova, Elena; Reisler, Emil; Kudryashov, Dmitri; Polenova, Tatyana

    2017-01-01

    Cellular actin dynamics is an essential element of numerous cellular processes, such as cell motility, cell division and endocytosis. Actin’s involvement in these processes is mediated by many actin-binding proteins, among which the cofilin family plays unique and essential role in accelerating actin treadmilling in filamentous actin (F-actin) in a nucleotide-state dependent manner. Cofilin preferentially interacts with older filaments by recognizing time-dependent changes in F-actin structure associated with the hydrolysis of ATP and release of inorganic phosphate (Pi) from the nucleotide cleft of actin. The structure of cofilin on F-actin and the details of the intermolecular interface remain poorly understood at atomic resolution. Here we report atomic-level characterization by magic angle spinning (MAS) NMR of the muscle isoform of human cofilin 2 (CFL2) bound to F-actin. We demonstrate that resonance assignments for the majority of atoms are readily accomplished and we derive the intermolecular interface between CFL2 and F-actin. The MAS NMR approach reported here establishes the foundation for atomic-resolution characterization of a broad range of actin-associated proteins bound to F-actin. PMID:28303963

  4. Three-dimensional structure of actin filaments and of an actin gel made with actin-binding protein.

    PubMed

    Niederman, R; Amrein, P C; Hartwig, J

    1983-05-01

    Purified muscle actin and mixtures of actin and actin-binding protein were examined in the transmission electron microscope after fixation, critical point drying, and rotary shadowing. The three-dimensional structure of the protein assemblies was analyzed by a computer-assisted graphic analysis applicable to generalized filament networks. This analysis yielded information concerning the frequency of filament intersections, the filament length between these intersections, the angle at which filaments branch at these intersections, and the concentration of filaments within a defined volume. Purified actin at a concentration of 1 mg/ml assembled into a uniform mass of long filaments which overlap at random angles between 0 degrees and 90 degrees. Actin in the presence of macrophage actin-binding protein assembled into short, straight filaments, organized in a perpendicular branching network. The distance between branch points was inversely related to the molar ratio of actin-binding protein to actin. This distance was what would be predicted if actin filaments grew at right angles off of nucleation sites on the two ends of actin-binding protein dimers, and then annealed. The results suggest that actin in combination with actin-binding protein self-assembles to form a three-dimensional network resembling the peripheral cytoskeleton of motile cells.

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

    PubMed

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

    2011-02-01

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

  6. Effect of recombinant growth hormone on expression of growth hormone receptor, insulin-like growth factor mRNA and serum level of leptin in growing pigs.

    PubMed

    Xu, Qingfu; Zhao, Zhihui; Ni, Yingdong; Zhao, Ruqian; Chen, Jie

    2003-04-01

    Sixteen Large White x Landrace castrated male pigs were allotted into treatment and control group. The treatment group was injected intramuscularly with recombinant porcine growth hormone (rpGH, 4 mg d(-1)) and the control group with vehicle for 28 days. Animals were slaughtered 4 h after final injection for liver, longissimus dorsi (LD) muscle and blood sampling. Serum concentration of insulin-like growth factor 1 (IGF-I) and leptin were determined by RIA. The total RNA was extracted from tissues to measure the abundance of growth hormone receptor (GHR), IGF-I mRNA by RT-PCR with 18S rRNA internal standard. Results showed that rpGH enhanced the average daily weight gain by 26.1% (P < 0.05), the serum IGF-I concentration by 70.94% (P < 0.01), decreased serum leptin by 34.8% (P < 0.01). The relative abundance of GHR and IGF-I mRNA in liver were increased by 24.45% (P < 0.05) and 45.30% (P < 0.01), respectively, but no difference of GHR (P > 0.05) and IGF-I mRNA (P > 0.05) in LD between GH treated and control group was found. These results suggest that rpGH can up-regulate hepatic GHR and IGF-I gene expression and improve animal growth. However the effect of rpGH on GHR and IGF-I gene expression are tissue-specific.

  7. Profiles of steady state levels of messenger RNAs coding for type I procollagen, elastin, and fibronectin in hamster lungs undergoing bleomycin-induced interstitial pulmonary fibrosis.

    PubMed Central

    Raghow, R; Lurie, S; Seyer, J M; Kang, A H

    1985-01-01

    We have characterized the messenger RNAs (mRNAs) coding for procollagen alpha 1(I), elastin, fibronectin, and actin in the lungs of Syrian golden hamsters by Northern blot analyses. While elastin, fibronectin, and beta-actin were each coded for by a single mRNA species of 4.1 kilobases (kb), 9.1 kb, and 2.1 kb in size, respectively, we identified a major (5.4 kb) and a minor (6.5 kb) procollagen alpha 1(I) mRNA species in the hamster lungs. The mRNAs for the three extracellular matrix proteins showed increased accumulation followed by steady decline in the bleomycin-treated lungs. There were significant differences among the three mRNAs in the relative increase and the time of maximum accumulation. After reaching the peak levels between 2-3 wk posttreatment, the levels of procollagen alpha 1(I) and elastin mRNAs declined to near normal values around the fourth week. In contrast, the accumulation of fibronectin mRNA was maximum in the first week after bleomycin treatment. The procollagen alpha 1(I) mRNA accumulated most dramatically (sevenfold above the levels in the untreated animals) compared with a five-fold increase in mRNA coding for fibronectin. Elastin mRNA increased approximately twofold above the control values. Nuclear runoff transcription experiments demonstrated a selective increase in the rates of transcription of genes coding for procollagen alpha 1(I), fibronectin, and elastin; the extent of transcriptional stimulation of procollagen alpha 1(I) and fibronectin genes was significantly greater than that of elastin. Since the amount of actin mRNA, as well as the rate of transcription of actin gene(s), varied only slightly after bleomycin treatment, we conclude that the metabolism of mRNAs coding for extracellular matrix proteins may be preferentially perturbed during pulmonary fibrosis. Images PMID:2414324

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

    SciTech Connect

    Wang, Yimeng; Zhou, Jianhong; Du, Yuchun

    2014-01-20

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

  9. Antibodies to Actin in Autoimmune Neutropenia

    DTIC Science & Technology

    1990-02-01

    protein as actin. Purified Acanthamoeba actin by anti-neutrophil antibodies in autoimmune neutropenia, comigrated with the protein and was specifically...anti-rabbit IgG were obtained from ICN Immunobiolog- formed using purified Acanthamoeba actin (gift of Dr Blair Bowers. icals, Naperville, IL. Cells...preparations𔃼 1 - was the protein recognized by these anti-neutrophil antibody 6 .2- positive sera, lgG, and F(ab’) 2. Purified Acanthamoeba actin

  10. An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins

    PubMed Central

    Paredez, Alexander R.; Assaf, Zoe June; Sept, David; Timofejeva, Ljudmilla; Dawson, Scott C.; Wang, Chung-Ju Rachel; Cande, W. Z.

    2011-01-01

    Giardia intestinalis, a human intestinal parasite and member of what is perhaps the earliest-diverging eukaryotic lineage, contains the most divergent eukaryotic actin identified to date and is the first eukaryote known to lack all canonical actin-binding proteins (ABPs). We sought to investigate the properties and functions of the actin cytoskeleton in Giardia to determine whether Giardia actin (giActin) has reduced or conserved roles in core cellular processes. In vitro polymerization of giActin produced filaments, indicating that this divergent actin is a true filament-forming actin. We generated an anti-giActin antibody to localize giActin throughout the cell cycle. GiActin localized to the cortex, nuclei, internal axonemes, and formed C-shaped filaments along the anterior of the cell and a flagella-bundling helix. These structures were regulated with the cell cycle and in encysting cells giActin was recruited to the Golgi-like cyst wall processing vesicles. Knockdown of giActin demonstrated that giActin functions in cell morphogenesis, membrane trafficking, and cytokinesis. Additionally, Giardia contains a single G protein, giRac, which affects the Giardia actin cytoskeleton independently of known target ABPs. These results imply that there exist ancestral and perhaps conserved roles for actin in core cellular processes that are independent of canonical ABPs. Of medical significance, the divergent giActin cytoskeleton is essential and commonly used actin-disrupting drugs do not depolymerize giActin structures. Therefore, the giActin cytoskeleton is a promising drug target for treating giardiasis, as we predict drugs that interfere with the Giardia actin cytoskeleton will not affect the mammalian host. PMID:21444821

  11. An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins.

    PubMed

    Paredez, Alexander R; Assaf, Zoe June; Sept, David; Timofejeva, Ljudmilla; Dawson, Scott C; Wang, Chung-Ju Rachel; Cande, W Z

    2011-04-12

    Giardia intestinalis, a human intestinal parasite and member of what is perhaps the earliest-diverging eukaryotic lineage, contains the most divergent eukaryotic actin identified to date and is the first eukaryote known to lack all canonical actin-binding proteins (ABPs). We sought to investigate the properties and functions of the actin cytoskeleton in Giardia to determine whether Giardia actin (giActin) has reduced or conserved roles in core cellular processes. In vitro polymerization of giActin produced filaments, indicating that this divergent actin is a true filament-forming actin. We generated an anti-giActin antibody to localize giActin throughout the cell cycle. GiActin localized to the cortex, nuclei, internal axonemes, and formed C-shaped filaments along the anterior of the cell and a flagella-bundling helix. These structures were regulated with the cell cycle and in encysting cells giActin was recruited to the Golgi-like cyst wall processing vesicles. Knockdown of giActin demonstrated that giActin functions in cell morphogenesis, membrane trafficking, and cytokinesis. Additionally, Giardia contains a single G protein, giRac, which affects the Giardia actin cytoskeleton independently of known target ABPs. These results imply that there exist ancestral and perhaps conserved roles for actin in core cellular processes that are independent of canonical ABPs. Of medical significance, the divergent giActin cytoskeleton is essential and commonly used actin-disrupting drugs do not depolymerize giActin structures. Therefore, the giActin cytoskeleton is a promising drug target for treating giardiasis, as we predict drugs that interfere with the Giardia actin cytoskeleton will not affect the mammalian host.

  12. Actinic cheilitis in dental practice.

    PubMed

    Savage, N W; McKay, C; Faulkner, C

    2010-06-01

    Actinic cheilitis is a potentially premalignant condition involving predominantly the vermilion of the lower lip. The aim of the current paper was to review the clinical presentation of actinic cheilitis and demonstrate the development of management plans using a series of cases. These are designed to provide immediate treatment where required but also to address the medium and long-term requirements of the patient. The authors suggest that the clinical examination of lips and the assessment of actinic cheilitis and other lip pathology become a regular part of the routine soft tissue examination undertaken as a part of the periodic examination of dental patients. Early recognition of actinic cheilitis can allow the development of strategies for individual patients that prevent progression. These are based on past sun exposure, future lifestyle changes and the daily use of emollient sunscreens, broad-brimmed hats and avoidance of sun exposure during the middle of the day. This is a service that is not undertaken as a matter of routine in general medical practice as patients are not seen with the regularity of dental patients and generally not under the ideal examination conditions available in the dental surgery.

  13. 1,25-Dihydroxyvitamin D3 and its analogues increase catalase at the mRNA, protein and activity level in a canine transitional carcinoma cell line.

    PubMed

    Middleton, R P; Nelson, R; Li, Q; Blanton, A; Labuda, J A; Vitt, J; Inpanbutr, N

    2015-12-01

    Antioxidant enzymes, such as catalase, superoxide dismutases (SOD), MnSOD and Cu/ZnSOD, protect cells by scavenging reactive oxygen species (ROS). Numerous studies have reported the anti-cancer effects of 1,25-dihydroxyvitamin D3 (calcitriol) and its related analogues, seocalcitol and analogue V. In this study, canine bladder transitional cell carcinoma (cbTCC) cells were used to determine effects of calcitriol and its related analogues on antioxidant enzyme gene expression, protein expression and activity. Catalase mRNA was increased in response to calcitriol (10(-7) M), and seocalcitol (10(-7) and 10(-9) M). MnSOD mRNA was decreased in response to calcitriol at 10(-7) M. Catalase was significantly increased in response to calcitriol (10(-7) and 10(-9) M), and seocalcitol (10(-9) M). Catalase enzymatic activity increased in response to calcitriol, seocalcitol and analogue V (10(-9) M). In addition, global gene expression analysis identified the involvement of mitogen-activated protein kinase (MAPK) signalling in cbTCC's response to calcitriol and seocalcitol treatment.

  14. Insulin-like growth factor II in human adrenal and pheochromocytomas and Wilms tumors: expression at the mRNA and protein level

    SciTech Connect

    Haselbacher, G.K.; Irminger, J.C.; Zapf, J.; Ziegler, W.H.; Humbel, R.E.

    1987-02-01

    Two forms of insulin-like growth factor (IGF) II with molecular masses of 10 and 7.5 kDa, respectively, were found in tumor tissue from human adrenal pheochromocytomas. The tumors contained 5.3-7.1 ..mu..g of immunoreactive IGF-II per g of tissue, which is about 20 times more than in adrenal medulla. The total bioactive IGF measured by radioimmunoassay in the pheochromocytomas exceeded that in normal liver or kidney, which contained only the 7.5-kDa IGF-II species, by a factor of approx.100. By contrast, the amount of IGF-I was just measurable and did not vary significantly between tumor and normal tissue. The high amounts of IGF-II in the pheochromocytomas were not reflected, however, by a corresponding increase of mRNA. The opposite situations was found in Wilms tumors, where IGF-II content was in the same range as in nontumor tissues despite increased expression of IGF-II mRNA.

  15. Dietary gamma-linolenic acid in the form of borage oil causes less body fat accumulation accompanying an increase in uncoupling protein 1 mRNA level in brown adipose tissue.

    PubMed

    Takahashi, Y; Ide, T; Fujita, H

    2000-10-01

    Rats were fed a low-fat diet containing 2% safflower oil or 20% fat diets containing either safflower oil rich in linoleic acid, borage oil containing 25% gamma (gamma)-linolenic acid or enzymatically prepared gamma-linolenic acid enriched borage oil containing 47% gamma-linolenic acid for 14 days. Energy intake and growth of animals were the same among groups. A high safflower oil diet compared with a low-fat diet caused significant increases in both epididymal and perirenal white adipose tissue weights. However, high-fat diets rich in gamma-linolenic acid failed to do so. Compared with a low-fat diet, all the high-fat diets increased mRNA levels of uncoupling protein 1 and lipoprotein lipase in brown adipose tissue. The extents of the increase were greater with high-fat diets rich in gamma-linolenic acid. Various high-fat diets, compared with a low-fat diet, decreased glucose transporter 4 mRNA in white adipose tissue to the same levels. The amount and types of dietary fat did not affect the leptin mRNA level in epididymal white adipose tissue. However, a high safflower oil diet, but not high-fat diets rich in gamma-linolenic acid relative to a low-fat diet, increased perirenal white adipose tissue leptin mRNA levels. All high-fat diets, relative to a low-fat diet, increased the hepatic mitochondrial fatty acid oxidation rate and fatty acid oxidation enzyme mRNA abundances to the same levels. High-fat diets also increased these parameters in the peroxisomal pathway, and the increases were greater with high-fat diets rich in gamma-linolenic acid. The physiological activity in increasing brown adipose tissue gene expression and peroxisomal fatty acid oxidation was similar between the two types of borage oil differing in gamma-linolenic acid content. It was suggested that dietary gamma-linolenic acid attenuates body fat accumulation through the increase in gene expressions of uncoupling protein 1 in brown adipose tissue. An increase in hepatic peroxisomal fatty acid

  16. Actin crosslinkers: repairing the sense of touch.

    PubMed

    Sun, Sean X; Walcott, Sam

    2010-10-26

    Cells use actin bundles infused with myosin to exert contractile forces on the extracellular environment. This active tension is essential for cellular mechanosensation. Now, the role of actin crosslinkers in stabilizing and repairing the actin bundles is coming into clearer view.

  17. Actin-cytoskeleton rearrangement modulates proton-induced uptake

    SciTech Connect

    Ben-Dov, Nadav; Korenstein, Rafi

    2013-04-15

    Recently it has been shown that elevating proton concentration at the cell surface stimulates the formation of membrane invaginations and vesicles accompanied by an enhanced uptake of macromolecules. While the initial induction of inward membrane curvature was rationalized in terms of proton-based increase of charge asymmetry across the membrane, the mechanisms underlying vesicle formation and its scission are still unknown. In light of the critical role of actin in vesicle formation during endocytosis, the present study addresses the involvement of cytoskeletal actin in proton-induced uptake (PIU). The uptake of dextran-FITC is used as a measure for the factual fraction of inward invaginations that undergo scission from the cell's plasma membrane. Our findings show that the rate of PIU in suspended cells is constant, whereas the rate of PIU in adherent cells is gradually increased in time, saturating at the level possessed by suspended cells. This is consistent with pH induced gradual degradation of stress-fibers in adherent cells. Wortmannin and calyculin-A are able to elevate PIU by 25% in adherent cells but not in suspended cells, while cytochalasin-D, rapamycin and latrunculin-A elevate PIU both in adherent and suspended cells. However, extensive actin depolymerization by high concentrations of latrunculin-A is able to inhibit PIU. We conclude that proton-induced membrane vesiculation is restricted by the actin structural resistance to the plasma membrane bending. Nevertheless, a certain degree of cortical actin restructuring is required for the completion of the scission process. - Highlights: ► Acidification of cells' exterior enhances uptake of macromolecules by the cells. ► Disruption of actin stress fibers leads to enhancement of proton induced uptake. ► Extensive depolymerization of cellular actin attenuates proton-induced uptake.

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

    PubMed