Sample records for amp responsive element

  1. The cyclic AMP response element modulator regulates transcription of the TCR zeta-chain

    NARCIS (Netherlands)

    Tenbrock, K; Kyttaris, VC; Ahlmann, M; Ehrchen, JA; Tolnay, M; Melkonyan, H; Mawrin, C; Roth, J; Sorg, C; Juang, YT; Tsokos, GC


    Systemic lupus erythematusus T cells display decreased amounts of TCR zeta mRNA that results in part from limited binding of the transcriptional enhancer Elf-1 to the TCR zeta promoter. We have identified a new cis-binding site for the cAMP response element (CRE) modulator (CREM) on the TCR zeta pro

  2. Glucose Enhances Basal or Melanocortin-Induced cAMP-Response Element Activity in Hypothalamic Cells. (United States)

    Breit, Andreas; Wicht, Kristina; Boekhoff, Ingrid; Glas, Evi; Lauffer, Lisa; Mückter, Harald; Gudermann, Thomas


    Melanocyte-stimulating hormone (MSH)-induced activation of the cAMP-response element (CRE) via the CRE-binding protein in hypothalamic cells promotes expression of TRH and thereby restricts food intake and increases energy expenditure. Glucose also induces central anorexigenic effects by acting on hypothalamic neurons, but the underlying mechanisms are not completely understood. It has been proposed that glucose activates the CRE-binding protein-regulated transcriptional coactivator 2 (CRTC-2) in hypothalamic neurons by inhibition of AMP-activated protein kinases (AMPKs), but whether glucose directly affects hypothalamic CRE activity has not yet been shown. Hence, we dissected effects of glucose on basal and MSH-induced CRE activation in terms of kinetics, affinity, and desensitization in murine, hypothalamic mHypoA-2/10-CRE cells that stably express a CRE-dependent reporter gene construct. Physiologically relevant increases in extracellular glucose enhanced basal or MSH-induced CRE-dependent gene transcription, whereas prolonged elevated glucose concentrations reduced the sensitivity of mHypoA-2/10-CRE cells towards glucose. Glucose also induced CRCT-2 translocation into the nucleus and the AMPK activator metformin decreased basal and glucose-induced CRE activity, suggesting a role for AMPK/CRTC-2 in glucose-induced CRE activation. Accordingly, small interfering RNA-induced down-regulation of CRTC-2 expression decreased glucose-induced CRE-dependent reporter activation. Of note, glucose also induced expression of TRH, suggesting that glucose might affect the hypothalamic-pituitary-thyroid axis via the regulation of hypothalamic CRE activity. These findings significantly advance our knowledge about the impact of glucose on hypothalamic signaling and suggest that TRH release might account for the central anorexigenic effects of glucose and could represent a new molecular link between hyperglycaemia and thyroid dysfunction.

  3. Regulation of Cox-2 by Cyclic AMP Response Element Binding Protein in Prostate Cancer: Potential Role for Nexrutine


    Rita Ghosh; Gretchen E. Garcia; Katherine Crosby; Hiroyasu Inoue; Thompson, Ian M.; Troyer, Dean A.; Kumar, Addanki P.


    We recently showed that NexrutineR, a Phellodendron amurense bark extract, suppresses proliferation of prostate cancer cell lines and tumor development in the transgenic adenocarcinoma of mouse prostate (TRAMP) model. Our data also indicate that the antiproliferative effects of NexrutineR are mediated in part by Akt and Cyclic AMP response element binding protein (CREB). Cyclooxygenase (Cox-2), a pro-inflammatory mediator, is a CREB target that induces prostaglandin E2 (PGE2) and suppresses a...

  4. Rolipram stimulates angiogenesis and attenuates neuronal apoptosis through the cAMP/cAMP-responsive element binding protein pathway following ischemic stroke in rats. (United States)

    Hu, Shouye; Cao, Qingwen; Xu, Peng; Ji, Wenchen; Wang, Gang; Zhang, Yuelin


    Rolipram, a phosphodiesterase-4 inhibitor, can activate the cyclic adenosine monophosphate (cAMP)/cAMP-responsive element binding protein (CREB) pathway to facilitate functional recovery following ischemic stroke. However, to date, the effects of rolipram on angiogenesis and cerebral ischemia-induced neuronal apoptosis are yet to be fully elucidated. In this study, the aim was to reveal the effect of rolipram on the angiogenesis and neuronal apoptosis following brain cerebral ischemia. Rat models of ischemic stroke were established following transient middle cerebral artery occlusion and rolipram was administered for three, seven and 14 days. The results were examined using behavioral tests, triphenyl tetrazolium chloride staining, immunostaining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) to evaluate the effects of rolipram therapy on functional outcome, angiogenesis and apoptosis. Western blot analysis was used to show the phosphorylated- (p-)CREB protein level in the ischemic hemisphere. The rolipram treatment group exhibited a marked reduction in infarct size and modified neurological severity score compared with the vehicle group, and rolipram treatment significantly promoted the microvessel density in the ischemic boundary region and increased p-CREB protein levels in the ischemic hemisphere. Furthermore, a significant reduction in the number of TUNEL-positive cells was observed in the rolipram group compared with the vehicle group. These findings suggest that rolipram has the ability to attenuate cerebral ischemic injury, stimulate angiogenesis and reduce neuronal apoptosis though the cAMP/CREB pathway.

  5. The catalytic subunit of cAMP-dependent protein kinase induces expression of genes containing cAMP-responsive enhancer elements. (United States)

    Riabowol, K T; Fink, J S; Gilman, M Z; Walsh, D A; Goodman, R H; Feramisco, J R


    Transcriptional regulation of eukaryotic genes by cyclic AMP requires a cAMP-dependent protein kinase (A kinase). Two hypotheses have been proposed to explain how the holoenzyme of the A kinase induces transcription. The regulatory subunits of the A kinase, which bind cAMP and DNA, and have amino-acid homology with the Escherichia coli catabolite activator protein could directly stimulate gene expression. Alternatively, phosphorylation by the catalytic subunits could induce transcription by activating proteins involved in gene transcription. To distinguish between these models, we microinjected purified preparations of the catalytic and regulatory subunits of A kinase into tissue culture cells and monitored expression of a stably integrated fusion gene containing a cAMP-responsive human promoter fused to a bacterial reporter gene, or of the endogenous c-fos gene. The catalytic subunit stimulated expression of these genes, whereas the regulatory subunit did not. These results indicate that the catalytic subunit of A kinase is sufficient to induce expression of two cAMP-responsive genes, without increasing levels of cAMP.

  6. Hypoxic regulation of lactate dehydrogenase A. Interaction between hypoxia-inducible factor 1 and cAMP response elements. (United States)

    Firth, J D; Ebert, B L; Ratcliffe, P J


    The oxygen-regulated control system responsible for the induction of erythropoietin (Epo) by hypoxia is present in most (if not all) cells and operates on other genes, including those involved in energy metabolism. To understand the organization of cis-acting sequences that are responsible for oxygen-regulated gene expression, we have studied the 5' flanking region of the mouse gene encoding the hypoxically inducible enzyme lactate dehydrogenase A (LDH). Deletional and mutational analysis of the function of mouse LDH-reporter fusion gene constructs in transient transfection assays defined three domains, between -41 and -84 base pairs upstream of the transcription initiation site, which were crucial for oxygen-regulated expression. The most important of these, although not capable of driving hypoxic induction in isolation, had the consensus of a hypoxia-inducible factor 1 (HIF-1) site, and cross-competed for the binding of HIF-1 with functionally active Epo and phosphoglycerate kinase-1 sequences. The second domain was positioned close to the HIF-1 site, in an analogous position to one of the critical regions in the Epo 3' hypoxic enhancer. The third domain had the motif of a cAMP response element (CRE). Activation of cAMP by forskolin had no effect on the level of LDH mRNA in normoxia, but produced a magnified response to hypoxia that was dependent upon the integrity of the CRE, indicating an interaction between inducible factors binding the HIF-1 and CRE sites.

  7. Transcriptional regulation of the bovine leukemia virus promoter by the cyclic AMP-response element modulator tau isoform. (United States)

    Nguyên, Thi Lien-Anh; de Walque, Stéphane; Veithen, Emmanuelle; Dekoninck, Ann; Martinelli, Valérie; de Launoit, Yvan; Burny, Arsène; Harrod, Robert; Van Lint, Carine


    Bovine leukemia virus (BLV) expression is controlled at the transcriptional level through three Tax(BLV)-responsive elements (TxREs) responsive to the viral transactivator Tax(BLV). The cAMP-responsive element (CRE)-binding protein (CREB) has been shown to interact with CRE-like sequences present in the middle of each of these TxREs and to play critical transcriptional roles in both basal and Tax(BLV)-transactivated BLV promoter activity. In this study, we have investigated the potential involvement of the cAMP-response element modulator (CREM) in BLV transcriptional regulation, and we have demonstrated that CREM proteins were expressed in BLV-infected cells and bound to the three BLV TxREs in vitro. Chromatin immunoprecipitation assays using BLV-infected cell lines demonstrated in the context of chromatin that CREM proteins were recruited to the BLV promoter TxRE region in vivo. Functional studies, in the absence of Tax(BLV), indicated that ectopic CREMtau protein had a CRE-dependent stimulatory effect on BLV promoter transcriptional activity. Cross-link of the B-cell receptor potentiated CREMtau transactivation of the viral promoter. Further experiments supported the notion that this potentiation involved CREMtau Ser-117 phosphorylation and recruitment of CBP/p300 to the BLV promoter. Although CREB and Tax(BLV) synergistically transactivated the BLV promoter, CREMtau repressed this Tax(BLV)/CREB synergism, suggesting that a modulation of the level of Tax(BLV) transactivation through opposite actions of CREB and CREMtau could facilitate immune escape and allow tumor development.

  8. cAMP response element binding protein (CREB activates transcription via two distinct genetic elements of the human glucose-6-phosphatase gene

    Directory of Open Access Journals (Sweden)

    Stefano Luisa


    Full Text Available Abstract Background The enzyme glucose-6-phosphatase catalyzes the dephosphorylation of glucose-6-phosphatase to glucose, the final step in the gluconeogenic and glycogenolytic pathways. Expression of the glucose-6-phosphatase gene is induced by glucocorticoids and elevated levels of intracellular cAMP. The effect of cAMP in regulating glucose-6-phosphatase gene transcription was corroborated by the identification of two genetic motifs CRE1 and CRE2 in the human and murine glucose-6-phosphatase gene promoter that resemble cAMP response elements (CRE. Results The cAMP response element is a point of convergence for many extracellular and intracellular signals, including cAMP, calcium, and neurotrophins. The major CRE binding protein CREB, a member of the basic region leucine zipper (bZIP family of transcription factors, requires phosphorylation to become a biologically active transcriptional activator. Since unphosphorylated CREB is transcriptionally silent simple overexpression studies cannot be performed to test the biological role of CRE-like sequences of the glucose-6-phosphatase gene. The use of a constitutively active CREB2/CREB fusion protein allowed us to uncouple the investigation of target genes of CREB from the variety of signaling pathways that lead to an activation of CREB. Here, we show that this constitutively active CREB2/CREB fusion protein strikingly enhanced reporter gene transcription mediated by either CRE1 or CRE2 derived from the glucose-6-phosphatase gene. Likewise, reporter gene transcription was enhanced following expression of the catalytic subunit of cAMP-dependent protein kinase (PKA in the nucleus of transfected cells. In contrast, activating transcription factor 2 (ATF2, known to compete with CREB for binding to the canonical CRE sequence 5'-TGACGTCA-3', did not transactivate reporter genes containing CRE1, CRE2, or both CREs derived from the glucose-6-phosphatase gene. Conclusions Using a constitutively active CREB2

  9. Mechanisms of extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor signaltransduction pathway in depressive disorder

    Institute of Scientific and Technical Information of China (English)

    Hongyan Wang; Yingquan Zhang; Mingqi Qiao


    The extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor signal transduction pathway plays an important role in the mechanism of action of antidepressant drugs and has dominated recent studies on the pathogenesis of depression. In the present review we summarize the known roles of extracellular signal-regulated kinase, cAMP response element-binding protein and brain-derived neurotrophic factor in the pathogenesis of depression and in the mechanism of action of antidepressant medicines. The extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor pathway has potential to be used as a biological index to help diagnose depression, and as such it is considered as an important new target in the treatment of depression.

  10. Mechanisms of extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor signal transduction pathway in depressive disorder. (United States)

    Wang, Hongyan; Zhang, Yingquan; Qiao, Mingqi


    The extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor signal transduction pathway plays an important role in the mechanism of action of antidepressant drugs and has dominated recent studies on the pathogenesis of depression. In the present review we summarize the known roles of extracellular signal-regulated kinase, cAMP response element-binding protein and brain-derived neurotrophic factor in the pathogenesis of depression and in the mechanism of action of antidepressant medicines. The extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor pathway has potential to be used as a biological index to help diagnose depression, and as such it is considered as an important new target in the treatment of depression.

  11. Hippocampal expression of synaptic structural proteins and phosphorylated cAMP response element-binding protein in a rat model of vascular dementia induced by chronic cerebral hypoperfusion

    Institute of Scientific and Technical Information of China (English)

    Hui Zhao; Zhiyong Li; Yali Wang; Qiuxia Zhang


    The present study established a rat model of vascular dementia induced by chronic cerebral hy-poperfusion through permanent ligation of bilateral common carotid arteries. At 60 days after mod-eling, escape latency and swimming path length during hidden-platform acquisition training in Morris water maze significantly increased in the model group. In addition, the number of accurate crossings over the original platform significantly decreased, hippocampal CA1 synaptophysin and growth-associated protein 43 expression significantly decreased, cAMP response element-binding protein expression remained unchanged, and phosphorylated cAMP response element-binding protein expression significantly decreased. Results suggested that abnormal expression of hippo-campal synaptic structural protein and cAMP response element-binding protein phosphorylation played a role in cognitive impairment following chronic cerebral hypoperfusion.

  12. cAMP response element binding protein is required for differentiation of respiratory epithelium during murine development.

    Directory of Open Access Journals (Sweden)

    A Daniel Bird

    Full Text Available The cAMP response element binding protein 1 (Creb1 transcription factor regulates cellular gene expression in response to elevated levels of intracellular cAMP. Creb1(-/- fetal mice are phenotypically smaller than wildtype littermates, predominantly die in utero and do not survive after birth due to respiratory failure. We have further investigated the respiratory defect of Creb1(-/- fetal mice during development. Lungs of Creb1(-/- fetal mice were pale in colour and smaller than wildtype controls in proportion to their reduced body size. Creb1(-/- lungs also did not mature morphologically beyond E16.5 with little or no expansion of airway luminal spaces, a phenotype also observed with the Creb1(-/- lung on a Crem(-/- genetic background. Creb1 was highly expressed throughout the lung at all stages examined, however activation of Creb1 was detected primarily in distal lung epithelium. Cell differentiation of E17.5 Creb1(-/- lung distal epithelium was analysed by electron microscopy and showed markedly reduced numbers of type-I and type-II alveolar epithelial cells. Furthermore, immunomarkers for specific lineages of proximal epithelium including ciliated, non-ciliated (Clara, and neuroendocrine cells showed delayed onset of expression in the Creb1(-/- lung. Finally, gene expression analyses of the E17.5 Creb1(-/- lung using whole genome microarray and qPCR collectively identified respiratory marker gene profiles and provide potential novel Creb1-regulated genes. Together, these results demonstrate a crucial role for Creb1 activity for the development and differentiation of the conducting and distal lung epithelium.

  13. Spatial Memory in the Morris Water Maze and Activation of Cyclic AMP Response Element-Binding (CREB) Protein within the Mouse Hippocampus (United States)

    Porte, Yves; Buhot, Marie Christine; Mons, Nicole E.


    We investigated the spatio-temporal dynamics of learning-induced cAMP response element-binding protein activation/phosphorylation (pCREB) in mice trained in a spatial reference memory task in the water maze. Using immunohistochemistry, we examined pCREB immunoreactivity (pCREB-ir) in hippocampal CA1 and CA3 and related brain structures. During the…

  14. Long-Term Memory for Place Learning Is Facilitated by Expression of cAMP Response Element-Binding Protein in the Dorsal Hippocampus (United States)

    Brightwell, Jennifer J.; Smith, Clayton A.; Neve, Rachael L.; Colombo, Paul J.


    Extensive research has shown that the hippocampus is necessary for consolidation of long-term spatial memory in rodents. We reported previously that rats using a place strategy to solve a cross maze task showed sustained phosphorylation of hippocampus cyclic AMP response element-binding protein (CREB), a transcription factor implicated in…

  15. Chronic antidepressant administration increases the expression of cAMP response element binding protein (CREB) in rat hippocampus. (United States)

    Nibuya, M; Nestler, E J; Duman, R S


    The present study demonstrates that chronic, but not acute, adminstration of several different classes of antidepressants, including serotonin- and norepinephrine-selective reuptake inhibitors, increases the expression of cAMP response element binding protein (CREB) mRNA in rat hippocampus. In contrast, chronic administration of several nonantidepressant psychotropic drugs did not influence expression of CREB mRNA, demonstrating the pharmacological specificity of this effect. In situ hybridization analysis demonstrates that antidepressant administration increases expression of CREB mRNA in CA1 and CA3 pyramidal and dentate gyrus granule cell layers of the hippocampus. In addition, levels of CRE immunoreactivity and of CRE binding activity were increased by chronic antidepressant administration, which indicates that expression and function of CREB protein are increased along with its mRNA. Chronic administration of the phosphodiesterase (PDE) inhibitors rolipram or papaverine also increased expression of CREB mRNA in hippocampus, demonstrating a role for the cAMP cascade. Moreover, coadministration of rolipram with imipramine resulted in a more rapid induction of CREB than with either treatment alone. Increased expression and function of CREB suggest that specific target genes may be regulated by these treatments. We have found that levels of brain-derived neurotrophic factor (BDNF) and trkB mRNA are also increased by administration of antidepressants or PDE inhibitors. These findings indicate that upregulation of CREB is a common action of chronic antidepressant treatments that may lead to regulation of specific target genes, such as BDNF and trkB, and to the long-term effects of these treatments on brain function.

  16. Regulation of Cox-2 by Cyclic AMP Response Element Binding Protein in Prostate Cancer: Potential Role for Nexrutine

    Directory of Open Access Journals (Sweden)

    Rita Ghosh


    Full Text Available We recently showed that NexrutineR, a Phellodendron amurense bark extract, suppresses proliferation of prostate cancer cell lines and tumor development in the transgenic adenocarcinoma of mouse prostate (TRAMP model. Our data also indicate that the antiproliferative effects of NexrutineR are mediated in part by Akt and Cyclic AMP response element binding protein (CREB. Cyclooxygenase (Cox-2, a pro-inflammatory mediator, is a CREB target that induces prostaglandin E2 (PGE2 and suppresses apoptosis. Treatment of LNCaP cells with NexrutineR reduced tumor necrosis factor α-induced enzymatic as well as promoter activities of Cox-2. NexrutineR also reduced the expression and promoter activity of Cox-2 in PC-3 cells that express high constitutive levels of Cox-2. Deletion analysis coupled with mutational analysis of the Cox-2 promoter identified CRE as being sufficient for mediating NexrutineR response. Immunohistochemical analysis of human prostate tumors show increased expression of CREB and DNA binding activity in high-grade tumors (three-fold higher in human prostate tumors compared to normal prostate; P = .01. We have identified CREB-mediated activation of Cox-2 as a potential signaling pathway in prostate cancer which can be blocked with a nontoxic, cost-effective dietary supplement like NexrutineR, demonstrating a prospective for development of NexrutineR for prostate cancer management.

  17. Cyclic AMP response element binding protein and brain-derived neurotrophic factor: Molecules that modulate our mood?

    Indian Academy of Sciences (India)

    A Nair; V A Vaidya


    Depression is the major psychiatric ailment of our times, afflicting ∼20% of the population. Despite its prevalence, the pathophysiology of this complex disorder is not well understood. In addition, although antidepressants have been in existence for the past several decades, the mechanisms that underlie their therapeutic effects remain elusive. Building evidence implicates a role for the plasticity of specific neuro-circuitry in both the pathophysiology and treatment of depression. Damage to limbic regions is thought to contribute to the etiology of depression and antidepressants have been reported to reverse such damage and promote adaptive plasticity. The molecular pathways that contribute to the damage associated with depression and antidepressant-mediated plasticity are a major focus of scientific enquiry. The transcription factor cyclic AMP response element binding protein (CREB) and the neurotrophin brain-derived neurotrophic factor (BDNF) are targets of diverse classes of antidepressants and are known to be regulated in animal models and in patients suffering from depression. Given their role in neuronal plasticity, CREB and BDNF have emerged as molecules that may play an important role in modulating mood. The purpose of this review is to discuss the role of CREB and BDNF in depression and as targets/mediators of antidepressant action.

  18. MicroRNA-181b targets cAMP responsive element binding protein 1 in gastric adenocarcinomas. (United States)

    Chen, Lin; Yang, Qian; Kong, Wei-Qing; Liu, Tao; Liu, Min; Li, Xin; Tang, Hua


    MicroRNAs are a class of small endogenous non-coding RNAs that function as post-transcriptional regulators. In our previous study, we found that miR-181b was significantly downregulated in human gastric adenocarcinoma tissue samples compared to the adjacent normal gastric tissues. In this study, we confirm the down-regulation of miR-181b in human gastric cancer cell lines versus the gastric epithelial cells. Overexpression of miR-181b suppressed the proliferation and colony formation rate of gastric cancer cells. miR-181b downregulated the expression of cAMP responsive element binding protein 1 (CREB1) by binding its 3' untranslated region. Overexpression of CREB1 counteracted the suppression of growth in gastric cancer cells caused by ectopic expression of miR-181b. These results indicate that miR-181b may function as a tumor suppressor in gastric adenocarcinoma cells through negative regulation of CREB1.

  19. Mechanisms of lung neutrophil activation after hemorrhage or endotoxemia: roles of reactive oxygen intermediates, NF-kappa B, and cyclic AMP response element binding protein. (United States)

    Shenkar, R; Abraham, E


    Acute inflammatory lung injury occurs frequently in the setting of severe infection or blood loss. Accumulation of activated neutrophils in the lungs and increased pulmonary proinflammatory cytokine levels are major characteristics of acute lung injury. In the present experiments, we examined mechanisms leading to neutrophil accumulation and activation in the lungs after endotoxemia or hemorrhage. Levels of IL-1 beta, TNF-alpha, and macrophage inflammatory protein-2 mRNA were increased in lung neutrophils from endotoxemic or hemorrhaged mice compared with those present in lung neutrophils from control mice or in peripheral blood neutrophils from endotoxemic, hemorrhaged, or control mice. The transcriptional regulatory factors NF-kappa B and cAMP response element binding protein were activated in lung but not blood neutrophils after hemorrhage or endotoxemia. Xanthine oxidase inhibition, achieved by feeding allopurinol or tungsten-containing diets, did not affect neutrophil trafficking to the lungs after hemorrhage or endotoxemia. Xanthine oxidase inhibition did prevent hemorrhage- but not endotoxemia-induced increases in proinflammatory cytokine expression among lung neutrophils. Hemorrhage- or endotoxemia-associated activation of NF-kappa B in lung neutrophils was not affected by inhibition of xanthine oxidase. cAMP response element binding protein activation was increased after hemorrhage, but not endotoxemia, in mice fed xanthine oxidase-inhibiting diets. Our results indicate that xanthine oxidase modulates cAMP response element binding protein activation and proinflammatory cytokine expression in lung neutrophils after hemorrhage, but not endotoxemia. These findings suggest that the mechanisms leading to acute inflammatory lung injury after hemorrhage differ from those associated with endotoxemia.


    NARCIS (Netherlands)



    Activation of the retinoic acid receptor (RAR) beta2 promoter is known to be mediated by a RA response element located in the proximity of the TATA-box. By deletion studies in P19 embryonal carcinoma cells we have analyzed the RARbeta2 promoter for the presence of additional regulatory elements. We

  1. Repression of protein kinase C and stimulation of cyclic AMP response elements by fumonisin, a fungal encoded toxin which is a carcinogen. (United States)

    Huang, C; Dickman, M; Henderson, G; Jones, C


    Fusarium moniliforme (FM) is a major fungal pathogen of corn and is involved with stalk rot disease. FM is widely spread throughout the world, including the United States. Most strains of FM produce several mycotoxins, the most prominent of which is called fumonisin. Recent epidemiological studies indicated that ingestion of fumonisin correlates with a higher incidence of esophageal cancer in Southern and Northern Africa and China. Furthermore, fumonisin causes a neurodegenerative disease in horses, induces hepatic cancer in rats, and induces pulmonary edema in swine. Considering that high levels of fumonisin have been detected in healthy and diseased corn grown in the United States, fumonisin may pose a health threat to humans and livestock animals. Structurally, fumonisin resembles sphingolipids which are present in the membranes of animal and plant cells. At the present time, very little is known concerning the mechanism by which fumonisin elicits its carcinogenic effect. Our studies indicate that fumonisin represses expression of protein kinase C and AP-1-dependent transcription. In contrast, fumonisin stimulated a simple promoter containing a single cyclic AMP response element. Since fumonisin did not alter protein kinase A activity, it appears that cyclic AMP response element activation was independent of protein kinase A. It is hypothesized that the ability of fumonisin to alter signal transduction pathways plays a role in carcinogenesis.

  2. Drosophila salt-inducible kinase (SIK) regulates starvation resistance through cAMP-response element-binding protein (CREB)-regulated transcription coactivator (CRTC). (United States)

    Choi, Sekyu; Kim, Wonho; Chung, Jongkyeong


    Salt-inducible kinase (SIK), one of the AMP-activated kinase (AMPK)-related kinases, has been suggested to play important functions in glucose homeostasis by inhibiting the cAMP-response element-binding protein (CREB)-regulated transcription coactivator (CRTC). To examine the role of SIK in vivo, we generated Drosophila SIK mutant and found that the mutant flies have higher amounts of lipid and glycogen stores and are resistant to starvation. Interestingly, SIK transcripts are highly enriched in the brain, and we found that neuron-specific expression of exogenous SIK fully rescued lipid and glycogen storage phenotypes as well as starvation resistance of the mutant. Using genetic and biochemical analyses, we demonstrated that CRTC Ser-157 phosphorylation by SIK is critical for inhibiting CRTC activity in vivo. Furthermore, double mutants of SIK and CRTC became sensitive to starvation, and the Ser-157 phosphomimetic mutation of CRTC reduced lipid and glycogen levels in the SIK mutant, suggesting that CRTC mediates the effects of SIK signaling. Collectively, our results strongly support the importance of the SIK-CRTC signaling axis that functions in the brain to maintain energy homeostasis in Drosophila.

  3. Loss of Kv3.1 tonotopicity and alterations in cAMP response element-binding protein signaling in central auditory neurons of hearing impaired mice. (United States)

    von Hehn, Christian A A; Bhattacharjee, Arin; Kaczmarek, Leonard K


    The promoter for the kv3.1 potassium channel gene is regulated by a Ca2+-cAMP responsive element, which binds the transcription factor cAMP response element-binding protein (CREB). Kv3.1 is expressed in a tonotopic gradient within the medial nucleus of the trapezoid body (MNTB) of the auditory brainstem, where Kv3.1 levels are highest at the medial end, which corresponds to high auditory frequencies. We have compared the levels of Kv3.1, CREB, and the phosphorylated form of CREB (pCREB) in a mouse strain that maintains good hearing throughout life, CBA/J (CBA), with one that suffers early cochlear hair cell loss, C57BL/6 (BL/6). A gradient of Kv3.1 immunoreactivity in the MNTB was detected in both young (6 week) and older (8 month) CBA mice. Although no gradient of CREB was detected, pCREB-immunopositive cells were grouped together in distinct clusters along the tonotopic axis. The same pattern of Kv3.1, CREB, and pCREB localization was also found in young BL/6 mice at a time (6 weeks) when hearing is normal. In contrast, at 8 months, when hearing is impaired, the gradient of Kv3.1 was abolished. Moreover, in the older BL/6 mice there was a decrease in CREB expression along the tonotopic axis, and the pattern of pCREB labeling appeared random, with no discrete clusters of pCREB-positive cells along the tonotopic axis. Our findings are consistent with the hypothesis that ongoing activity in auditory brainstem neurons is necessary for the maintenance of Kv3.1 tonotopicity through the CREB pathway.

  4. Differential regulation of phosphorylation of the cAMP response element-binding protein after activation of EP2 and EP4 prostanoid receptors by prostaglandin E2. (United States)

    Fujino, Hiromichi; Salvi, Sambhitab; Regan, John W


    The EP2 and EP4 prostanoid receptors are G-protein-coupled receptors whose activation by their endogenous ligand, prostaglandin (PG) E2, stimulates the formation of intracellular cAMP. We have previously reported that the stimulation of cAMP formation in EP4-expressing cells is significantly less than in EP2-expressing cells, despite nearly identical levels of receptor expression (J Biol Chem 277:2614-2619, 2002). In addition, a component of EP4 receptor signaling, but not of EP2 receptor signaling, was found to involve the activation of phosphatidylinositol 3-kinase (PI3K). In this study, we report that PGE2 stimulation of cells expressing either the EP2 or EP4 receptor results in the phosphorylation of the cAMP response element binding protein (CREB) at serine-133. Pretreatment of cells with N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H-89), an inhibitor of protein kinase A (PKA), attenuated the PGE2-mediated phosphorylation of CREB in EP2-expressing cells, but not in EP4-expressing cells. Pretreatment of cells with wortmannin, an inhibitor of PI3K, had no effects on the PGE2-mediated phosphorylation of CREB in either EP2- or EP4-expressing cells, although it significantly increased the PGE2-mediated activation of PKA in EP4-expressing cells. However, combined pretreatment with H-89 and wortmannin blocked PGE2-mediated phosphorylation in EP2-expressing cells as well as in EP2-expressing cells. PGE2-mediated intracellular cAMP formation was not affected by pretreatment with wortmannin, or combined treatment with wortmannin and H-89, in either the EP2- or EP4-expressing cells. These findings suggest that PGE2 stimulation of EP4 receptors, but not EP2 receptors, results in the activation of a PI3K signaling pathway that inhibits the activity of PKA and that the PGE2-mediated phosphorylation of CREB by these receptors occurs through different signaling pathways

  5. Pin1 associates with and induces translocation of CRTC2 to the cytosol, thereby suppressing cAMP-responsive element transcriptional activity. (United States)

    Nakatsu, Yusuke; Sakoda, Hideyuki; Kushiyama, Akifumi; Ono, Hiraku; Fujishiro, Midori; Horike, Nanao; Yoneda, Masayasu; Ohno, Haruya; Tsuchiya, Yoshihiro; Kamata, Hideaki; Tahara, Hidetoshi; Isobe, Toshiaki; Nishimura, Fusanori; Katagiri, Hideki; Oka, Yoshitomo; Fukushima, Toshiaki; Takahashi, Shin-Ichiro; Kurihara, Hiroki; Uchida, Takafumi; Asano, Tomoichiro


    Pin1 is a unique regulator, which catalyzes the conversion of a specific phospho-Ser/Thr-Pro-containing motif in target proteins. Herein, we identified CRTC2 as a Pin1-binding protein by overexpressing Pin1 with Myc and FLAG tags in mouse livers and subsequent purification of the complex containing Pin1. The association between Pin1 and CRTC2 was observed not only in overexpression experiments but also endogenously in the mouse liver. Interestingly, Ser(136) in the nuclear localization signal of CRTC2 was shown to be involved in the association with Pin1. Pin1 overexpression in HepG2 cells attenuated forskolin-induced nuclear localization of CRTC2 and cAMP-responsive element (CRE) transcriptional activity, whereas gene knockdown of Pin1 by siRNA enhanced both. Pin1 also associated with CRTC1, leading to their cytosol localization, essentially similar to the action of CRTC2. Furthermore, it was shown that CRTC2 associated with Pin1 did not bind to CREB. Taken together, these observations indicate the association of Pin1 with CRTC2 to decrease the nuclear CBP·CRTC·CREB complex. Indeed, adenoviral gene transfer of Pin1 into diabetic mice improved hyperglycemia in conjunction with normalizing phosphoenolpyruvate carboxykinase mRNA expression levels, which is regulated by CRE transcriptional activity. In conclusion, Pin1 regulates CRE transcriptional activity, by associating with CRTC1 or CRTC2.

  6. Protein kinases mediate increment of the phosphorylation of cyclic AMP -responsive element binding protein in spinal cord of rats following capsaicin injection

    Directory of Open Access Journals (Sweden)

    Li Junfa


    Full Text Available Abstract Background Strong noxious stimuli cause plastic changes in spinal nociceptive neurons. Intracellular signal transduction pathways from cellular membrane to nucleus, which may further regulate gene expression by critical transcription factors, convey peripheral stimulation. Cyclic AMP-responsive element binding protein (CREB is a well-characterized stimulus-induced transcription factor whose activation requires phosphorylation of the Serine-133 residue. Phospho-CREB can further induce gene transcription and strengthen synaptic transmission by the activation of the protein kinase cascades. However, little is known about the mechanisms by which CREB phosphorylation is regulated by protein kinases during nociception. This study was designed to use Western blot analysis to investigate the role of mitogen-activated protein (MAP/extracellular signal-regulated kinase (ERK kinase (MEK 1/2, PKA and PKC in regulating the phosphorylation of CREB in the spinal cord of rats following intraplantar capsaicin injection. Results We found that capsaicin injection significantly increased the phosphorylation level of CREB in the ipsilateral side of the spinal cord. Pharmacological manipulation of MEK 1/2, PKA and PKC with their inhibitors (U0126, H89 and NPC 15473, respectively significantly blocked this increment of CREB phosphorylation. However, the expression of CREB itself showed no change in any group. Conclusion These findings suggest that the activation of intracellular MAP kinase, PKA and PKC cascades may contribute to the regulation of phospho-CREB in central nociceptive neurons following peripheral painful stimuli.

  7. Prophylactic Melatonin Attenuates Isoflurane-Induced Cognitive Impairment in Aged Rats through Hippocampal Melatonin Receptor 2 - cAMP Response Element Binding Signalling. (United States)

    Liu, Yajie; Ni, Cheng; Li, Zhengqian; Yang, Ning; Zhou, Yang; Rong, Xiaoying; Qian, Min; Chui, Dehua; Guo, Xiangyang


    Melatonin exerts many physiological effects via melatonin receptors, among which the melatonin-2 receptor (MT2 ) plays a critical role in circadian rhythm disorders, Alzheimer's disease and other neurological disorders. A melatonin replacement strategy has been tested previously, and MT2 was a critical target during the process. cAMP response element binding (CREB) is an essential transcription factor for memory formation and could be involved in MT2 signalling. Therefore, the present study was designed to investigate the effects of prophylactic melatonin on inhaled anaesthetic isoflurane-induced cognitive impairment, and to determine whether the protective effects of melatonin are dependent on MT2 and downstream CREB signalling in the hippocampus of aged rats. The results showed that prophylactic melatonin attenuated isoflurane-induced decreases in plasma/hippocampal melatonin levels and cognitive impairment in aged rats. Furthermore, 4P-PDOT, a selective MT2 antagonist, blocked the protective effects of melatonin on isoflurane-induced decreases in both hippocampal MT2 expression and downstream CREB phosphorylation. And 4P-PDOT blocked the attenuation of melatonin on isoflurane-induced memory impairment. Collectively, the results suggest that the protective effects of prophylactic melatonin are dependent on hippocampal MT2 -CREB signalling, which could be a potential therapeutic target for anaesthetic-induced cognitive impairment.

  8. Detection of novelty, but not memory of spatial habituation, is associated with an increase in phosphorylated cAMP response element-binding protein levels in the hippocampus. (United States)

    Winograd, Milena; Viola, Haydée


    There is a growing body of evidence showing that the formation of associative memories is associated with an increase in phosphorylated cAMP response element-binding protein (pCREB) levels. We recently reported increased pCREB levels in the rat hippocampus after an exploration to a novel environment. In the present work, we studied whether this increment in CREB activation is associated with the formation of memory of habituation to a novel environment or with the detection of novelty. Rats were submitted to consecutive open field sessions at 3-h intervals. Measurement of the hippocampal pCREB level, carried out 1 h after each training session, showed that (1) it did not increase when rats explored a familiar environment; (2) it did not increase after a reexposure that improves the memory of habituation; (3) it increased after a brief novel exploration unable to form memory of habituation; and (4) it increased in amnesic rats for spatial habituation. Taken as a whole, our results suggest that the elevated pCREB level after a single open field exploration is not associated with the memory formation of habituation. It is indeed associated with the detection of a novel environment.

  9. 1,25-dihydroxyvitamin D3 suppresses renin gene transcription by blocking the activity of the cyclic AMP response element in the renin gene promoter. (United States)

    Yuan, Weihua; Pan, Wei; Kong, Juan; Zheng, Wei; Szeto, Frances L; Wong, Kari E; Cohen, Ronald; Klopot, Anna; Zhang, Zhongyi; Li, Yan Chun


    We have shown that 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) down-regulates renin expression. To explore the molecular mechanism, we analyzed the mouse Ren-1c gene promoter by luciferase reporter assays. Deletion analysis revealed two DNA fragments from -2,725 to -2,647 (distal fragment) and from -117 to +6 (proximal fragment) that are sufficient to mediate the repression. Mutation of the cAMP response element (CRE) in the distal fragment blunted forskolin stimulation as well as 1,25(OH)(2)D(3) inhibition of the transcriptional activity, suggesting the involvement of CRE in 1,25(OH)(2)D(3)-induced suppression. EMSA revealed that 1,25(OH)(2)D(3) markedly inhibited nuclear protein binding to the CRE in the promoter. ChIP and GST pull-down assays demonstrated that liganded VDR blocked the binding of CREB to the CRE by directly interacting with CREB with the ligand-binding domain, and the VDR-mediated repression can be rescued by CREB, CBP, or p300 overexpression. These data indicate that 1,25(OH)(2)D(3) suppresses renin gene expression at least in part by blocking the formation of CRE-CREB-CBP complex.

  10. Changes of phosphorylation of cAMP response element binding protein in rat nucleus accumbens after chronic ethanol intake: naloxone reversal

    Institute of Scientific and Technical Information of China (English)

    LIJing; LIYue-Hua; YUANXiao-Ru


    AIM: To study the changes in the expression and phosphorylation of cAMP response element binding protein(CREB) in the rat nucleus accumbens after chronic ethanol intake and its withdrawal. METHODS: Ethanol wasgiven in drinking water at the concentration of 6 % (v/v), for one month. Changes in the levels of CREB andphospho-CREB (p-CREB) protein in the nucleus accumbens were measured by immunohistochemistry methods.RESULTS: Ethanol given to rats in drinking water decreased the level of p-CREB protein in the nucleus accumbens(-75 %) at the time of exposure to ethanol. The decrement of p-CREB protein in the nucleus accumbens remainedat 24 h (-35 %) and 72 h (-28 %) of ethanol withdrawal, which recovered toward control level after 7 d of ethanolwithdrawal. However, chronic ethanol, as well as ethanol withdrawal failed to produce any significant alteration inthe level of CREB protein in the nucleus accumbens. Naloxone (alone) treatment of rats had no effect on the levelsof CREB and p-CREB protein in the nucleus accumbens. However, when naloxone was administered concurrentlywith ethanol treatment, it antagonized the down-regulation of p-CREB protein in the nucleus accumbens (142 %) ofrats exposed to ethanol. CONCLUSION: A long-term intake of ethanol solution down-regulates the phosphoryla-tion of CREB in the nucleus accumbens, and those changes can be reversed by naloxone, which may be one kindof the molecular mechanisms associated with ethano1 dependence.

  11. Regulation of insulin-like growth factor I transcription by cyclic adenosine 3',5'-monophosphate (cAMP) in fetal rat bone cells through an element within exon 1: protein kinase A-dependent control without a consensus AMP response element (United States)

    McCarthy, T. L.; Thomas, M. J.; Centrella, M.; Rotwein, P.


    Insulin-like growth factor I (IGF-I) is a locally synthesized anabolic growth factor for bone. IGF-I synthesis by primary fetal rat osteoblasts (Ob) is stimulated by agents that increase the intracellular cAMP concentration, including prostaglandin E2 (PGE2). Previous studies with Ob cultures demonstrated that PGE2 enhanced IGF-I transcription through selective use of IGF-I promoter 1, with little effect on IGF-I messenger RNA half-life. Transient transfection of Ob cultures with an array of promoter 1-luciferase reporter fusion constructs has now allowed localization of a potential cis-acting promoter element(s) responsible for cAMP-stimulated gene expression to the 5'-untranslated region (5'-UTR) of IGF-I exon 1, within a segment lacking a consensus cAMP response element. Our evidence derives from three principal observations: 1) a transfection construct containing only 122 nucleotides (nt) of promoter 1 and 328 nt of the 5'-UTR retained full PGE2-stimulated reporter expression; 2) maximal PGE2-driven reporter expression required the presence of nt 196 to 328 of exon 1 when tested within the context of IGF-I promoter 1; 3) cotransfection of IGF-I promoter-luciferase-reporter constructs with a plasmid encoding the alpha-isoform of the catalytic subunit of murine cAMP-dependent protein kinase (PKA) produced results comparable to those seen with PGE2 treatment, whereas cotransfection with a plasmid encoding a mutant regulatory subunit of PKA that cannot bind cAMP blocked PGE2-induced reporter expression. Deoxyribonuclease I footprinting of the 5'-UTR of exon 1 demonstrated protected sequences at HS3A, HS3B, and HS3D, three of six DNA-protein binding sites previously characterized with rat liver nuclear extracts. Of these three regions, only the HS3D binding site is located within the functionally identified hormonally responsive segment of IGF-I exon 1. These results directly implicate PKA in the control of IGF-I gene transcription by PGE2 and identify a segment of

  12. Aldose Reductase Regulates Microglia/Macrophages Polarization Through the cAMP Response Element-Binding Protein After Spinal Cord Injury in Mice. (United States)

    Zhang, Qian; Bian, Ganlan; Chen, Peng; Liu, Ling; Yu, Caiyong; Liu, Fangfang; Xue, Qian; Chung, Sookja K; Song, Bing; Ju, Gong; Wang, Jian


    Inflammatory reactions are the most critical pathological processes occurring after spinal cord injury (SCI). Activated microglia/macrophages have either detrimental or beneficial effects on neural regeneration based on their functional polarized M1/M2 subsets. However, the mechanism of microglia/macrophage polarization to M1/M2 at the injured spinal cord environment remains unknown. In this study, wild-type (WT) or aldose reductase (AR)-knockout (KO) mice were subjected to SCI by a spinal crush injury model. The expression pattern of AR, behavior tests for locomotor activity, and lesion size were assessed at between 4 h and 28 days after SCI. We found that the expression of AR is upregulated in microglia/macrophages after SCI in WT mice. In AR KO mice, SCI led to smaller injury lesion areas compared to WT. AR deficiency-induced microglia/macrophages induce the M2 rather than the M1 response and promote locomotion recovery after SCI in mice. In the in vitro experiments, microglia cell lines (N9 or BV2) were treated with the AR inhibitor (ARI) fidarestat. AR inhibition caused 4-hydroxynonenal (HNE) accumulation, which induced the phosphorylation of the cAMP response element-binding protein (CREB) to promote Arg1 expression. KG501, the specific inhibitor of phosphorylated CREB, could cancel the upregulation of Arg1 by ARI or HNE stimulation. Our results suggest that AR works as a switch which can regulate microglia by polarizing cells to either the M1 or the M2 phenotype under M1 stimulation based on its states of activity. We suggest that inhibiting AR may be a promising therapeutic method for SCI in the future.

  13. Functional cyclic AMP response element in the breast cancer resistance protein (BCRP/ABCG2) promoter modulates epidermal growth factor receptor pathway- or androgen withdrawal-mediated BCRP/ABCG2 transcription in human cancer cells. (United States)

    Xie, Yi; Nakanishi, Takeo; Natarajan, Karthika; Safren, Lowell; Hamburger, Anne W; Hussain, Arif; Ross, Douglas D


    Phosphorylated cyclic-AMP (cAMP) response element binding protein (p-CREB) is a downstream effector of a variety of important signaling pathways. We investigated whether the human BCRP promoter contains a functional cAMP response element (CRE). 8Br-cAMP, a cAMP analogue, increased the activity of a BCRP promoter reporter construct and BCRP mRNA in human carcinoma cells. Epidermal growth factor receptor (EGFR) pathway activation also led to an increase in p-CREB and in BCRP promoter reporter activity via two major downstream EGFR signaling pathways: the phosphotidylinositol-3-kinase (PI3K)/AKT pathway and the mitogen-activated protein kinase (MAPK) pathway. EGF treatment increased the phosphorylation of EGFR, AKT, ERK and CREB, while simultaneously enhancing BCRP mRNA and functional protein expression. EGF-stimulated CREB phosphorylation and BCRP induction were diminished by inhibition of EGFR, PI3K/AKT or RAS/MAPK signaling. CREB silencing using RNA interference reduced basal levels of BCRP mRNA and diminished the induction of BCRP by EGF. Chromatin immunoprecipitation assays confirmed that a putative CRE site on the BCRP promoter bound p-CREB by a point mutation of the CRE site abolished EGF-induced stimulation of BCRP promoter reporter activity. Furthermore, the CREB co-activator, cAMP-regulated transcriptional co-activator (CRTC2), is involved in CREB-mediated BCRP transcription: androgen depletion of LNCaP human prostate cancer cells increased both CREB phosphorylation and CRTC2 nuclear translocation, and enhanced BCRP expression. Silencing CREB or CRTC2 reduced basal BCRP expression and BCRP induction under androgen-depletion conditions. This novel CRE site plays a central role in mediating BCRP gene expression in several human cancer cell lines following activation of multiple cancer-relevant signaling pathways.

  14. cAMP-independent role of PKA in tonicity-induced transactivation of tonicity-responsive enhancer/ osmotic response element-binding protein



    Hypertonicity-induced increase in activity of the transcription factor tonicity-responsive enhancer/osmotic response element-binding protein (TonEBP/OREBP) protects renal cells by increasing transcription of genes, including those involved in increased accumulation of organic osmolytes. We previously showed that hypertonicity increases transactivating activity of TonEBP/OREBP. Assay with a binary GAL4 transactivation system showed that the 984 C-terminal amino acids of TonEBP/OREBP (amino aci...

  15. Bidirectional regulation of the cAMP response element binding protein encodes spatial map alignment in prism-adapting barn owls. (United States)

    Nichols, Grant S; DeBello, William M


    The barn owl midbrain contains mutually aligned maps of auditory and visual space. Throughout life, map alignment is maintained through the actions of an instructive signal that encodes the magnitude of auditory-visual mismatch. The intracellular signaling pathways activated by this signal are unknown. Here we tested the hypothesis that CREB (cAMP response element-binding protein) provides a cell-specific readout of instructive information. Owls were fitted with prismatic or control spectacles and provided rich auditory-visual experience: hunting live mice. CREB activation was analyzed within 30 min of hunting using phosphorylation state-specific CREB (pCREB) and CREB antibodies, confocal imaging, and immunofluorescence measurements at individual cell nuclei. In control owls or prism-adapted owls, which experience small instructive signals, the frequency distributions of pCREB/CREB values obtained for cell nuclei within the external nucleus of the inferior colliculus (ICX) were unimodal. In contrast, in owls adapting to prisms or readapting to normal conditions, the distributions were bimodal: certain cells had received a signal that positively regulated CREB and, by extension, transcription of CREB-dependent genes, whereas others received a signal that negatively regulated it. These changes were restricted to the subregion of the inferior colliculus that received optically displaced input, the rostral ICX, and were not evident in the caudal ICX or central nucleus. Finally, the topographic pattern of CREB regulation was patchy, not continuous, as expected from the actions of a topographically precise signal encoding discrete events. These results support a model in which the magnitude of CREB activation within individual cells provides a readout of the instructive signal that guides plasticity and learning.

  16. Presenilins regulate neurotrypsin gene expression and neurotrypsin-dependent agrin cleavage via cyclic AMP response element-binding protein (CREB) modulation. (United States)

    Almenar-Queralt, Angels; Kim, Sonia N; Benner, Christopher; Herrera, Cheryl M; Kang, David E; Garcia-Bassets, Ivan; Goldstein, Lawrence S B


    Presenilins, the catalytic components of the γ-secretase complex, are upstream regulators of multiple cellular pathways via regulation of gene transcription. However, the underlying mechanisms and the genes regulated by these pathways are poorly characterized. In this study, we identify Tequila and its mammalian ortholog Prss12 as genes negatively regulated by presenilins in Drosophila larval brains and mouse embryonic fibroblasts, respectively. Prss12 encodes the serine protease neurotrypsin, which cleaves the heparan sulfate proteoglycan agrin. Altered neurotrypsin activity causes serious synaptic and cognitive defects; despite this, the molecular processes regulating neurotrypsin expression and activity are poorly understood. Using γ-secretase drug inhibitors and presenilin mutants in mouse embryonic fibroblasts, we found that a mature γ-secretase complex was required to repress neurotrypsin expression and agrin cleavage. We also determined that PSEN1 endoproteolysis or processing of well known γ-secretase substrates was not essential for this process. At the transcriptional level, PSEN1/2 removal induced cyclic AMP response element-binding protein (CREB)/CREB-binding protein binding, accumulation of activating histone marks at the neurotrypsin promoter, and neurotrypsin transcriptional and functional up-regulation that was dependent on GSK3 activity. Upon PSEN1/2 reintroduction, this active epigenetic state was replaced by a methyl CpG-binding protein 2 (MeCP2)-containing repressive state and reduced neurotrypsin expression. Genome-wide analysis revealed hundreds of other mouse promoters in which CREB binding is similarly modulated by the presence/absence of presenilins. Our study thus identifies Tequila and neurotrypsin as new genes repressed by presenilins and reveals a novel mechanism used by presenilins to modulate CREB signaling based on controlling CREB recruitment.

  17. Bicarbonate-responsive “soluble” adenylyl cyclase defines a nuclear cAMP microdomain (United States)

    Zippin, Jonathan H.; Farrell, Jeanne; Huron, David; Kamenetsky, Margarita; Hess, Kenneth C.; Fischman, Donald A.; Levin, Lonny R.; Buck, Jochen


    Bicarbonate-responsive “soluble” adenylyl cyclase resides, in part, inside the mammalian cell nucleus where it stimulates the activity of nuclear protein kinase A to phosphorylate the cAMP response element binding protein (CREB). The existence of this complete and functional, nuclear-localized cAMP pathway establishes that cAMP signals in intracellular microdomains and identifies an alternate pathway leading to CREB activation. PMID:14769862

  18. Bicarbonate-responsive “soluble” adenylyl cyclase defines a nuclear cAMP microdomain



    Bicarbonate-responsive “soluble” adenylyl cyclase resides, in part, inside the mammalian cell nucleus where it stimulates the activity of nuclear protein kinase A to phosphorylate the cAMP response element binding protein (CREB). The existence of this complete and functional, nuclear-localized cAMP pathway establishes that cAMP signals in intracellular microdomains and identifies an alternate pathway leading to CREB activation.

  19. Regulation of Dehydroepiandrosterone(DHEA) on cAMP/PKA Signalling System and cAMP-response Element Binding Protein (CREB) in Cultured Primary Chicken Hepatocytes%脱氢表雄酮(DHEA)对鸡胚原代肝细胞cAMP/PKA信号通路和cAMP反应元件结合蛋白(CREB)的影响与调节

    Institute of Scientific and Technical Information of China (English)

    唐雪; 马海田; 邹思湘


    Considerable research efforts have been expended to study the factors that are associated with fat deposition in poultry production. Dehydroepiandrosterone (DHEA, 3b-hydroxy-5-androsterone-17-one) is a steroidal compound that is secreted by the mammalian adrenal cortex gland. It is known to be a fat-reducing agent by activation of the steroid hormone receptors. In the present study, cultured primary chicken hepatocytes exposed to DHEA (0,0.01,0.1,1.0 10 and 100 μmol/L, respectively) dissolved in medium were left for 20 min for cAMP assay by RlA(radioimmunoassay) kit. We found that the levels of cAMP were significantly higher in 0.1~100 μmol/L DHEA groups(P0.05). As expected, a full stimulation was achieved at 20 μmol/L forskolin (f0.05). The results suggest that DHEA can activate the cAMP/PKA signaling pathway and regulate lipid metabolism by enhancing CREB phosphorylation level, and provide a better understanding for improvements proposed for DHEA in decrease of body fat in broiler chickens.%控制肉鸡脂肪过多沉积是肉鸡生产中亟待解决的问题.脱氢表雄酮(dehydroepiandrosterone,DHEA)是人体分泌最为丰富的肾上腺类固醇激素,可经由类固醇激素受体介导发挥生理功能,降低机体生脂能力.本研究以鸡胚原代肝细胞为研究对象,选用含DHEA终浓度为0(对照)、0.01、0.1、1.0、10和100μmol/L的培养液孵育肝细胞20 min后收集细胞,放射免疫测定法(RIA)检测胞内cAMP水平.结果发现,0.1~100 μmol/L DHEA孵育肝细胞均可显著提高胞内cAMP水平(P<0.05),其中0.1 μmnol/L DHEA效果最为显著(P<0.01).0.1 μmol/L DHEA孵育肝细胞20 min或1h后收集细胞,RIA法分别检测胞内腺苷酸环化酶(adenylate cyclase,AC)和磷酸二酯酶(phosphodiesterase,PDE)活性,(γ-32P)ATP掺入法测定cAMP依赖性蛋白激酶A(protein kinase A,PKA)活性,Western blot法测定cAMP反应元件结合蛋白(cAMP-response element binding protein,CREB)磷酸化水平.结果显示,0

  20. Role of cAMP-responsive element-binding protein (CREB)-regulated transcription coactivator 3 (CRTC3) in the initiation of mitochondrial biogenesis and stress response in liver cells. (United States)

    Than, Tin Aung; Lou, Huan; Ji, Cheng; Win, Sanda; Kaplowitz, Neil


    Peroxisome proliferator-activated receptor α, coactivator 1α (PGC-1α) is the master regulator of mitochondrial biogenesis. PGC-1α expression is under the control of the transcription factor, cAMP-responsive element-binding protein (CREB). In searching for candidate transcription factors that mediate mitochondrial stress-initiated mitochondria-to-nucleus signaling in the regulation of mitochondrial biogenesis, we assessed the effect of silencing CREB-regulated transcription co-activators (CRTC). CRTC isoforms are co-activators of CREB-regulated transcription by a CREB phosphorylation-independent pathway. Using cultured HepG2 cells and primary mouse hepatocytes, we determined that mitochondrial stress imposed by the complex I inhibitor rotenone elicited mitochondrial biogenesis, which was dependent on an induction of PGC-1α, which was inhibited by silencing PGC-1α. PGC-1α induction in response to rotenone was inhibited by silencing the expression of CRTC3, which blocked downstream mitochondria biogenesis. In contrast, silencing CRTC2 did not affect the induction of this pathway in response to rotenone. Thus, CRTC3 plays a selective role in mitochondrial biogenesis in response to rotenone.

  1. Reactive oxygen species decrease cAMP response element binding protein expression in cardiomyocytes via a protein kinase D1-dependent mechanism that does not require Ser133 phosphorylation. (United States)

    Ozgen, Nazira; Guo, Jianfen; Gertsberg, Zoya; Danilo, Peter; Rosen, Michael R; Steinberg, Susan F


    Reactive oxygen species (ROS) exert pleiotropic effects on a wide array of signaling proteins that regulate cellular growth and apoptosis. This study shows that long-term treatment with a low concentration of H2O2 leads to the activation of signaling pathways involving extracellular signal-regulated kinase, ribosomal protein S6 kinase, and protein kinase D (PKD) that increase cAMP binding response element protein (CREB) phosphorylation at Ser(133) in cardiomyocytes. Although CREB-Ser(133) phosphorylation typically mediates cAMP-dependent increases in CREB target gene expression, the H2O2-dependent increase in CREB-Ser(133) phosphorylation is accompanied by a decrease in CREB protein abundance and no change in Cre-luciferase reporter activity. Mutagenesis studies indicate that H2O2 decreases CREB protein abundance via a mechanism that does not require CREB-Ser(133) phosphorylation. Rather, the H2O2-dependent decrease in CREB protein is prevented by the proteasome inhibitor lactacystin, by inhibitors of mitogen-activated protein kinase kinase or protein kinase C activity, or by adenoviral-mediated delivery of a small interfering RNA that decreases PKD1 expression. A PKD1-dependent mechanism that links oxidative stress to decreased CREB protein abundance is predicted to contribute to the pathogenesis of heart failure by influencing cardiac growth and apoptosis responses.

  2. Complexes containing activating transcription factor (ATF)/cAMP-responsive-element-binding protein (CREB) interact with the CCAAT/enhancer-binding protein (C/EBP)-ATF composite site to regulate Gadd153 expression during the stress response. (United States)

    Fawcett, T W; Martindale, J L; Guyton, K Z; Hai, T; Holbrook, N J


    Gadd153, also known as chop, encodes a member of the CCAAT/enhancer-binding protein (C/EBP) transcription factor family and is transcriptionally activated by cellular stress signals. We recently demonstrated that arsenite treatment of rat pheochromocytoma PC12 cells results in the biphasic induction of Gadd153 mRNA expression, controlled in part through binding of C/EBPbeta and two uncharacterized protein complexes to the C/EBP-ATF (activating transcription factor) composite site in the Gadd153 promoter. In this report, we identified components of these additional complexes as two ATF/CREB (cAMP-responsive-element-binding protein) transcription factors having differential binding activities dependent upon the time of arsenite exposure. During arsenite treatment of PC12 cells, we observed enhanced binding of ATF4 to the C/EBP-ATF site at 2 h as Gadd153 mRNA levels increased, and enhanced binding of ATF3 complexes at 6 h as Gadd153 expression declined. We further demonstrated that ATF4 activates, while ATF3 represses, Gadd153 promoter activity through the C/EBP-ATF site. ATF3 also repressed ATF4-mediated transactivation and arsenite-induced activation of the Gadd153 promoter. Our results suggest that numerous members of the ATF/CREB family are involved in the cellular stress response, and that regulation of stress-induced biphasic Gadd153 expression in PC12 cells involves the ordered, sequential binding of multiple transcription factor complexes to the C/EBP-ATF composite site. PMID:10085237

  3. Cyclic AMP enhances TGFβ responses of breast cancer cells by upregulating TGFβ receptor I expression.

    Directory of Open Access Journals (Sweden)

    Ilka Oerlecke

    Full Text Available Cellular functions are regulated by complex networks of many different signaling pathways. The TGFβ and cAMP pathways are of particular importance in tumor progression. We analyzed the cross-talk between these pathways in breast cancer cells in 2D and 3D cultures. We found that cAMP potentiated TGFβ-dependent gene expression by enhancing Smad3 phosphorylation. Higher levels of total Smad3, as observed in 3D-cultured cells, blocked this effect. Two Smad3 regulating proteins, YAP (Yes-associated protein and TβRI (TGFβ receptor 1, were responsive to cAMP. While YAP had little effect on TGFβ-dependent expression and Smad3 phosphorylation, a constitutively active form of TβRI mimicked the cAMP effect on TGFβ signaling. In 3D-cultured cells, which show much higher levels of TβRI and cAMP, TβRI was unresponsive to cAMP. Upregulation of TβRI expression by cAMP was dependent on transcription. A proximal TβRI promoter fragment was moderately, but significantly activated by cAMP suggesting that cAMP increases TβRI expression at least partially by activating TβRI transcription. Neither the cAMP-responsive element binding protein (CREB nor the TβRI-regulating transcription factor Six1 was required for the cAMP effect. An inhibitor of histone deacetylases alone or together with cAMP increased TβRI expression by a similar extent as cAMP alone suggesting that cAMP may exert its effect by interfering with histone acetylation. Along with an additive stimulatory effect of cAMP and TGFβ on p21 expression an additive inhibitory effect of these agents on proliferation was observed. Finally, we show that mesenchymal stem cells that interact with breast cancer cells can simultaneously activate the cAMP and TGFβ pathways. In summary, these data suggest that combined effects of cAMP and TGFβ, as e.g. induced by mesenchymal stem cells, involve the upregulation of TβRI expression on the transcriptional level, likely due to changes in histone acetylation

  4. The selective glucocorticoid receptor antagonist ORG 34116 decreases immobility time in the forced swim test and affects cAMP-responsive element-binding protein phosphorylation in rat brain. (United States)

    Bachmann, Cornelius G; Bilang-Bleuel, Alicia; De Carli, Sonja; Linthorst, Astrid C E; Reul, Johannes M H M


    Glucocorticoid receptor (GR) antagonists can block the retention of the immobility response in the forced swimming test. Recently, we showed that forced swimming evokes a distinct spatiotemporal pattern of cAMP-responsive element-binding protein (CREB) phosphorylation in the dentate gyrus (DG) and neocortex. In the present study, we found that chronic treatment of rats with the selective GR antagonist ORG 34116 decreased the immobility time in the forced swim test, increased baseline levels of phosphorylated CREB (P-CREB) in the DG and neocortex and affected the forced swimming-induced changes in P-CREB levels in a time- and site-specific manner. Overall, we observed that, in control rats, forced swimming evoked increases in P-CREB levels in the DG and neocortex, whereas in ORG 34116-treated animals a major dephosphorylation of P-CREB was observed. These observations underscore an important role of GRs in the control of the phosphorylation state of CREB which seems to be of significance for the immobility response in the forced swim test and extend the molecular mechanism of action of GRs in the brain.

  5. cAMP response element binding protein1 is essential for activation of steroyl co-enzyme a desaturase 1 (Scd1 in mouse lung type II epithelial cells.

    Directory of Open Access Journals (Sweden)

    Nisha Antony

    Full Text Available Cyclic AMP Response Element-Binding Protein 1 (Creb1 is a transcription factor that mediates cyclic adenosine 3', 5'-monophosphate (cAMP signalling in many tissues. Creb1(-/- mice die at birth due to respiratory failure and previous genome-wide microarray analysis of E17.5 Creb1(-/- fetal mouse lung identified important Creb1-regulated gene targets during lung development. The lipogenic enzymes stearoyl-CoA desaturase 1 (Scd1 and fatty acid synthase (Fasn showed highly reduced gene expression in Creb1(-/- lungs. We therefore hypothesized that Creb1 plays a crucial role in the transcriptional regulation of genes involved in pulmonary lipid biosynthetic pathways during lung development. In this study we confirmed that Scd1 and Fasn mRNA levels were down regulated in the E17.5 Creb1(-/- mouse lung while the lipogenic-associated transcription factors SrebpF1, C/ebpα and Pparγ were increased. In vivo studies using germline (Creb1(-/- and lung epithelial-specific (Creb1(EpiΔ/Δ Creb1 knockout mice showed strongly reduced Scd1, but not Fasn gene expression and protein levels in lung epithelial cells. In vitro studies using mouse MLE-15 epithelial cells showed that forskolin-mediated activation of Creb1 increased both Scd1 gene expression and protein synthesis. Additionally, MLE15 cells transfected with a dominant-negative ACreb vector blocked forskolin-mediated stimulation of Scd1 gene expression. Lipid profiling in MLE15 cells showed that dominant-negative ACreb suppressed forskolin-induced desaturation of ether linked lipids to produce plasmalogens, as well as levels of phosphatidylethanolamine, ceramide and lysophosphatidylcholine. Taken together these results demonstrate that Creb1 is essential for the induction and maintenance of Scd1 in developing fetal mouse lung epithelial cells.

  6. Regulation of cyclooxygenase-2 expression by cAMP response element and mRNA stability in a human airway epithelial cell line exposed to zinc (United States)

    Exposure to zinc-laden particulate matter in ambient and occupational settings has been associated with proinflammatory responses in the lung. Cyclooxygenase 2-derived eicosanoids are important modulators of airway inflammation. In this study, we characterized the transcriptional...

  7. Minocycline upregulates cyclic AMP response element binding protein and brain-derived neurotrophic factor in the hippocampus of cerebral ischemia rats and improves behavioral deficits

    Directory of Open Access Journals (Sweden)

    Zhao Y


    Full Text Available Yu Zhao,1 Ming Xiao,2 Wenbo He,3 Zhiyou Cai3 1Department of Neurology, the Fourth Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province, People’s Republic of China; 2Department of Anatomy, Nanjing Medical University, Nanjing, Jiangsu, People’s Republic of China; 3Department of Neurology, Renmin Hospital, Hubei University of Medicine, Shiyan Renmin Hospital, Shiyan, Hubei Province, People’s Republic of China Background and purpose: The cAMP response element binding protein (CREB plays an important role in the mechanism of cognitive impairment and is also pivotal in the switch from short-term to long-term memory. Brain-derived neurotrophic factor (BDNF seems a promising avenue in the treatment of cerebral ischemia injury since this neurotrophin could stimulate structural plasticity and repair cognitive impairment. Several findings have displayed that the dysregulation of the CREB–BDNF cascade has been involved in cognitive impairment. The aim of this study was to investigate the effect of cerebral ischemia on learning and memory as well as on the levels of CREB, phosphorylated CREB (pCREB, and BDNF, and to determine the effect of minocycline on CREB, pCREB, BDNF, and behavioral functional recovery after cerebral ischemia. Methods: The animal model was established by permanent bilateral occlusion of both common carotid arteries. Behavior was evaluated 5 days before decapitation with Morris water maze and open-field task. Four days after permanent bilateral occlusion of both common carotid arteries, minocycline was administered by douche via the stomach for 4 weeks. CREB and pCREB were examined by Western blotting, reverse transcription polymerase chain reaction, and immunohistochemistry. BDNF was measured by immunohistochemistry and Western blotting. Results: The model rats after minocycline treatment swam shorter distances than control rats before finding the platform (P=0.0007. The number of times the

  8. Brain-derived neurotrophic factor, phosphorylated cyclic AMP response element binding protein and neuropeptide Y decline as early as middle age in the dentate gyrus and CA1 and CA3 subfields of the hippocampus. (United States)

    Hattiangady, Bharathi; Rao, Muddanna S; Shetty, Geetha A; Shetty, Ashok K


    The hippocampus is very susceptible to aging. Severely diminished dentate neurogenesis at middle age is one of the most conspicuous early changes in the aging hippocampus, which is likely linked to an early decline in the concentration of neurotrophic factors and signaling proteins that influence neurogenesis. We analyzed three proteins that are well-known to promote dentate neurogenesis and learning and memory function in the dentate gyrus and the hippocampal CA1 and CA3 subfields of young, middle-aged and aged F344 rats. These include the brain-derived neurotrophic factor (BDNF), the transcription factor phosphorylated cyclic AMP response element binding protein (p-CREB) and the neuropeptide neuropeptide Y (NPY). The BDNF was analyzed via ELISA and BDNF immunohistochemistry, the p-CREB through densitometric analysis of p-CREB immunopositive cells, and the NPY via stereological counting of NPY-immunopositive interneurons. We provide new evidence that the BDNF concentration, the p-CREB immunoreactivity and the number of NPY immunopositive interneurons decline considerably by middle age in both dentate gyrus and CA1 and CA3 subfields of the hippocampus. However, both BDNF concentration and NPY immunopositive interneuron numbers exhibit no significant decrease between middle age and old age. In contrast, the p-CREB immunoreactivity diminishes further during this period, which is also associated with reduced BDNF immunoreaction within the soma of dentate granule cells and hippocampal pyramidal neurons. Collectively, these results suggest that severely dampened dentate neurogenesis observed at middle age is linked at least partially to reduced concentrations of BDNF, p-CREB and NPY, as each of these proteins is a positive regulator of dentate neurogenesis. Dramatically diminished CREB phosphorylation (and persistently reduced levels of BDNF and NPY) at old age may underlie the learning and memory impairments observed during senescence.

  9. Effects of estrogen treatment on expression of brain-derived neurotrophic factor and cAMP response element-binding protein expression and phosphorylation in rat amygdaloid and hippocampal structures. (United States)

    Zhou, Jin; Zhang, Huaibo; Cohen, Rochelle S; Pandey, Subhash C


    Clinical studies indicate an effect of estrogen (E2) on affect and cognition, which may be mediated by the cAMP response element-binding protein (CREB) pathway and CREB-related gene target brain-derived neurotrophic factor (BDNF). We investigated the effect of E2 on CREB expression and phosphorylation and BDNF expression in the amygdala and hippocampus, areas involved in emotional processing. Ovariectomized rats were given 10 microg 17beta-estradiol or vehicle for 14 days and expression of components of the CREB signaling pathway, i.e., CREB, phosphorylated CREB (pCREB), and BDNF in amygdala and hippocampus were investigated using immunogold labeling. Levels of BDNF mRNA were determined by in situ reverse-transcriptase polymerase chain reaction. We also examined the effect of E2 on calcium/calmodulin kinase (CaMK IV) immunolabeling in the hippocampus. E2 increased immunolabeling and mRNA levels of BDNF in the medial and basomedial amygdala and CA1 and CA3 regions of the hippocampus, but not in any other amygdaloid or hippocampal regions examined. E2 increased immunolabeling of CREB and pCREB in the medial and basomedial, but not central or basolateral amygdala. E2 also increased CaMK IV and pCREB immunolabeling in the CA1 and CA3 regions, but not CA2 region or dentate gyrus, of the hippocampus. There was no change in immunolabeling of CREB in any hippocampal region. These data identify a signaling pathway through which E2 increases BDNF expression that may underlie some actions of E2 on affective behavior and indicate neuroanatomical heterogeneity in the E2 effect within the amygdala and hippocampus.

  10. [Blockade of NMDA receptor enhances corticosterone-induced downregulation of brain-derived neurotrophic factor gene expression in the rat hippocampus through cAMP response element binding protein pathway]. (United States)

    Feng, Hao; Lu, Li-Min; Huang, Ying; Zhu, Yi-Chun; Yao, Tai


    High concentration of corticosterone leads to morphological and functional impairments in hippocampus, ranging from a reversible atrophy of pyramidal CA3 apical dendrites to the impairment of long-term potentiation (LTP) and hippocampus-dependent learning and memory. Glutamate and N-methyl-D-aspartate (NMDA) receptor play an important role in this effect. Because of the importance of brain-derived neurotrophic factor (BDNF) in the functions of the hippocampal neurons, alteration of the expression of BDNF is thought to be involved in the corticosterone effect on the hippocampus. To determine whether change in BDNF in the hippocampus is involved in the corticosterone effect, we injected corticosterone (2 mg/kg, s.c.) to Sprague-Dawley rats and measured the mRNA, proBDNF and mature BDNF protein levels in the hippocampus. We also measured the phosphorylation level of the transcription factor cAMP response element binding protein (CREB). Furthermore, we intraperitoneally injected NMDA receptor antagonist MK801 (0.1 mg/kg) 30 min before corticosterone administration to investigate whether and how MK801 affected the regulation of BDNF gene expression by corticosterone. Our results showed that 3 h after single s.c. injection of corticsterone, the expression of BDNF mRNA, proBDNF and mature BDNF protein decreased significantly (PBDNF gene expression in the rat hippocampus by corticosterone. We also found that either applying corticosterone or co-applying corticosterone with MK801 downregulated the phosphoration level of CREB, the latter (corticosterone plus MK801) being more effective (PBDNF gene expression in the rat hippocampus through CREB pathway and that blockade of NMDA receptor enhances this effect of corticosterone in reducing BDNF expression.


    Kesh, Kousik; Subramanian, Lakshmi; Ghosh, Nillu; Gupta, Vinayak; Gupta, Arnab; Bhattacharya, Samir; Mahapatra, Nitish R; Swarnakar, Snehasikta


    Elevated expression of matrix metalloproteinase7 (MMP7) has been demonstrated to play a pivotal role in cancer invasion. The -181A→G (rs11568818) polymorphism in the MMP7 promoter modulates gene expression and possibly affects cancer progression. Here, we evaluated the impact of -181A→G polymorphism on MMP7 promoter activity and its association with gastric cancer risk in eastern Indian case-control cohorts (n = 520). The GG genotype as compared with the AA genotype was predisposed (p = 0.02; odds ratio = 1.9, 95% confidence interval = 1.1-3.3) to gastric cancer risk. Stratification analysis showed that tobacco addiction enhanced gastric cancer risk in GG subjects when compared with AA subjects (p = 0.03, odds ratio = 2.46, and 95% confidence interval = 1.07-5.68). Meta-analysis revealed that tobacco enhanced the risk for cancer more markedly in AG and GG carriers. Activity and expression of MMP7 were significantly higher in GG than in AA carriers. In support, MMP7 promoter-reporter assays showed greater transcriptional activity toward A to G transition under basal/nicotine-induced/cAMP-response element-binding protein (CREB) overexpressed conditions in gastric adenocarcinoma cells. Moreover, nicotine (a major component of tobacco) treatment significantly up-regulated MMP7 expression due to enhanced CREB phosphorylation followed by its nuclear translocation in gastric adenocarcinoma cells. Furthermore, chromatin immunoprecipitation experiments revealed higher binding of phosphorylated CREB with the -181G than the -181A allele. Altogether, specific binding of phosphorylated CREB to the G allele-carrying promoter enhances MMP7 gene expression that is further augmented by nicotine due to increased CREB phosphorylation and thereby increases the risk for gastric cancer.

  12. Enhancing T3 and cAMP responsive gene participation in the thermogenic regulation of fuel oxidation pathways



    OBJECTIVE: We sought to identify glycolysis, glycogenolysis, lipolysis, Krebs cycle, respiratory chain, and oxidative phosphorylation enzymes simultaneously regulated by T3 and cAMP. MATERIALS AND METHODS: We performed in silico analysis of 56 promoters to search for cis-cAMP (CREB) and cis-thyroid (TRE) response elements, considering UCP1, SERCA2 and glyceraldehyde 3-phosphate dehydrogenase as reference. Only regulatory regions with prior in vitro validation were selected. RESULTS: 29/56 enz...

  13. Effects of ohmefentanyl stereoisomers on phosphorylation of cAMP- response element binding protein in cultured rat hippocampal neurons%羟甲芬太尼立体异构体对培养的海马神经元cAMP-反应元件结合蛋白磷酸化的影响

    Institute of Scientific and Technical Information of China (English)

    高灿; 谌立伟; 陶亦敏; 陈洁; 徐学军; 池志强


    AIM: To define the effects and signal pathways of ohmefentanyl stereoisomers [(-)-cis-(3R,4S,2'R) OMF (F9202), (+)-cis-(3R, 4S, 2′S) OMF (F9204), and (-)-cis-(3S, 4S, 2′R) OMF (F9203)] on the phosphorylation of cAMP-re sponse element binding protein (CREB) in cultured rat hippocampal neurons. METHODS: The effects of the three OMF stereoisomers and morphine (Mor) on cAMP accumulation and CREB phosphorylation were monitored by radioimmunoassay and Western blot analysis, respectively. RESULTS: The three OMF stereoisomers and Mor could all partially inhibit forskolin-stimulated (25 μmol/L, 15 min) cAMP accumulation in a dose-dependent manner and this effect could be reversed by naloxone. F9202, F9204, and Mor could significantly increase CREB phospho rylation from 2.88 to 3.59 folds over control levels after 30-min exposure. This effect was reversed by naloxone,but F9203 failed to increase CREB phosphorylation. KN-62 and staurosporine significantly blocked the opioidsinduced CREB phosphorylation, while H-89 and PD 98059 had no effect on the actions. CONCLUSION: Mor,F9202, and F9204, which could induce psychological dependence affected via the μ-opioid receptor, stimulated intracellular signal pathways involving Ca2+/calmodulin-dependent protein kinases (CCDPK) and protein kinase C (PKC) pathways, which in turn initiated CREB phosphorylation. F9203, which could not induce dependence, had no effect on CREB phosphorylation in hippocampal neurons. The increased CREB phosphorylation in hippocampal neurons may play a role in opioids dependence.

  14. 丙泊酚对大鼠海马cAMP效应元件结合蛋白磷酸化和cAMP效应元件结合蛋白mRNA表达水平的影响%Effects of propofol on cyclic AMP response element binding protein phosphorylation and cyclic AMP response element binding protein mRNA expression in hippocampus of rats

    Institute of Scientific and Technical Information of China (English)

    张英; 吴新海; 郑利民


    Objective to investigate the effects of propofol on cyclic AMP response element binding protein (CREB) phosphorylation and CREB mRNA expression in hippocampus of rats. Methods Sixty -four male Sprague -Dawley (SD) rats weighting 250 g-280 g were randomly divided into 2 groups (n=32) with Randomization Adviser 1.0 software: normal saline treated (Group S) and propofol treated (Group P). Animals of both groups underwent a continuous multiple -trial inhibitory avoidance training. The times of trial needed for each animal to attain the learning criterion( 100 seconds) were recorded. Each animal was given intraperitoneal propofol 9 mg/kg or normal saline 2 ml/kg at 15 min before training. The memory retention was tested at 1, 3 and 24 h( n=8, at each time point) after the training session and the memory latency was recorded. Animals were sacrificed at 15 min after reagent administration (T0) and after the memory testing (T1~3). Hippocampus was obtained to determine the phosphorylation of CRKB (pCREB) and CREB mRNA expression. Results The times of trials required for the rats to learn the task in Group S and Group P is 1 (0.25) and 3 (1.25), respectively. Learning ability was significantly impaired in rats of Group P(P<0.01 ). Rats of Group S had a median retention latency more than 300 s at each memory testing. The median retention latency of rats in Group P at T1, T2and T3 was 319( 144) s, 131( 114) s and 56(30) s, respectively. Compared with group S, there was a decrease of CREB phosphorylation expression at T0-3 in Group P (P<0.01). The CREB mRNA expression in Group P was decreased at T3 (P<0.01). Conclusion Propofol can inhibit CREB phosphorylation, and downregulate CREB mRNA expression in hippocampus of rats.%目的 探讨丙泊酚对大鼠海马cAMP效应元件结合蛋白(cAMP response element binding protein,CREB)磷酸化和CREB mRNA表达水平的影响.方法 成年雄性SD大鼠64只,体重250 g~280 g,采用RandA1.0随机分组软件

  15. Effect of Tiantai No.1(天泰1号) on β-Amyloid-induced Neurotoxicity and NF- к B and cAMP Responsive Element-binding Protein

    Institute of Scientific and Technical Information of China (English)

    WU Zheng-zhi; Andrew C. J. Huang; Jean de Vellis; LI Ying-hong


    To investigate the effect and molecular mechanism of Tiantai No.1 (天泰1号), a compound Chinese herbal preparation, for the prevention and reduction of neurotoxicity induced by beta-element-binding protein (CREB) pathways using the gene transfection technique. Methods: B104 neuronal cells were used to examine the effects of Tiantai No.1 on lowering the neurotoxicity induced by Abeta. The cells were pre-treated with Tiantai No.1 at doses of 50, 100, 150, or 200 μg/mL respectively for 3 days and co-treated with Tiantai No.1 and beta-amyloid peptidel-40 (Aβ 1-40, 10 μmol/L) for 48 h or post-treated with Tiantai No.1 for 48 h after the cells were exposed to beta-amyloid peptides25-35 (A β 25-35) for 8 h. In gene transfection assays, cells were treated with Tiantai No.1 at 50 μg/m/and 150 μg/mL for 5 days or co-treated with Tiantai No.1 and A β 1-40 (5 μ mo/L) for 3 days after electroporation for the with Tiantai No.1 lowered the neurotoxicity induced by Abeta, and post-treating with Tiantai No.1 reduced or blocked B104 neuronal apoptotic death induced by Abeta (PAMP cellular signal pathways.

  16. Evidences for involvement of endogenous cAMP in Arabidopsis defense responses to Verticillium toxins

    Institute of Scientific and Technical Information of China (English)

    Jing JIANG; Ling Wen FAN; Wei Hua WU


    Although there were reports suggesting the involvement of endogenous cAMP in plant defense signaling cascades,there is no direct evidence supporting this notion yet and the detailed mechanism is unclear. In the present study, we have used pathogenic fungi Verticillium dahliae and Arabidopsis plants as a model system of plant-microb interaction to demonstrate the function of endogenous cAMP in Arabidopsis defense responses. Both V. dahliae inoculation and Verticillium toxins injection induced typical "wilt" symptoms in Arabidopsis seedlings. When either 8-Br-AMP (a membrane permeable cAMP analogue) or salicylic acid (SA) was applied to Arabidopsis, the plants became resistant to V. dahliae toxins. However, addition of 8-Br-AMP did not increase the resistance of Arabidopsis transgenic plants deficient in SA to the toxins, suggesting that cAMP might act upstream of SA in plant defense signaling pathway.Indeed, 8-Br-cAMP and forskolin, an activator of adenylyl cyclase, significantly stimulated the endogenous SA level in plants, whereas DDA, an inhibitor of adenylyl cyclase dramatically reduced toxin-induced SA increase. Both the endogenous cAMP and SA increased significantly in Arabidopsis seedlings treated with toxins. Furthermore, transcription level of pathogenesis-related protein 1 gene (PR1) was strongly induced by both 8-Br-cAMP and the toxin treatment. Taken together, our data demonstrate that endogenous cAMP is involved in plant defense responses against Verticilliumsecreted toxins by regulating the production of the known signal SA in plant defense pathway.

  17. Proteomic signatures implicate cAMP in light and temperature responses in Arabidopsis thaliana

    KAUST Repository

    Thomas, Ludivine


    The second messenger 3\\'-5\\'-cyclic adenosine monophosphate (cAMP) and adenylyl cyclases (ACs), enzymes that catalyse the formation of cAMP from ATP, are increasingly recognized as important signaling molecules in a number of physiological responses in higher plants. Here we used proteomics to identify cAMP-dependent protein signatures in Arabidopsis thaliana and identify a number of differentially expressed proteins with a role in light- and temperature-dependent responses, notably photosystem II subunit P-1, plasma membrane associated cation-binding protein and chaperonin 60 β. Based on these proteomics results we conclude that, much like in cyanobacteria, algae and fungi, cAMP may have a role in light signaling and the regulation of photosynthesis as well as responses to temperature and we speculate that ACs could act as light and/or temperature sensors in higher plants. Biological significance: This current study is significant since it presents the first proteomic response to cAMP, a novel and key second messenger in plants. It will be relevant to researchers in plant physiology and in particular those with an interest in second messengers and their role in biotic and abiotic stress responses. © 2013 Elsevier B.V.

  18. Neuron-restrictive silencer factor (NRSF) represses cocaine- and amphetamine-regulated transcript (CART) transcription and antagonizes cAMP-response element-binding protein signaling through a dual NRSE mechanism. (United States)

    Zhang, Jing; Wang, Sihan; Yuan, Lin; Yang, Yinxiang; Zhang, Bowen; Liu, Qingbin; Chen, Lin; Yue, Wen; Li, Yanhua; Pei, Xuetao


    Cocaine- and amphetamine-regulated transcript (CART) peptide plays a pivotal role in neuroprotection against stroke-related brain injury. However, the regulatory mechanism on CART transcription, especially the repression mechanism, is not fully understood. Here, we show that the transcriptional repressor neuron-restrictive silencer elements (NRSF, also known as REST) represses CART expression through direct binding to two NRSF-binding elements (NRSEs) in the CART promoter and intron 1 (named pNRSE and iNRSE, respectively). EMSA show that NRSF binds to pNRSE and iNRSE directly in vitro. ChIP assays show that NRSF recruits differential co-repressor complexes including CoREST and HDAC1 to these NRSEs. The presence of both NRSEs is required for efficient repression of CART transcription as indicated by reporter gene assays. NRSF overexpression antagonizes forskolin-mediated up-regulation of CART mRNA and protein. Ischemia insult triggered by oxygen-glucose deprivation (OGD) enhances NRSF mRNA levels and then NRSF antagonizes the CREB signaling on CART activation, leading to augmented cell death. Depletion of NRSF in combination with forskolin treatment increases neuronal survival after ischemic insult. These findings reveal a novel dual NRSE mechanism by which NRSF represses CART expression and suggest that NRSF may serve as a therapeutic target for stroke treatment.

  19. Innate hemocyte responses of Malacosoma disstria larvae (C. Insecta) to antigens are modulated by intracellular cyclic AMP. (United States)

    Gulii, Vladislav; Dunphy, Gary B; Mandato, Craig A


    Invertebrate intracellular hemocyte signaling pathways affecting cellular-antigen responses, although defined for molluscs and some arthropods including dipteran insects, is less known for lepidopterans. Hemocytic-antigen responses of the arboreal pest lepidopteran Malacosoma disstria are linked to cAMP-dependent protein kinase A implicating cAMP in cellular hemocyte immune responses. The purpose in the present study was to determine intracellular cAMP effects on larval M. disstria hemocytes adhering to slides and bacteria. Altering adenylate cyclase and phosphodiesterase activities as well as cAMP levels in vitro and in vivo changed hemocyte responses to antigens. Quiescent hemocytes had high cAMP levels due to adenylate cyclase activity and possibly low phosphodiesterase (type 4) activity. Antigen contact diminished hemocytic cAMP levels. Inhibiting adenylate cyclase increased hemocyte-antigen and hemocyte-hemocyte adhesion, the latter producing nodules in vivo without bacterial antigens. Inhibiting phosphodiesterase type 4 produced the reverse effects. Pharmacologically increasing intracellular cAMP in attached hemocytes caused many of the cells to detach. Diminished intracellular cAMP changed hemograms in vivo in bacteria-free larvae comparable to changes induced by the bacterium, Bacillus subtilis, by producing nodules. Lowering cAMP enhanced also the removal of Xenorhabdus nematophila and B. subtilisin vivo.

  20. Relationship between Adaptation of the Folic Acid and the cAMP Mediated cGMP Response in Dictyostelium

    NARCIS (Netherlands)

    Haastert, Peter J.M. van


    Chemotactic stimulation of post-vegetative Dictyostelium cells with folic acid or aggregative cells with cAMP results in a fast transient cGMP response which peaks at 10 s; basal levels are recovered in about 30-40 s. Stimulation with folic acid or cAMP rapidly desensitizes the cells for equal or lo

  1. Responsiveness of glycogen breakdown to cyclic AMP in perfused liver from rats with insulin-induced hypoglycemia

    Directory of Open Access Journals (Sweden)

    M. Vardanega-Peicher


    Full Text Available The responsiveness of glycogen breakdown to cAMP was investigated in isolated perfused liver from male Wistar fed rats (200-220 g with insulin-induced hypoglycemia. The activation of glycogenolysis by 3 µM cAMP was decreased (P<0.05 in livers from rats with hypoglycemia induced by the administration of insulin or during the direct infusion of insulin into the isolated liver. The direct effect of insulin on glycogen catabolism promoted by 3 µM cAMP occurred as early as 3 min after starting insulin infusion. In contrast, the cAMP agonists resistant to phosphodiesterases, 8Br-cAMP and 6MB-cAMP, used at the same concentration as cAMP, i.e., 3 µM, did not modify the effect of insulin. The data suggest that the decreased hepatic responsiveness of glycogen breakdown during insulin-induced hypoglycemia is a direct effect of insulin decreasing the intracellular levels of cAMP.

  2. Aging of the rat adrenocortical cell: response to ACTH and cyclic AMP in vitro. (United States)

    Malamed, S; Carsia, R V


    To study intrinsic age-related changes in adrenocortical steroid production, cells isolated from rats of different ages (3 to 24 months) were used. Acute (2 hour) corticosterone production in response to stimulation by adrenocorticotrophic hormone (ACTH) and adenosine 3':5'-cyclic monophosphate (cAMP) was measured by radioimmunoassay. With age, adrenocortical cells lose much of their ability to produce corticosterone in the absence or presence of ACTH or cAMP. The loss is progressive from 6 to 24 months of age. Analysis of the data suggests that from 6 to 12 months, an intracellular steroidogenic lesion develops; in addition there may be a loss in ACTH receptors on the plasma membrane. After 12 months these defects increase and are accompanied by a decrease in receptor sensitivity to ACTH.

  3. Cyclic-AMP regulates postnatal development of neural and behavioral responses to NaCl in rats (United States)

    Qian, Jie; Mummalaneni, Shobha; Phan, Tam-Hao T.; Heck, Gerard L.; DeSimone, John A.; West, David; Mahavadi, Sunila; Hojati, Deanna; Murthy, Karnam S.; Rhyu, Mee-Ra; Spielman, Andrew I.; Özdener, Mehmet Hakan


    During postnatal development rats demonstrate an age-dependent increase in NaCl chorda tympani (CT) responses and the number of functional apical amiloride-sensitive epithelial Na+ channels (ENaCs) in salt sensing fungiform (FF) taste receptor cells (TRCs). Currently, the intracellular signals that regulate the postnatal development of salt taste have not been identified. We investigated the effect of cAMP, a downstream signal for arginine vasopressin (AVP) action, on the postnatal development of NaCl responses in 19–23 day old rats. ENaC-dependent NaCl CT responses were monitored after lingual application of 8-chlorophenylthio-cAMP (8-CPT-cAMP) under open-circuit conditions and under ±60 mV lingual voltage clamp. Behavioral responses were tested using 2 bottle/24h NaCl preference tests. The effect of [deamino-Cys1, D-Arg8]-vasopressin (dDAVP, a specific V2R agonist) was investigated on ENaC subunit trafficking in rat FF TRCs and on cAMP generation in cultured adult human FF taste cells (HBO cells). Our results show that in 19–23 day old rats, the ENaC-dependent maximum NaCl CT response was a saturating sigmoidal function of 8-CPT-cAMP concentration. 8-CPT-cAMP increased the voltage-sensitivity of the NaCl CT response and the apical Na+ response conductance. Intravenous injections of dDAVP increased ENaC expression and γ-ENaC trafficking from cytosolic compartment to the apical compartment in rat FF TRCs. In HBO cells dDAVP increased intracellular cAMP and cAMP increased trafficking of γ- and δ-ENaC from cytosolic compartment to the apical compartment 10 min post-cAMP treatment. Control 19–23 day old rats were indifferent to NaCl, but showed clear preference for appetitive NaCl concentrations after 8-CPT-cAMP treatment. Relative to adult rats, 14 day old rats demonstrated significantly less V2R antibody binding in circumvallate TRCs. We conclude that an age-dependent increase in V2R expression produces an AVP-induced incremental increase in cAMP that

  4. AMPS/AAM凝胶响应性能的研究%Study on response performance of AMPS/AAM hydrogels

    Institute of Scientific and Technical Information of China (English)

    冯霞; 陈莉; 周军杰



  5. The effects of high fat diet and exercise on cAMP response element binding protein gene expression in brain of mice%高脂膳食与运动对小鼠脑内cAMP反应元件结合蛋白基因表达的影响

    Institute of Scientific and Technical Information of China (English)

    云少君; 乔欣; 李蔓; 魏守刚


    目的:通过观察cAMP反应元件结合蛋白( CREB)基因表达探讨高脂膳食与运动对脑老化的影响。方法70只ICR小鼠按体重随机分为普通对照组、高脂对照组、运动对照组、普通脑老化组、脑老化+高脂组、脑老化+运动组及脑老化+高脂+运动组。造模10 w后,以 Morris水迷宫实验检测小鼠的学习记忆能力, RT-PCR检测小鼠大脑CREB基因的表达水平。结果与普通对照组相比,普通脑老化组、脑老化+高脂组、脑老化+高脂+运动组及高脂对照组小鼠脑CREB基因表达水平明显降低(P<0.05),运动对照组CREB 基因表达水平无明显变化。脑老化+运动组小鼠脑CREB基因表达水平明显高于脑老化组( P<0.05)。结论脑老化小鼠 CREB基因表达水平降低。高脂膳食下调CREB 基因的表达,具有促脑老化效应。运动可以防止脑老化小鼠CREB基因的表达下降,具有抗脑老化效应。%Objective To explore the effects of high fat diet and exercise on the expression of cAMP response element binding pro-tein( CREB) gene in brain of mice.Methods Seventy mice were randomly divided into control , high fat diet, exercise, brain aging, brain aging +high fat diet, brain aging +exercise, brain aging +high fat diet +exercise groups.The experimental duration was 10 weeks.Morris water maze test was used to measure the learning and memory ability .Reverse transcription polymerase chain reaction ( RT-PCR) was used to detect the brain CREB gene expression .Results Compared with that of control group , the CREB gene expression was decreased in brain aging mice and brain aging +high fat diet group (P<0.05).There was not significant difference of CREB gene expression between brain ag-ing+exercise group and control group .The CREB gene expression in brain aging +high fat diet +exercise group was decreased significantly compared with that of normal group , but compared with that of brain

  6. Differential control by IHF and cAMP of two oppositely oriented genes, hpt and gcd, in Escherichia coli: significance of their partially overlapping regulatory elements. (United States)

    Izu, H; Ito, S; Elias, M D; Yamada, M


    The hpt gene, which encodes hypoxanthine phosphoribosyltransferase, is located next to, but transcribed in the opposite direction to, the gcd gene, which codes for a membrane-bound glucose dehydrogenase, at 3.1 min on the Escherichia coli genome. In their promoter-operator region, putative regulatory elements for integration host factor (IHF) and for the complex comprising 3', 5'-cyclic AMP (cAMP) and its receptor protein (CRP) are present, and they overlap the promoters for hpt and gcd, respectively. The involvement of IHF and cAMP-CRP, as well as the corresponding putative cis-acting elements, in the expression of the two genes was investigated by using lacZ operon fusions. In an adenylate cyclase-deficient strain, addition of cAMP increased the expression of hpt and reduced the expression of gcd. In agreement with this observation, the introduction of mutations into the putative binding element for the cAMP-CRP complex enhanced the expression of gcd. In contrast, mutations introduced into the putative IHF-binding elements increased the level of hpt expression. Similar results were obtained with IHF-defective strains. Thus, the expression of the two genes is regulated in a mutually exclusive manner. Additional experiments with mutations at the -10 sequence of the gcd promoter suggest that the binding of RNA polymerase to the hpt promoter interferes with the interaction of RNA polymerase with the gcd promoter, and vice versa.

  7. Cyclic AMP affects the haemocyte responses of larval Galleria mellonella to selected antigens. (United States)

    Marin, David; Dunphy, Gary B; Mandato, Craig A


    Signal transduction of the innate immediate responses of insect haemocytes to foreign matter is rarely considered. Herein using a combination of adenylate cyclase inhibitors and activators and phosphodiesterase inhibitors we determined that cyclic adenosine monophosphate (cAMP) at high levels normally impairs non-self response. Haemocyte contact with glass and bacteria lowered cAMP in vitro. Inactive phosphodiesterases, including type 4, impaired haemocyte reactions in vitro. Using the drugs in vivo to modulate adenylate cyclase and phosphodiesterases altered the total and types of haemocytes. Adenylate cyclase inhibitors and etazolate (a type 4 phosphodiesterase inhibitor) alone produced changes in the haemograms similar to those caused by Bacillus subtilis. Sequential injections of an enzyme modulator followed by B. subtilis impaired bacterial removal due (1) in the case of enzyme inhibitors, to the removal of haemocytes prior to bacterial challenge and (2) in the case of forskolin and IBMX to the shut-down of the haemocytes. Activating adenylate cyclase or inhibiting phosphodiesterase impaired bacterial removal when co-injecting the compounds and bacteria.

  8. Cyclic AMP efflux inhibitors as potential therapeutic agents for leukemia (United States)

    Perez, Dominique R.; Smagley, Yelena; Garcia, Matthew; Carter, Mark B.; Evangelisti, Annette; Matlawska-Wasowska, Ksenia; Winter, Stuart S.; Sklar, Larry A.; Chigaev, Alexandre


    Apoptotic evasion is a hallmark of cancer. We propose that some cancers may evade cell death by regulating 3′-5′-cyclic adenosine monophosphate (cAMP), which is associated with pro-apoptotic signaling. We hypothesize that leukemic cells possess mechanisms that efflux cAMP from the cytoplasm, thus protecting them from apoptosis. Accordingly, cAMP efflux inhibition should result in: cAMP accumulation, activation of cAMP-dependent downstream signaling, viability loss, and apoptosis. We developed a novel assay to assess cAMP efflux and performed screens to identify inhibitors. In an acute myeloid leukemia (AML) model, several identified compounds reduced cAMP efflux, appropriately modulated pathways that are responsive to cAMP elevation (cAMP-responsive element-binding protein phosphorylation, and deactivation of Very Late Antigen-4 integrin), and induced mitochondrial depolarization and caspase activation. Blocking adenylyl cyclase activity was sufficient to reduce effects of the most potent compounds. These compounds also decreased cAMP efflux and viability of B-lineage acute lymphoblastic leukemia (B-ALL) cell lines and primary patient samples, but not of normal primary peripheral blood mononuclear cells. Our data suggest that cAMP efflux is a functional feature that could be therapeutically targeted in leukemia. Furthermore, because some of the identified drugs are currently used for treating other illnesses, this work creates an opportunity for repurposing. PMID:27129155

  9. Ca2+ participates in α1B-adrenoceptor-mediated cAMP response in HEK293 cells

    Institute of Scientific and Technical Information of China (English)

    Yao SONG; Yun-fang LI; Er-dan DONG; Qi-de HAN; You-yi ZHANG


    Aim: To investigate the α1B-adrenoceptor (α1B-AR)-mediated cAMP response and underlying mechanisms in HEK293 cells. Methods: Full-length cDNA encoding α1B-AR was transfected into HEK293 cells using the calcium phosphate precipitation method, and α1B-AR expression and cAMP accumulation were determined by using the saturation radioligand binding assay and ion-exchange chromatography, respectively. Results: Under agonist stimulation, α1B-AR mediated cAMP synthesis in HEK293 cells, and blockade by PLC-PKC or tyrosine kinase did not reduce cAMP accumulation induced by NE. Pretreatment with pertussis toxin(PTX) had little effect on basal cAMP accumulation as well as norepinephrine(NE)-stimulated cAMP accumulation. In addition, pretreatment with cholera toxin(CTX) neither mimicked nor blocked the effect induced by NE. The extracellular Ca2+ chelator egtazic acid (EGTA), nonselective Ca2+ channel blocker CdC12 and calmodulin (CaM) inhibitor W-7 significantly reduced NE-induced cAMP accumulation from 1.59%±0.47% to 1.00%±0.31%, 0.78%±0.23%, and 0.90%±0.40%,respectively. Conclusion: By coupling with a PTX-insensitive G protein, α1B-AR promotes Ca2+ influx via receptor-dependent Ca2+ channels, then Ca2+ is linked to CaM to form a Ca2+-CaM complex, which stimulates adenylyl cyclase (AC),thereby increasing the cAMP production in HEK293 cell lines.

  10. Metabolism. AMP-activated protein kinase mediates mitochondrial fission in response to energy stress. (United States)

    Toyama, Erin Quan; Herzig, Sébastien; Courchet, Julien; Lewis, Tommy L; Losón, Oliver C; Hellberg, Kristina; Young, Nathan P; Chen, Hsiuchen; Polleux, Franck; Chan, David C; Shaw, Reuben J


    Mitochondria undergo fragmentation in response to electron transport chain (ETC) poisons and mitochondrial DNA-linked disease mutations, yet how these stimuli mechanistically connect to the mitochondrial fission and fusion machinery is poorly understood. We found that the energy-sensing adenosine monophosphate (AMP)-activated protein kinase (AMPK) is genetically required for cells to undergo rapid mitochondrial fragmentation after treatment with ETC inhibitors. Moreover, direct pharmacological activation of AMPK was sufficient to rapidly promote mitochondrial fragmentation even in the absence of mitochondrial stress. A screen for substrates of AMPK identified mitochondrial fission factor (MFF), a mitochondrial outer-membrane receptor for DRP1, the cytoplasmic guanosine triphosphatase that catalyzes mitochondrial fission. Nonphosphorylatable and phosphomimetic alleles of the AMPK sites in MFF revealed that it is a key effector of AMPK-mediated mitochondrial fission.

  11. Methacholine and adenosine 5'-monophosphate (AMP) responsiveness, and the presence and degree of atopy in children with asthma. (United States)

    Suh, Dong I; Lee, Ju K; Kim, Chang K; Koh, Young Y


    The relationship between atopy and bronchial hyperresponsiveness (BHR), both key features of asthma, remains to be clarified. BHR is commonly evaluated by bronchial challenges using direct and indirect stimuli. The aim of this study was to investigate the degree of BHR to methacholine (direct stimulus) and adenosine 5'-monophosphate (AMP) (indirect stimulus) according to the presence and degree of atopy in children with asthma. We performed a retrospective analysis of data from 120 children presenting with a diagnosis of asthma. These children were characterized by skin-prick tests (SPTs), spirometry and bronchial challenges with methacholine and AMP. Atopy was defined by at least one positive reaction to SPTs, and its degree was measured using serum total IgE levels, number of positive SPTs and atopic scores (sum of graded wheal size). A provocative concentration causing a 20% decline in FEV(1) (PC(20) ) was determined for each challenge. Patients with atopy(n=94) had a significantly lower AMP PC(20) than non-atopic patients (n=26), whereas methacholine PC(20) was not different between the two groups. Among the patients with atopy, there was no association between methacholine PC(20) and any atopy parameter. In contrast, a significant association was found between AMP PC(20) and the degree of atopy reflected in serum total IgE, number of positive SPTs and atopic scores (anova trend test, p=0.002, 0.001, 0.003, respectively). AMP responsiveness was associated with the presence and degree of atopy, whereas such a relationship was not observed for methacholine responsiveness. These findings suggest that atopic status may be better reflected by bronchial responsiveness assessed by AMP than by methacholine.

  12. Aspergillus fumigatus allergen expression is coordinately regulated in response to hydrogen peroxide and cyclic AMP

    Directory of Open Access Journals (Sweden)

    Bowyer Paul


    Full Text Available Abstract Background A. fumigatus has been associated with a wide spectrum of allergic disorders such as ABPA or SAFS. It is poorly understood what allergens in particular are being expressed during fungal invasion and which are responsible for stimulation of immune responses. Study of the dynamics of allergen production by fungi may lead to insights into how allergens are presented to the immune system. Methods Expression of 17 A. fumigatus allergen genes was examined in response to various culture conditions and stimuli as well as in the presence of macrophages in order to mimic conditions encountered in the lung. Results Expression of 14/17 allergen genes was strongly induced by oxidative stress caused by hydrogen peroxide (Asp f 1, -2, -4, -5, -6, -7, -8, -10, -13, -17 and -18, all >10-fold and Asp f 11, -12, and -22, 5-10-fold and 16/17 allergen genes were repressed in the presence of cAMP. The 4 protease allergen genes (Asp f -5, -10, -13 and -18 were expressed at very low levels compared to the comparator (β-tubulin under all other conditions examined. Mild heat shock, anoxia, lipid and presence of macrophages did not result in coordinated changes in allergen gene expression. Growth on lipid as sole carbon source contributed to the moderate induction of most of the allergen genes. Heat shock (37°C > 42°C caused moderate repression in 11/17 genes (Asp f 1, -2, -4, -5, -6, -9, -10, -13, -17, -18 and -23 (2- to 9-fold, which was mostly evident for Asp f 1 and -9 (~9-fold. Anaerobic stress led to moderate induction of 13/17 genes (1.1 to 4-fold with one, Asp f 8 induced over 10-fold when grown under mineral oil. Complex changes were seen in gene expression during co-culture of A. fumigatus with macrophages. Conclusions Remarkable coordination of allergen gene expression in response to a specific condition (oxidative stress or the presence of cAMP has been observed, implying that a single biological stimulus may play a role in allergen gene

  13. Selective down-regulation of cell surface cAMP-binding sites and cAMP-induced responses in Dictyostelium discoideum

    NARCIS (Netherlands)

    Kesbeke, Fanja; Haastert, Peter J.M. van


    Extracellular cAMP induces an intracellular accumulation of cAMP and cGMP levels in Dictyostelium discoideum, cAMP is detected by cell-surface receptors which are composed of a class of fast-dissociating sites (t1/2 = 1-2 s) and a class of slow-dissociating sites (t1/2 = 15-150 s). Exposure of D. di

  14. Integrating Responsive Building Elements in Buildings

    DEFF Research Database (Denmark)

    Haase, Matthias; Amato, Alex; Heiselberg, Per


    There is a global need for a more sustainable building development. About 50% of energy is used in buildings indicating that buildings provide a considerable potential for operational energy savings. Studies were conducted with the following objectives: to perform a state-of-the-art review...... energy strategies to develop guidelines and procedures for estimation of environmental performance of responsive building elements and integrated building concepts This paper introduces the ideas of this collaborative work and discusses its usefulness for Hong Kong and China. Special focus was put...... of responsive building elements, of integrated building concepts and of environmental performance assessment methods to improve and optimize responsive building elements to develop and optimize new building concepts with integration of responsive building elements, HVAC-systems as well as natural and renewable...

  15. 高脂膳食和跑台运动构建模型大鼠腓肠肌葡萄糖转运体4和 cAMP反应元件结合蛋白的变化%Effects of high-fat diet and treadmill exercise on glucose transporter 4 and cAMP response element binding protein in rat gastrocnemius muscle

    Institute of Scientific and Technical Information of China (English)

    张云丽; 蔡明; 李静静; 辛磊; 娄淑杰


    BACKGROUND:Some studies indicate that PI3K/Akt signaling pathway is associated with the expression of glucose transporter 4 (GLUT4) and the function of cAMP response element binding protein (CREB) in skeletal muscle. However, it is stil unclear whether PI3K/Akt signaling pathway has the effects on CREB and GLUT4 in skeletal muscle of the rats with high-fat diet and treadmil exercise. OBJECTIVE:To investigate whether PI3K/Akt signaling pathway has the effects on CREB and GLUT4 in gastrocnemius muscle of the rats with high-fat diet and treadmil exercise. METHODS:A total of 70 rats were fed with normal diet for 2 weeks, and randomly divided into common feed group (n=20) and high-fat feed group (n=50). Rats in both groups were respectively fed with common feed and high-fat feed for 8 weeks. The rats in the common feed group were equaly assigned to common feed quiet group and common feed exercise group. 20 rats from the high-fat feed group whose body weight was 1.4 times of common rats were randomly and equaly assigned to obese quiet group and obese exercise group. Rats in the quiet groups did not do exercises. Rats in the exercise groups received adaptive sports for 1 week and medium-intensity treadmil exercise for 8 weeks. RESULTS AND CONCLUSION:(1) Impairments of PI3K/Akt signaling pathway appeared in obese rats, however, the quantity of GLUT4 expression did not change obviously in gastrocnemius muscles of obese rats. The reasons for the decrease of the nuclear protein CREB level of gastrocnemius muscles of obese rats might be related to the decrease of pAkt-Ser473 level. (2) The increase of the quantity of GLUT4 expression was accompanied by significantly up-regulated pAkt-Ser473 level by exercise intervention in gastrocnemius muscles of obese rats. Exercise intervention significantly increased the expression of nuclear protein CREB in gastrocnemius muscles of chow-fed rats and obese rats, which was consistent with the changes of pAkt-Ser473. These findings suggest

  16. Cyclic AMP Receptor Protein Acts as a Transcription Regulator in Response to Stresses in Deinococcus radiodurans.

    Directory of Open Access Journals (Sweden)

    Su Yang

    Full Text Available The cyclic AMP receptor protein family of transcription factors regulates various metabolic pathways in bacteria, and also play roles in response to environmental changes. Here, we identify four homologs of the CRP family in Deinococcus radiodurans, one of which tolerates extremely high levels of oxidative stress and DNA-damaging reagents. Transcriptional levels of CRP were increased under hydrogen peroxide (H2O2 treatment during the stationary growth phase, indicating that CRPs function in response to oxidative stress. By constructing all CRP single knockout mutants, we found that the dr0997 mutant showed the lowest tolerance toward H2O2, ultraviolet radiation, ionizing radiation, and mitomycin C, while the phenotypes of the dr2362, dr0834, and dr1646 mutants showed slight or no significant differences from those of the wild-type strain. Taking advantage of the conservation of the CRP-binding site in many bacteria, we found that transcription of 18 genes, including genes encoding chromosome-partitioning protein (dr0998, Lon proteases (dr0349 and dr1974, NADH-quinone oxidoreductase (dr1506, thiosulfate sulfurtransferase (dr2531, the DNA repair protein UvsE (dr1819, PprA (dra0346, and RecN (dr1447, are directly regulated by DR0997. Quantitative real-time polymerase chain reaction (qRT-PCR analyses showed that certain genes involved in anti-oxidative responses, DNA repair, and various cellular pathways are transcriptionally attenuated in the dr0997 mutant. Interestingly, DR0997 also regulate the transcriptional levels of all CRP genes in this bacterium. These data suggest that DR0997 contributes to the extreme stress resistance of D. radiodurans via its regulatory role in multiple cellular pathways, such as anti-oxidation and DNA repair pathways.

  17. Composite structure of auxin response elements. (United States)

    Ulmasov, T; Liu, Z B; Hagen, G; Guilfoyle, T J


    The auxin-responsive soybean GH3 gene promoter is composed of multiple auxin response elements (AuxREs), and each AuxRE contributes incrementally to the strong auxin inducibility to the promoter. Two independent AuxREs of 25 bp (D1) and 32 bp (D4) contain the sequence TGTCTC. Results presented here show that the TGTCTC element in D1 and D4 is required but not sufficient for auxin inducibility in carrot protoplast transient expression assays. Additional nucleotides upstream of TGTCTC are also required for auxin inducibility. These upstream sequences showed constitutive activity and no auxin inducibility when part or all of the TGTCTC element was mutated or deleted. In D1, the constitutive element overlaps the 5' portion of TGTCTC; in D4, the constitutive element is separated from TGTCTC. An 11-bp element in D1, CCTCGTGTCTC, conferred auxin inducibility to a minimal cauliflower mosaic virus 35S promoter in transgenic tobacco seedlings as well as in carrot protoplasts (i.e., transient expression assays). Both constitutive elements bound specifically to plant nuclear proteins, and the constitutive element in D1 bound to a recombinant soybean basic leucine zipper transcription factor with G-box specificity. To demonstrate further the composite nature of AuxREs and the ability of the TGTCTC element to confer auxin inducibility, we created a novel AuxRE by placing a yeast GAL4 DNA binding site adjacent to the TGTCTC element. Expression of a GAL4-c-Rel transactivator in the presence of this novel AuxRE resulted in auxin-inducible expression. Our results indicate that at least some AuxREs have a composite structure consisting of a constitutive element adjacent to a conserved TGTCTC element that confers auxin inducibility.

  18. 坐骨神经结扎对大鼠背根神经节和脊髓背角神经元磷酸化环酸腺苷反应元件结合蛋白表达的影响%Increased phosphorylation of cyclic AMP response element binding protein (CREB) in the dorsal root ganglia and superficial dorsal hornneurons following chronic constriction injury

    Institute of Scientific and Technical Information of China (English)

    姚永兴; 祝继洪; 宋学军; 张励才; 曾因明


    Objective To investigate whether chronic constriction injury (CCI) of the sciatic nerve of rats could produce alterations in the phosphorylation of cyclic AMP response element binding(CREB) protein in dorsal root ganglia (DRG) and superficial dorsal horn neurons of the spinal cord. Methods Chronic constriction injury (CCI) of the sciatic nerve was employed as a model of neuropathic pain. Thirty-two Sprague-Dawley rats were randomly divided into Naive, Sham, CCI 2w(received CCI for 2 weeks) and CCI 4w(received CCI for 4 weeks)groups. Hind paw withdrawal threshold to mechanical stimuli and withdrawal latency to thermal stimuli were used to determine the mechanical and thermal hyperalgesia. Then all the rats were deeply anesthetized and perfused intracardially with paraformaldehyde. The fixed L4-5 spinal cord and the L5 DRG ipsilateral to CCI were harvested for fixation. The pCREB-immunoreactive(pCREB-IR) cells in both DRG and superficial dorsal horn neurons were quantified for analysis using immunohistochemistry methods. Results On the 14th day after sciatic nerve injury, all the rats exhibited significant mechanical and thermal hyperalgesia. The mechanical withdrawal thresholds to yon Frey filament from CCI 2w group decreased significantly compared to both baseline values and those of Sham group( P < 0.01); Thermal withdwal latencies from CCI 2w group decreased significantly compared to both baseline values and those of Sham group( P <0.01). Some rats from Sham group also showed mechanical hyperalgesia compared to hoth baseline values and those of Naive group( P < 0.01 ). 28 days after CCI, both mechanical and thermal hypersensitivity were significantly alleviated, with no statistical significance compared to those of Sham group. On the 14th day after CCI, the number of pCREB-IR cells significantly increased in ipsilateral L5 DRGs and superficial dorsal horns( P <0.01) compared to Sham group. The number of phosphorylated CREB-IR cells in the ipsilateral DRGs

  19. cAMP-synthesis in a medullary thyroid carcinoma cell line: response to adrenergic agents and prostaglandines. (United States)

    Mertens, P R; Goretzki, P E; Keck, E


    Calcitonin secretion by C-cells is mediated through intracellular 3'5'-cyclic adenosine monophosphate (cAMP) and calcium signaling. Calcitonin release stimulation tests may take advantage of both signaling cascades in screening for medullary thyroid carcinomas (MTC). To elucidate the regulation of the adenylyl cyclase system we have determined cAMP levels of a calcitonin-expressing MTC cell line (RG) after exposure to adrenergic agents and prostaglandines. In early passages (20-30) cAMP concentrations were significantly elevated in RG cells after exposure to beta-adrenergic agents and prostaglandines E1 and E2. In advanced passages (60-80) the beta-adrenergic response was no longer detectable and adrenergic receptors were uncoupled from the adenylyl cyclase complex; while the effect of prostaglandines E1 and E2 remained unaffected. Preincubation with dexamethasone, in a process requiring protein new synthesis, re-established the adrenergic response in later passages, indicating that RG cells dedifferentiated in culture over time. Our in vitro findings suggest that MTC cell dedifferentiation may be accompanied by adrenergic receptor-uncoupling from the adenylate cyclase system and that this process may be reversed by dexamethasone incubation.

  20. Can packaging elements elicit consumers’ emotional responses?

    DEFF Research Database (Denmark)

    Liao, Lewis; Corsi, Armando; Lockshin, Larry;

    Emotion has been an important concept in many areas of consumer research such as judgment, decision-making and advertising. Little research has been done on emotion in packaging adopting the physiological measures used in other areas. This paper draws on past studies in advertising that measure....... The results show that packaging can elicit an emotional response via different elements. The paper also raises concerns about the accuracy of using selfreport measures of emotional responses to packaging research....

  1. 大麻素受体2激动剂JWH-015对骨癌痛大鼠脊髓背角磷酸化环磷酸腺苷反应元件结合蛋白的影响%The effect of intraperitoneal injection cannabinoid 2 receptor agonist JWH-015 on the expression of phosphorylated cyclic AMP response element binding protein in spinal dorsal horn in a rat model of bone cancer pain

    Institute of Scientific and Technical Information of China (English)

    孙蓓; 张羽; 冷鑫; 顾小萍; 马正良


    目的 探讨腹腔注射大麻素受体(cannabinoid receptor,CB)2激动剂对骨癌痛大鼠脊髓背角磷酸化环磷酸腺苷反应元件结合蛋白(phosphorylated cyclic AMP response element binding protein,pCREB)表达的影响. 方法 运用随机数字表法将63只雌性SD大鼠分为3组:肿瘤给药组(J组,15只)、肿瘤对照组(D组,24只)和假手术对照组(S组,24只).J组、D组的大鼠左侧胫骨上端骨髓腔被注入5μlWalker256大鼠乳腺癌细胞制备骨癌痛模型;S组则注入等量的生理盐水.在造模后第10天,J组腹腔注射JWH-015(100 μg/500μl),D组、S组注射等量JWH-015溶剂二甲基亚髓砜(dimethylsulfoxide,DMSO).每组大鼠于造模前1d,造模后4、7、10 d,腹腔注射后2、6、24、48、72 h,检测手术侧机械刺激缩足阈值(paw withdrawal mechanicalthreshold,PWMT)和行走痛行为学评分.D组和S组大鼠于造模后4、7d,J组、D组和S组大鼠于造模后10 d及腹腔注射后6、24、72 h,取脊髓腰膨大进行免疫印迹分析. 结果 与S组比较,J组和D组大鼠造模后7 d PWMT开始降低(P<0.05),造模后10 d行走痛行为学评分增加(P<0.05),脊髓背角pCREB表达水平于7、10 d上调(P<0.05).与D组比较,腹腔注射JWH-015后24 h,J组PWMT(8.7±1.6)g显著上升(P<0.05),行走痛行为学评分(1.0±0.6)分和pCREB的表达(0.56±0.10)明显下降(P<0.05). 结论 腹腔注射JWH-015可能通过下调脊髓背角pCREB的表达改善骨癌痛大鼠的痛行为.%Objective To investigate the change of phosphorylated cyclic AMP response element binding protein (pCREB) in spinal dorsal horn in a rat model of bone cancer pain,after intraperitoneal injection JWH-015.Methods Sixty-three female SD rats were randomly divided into 3 group:medication administration of JWH-015 group (group J,n=15),medication administration of dimethylsulfoxide (DMSO) group (group D,n=15) and sham group (group S,n=21).Group J,D:5 μl Walker256 breast cancer cells of rat were implanted

  2. Reduced lipolysis response to adipose afferent reflex involved in impaired activation of adrenoceptor-cAMP-PKA-hormone sensitive lipase pathway in obesity (United States)

    Ding, Lei; Zhang, Feng; Zhao, Ming-Xia; Ren, Xing-Sheng; Chen, Qi; Li, Yue-Hua; Kang, Yu-Ming; Zhu, Guo-Qing


    Chemical stimulation of white adipose tissue (WAT) causes adipose afferent reflex (AAR) and sympathetic activation. This study is to investigate the effects of AAR on lipolysis and the mechanisms of attenuated lipolysis response to enhanced AAR in obesity. Obesity was caused by high-fat diet for 12 weeks in rats. AAR was induced by injection of capsaicin into inguinal WAT or electrical stimulation of epididymal WAT afferent nerve. AAR caused sympathetic activation, which was enhanced in obesity rats. AAR increased cAMP levels and PKA activity, promoted hormone sensitive lipase (HSL) and perilipin phosphorylation, and increased lipolysis in WAT, which were attenuated in obesity rats. PKA activity, cAMP, perilipin and β-adrenoceptor levels were reduced, while HSL was upregulated in adipocytes from obesity rats. In primary adipocytes, isoproterenol increased cAMP levels and PKA activity, promoted HSL and perilipin phosphorylation, and increased lipolysis, which were attenuated in obesity rats. The attenuated effects of isoproterenol in adipocytes from obesity rats were prevented by a cAMP analogue dbcAMP. The results indicate that reduced lipolysis response to enhanced AAR in obesity is attributed to the impaired activation of β-adrenoceptor-cAMP-PKA-HSL pathway. Increased cAMP level in adipocytes rectifies the attenuated lipolysis in obesity. PMID:27694818

  3. Gene Expression Patterns Define Key Transcriptional Events InCell-Cycle Regulation By cAMP And Protein Kinase A

    Energy Technology Data Exchange (ETDEWEB)

    Zambon, Alexander C.; Zhang, Lingzhi; Minovitsky, Simon; Kanter, Joan R.; Prabhakar, Shyam; Salomonis, Nathan; Vranizan, Karen; Dubchak Inna,; Conklin, Bruce R.; Insel, Paul A.


    Although a substantial number of hormones and drugs increase cellular cAMP levels, the global impact of cAMP and its major effector mechanism, protein kinase A (PKA), on gene expression is not known. Here we show that treatment of murine wild-type S49 lymphoma cells for 24 h with 8-(4-chlorophenylthio)-cAMP (8-CPTcAMP), a PKA-selective cAMP analog, alters the expression of approx equal to 4,500 of approx. equal to 13,600 unique genes. By contrast, gene expression was unaltered in Kin- S49 cells (that lack PKA) incubated with 8-CPTcAMP. Changes in mRNA and protein expression of several cell cycle regulators accompanied cAMP-induced G1-phase cell-cycle arrest of wild-type S49 cells. Within 2h, 8-CPT-cAMP altered expression of 152 genes that contain evolutionarily conserved cAMP-response elements within 5 kb of transcriptional start sites, including the circadian clock gene Per1. Thus, cAMP through its activation of PKA produces extensive transcriptional regulation in eukaryotic cells. These transcriptional networks include a primary group of cAMP-response element-containing genes and secondary networks that include the circadian clock.

  4. PKA and cAMP/CNG Channels Independently Regulate the Cholinergic Ca2+-Response of Drosophila Mushroom Body Neurons1,2,3 (United States)

    Pavot, Pierre; Carbognin, Elena


    Abstract The mushroom bodies (MBs), one of the main structures in the adult insect brain, play a critical role in olfactory learning and memory. Though historical genes such as dunce and rutabaga, which regulate the level of cAMP, were identified more than 30 years ago, their in vivo effects on cellular and physiological mechanisms and particularly on the Ca2+-responses still remain largely unknown. In this work, performed in Drosophila, we took advantage of in vivo bioluminescence imaging, which allowed real-time monitoring of the entire MBs (both the calyx/cell-bodies and the lobes) simultaneously. We imaged neuronal Ca2+-activity continuously, over a long time period, and characterized the nicotine-evoked Ca2+-response. Using both genetics and pharmacological approaches to interfere with different components of the cAMP signaling pathway, we first show that the Ca2+-response is proportional to the levels of cAMP. Second, we reveal that an acute change in cAMP levels is sufficient to trigger a Ca2+-response. Third, genetic manipulation of protein kinase A (PKA), a direct effector of cAMP, suggests that cAMP also has PKA-independent effects through the cyclic nucleotide-gated Ca2+-channel (CNG). Finally, the disruption of calmodulin, one of the main regulators of the rutabaga adenylate cyclase (AC), yields different effects in the calyx/cell-bodies and in the lobes, suggesting a differential and regionalized regulation of AC. Our results provide insights into the complex Ca2+-response in the MBs, leading to the conclusion that cAMP modulates the Ca2+-responses through both PKA-dependent and -independent mechanisms, the latter through CNG-channels. PMID:26464971

  5. Effects of valproate sodium on phosphorylatted cAMP responsive element binding protein after hippocampal neuronal epileptiform discharge in rat%丙戊酸钠对大鼠海马神经元癫痫样放电后P-CREB1表达的影响

    Institute of Scientific and Technical Information of China (English)

    刘华; 徐祖才; 陈阳美


    目的:研究丙戊酸钠对大鼠海马神经元癫痫样放电后磷酸化腺感苷反应元件结合蛋白1(Phosphorylated cAMP responsiveelement binding protein1,P-CREB1)表达的影响.方法:wistar新生鼠,迅速断头取脑,体外培养海马神经元,建立神经元癫痫样放电模型.将神经元分为空白组、模型组、丙戊酸钠低剂量(50 mg/L)组、丙戊酸钠高剂量(100 mg/L)组,运用免疫荧光技术观察P-CREB1在神经元癫痫样放电后在细胞内的表达部位,采用wester blot技术测定P-CREB1在不同分组中的表达强度.结果:通过免疫荧光技术,在各组中都可以看到P-CREB1在细胞核内表达,以模型组最明显;运用Western blot,发现表达趋势与免疫荧光一致,并且,给予丙戊酸钠后,P-CREB1表达减弱,且高剂量组与低剂量组之间差异有统计学意义(P<0.05).结论:海马神经元无镁处理后呈癫痫样放电,同时P-CREB1被过度激活,而有效浓度的丙戊酸钠可抑制此反应P-CREB1的磷酸化水平.

  6. Antimicrobial Peptides (AMPs

    Directory of Open Access Journals (Sweden)

    Mehrzad Sadredinamin


    Full Text Available Antimicrobial peptides (AMPs are extensive group of molecules that produced by variety tissues of invertebrate, plants, and animal species which play an important role in their immunity response. AMPs have different classifications such as; biosynthetic machines, biological sources, biological functions, molecular properties, covalent bonding patterns, three dimensional structures, and molecular targets.These molecules have multidimensional properties including antimicrobial activity, antiviral activity, antifungal activity, anti-parasite activity, biofilm control, antitumor activity, mitogens activity and linking innate to adaptive immunity that making them promising agents for therapeutic drugs. In spite of this advantage of AMPs, their clinical developments have some limitation for commercial development. But some of AMPs are under clinical trials for the therapeutic purpose such as diabetic foot ulcers, different bacterial infections and tissue damage. In this review, we emphasized on the source, structure, multidimensional properties, limitation and therapeutic applications of various antimicrobial peptides.

  7. Increases in cAMP, MAPK Activity and CREB Phosphorylation during REM Sleep: Implications for REM Sleep and Memory Consolidation


    Luo, Jie; Phan, Trongha X.; Yang, Yimei; Garelick, Michael G.; Storm, Daniel R.


    The cyclic adenosine monophosphate (cAMP), mitogen-activated protein kinase (MAPK) and cAMP response element-binding protein (CREB) transcriptional pathway is required for consolidation of hippocampus-dependent memory. In mice, this pathway undergoes a circadian oscillation required for memory persistence that reaches a peak during the daytime. Since mice exhibit polyphasic sleep patterns during the day, this suggested the interesting possibility that cAMP, MAPK activity and CREB phosphorylat...

  8. Changes in the Arabidopsis thaliana Proteome Implicate cAMP in Biotic and Abiotic Stress Responses and Changes in Energy Metabolism

    Directory of Open Access Journals (Sweden)

    May Alqurashi


    Full Text Available The second messenger 3′,5′-cyclic adenosine monophosphate (cAMP is increasingly recognized as having many different roles in plant responses to environmental stimuli. To gain further insights into these roles, Arabidopsis thaliana cell suspension culture was treated with 100 nM of cell permeant 8-bromo-cAMP for 5 or 10 min. Here, applying mass spectrometry and comparative proteomics, 20 proteins were identified as differentially expressed and we noted a specific bias in proteins with a role in abiotic stress, particularly cold and salinity, biotic stress as well as proteins with a role in glycolysis. These findings suggest that cAMP is sufficient to elicit specific stress responses that may in turn induce complex changes to cellular energy homeostasis.

  9. Changes in the Arabidopsis thaliana Proteome Implicate cAMP in Biotic and Abiotic Stress Responses and Changes in Energy Metabolism

    KAUST Repository

    Alquraishi, May Majed


    The second messenger 3′,5′-cyclic adenosine monophosphate (cAMP) is increasingly recognized as having many different roles in plant responses to environmental stimuli. To gain further insights into these roles, Arabidopsis thaliana cell suspension culture was treated with 100 nM of cell permeant 8-bromo-cAMP for 5 or 10 min. Here, applying mass spectrometry and comparative proteomics, 20 proteins were identified as differentially expressed and we noted a specific bias in proteins with a role in abiotic stress, particularly cold and salinity, biotic stress as well as proteins with a role in glycolysis. These findings suggest that cAMP is sufficient to elicit specific stress responses that may in turn induce complex changes to cellular energy homeostasis.

  10. Delineating the core regulatory elements crucial for directed cell migration by examining folic-acid-mediated responses. (United States)

    Srinivasan, Kamalakkannan; Wright, Gus A; Hames, Nicole; Housman, Max; Roberts, Alayna; Aufderheide, Karl J; Janetopoulos, Chris


    Dictyostelium discoideum shows chemotaxis towards folic acid (FA) throughout vegetative growth, and towards cAMP during development. We determined the spatiotemporal localization of cytoskeletal and signaling molecules and investigated the FA-mediated responses in a number of signaling mutants to further our understanding of the core regulatory elements that are crucial for cell migration. Proteins enriched in the pseudopods during chemotaxis also relocalize transiently to the plasma membrane during uniform FA stimulation. In contrast, proteins that are absent from the pseudopods during migration redistribute transiently from the PM to the cytosol when cells are globally stimulated with FA. These chemotactic responses to FA were also examined in cells lacking the GTPases Ras C and G. Although Ras and phosphoinositide 3-kinase activity were significantly decreased in Ras G and Ras C/G nulls, these mutants still migrated towards FA, indicating that other pathways must support FA-mediated chemotaxis. We also examined the spatial movements of PTEN in response to uniform FA and cAMP stimulation in phospholipase C (PLC) null cells. The lack of PLC strongly influences the localization of PTEN in response to FA, but not cAMP. In addition, we compared the gradient-sensing behavior of polarized cells migrating towards cAMP to that of unpolarized cells migrating towards FA. The majority of polarized cells make U-turns when the cAMP gradient is switched from the front of the cell to the rear. Conversely, unpolarized cells immediately extend pseudopods towards the new FA source. We also observed that plasma membrane phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] levels oscillate in unpolarized cells treated with Latrunculin-A, whereas polarized cells had stable plasma membrane PtdIns(3,4,5)P3 responses toward the chemoattractant gradient source. Results were similar for cells that were starved for 4 hours, with a mixture of polarized and unpolarized cells responding

  11. Neuromodulatory effect of Gαs- or Gαq-coupled G-protein-coupled receptor on NMDA receptor selectively activates the NMDA receptor/Ca2+/calcineurin/cAMP response element-binding protein-regulated transcriptional coactivator 1 pathway to effectively induce brain-derived neurotrophic factor expression in neurons. (United States)

    Fukuchi, Mamoru; Tabuchi, Akiko; Kuwana, Yuki; Watanabe, Shinjiro; Inoue, Minami; Takasaki, Ichiro; Izumi, Hironori; Tanaka, Ayumi; Inoue, Ran; Mori, Hisashi; Komatsu, Hidetoshi; Takemori, Hiroshi; Okuno, Hiroyuki; Bito, Haruhiko; Tsuda, Masaaki


    Although coordinated molecular signaling through excitatory and modulatory neurotransmissions is critical for the induction of immediate early genes (IEGs), which lead to effective changes in synaptic plasticity, the intracellular mechanisms responsible remain obscure. Here we measured the expression of IEGs and used bioluminescence imaging to visualize the expression of Bdnf when GPCRs, major neuromodulator receptors, were stimulated. Stimulation of pituitary adenylate cyclase-activating polypeptide (PACAP)-specific receptor (PAC1), a Gαs/q-protein-coupled GPCR, with PACAP selectively activated the calcineurin (CN) pathway that is controlled by calcium signals evoked via NMDAR. This signaling pathway then induced the expression of Bdnf and CN-dependent IEGs through the nuclear translocation of CREB-regulated transcriptional coactivator 1 (CRTC1). Intracerebroventricular injection of PACAP and intraperitoneal administration of MK801 in mice demonstrated that functional interactions between PAC1 and NMDAR induced the expression of Bdnf in the brain. Coactivation of NMDAR and PAC1 synergistically induced the expression of Bdnf attributable to selective activation of the CN pathway. This CN pathway-controlled expression of Bdnf was also induced by stimulating other Gαs- or Gαq-coupled GPCRs, such as dopamine D1, adrenaline β, CRF, and neurotensin receptors, either with their cognate agonists or by direct stimulation of the protein kinase A (PKA)/PKC pathway with chemical activators. Thus, the GPCR-induced expression of IEGs in coordination with NMDAR might occur via the selective activation of the CN/CRTC1/CREB pathway under simultaneous excitatory and modulatory synaptic transmissions in neurons if either the Gαs/adenylate cyclase/PKA or Gαq/PLC/PKC-mediated pathway is activated.

  12. Integrating Environmentally Responsive Elements in Buildings

    DEFF Research Database (Denmark)

    Heiselberg, Per; Andresen, Inger; Perino, Marco;


    Significant improvement have been achieved on efficiency improvements of specific building elements like the building envelope and building equipment and services and whilst most building elements still offer opportunities for efficiency improvements, the greatest future potential lie with techno...

  13. Application and optimization of the tenderization of pig Longissimus dorsi muscle by adenosine 5'-monophosphate (AMP) using the response surface methodology. (United States)

    Deng, Shaoying; Wang, Daoying; Zhang, Muhan; Geng, Zhiming; Sun, Chong; Bian, Huan; Xu, Weimin; Zhu, Yongzhi; Liu, Fang; Wu, Haihong


    Based on single factor experiments, NaCl concentration, adenosine 5'-monophosphate (AMP) concentration and temperature were selected as independent variables for a three-level Box-Behnken experimental design, and the shear force and cooking loss were response values for regression analysis. According to the statistical models, it showed that all independent variables had significant effects on shear force and cooking loss, and optimal values were at the NaCl concentration of 4.15%, AMP concentration of 22.27 mmol/L and temperature of 16.70°C, which was determined with three-dimensional response surface diagrams and contour plots. Under this condition, the observed shear force and cooking loss were 0.625 kg and 8.07%, respectively, exhibiting a good agreement with their predicted values, showing the good applicability and feasibility of response surface methodology (RSM) for improving pork tenderness. Compared with control pig muscles, AMP combined with NaCl treatment demonstrated significant effects on improvement of meat tenderness and reduction of cooking loss. Therefore, AMP could be regarded as an effective tenderization agent for pork.

  14. Synthesis, characterization, and swelling behaviors of a pH-responsive CMC-g-poly(AA-co-AMPS) superabsorbent hydrogel



    New superabsorbent hydrogels were synthesized through free radical graft copolymerization of partially neutralized acrylic acid (AA) and 2-acrylamido-2-methylpropanesulfonic acid (AMPS) onto carboxymethyl cellulose (CMC) backbones. The structure and morphology of the synthesized superabsorbent hydrogels were characterized by Fourier transform infrared spectroscopy and scanning electron microscope. The effect of the molar ratio of AMPS to AA, the APS concentration, and the CMC content on ...

  15. Thresholds in shock response across the elements (United States)

    Bourne, F. L.; Bourne, N. K.


    Compendia of shock data have been assembled across national laboratories across the world. Previous work has shown a threshold in behaviour for materials; the weak shock limit. This corresponds to the stress state at which the shock is overdriven in a single front. Here the shock velocity-particle velocity data for elements and compounds has been systematically analysed to note discontinuities in the data. A range of materials show such features and the form of the discontinuity in each case is analysed. Some of these are found to correspond to martensitic phase transformations as expected whilst others are more difficult to classify. Particular groups within the elements show characteristic forms according to their groupings within the periodic table. Typical datasets are presented and trends in behaviour are noted for a range of elements.

  16. Ablative Thermal Response Analysis Using the Finite Element Method (United States)

    Dec John A.; Braun, Robert D.


    A review of the classic techniques used to solve ablative thermal response problems is presented. The advantages and disadvantages of both the finite element and finite difference methods are described. As a first step in developing a three dimensional finite element based ablative thermal response capability, a one dimensional computer tool has been developed. The finite element method is used to discretize the governing differential equations and Galerkin's method of weighted residuals is used to derive the element equations. A code to code comparison between the current 1-D tool and the 1-D Fully Implicit Ablation and Thermal Response Program (FIAT) has been performed.

  17. cAMP and EPAC are key players in the regulation of the signal transduction pathway involved in the α-hemolysin autophagic response.

    Directory of Open Access Journals (Sweden)

    María Belén Mestre

    Full Text Available Staphylococcus aureus is a microorganism that causes serious diseases in the human being. This microorganism is able to escape the phagolysosomal pathway, increasing intracellular bacterial survival and killing the eukaryotic host cell to spread the infection. One of the key features of S. aureus infection is the production of a series of virulence factors, including secreted enzymes and toxins. We have shown that the pore-forming toxin α-hemolysin (Hla is the S. aureus-secreted factor responsible for the activation of the autophagic pathway and that this response occurs through a PI3K/Beclin1-independent form. In the present report we demonstrate that cAMP has a key role in the regulation of this autophagic response. Our results indicate that cAMP is able to inhibit the autophagy induced by Hla and that PKA, the classical cAMP effector, does not participate in this regulation. We present evidence that EPAC and Rap2b, through calpain activation, are the proteins involved in the regulation of Hla-induced autophagy. Similar results were obtained in cells infected with different S. aureus strains. Interestingly, in this report we show, for the first time to our knowledge, that both EPAC and Rap2b are recruited to the S. aureus-containing phagosome. We believe that our findings have important implications in understanding innate immune processes involved in intracellular pathogen invasion of the host cell.

  18. The μ opioid agonist morphine modulates potentiation of capsaicin-evoked TRPV1 responses through a cyclic AMP-dependent protein kinase A pathway

    Directory of Open Access Journals (Sweden)

    Roberts-Thomson Sarah J


    Full Text Available Abstract Background The vanilloid receptor 1 (TRPV1 is critical in the development of inflammatory hyperalgesia. Several receptors including G-protein coupled prostaglandin receptors have been reported to functionally interact with the TRPV1 through a cAMP-dependent protein kinase A (PKA pathway to potentiate TRPV1-mediated capsaicin responses. Such regulation may have significance in inflammatory pain. However, few functional receptor interactions that inhibit PKA-mediated potentiation of TRPV1 responses have been described. Results In the present studies we investigated the hypothesis that the μ opioid receptor (MOP agonist morphine can modulate forskolin-potentiated capsaicin responses through a cAMP-dependent PKA pathway. HEK293 cells were stably transfected with TRPV1 and MOP, and calcium (Ca2+ responses to injection of the TRPV1 agonist capsaicin were monitored in Fluo-3-loaded cells. Pre-treatment with morphine did not inhibit unpotentiated capsaicin-induced Ca2+ responses but significantly altered capsaicin responses potentiated by forskolin. TRPV1-mediated Ca2+ responses potentiated by the direct PKA activator 8-Br-cAMP and the PKC activator Phorbol-12-myristate-13-acetatewere not modulated by morphine. Immunohistochemical studies confirmed that the TRPV1 and MOP are co-expressed on cultured Dorsal Root Ganglion neurones, pointing towards the existence of a functional relationship between the G-protein coupled MOP and nociceptive TRPV1. Conclusion The results presented here indicate that the opioid receptor agonist morphine acts via inhibition of adenylate cyclase to inhibit PKA-potentiated TRPV1 responses. Targeting of peripheral opioid receptors may therefore have therapeutic potential as an intervention to prevent potentiation of TRPV1 responses through the PKA pathway in inflammation.

  19. The relationship between some beta-adrenergic mediated responses and plasma concentrations of adrenaline and cyclic AMP in man

    DEFF Research Database (Denmark)

    Philipsen, E K; Myhre, John Gabriel; Larsen, S;


    concentrations at low adrenaline infusion rates was prevented, whereas a small increase in cyclic AMP was found at high adrenaline infusion rates, probably owing to incomplete beta-receptor blockade. Likewise, the adrenaline-induced increments in blood substrates (glucose, lactate, glycerol and beta......To test the hypothesis that increments in plasma cyclic AMP during beta-adrenergic stimulation reflect integrated second messenger function of the tissues activated by the agonist, graded adrenaline infusion resulting in plasma adrenaline concentrations within the physiological range was performed...... hydroxybutyric acid) were significantly reduced but not completely prevented by beta-blockade. We conclude that an altered relationship between beta-agonist concentrations and plasma cyclic AMP may provide evidence for the existence of differences in beta-adrenergic sensitivity in man....

  20. Finite Element Modeling of the Buckling Response of Sandwich Panels (United States)

    Rose, Cheryl A.; Moore, David F.; Knight, Norman F., Jr.; Rankin, Charles C.


    A comparative study of different modeling approaches for predicting sandwich panel buckling response is described. The study considers sandwich panels with anisotropic face sheets and a very thick core. Results from conventional analytical solutions for sandwich panel overall buckling and face-sheet-wrinkling type modes are compared with solutions obtained using different finite element modeling approaches. Finite element solutions are obtained using layered shell element models, with and without transverse shear flexibility, layered shell/solid element models, with shell elements for the face sheets and solid elements for the core, and sandwich models using a recently developed specialty sandwich element. Convergence characteristics of the shell/solid and sandwich element modeling approaches with respect to in-plane and through-the-thickness discretization, are demonstrated. Results of the study indicate that the specialty sandwich element provides an accurate and effective modeling approach for predicting both overall and localized sandwich panel buckling response. Furthermore, results indicate that anisotropy of the face sheets, along with the ratio of principle elastic moduli, affect the buckling response and these effects may not be represented accurately by analytical solutions. Modeling recommendations are also provided.

  1. Decreased response to cAMP in the glucose and glycogen catabolism in perfused livers of Walker-256 tumor-bearing rats. (United States)

    de Morais, Hely; Cassola, Priscila; Moreira, Carolina Campos Lima; Bôas, Suéllen Kathiane Fernandes Vilas; Borba-Murad, Glaucia Regina; Bazotte, Roberto Barbosa; de Souza, Helenir Medri


    The hepatic response to cyclic adenosine monophosphate (cAMP) and N6-monobutyryl-cAMP (N6-MB-cAMP) in the glucose and glycogen catabolism and hepatic glycogen levels were evaluated in Walker-256 tumor-bearing rats, on days 5 (WK5), 8 (WK8), and 11 (WK11) after the implantation of tumor. Rats without tumor fed ad libitum (fed control rats) or that received the same daily amount of food ingested by anorexics tumor-bearing rats (pair-fed control rats) or 24 h fasted (fasted control rats) were used as controls. Glucose and glycogen catabolism were measured in perfused liver. Hepatic glycogen levels were lower (p catabolism was lower (p catabolism, under condition of depletion of hepatic glycogen (24 h fast), was lower (p catabolism was lower (p catabolism in various stages of tumor development (days 5, 8 and 11), which was probably not due to the lower hepatic glycogen levels nor due to the increased activity of PDE3B.

  2. CREB modulates calcium signaling in cAMP-induced bone marrow stromal cells (BMSCs). (United States)

    Zhang, Linxia; Liu, Li; Thompson, Ryan; Chan, Christina


    Calcium signaling has a versatile role in many important cellular functions. Despite its importance, regulation of calcium signaling in bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) has not been explored extensively. Our previous study revealed that cyclic adenosine monophosphate (cAMP) enabled BMSCs to generate calcium signal upon stimulation by dopamine, KCl and glutamate. Concurrently, cAMP transiently activated the transcription factor cAMP response element binding protein (CREB) in BMSCs. Activity of CREB can be modulated by the calcium/calmodulin-dependent kinase signaling pathway, however, whether the calcium signaling observed in cAMP-induced BMSCs requires CREB has not been investigated. In an effort to uncover the role of CREB in the generation of calcium signaling in response to modulators such as dopamine and KCl, we knocked down CREB activity in BMSCs. Our study indicated that BMSCs, but not its close relative fibroblasts, are responsive to dopamine and KCl after cAMP treatment. Calcium signal elicited by dopamine depends, in part, on calcium influx whereas that elicited by KCl depends completely on calcium influx. Knock-down of CREB activity significantly reduced or abolished the cAMP-induced calcium response, and reintroducing a constitutively active CREB partially restored the calcium response.

  3. Finite Element Model Updating Using Response Surface Method

    CERN Document Server

    Marwala, Tshilidzi


    This paper proposes the response surface method for finite element model updating. The response surface method is implemented by approximating the finite element model surface response equation by a multi-layer perceptron. The updated parameters of the finite element model were calculated using genetic algorithm by optimizing the surface response equation. The proposed method was compared to the existing methods that use simulated annealing or genetic algorithm together with a full finite element model for finite element model updating. The proposed method was tested on an unsymmetri-cal H-shaped structure. It was observed that the proposed method gave the updated natural frequen-cies and mode shapes that were of the same order of accuracy as those given by simulated annealing and genetic algorithm. Furthermore, it was observed that the response surface method achieved these results at a computational speed that was more than 2.5 times as fast as the genetic algorithm and a full finite element model and 24 ti...

  4. Eviprostat Activates cAMP Signaling Pathway and Suppresses Bladder Smooth Muscle Cell Proliferation

    Directory of Open Access Journals (Sweden)

    Masayuki Takeda


    Full Text Available Eviprostat is a popular phytotherapeutic agent for the treatment of lower urinary tract symptoms (LUTS. At present, the signaling mechanisms underlying its therapeutic effects are still poorly understood. Given that cAMP has been reported to suppress cell hyperplasia and hypertrophy in various pathological situations, we asked whether the effect of Eviprostat could be ascribed to the activation of the cAMP signaling pathway. In the study, exposure of cAMP response element (CRE-secreted alkaline phosphatase (SEAP (CRE-SEAP-reporter cells to Eviprostat elevated SEAP secretion, which was associated with an increased phosphorylation of vasodilator-stimulated phosphoprotein (VASP and cAMP-response element-binding protein (CREB, as well as enhanced expression of CRE-regulated protein connexin43, indicating an activation of the cAMP signaling pathway. Consistent with these observations, Eviprostat-induced expression of Cx43 was abolished in the presence of adenylyl cyclase inhibitor SQ22536 or PKA inhibitor H89, whereas it was mimicked by adenylyl cyclase activator, forskolin. Further analysis demonstrated that Eviprostat significantly potentiated the effect of phosphodiesterase 3 (PDE3 inhibitor, but not that of PDE4 inhibitor, on CRE activation. Moreover, Eviprostat suppressed PDGF-induced activation of ERK and Akt and inhibited cell proliferation and hillock formation in both mesangial cells and bladder smooth muscle cells. Collectively, activation of the cAMP signaling pathway could be an important mechanism by which Eviprostat exerts its therapeutic effects for LUTS.

  5. Crystal structure of a c-di-AMP riboswitch reveals an internally pseudo-dimeric RNA. (United States)

    Jones, Christopher P; Ferré-D'Amaré, Adrian R


    Cyclic diadenosine monophosphate (c-di-AMP) is a second messenger that is essential for growth and homeostasis in bacteria. A recently discovered c-di-AMP-responsive riboswitch controls the expression of genes in a variety of bacteria, including important pathogens. To elucidate the molecular basis for specific binding of c-di-AMP by a gene-regulatory mRNA domain, we have determined the co-crystal structure of this riboswitch. Unexpectedly, the structure reveals an internally pseudo-symmetric RNA in which two similar three-helix-junction elements associate head-to-tail, creating a trough that cradles two c-di-AMP molecules making quasi-equivalent contacts with the riboswitch. The riboswitch selectively binds c-di-AMP and discriminates exquisitely against other cyclic dinucleotides, such as c-di-GMP and cyclic-AMP-GMP, via interactions with both the backbone and bases of its cognate second messenger. Small-angle X-ray scattering experiments indicate that global folding of the riboswitch is induced by the two bound cyclic dinucleotides, which bridge the two symmetric three-helix domains. This structural reorganization likely couples c-di-AMP binding to gene expression.

  6. DNA-Mediated Cyclic GMP-AMP Synthase-Dependent and -Independent Regulation of Innate Immune Responses. (United States)

    Motani, Kou; Ito, Shinji; Nagata, Shigekazu


    Cytoplasmic DNA activates cyclic GMP-AMP synthase (cGAS) to produce cyclic 2'-5'3'-5'GMP-AMP dinucleotide (2'5 'cGAMP). The binding of 2'5'cGAMP to an adaptor protein, stimulator of IFN genes (STING), activates a transcription factor, IFN regulatory factor 3, leading to the induction of IFN and chemokine gene expression. In this study, we found that the 2'5'cGAMP-dependent STING activation induced highly upregulated CXCL10 gene expression. Formation of a distinct STING dimer, which was detected by native PAGE, was induced by 2'5'cGAMP, but not 3'-5'3'-5'cGAMP. Analysis of DNase II(-/-) mice, which constitutively produce IFN-β and CXCL10, showed the accumulation of 2'5'cGAMP in their fetal livers and spleens, suggesting that the undigested DNA accumulating in DNase II(-/-) cells may have leaked from the lysosomes into the cytoplasm. The DNase II(-/-) mouse embryonic fibroblasts produced 2'5'cGAMP in a cGAS-dependent manner during apoptotic cell engulfment. However, cGAS deficiency did not impair the STING-dependent upregulation of CXCL10 in DNase II(-/-) mouse embryonic fibroblasts that was induced by apoptotic cell engulfment or DNA lipofection. These results suggest the involvement of a cGAS-independent additional DNA sensor(s) that induces the STING-dependent activation of innate immunity.

  7. Nonlinear analysis of the forced response of structural elements (United States)

    Nayfeh, A. H.; Mook, D. T.; Sridhar, S.


    A general procedure is presented for the nonlinear analysis of the forced response of structural elements to harmonic excitations. Internal resonances (i.e., modal interactions) are taken into account. All excitations are considered, with special consideration given to resonant excitations. The general procedure is applied to clamped-hinged beams. The results reveal that exciting a higher mode may lead to a larger response in a lower interacting mode, contrary to the results of linear analyses.

  8. Finite element estimation of acoustical response functions in HID lamps

    Energy Technology Data Exchange (ETDEWEB)

    Baumann, Bernd; Wolff, Marcus [Department of Mechanical Engineering and Production, School of Engineering and Computer Science, Hamburg University of Applied Sciences, Berliner Tor 21, 20099 Hamburg (Germany); Hirsch, John; Antonis, Piet [Philips Lighting BV, Lightlabs, Mathildelaan 1, 5600 JM Eindhoven (Netherlands); Bhosle, Sounil [Universite de Toulouse (United States); Barrientos, Ricardo Valdivia, E-mail: bernd.baumann@haw-hamburg.d [National Nuclear Research Institute, Highway Mexico-Toluca s/n, La Marquesa, Ocoyoacac, CP 52750 (Mexico)


    High intensity discharge lamps can experience flickering and even destruction when operated at high frequency alternating current. The cause of these problems has been identified as acoustic resonances inside the lamp's arc tube. Here, a finite element approach for the calculation of the acoustic response function is described. The developed model does not include the plasma dynamics.

  9. Finite Element Vibration and Dynamic Response Analysis of Engineering Structures

    Directory of Open Access Journals (Sweden)

    Jaroslav Mackerle


    Full Text Available This bibliography lists references to papers, conference proceedings, and theses/dissertations dealing with finite element vibration and dynamic response analysis of engineering structures that were published from 1994 to 1998. It contains 539 citations. The following types of structures are included: basic structural systems; ground structures; ocean and coastal structures; mobile structures; and containment structures.

  10. Novel mechanisms and signaling pathways of esophageal ulcer healing: the role of prostaglandin EP2 receptors, cAMP, and pCREB. (United States)

    Ahluwalia, Amrita; Baatar, Dolgor; Jones, Michael K; Tarnawski, Andrzej S


    Clinical studies indicate that prostaglandins of E class (PGEs) may promote healing of tissue injury e.g., gastroduodenal and dermal ulcers. However, the precise roles of PGEs, their E-prostanoid (EP) receptors, signaling pathways including cAMP and cAMP response element-binding protein (CREB), and their relation to VEGF and angiogenesis in the tissue injury healing process remain unknown, forming the rationale for this study. Using an esophageal ulcer model in rats, we demonstrated that esophageal mucosa expresses predominantly EP2 receptors and that esophageal ulceration triggers an increase in expression of the EP2 receptor, activation of CREB (the downstream target of the cAMP signaling), and enhanced VEGF gene expression. Treatment of rats with misoprostol, a PGE1 analog capable of activating EP receptors, enhanced phosphorylation of CREB, stimulated VEGF expression and angiogenesis, and accelerated esophageal ulcer healing. In cultured human esophageal epithelial (HET-1A) cells, misoprostol increased intracellular cAMP levels (by 163-fold), induced phosphorylation of CREB, and stimulated VEGF expression. A cAMP analog (Sp-cAMP) mimicked, whereas an inhibitor of cAMP-dependent protein kinase A (Rp-cAMP) blocked, these effects of misoprostol. These results indicate that the EP2/cAMP/protein kinase A pathway mediates the stimulatory effect of PGEs on angiogenesis essential for tissue injury healing via the induction of CREB activity and VEGF expression.

  11. Parallel finite element modeling of earthquake ground response and liquefaction

    Institute of Scientific and Technical Information of China (English)

    Jinchi Lu(陆金池); Jun Peng(彭军); Ahmed Elgamal; Zhaohui Yang(杨朝晖); Kincho H. Law


    Parallel computing is a promising approach to alleviate the computational demand in conducting large-scale finite element analyses. This paper presents a numerical modeling approach for earthquake ground response and liquefaction using the parallel nonlinear finite element program, ParCYCLIC, designed for distributed-memory message-passing parallel computer systems. In ParCYCLIC, finite elements are employed within an incremental plasticity, coupled solid-fluid formulation. A constitutive model calibrated by physical tests represents the salient characteristics of sand liquefaction and associated accumulation of shear deformations. Key elements of the computational strategy employed in ParCYCLIC include the development of a parallel sparse direct solver, the deployment of an automatic domain decomposer, and the use of the Multilevel Nested Dissection algorithm for ordering of the finite element nodes. Simulation results of centrifuge test models using ParCYCLIC are presented. Performance results from grid models and geotechnical simulations show that ParCYCLIC is efficiently scalable to a large number of processors.

  12. Finite Element Response Sensitivity Analysis: a comparison between force-based and Displacement-Based Frame Element Models


    Barbato, Michele; Conte, J P


    This paper focuses on a comparison between displacement-based and force-based elements for static and dynamic response sensitivity analysis of frame type structures. Previous research has shown that force-based frame elements are superior to classical displacement-based elements enabling, at no significant additional computational costs, a drastic reduction in the number of elements required for a given level of accuracy in the simulated response. The present work shows that this advantage of...

  13. Cyclic AMP intoxication of macrophages by a Mycobacterium tuberculosis adenylate cyclase. (United States)

    Agarwal, Nisheeth; Lamichhane, Gyanu; Gupta, Radhika; Nolan, Scott; Bishai, William R


    With 8.9 million new cases and 1.7 million deaths per year, tuberculosis is a leading global killer that has not been effectively controlled. The causative agent, Mycobacterium tuberculosis, proliferates within host macrophages where it modifies both its intracellular and local tissue environment, resulting in caseous granulomas with incomplete bacterial sterilization. Although infection by various mycobacterial species produces a cyclic AMP burst within macrophages that influences cell signalling, the underlying mechanism for the cAMP burst remains unclear. Here we show that among the 17 adenylate cyclase genes present in M. tuberculosis, at least one (Rv0386) is required for virulence. Furthermore, we demonstrate that the Rv0386 adenylate cyclase facilitates delivery of bacterial-derived cAMP into the macrophage cytoplasm. Loss of Rv0386 and the intramacrophage cAMP it delivers results in reductions in TNF-alpha production via the protein kinase A and cAMP response-element-binding protein pathway, decreased immunopathology in animal tissues, and diminished bacterial survival. Direct intoxication of host cells by bacterial-derived cAMP may enable M. tuberculosis to modify both its intracellular and tissue environments to facilitate its long-term survival.

  14. Spectral response of multi-element silicon detectors

    Energy Technology Data Exchange (ETDEWEB)

    Ludewigt, B.A.; Rossington, C.S.; Chapman, K. [Univ. of California, Berkeley, CA (United States)


    Multi-element silicon strip detectors, in conjunction with integrated circuit pulse-processing electronics, offer an attractive alternative to conventional lithium-drifted silicon Si(Li) and high purity germanium detectors (HPGe) for high count rate, low noise synchrotron x-ray fluorescence applications. One of the major differences between the segmented Si detectors and the commercially available single-element Si(Li) or HPGe detectors is that hundreds of elements can be fabricated on a single Si substrate using standard silicon processing technologies. The segmentation of the detector substrate into many small elements results in very low noise performance at or near, room temperature, and the count rate of the detector is increased many-fold due to the multiplication in the total number of detectors. Traditionally, a single channel of detector with electronics can handle {approximately}100 kHz count rates while maintaining good energy resolution; the segmented detectors can operate at greater than MHz count rates merely due to the multiplication in the number of channels. One of the most critical aspects in the development of the segmented detectors is characterizing the charge sharing and charge loss that occur between the individual detector strips, and determining how these affect the spectral response of the detectors.

  15. AMP is an adenosine A1 receptor agonist. (United States)

    Rittiner, Joseph E; Korboukh, Ilia; Hull-Ryde, Emily A; Jin, Jian; Janzen, William P; Frye, Stephen V; Zylka, Mark J


    Numerous receptors for ATP, ADP, and adenosine exist; however, it is currently unknown whether a receptor for the related nucleotide adenosine 5'-monophosphate (AMP) exists. Using a novel cell-based assay to visualize adenosine receptor activation in real time, we found that AMP and a non-hydrolyzable AMP analog (deoxyadenosine 5'-monophosphonate, ACP) directly activated the adenosine A(1) receptor (A(1)R). In contrast, AMP only activated the adenosine A(2B) receptor (A(2B)R) after hydrolysis to adenosine by ecto-5'-nucleotidase (NT5E, CD73) or prostatic acid phosphatase (PAP, ACPP). Adenosine and AMP were equipotent human A(1)R agonists in our real-time assay and in a cAMP accumulation assay. ACP also depressed cAMP levels in mouse cortical neurons through activation of endogenous A(1)R. Non-selective purinergic receptor antagonists (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid and suramin) did not block adenosine- or AMP-evoked activation. Moreover, mutation of His-251 in the human A(1)R ligand binding pocket reduced AMP potency without affecting adenosine potency. In contrast, mutation of a different binding pocket residue (His-278) eliminated responses to AMP and to adenosine. Taken together, our study indicates that the physiologically relevant nucleotide AMP is a full agonist of A(1)R. In addition, our study suggests that some of the physiological effects of AMP may be direct, and not indirect through ectonucleotidases that hydrolyze this nucleotide to adenosine.

  16. Actin induction during PMA and cAMP-dependent signal pathway activation in Entamoeba histolytica trophozoites. (United States)

    Ortiz, D; del Carmen Dominguez-Robles, M; Villegas-Sepúlveda, N; Meza, I


    Activation of PKC or cAMP-dependent signalling pathways in Entamoeba histolytica triggers the phosphorylation of proteins involved in actin rearrangements necessary for adhesion and locomotion. Analogous motifs to SRE and CRE sequences--known to respond to PMA and cAMP--were identified within the 5' regulatory region (5'RR) of one of the parasite actin genes. These sequences could be involved in the actin transcriptional upregulation reported during signalling. To test this hypothesis, a plasmid containing the 5'RR of the actin gene fused to the bacterial neomycin gene (neo) was used for stable transfection. Expression of neo and endogenous actin was measured after stimulation of transfected amoebae by PMA and dcAMP. It was found that both compounds induced neo and actin expression and showed a co-operative effect in the induction of neo. Induction by PMA or dcAMP failed if the directing amoebic 5'RR lacked SRE and CRE motifs. Transfection of amoebae with plasmid constructs, containing either progressive deletions of the actin 5'RR or site-directed mutations of the SRE and CRE-like motifs, corroborated that these sequences and a co-ordinated participation of PKC- and PKA-activated transcription factors are responsible for the increments in neo and actin mRNAs. In vivo, these PMA and cAMP-response elements could play an important role in regulating actin expression and organization in signalling processes activated during tissue invasion.

  17. IP{sub 3}-dependent intracellular Ca{sup 2+} release is required for cAMP-induced c-fos expression in hippocampal neurons

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Wenting; Tingare, Asmita; Ng, David Chi-Heng [Department of Pharmacology, University of Cambridge (United Kingdom); Johnson, Hong W.; Schell, Michael J. [Department of Pharmacology, Uniformed Services University, Bethesda (United States); Lord, Rebecca L. [Department of Biology, University of York (United Kingdom); Chawla, Sangeeta, E-mail: [Department of Pharmacology, University of Cambridge (United Kingdom); Department of Biology, University of York (United Kingdom)


    Highlights: Black-Right-Pointing-Pointer cAMP-induced c-fos expression in hippocampal neurons requires a submembraneous Ca{sup 2+} pool. Black-Right-Pointing-Pointer The submembraneous Ca{sup 2+} pool derives from intracellular ER stores. Black-Right-Pointing-Pointer Expression of IP{sub 3}-metabolizing enzymes inhibits cAMP-induced c-fos expression. Black-Right-Pointing-Pointer SRE-mediated and CRE-mediated gene expression is sensitive to IP{sub 3}-metabolizing enzymes. Black-Right-Pointing-Pointer Intracellular Ca{sup 2+} release is required for cAMP-induced nuclear translocation of TORC1. -- Abstract: Ca{sup 2+} and cAMP are widely used in concert by neurons to relay signals from the synapse to the nucleus, where synaptic activity modulates gene expression required for synaptic plasticity. Neurons utilize different transcriptional regulators to integrate information encoded in the spatiotemporal dynamics and magnitude of Ca{sup 2+} and cAMP signals, including some that are Ca{sup 2+}-responsive, some that are cAMP-responsive and some that detect coincident Ca{sup 2+} and cAMP signals. Because Ca{sup 2+} and cAMP can influence each other's amplitude and spatiotemporal characteristics, we investigated how cAMP acts to regulate gene expression when increases in intracellular Ca{sup 2+} are buffered. We show here that cAMP-mobilizing stimuli are unable to induce expression of the immediate early gene c-fos in hippocampal neurons in the presence of the intracellular Ca{sup 2+} buffer BAPTA-AM. Expression of enzymes that attenuate intracellular IP{sub 3} levels also inhibited cAMP-dependent c-fos induction. Synaptic activity induces c-fos transcription through two cis regulatory DNA elements - the CRE and the SRE. We show here that in response to cAMP both CRE-mediated and SRE-mediated induction of a luciferase reporter gene is attenuated by IP{sub 3} metabolizing enzymes. Furthermore, cAMP-induced nuclear translocation of the CREB coactivator TORC1 was inhibited

  18. PVP/(AA-co-AMPS)互穿网络水凝胶的制备及其电场响应性研究%Study on Preparation and Electronic Responsivity of PVP/(AA-co-AMPS) Novel IPN Hydrogels

    Institute of Scientific and Technical Information of China (English)

    马驰; 陈尔凡; T Vladlkova; 刘艺



  19. Response of structural concrete elements to severe impulsive loads (United States)

    Krauthammer, T.; Shanaa, H. M.; Assadi, A.


    The behavior and response of structural concrete elements under severe short duration dynamic loads was investigated numerically. The analytical approach utilized the Timoshenko beam theory for the analysis of reinforced concrete beams and one-way slabs. Nonlinear material models were used to derive the flexural and shear resistances, and the differential equations of the Timoshenko beam theory were solved numerically by applying the finite difference technique. A simplified approach was developed for estimating the strain rate in structural concrete members, and the corresponding strain rate effects on the strength of the steel and concrete were incorporated into the analysis. Detailed failure criteria were established for predicting the collapse of structural concrete members. Five cases subjected to localized impact loads and eleven cases subjected to distributed explosive loads were analyzed, and the results were compared to experimental data obtained by other investigators.

  20. Phosphodiesterase 4 and compartmentalization of cyclic AMP signaling

    Institute of Scientific and Technical Information of China (English)

    WANG ZhengChao; SHI FangXiong


    Cyclic AMP (cAMP), as a second messenger, plays a critical role in cellular signaling transduction. However, it is not clear how this apparently identical cAMP signal induces divergent physiological responses. The potential explanation that cAMP signaling is compartmentalized was proposed by Buxton and Brunton twenty years ago. Compartmentalization of cAMP signaling allows spatially distinct pools of protein kinase A (PKA) to be differently activated. Research on cAMP signaling has regained impetus in many fields of life sciences due to the progress in understanding cAMP signaling complexity and functional diversity. The cAMP/PKA signaling compartments are maintained by A-kinase anchoring proteins (AKAPs) which bind PKA and other signaling proteins, and by PDEs which hydrolyse cAMP and thus terminate PKA activity. PDE4 enzymes belong to PDE superfamily and stand at a crossroad that allows them to integrate various signaling pathways with that of cAMP in spatially distinct compartments. In the current review, the nomenclature, taxonomy and gene expression of PDE4, and the system and region of its effect are described. In addition, the idiographic molecules, mechanisms, and regulation models of PDE4 are summarized. Furthermore, the important roles PDE4 plays in the maturation of rat granulosa cells and cAMP signaling compartmentalization are discussed.

  1. The Role of Cyclic AMP and Its Relationship to Parathyroid Hormone Response in an In Vitro Model of Chondrogenesis. (United States)


    meaningful and complete information. When a report is prepared in more than one volume , repeat the primary title, add volume number, and include al. (1990) found human PTH (hPTH) 1-34 inhibited ALPase activity and stimulated mitogenesis in mandibular condylar cartilage in vitro. of cartilage cells in vivo. Copray et al. (1988) observed prominent differences in the response to PTH by rat secondary mandibular condylar and

  2. Manassantin A inhibits cAMP-induced melanin production by down-regulating the gene expressions of MITF and tyrosinase in melanocytes. (United States)

    Lee, Hwa Dong; Lee, Won-Hee; Roh, Eunmiri; Seo, Chang-Seob; Son, Jong-Keun; Lee, Seung Ho; Hwang, Bang Yeon; Jung, Sang-Hun; Han, Sang-Bae; Kim, Youngsoo


    Microphthalmia-associated transcription factor (MITF) is inducible in response to cAMP through the cAMP-responsive element-binding protein (CREB) and plays a pivotal role in the melanocyte-specific expression of tyrosinase or tyrosinase-related proteins (TRPs) for melanin biosynthesis. Manassantin A from Saururus chinensis inhibits cAMP-induced melanin production in B16 melanoma cells. Here, we focused on molecular basis of the antimelanogenic activity. Manassantin A consistently inhibited the cAMP elevator 3-isobutyl-1-methylxanthine (IBMX)- or dibutyryl cAMP-induced melanin production in B16 cells or in melan-a melanocytes by down-regulating the expression of tyrosinase or TRP1 gene. Moreover, manassantin A suppressed MITF induction through IBMX-activated CREB pathway, directly inhibiting the Ser-133 phosphorylation of CREB. However, manassantin A did not affect IBMX-increased cAMP levels in these cells but also other cAMP-dependent melanogenic pathways through post-translational modifications of MITF. This putative molecular mechanism of manassantin A in the inhibition of melanin production suggests its pharmacological potential in skin hyperpigmentation.

  3. Activation of the adenylyl cyclase/cyclic AMP/protein kinase A pathway in endothelial cells exposed to cyclic strain (United States)

    Cohen, C. R.; Mills, I.; Du, W.; Kamal, K.; Sumpio, B. E.


    The aim of this study was to assess the involvement of the adenylyl cyclase/cyclic AMP/protein kinase A pathway (AC) in endothelial cells (EC) exposed to different levels of mechanical strain. Bovine aortic EC were seeded to confluence on flexible membrane-bottom wells. The membranes were deformed with either 150 mm Hg (average 10% strain) or 37.5 mm Hg (average 6% strain) vacuum at 60 cycles per minute (0.5 s strain; 0.5 s relaxation) for 0-60 min. The results demonstrate that at 10% average strain (but not 6% average strain) there was a 1.5- to 2.2-fold increase in AC, cAMP, and PKA activity by 15 min when compared to unstretched controls. Further studies revealed an increase in cAMP response element binding protein in EC subjected to the 10% average strain (but not 6% average strain). These data support the hypothesis that cyclic strain activates the AC/cAMP/PKA signal transduction pathway in EC which may occur by exceeding a strain threshold and suggest that cyclic strain may stimulate the expression of genes containing cAMP-responsive promoter elements.

  4. The interplay between cyclic AMP and insulin during obesity development

    DEFF Research Database (Denmark)

    Borkowski, Kamil

    Insulin and cAMP signalling are related to two opposite metabolic responses. Insulin secretion is elicited in response to food availability and trigger catabolic processes like lipogenesis and glycogen synthesis with a purpose of energy storage. On the other hand cAMP signalling is associated wit...

  5. Analogs of cyclic AMP as chemoattractants and inhibitors of Dictyostelium chemotaxis. (United States)

    Van Haastert, P J; Jastorff, B; Pinas, J E; Konijn, T M


    Aggregative amoebae of Dictyostelium discoideum, D. mucoroides, D. purpureum, and D. rosarium react chemotactically to cyclic AMP (cAMP). We measured the chemotactic activity of 14 cAMP analogs and found that these four species have a similar sensitivity to chemical modifications of cAMP; this suggests that the cAMP receptor is identical in all of these species. Besides the induction of a chemotactic response, cAMP analogs also may delay or prevent cell aggregation. cAMP analogs like N1-O-cAMP, 2'-H-cAMP, and 5'NH-cAMP are chemotactically nearly as active as cAMP and induced no, or only a short, delay of cell aggregation. Other cAMP derivatives, such as 6-Cl-cPMP and 8-Br-cAMP, are chemotactically active only at high concentrations and delayed cell aggregation for several hours. Still other cAMP analogs, which do not induce a chemotactic reaction in D. mucoroides, D. purpureum, and D. rosarium, either prevented cell aggregation [cAMPS(S), cAMPS(R), and 3'-NH-cAMP[ or had no effect on cell aggregation [cAMPN(CH3)2(S) and cAMPN(CH3)2(R)]. cAMP analog 3'-NH-cAMP prevented cell aggregation by the inhibition of chemotaxis, whereas cell locomotion was not affected. Although we cannot provide a satisfactory explantation for these observations, our data suggest that occupation and activation of the cAMP receptors do not always induced a chemotactic response.

  6. Directed evolution of the Escherichia coli cAMP receptor protein at the cAMP pocket. (United States)

    Gunasekara, Sanjiva M; Hicks, Matt N; Park, Jin; Brooks, Cory L; Serate, Jose; Saunders, Cameron V; Grover, Simranjeet K; Goto, Joy J; Lee, Jin-Won; Youn, Hwan


    The Escherichia coli cAMP receptor protein (CRP) requires cAMP binding to undergo a conformational change for DNA binding and transcriptional regulation. Two CRP residues, Thr(127) and Ser(128), are known to play important roles in cAMP binding through hydrogen bonding and in the cAMP-induced conformational change, but the connection between the two is not completely clear. Here, we simultaneously randomized the codons for these two residues and selected CRP mutants displaying high CRP activity in a cAMP-producing E. coli. Many different CRP mutants satisfied the screening condition for high CRP activity, including those that cannot form any hydrogen bonds with the incoming cAMP at the two positions. In vitro DNA-binding analysis confirmed that these selected CRP mutants indeed display high CRP activity in response to cAMP. These results indicate that the hydrogen bonding ability of the Thr(127) and Ser(128) residues is not critical for the cAMP-induced CRP activation. However, the hydrogen bonding ability of Thr(127) and Ser(128) was found to be important in attaining high cAMP affinity. Computational analysis revealed that most natural cAMP-sensing CRP homologs have Thr/Ser, Thr/Thr, or Thr/Asn at positions 127 and 128. All of these pairs are excellent hydrogen bonding partners and they do not elevate CRP activity in the absence of cAMP. Taken together, our analyses suggest that CRP evolved to have hydrogen bonding residues at the cAMP pocket residues 127 and 128 for performing dual functions: preserving high cAMP affinity and keeping CRP inactive in the absence of cAMP.

  7. The role of cAMP in synaptic homeostasis in response to environmental temperature challenges and hyperexcitability mutations

    Directory of Open Access Journals (Sweden)

    Atsushi eUeda


    Full Text Available Homeostasis is the ability of physiological systems to regain functional balance following environment or experimental insults and synaptic homeostasis has been demonstrated in various species following genetic or pharmacological disruptions. Among environmental challenges, homeostatic responses to temperature extremes are critical to animal survival under natural conditions. We previously reported that axon terminal arborization in Drosophila larval neuromuscular junctions is enhanced at elevated temperatures; however, the amplitude of excitatory junctional potentials (EJPs remains unaltered despite the increase in synaptic bouton numbers. Here we determine the cellular basis of this homeostatic adjustment in larvae reared at high temperature (HT, 29 ˚C. We found that synaptic current focally recorded from individual synaptic boutons was unaffected by rearing temperature (30 ˚C. However, HT rearing decreased the quantal size (amplitude of spontaneous miniature EJPs, or mEJPs, which compensates for the increased number of synaptic releasing sites to retain a normal EJP size. The quantal size decrease is accounted for by a decrease in input resistance of the postsynaptic muscle fiber, indicating an increase in membrane area that matches the synaptic growth at HT. Interestingly, a mutation in rutabaga (rut encoding adenylyl cyclase (AC exhibited no obvious changes in quantal size or input resistance of postsynaptic muscle cells after HT rearing, suggesting an important role for rut AC in temperature-induced synaptic homeostasis in Drosophila. This extends our previous finding of rut-dependent synaptic homeostasis in hyperexcitable mutants, e.g. slowpoke (slo. In slo larvae, the lack of BK channel function is partially ameliorated by upregulation of presynaptic Sh IA current to limit excessive transmitter release in addition to postsynaptic glutamate receptor recomposition that reduces the quantal size.

  8. Vibration Response of Multi Storey Building Using Finite Element Modelling (United States)

    Chik, T. N. T.; Zakaria, M. F.; Remali, M. A.; Yusoff, N. A.


    Interaction between building, type of foundation and the geotechnical parameter of ground may trigger a significant effect on the building. In general, stiffer foundations resulted in higher natural frequencies of the building-soil system and higher input frequencies are often associated with other ground. Usually, vibrations transmitted to the buildings by ground borne are often noticeable and can be felt. It might affect the building and become worse if the vibration level is not controlled. UTHM building is prone to the ground borne vibration due to closed distance from the main road, and the construction activities adjacent to the buildings. This paper investigates the natural frequency and vibration mode of multi storey office building with the presence of foundation system and comparison between both systems. Finite element modelling (FEM) package software of LUSAS is used to perform the vibration analysis of the building. The building is modelled based on the original plan with the foundation system on the structure model. The FEM results indicated that the structure which modelled with rigid base have high natural frequency compare to the structure with foundation system. These maybe due to soil structure interaction and also the damping of the system which related to the amount of energy dissipated through the foundation soil. Thus, this paper suggested that modelling with soil is necessary to demonstrate the soil influence towards vibration response to the structure.

  9. Postaggregative differentiation induction by cyclic AMP in Dictyostelium: intracellular transduction pathway and requirement for additional stimuli. (United States)

    Schaap, P; Van Lookeren Campagne, M M; Van Driel, R; Spek, W; Van Haastert, P J; Pinas, J


    Cyclic AMP induces postaggregative differentiation in aggregation competent cells of Dictyostelium by interacting with cell surface cAMP receptors. We investigated the transduction pathway of this response and additional requirements for the induction of postaggregative differentiation. Optimal induction of postaggregative gene expression requires that vegetative cells are first exposed to 2-4 hr of nanomolar cAMP pulses, and subsequently for 4-6 hr to steady-state cAMP concentrations in the micromolar range. Cyclic AMP pulses, which are endogenously produced before and during aggregation, induce full responsiveness to cAMP as a morphogen. The transduction pathway from the cell surface cAMP receptor to postaggregative gene expression may involve Ca2+ ions as intracellular messengers. A cAMP-induced increase in intracellular cAMP or cGMP levels is not involved in the transduction pathway.

  10. Postaggregative Differentiation Induction by Cyclic AMP in Dictyostelium : Intracellular Transduction Pathway and Requirement for Additional Stimuli

    NARCIS (Netherlands)

    Schaap, Pauline; Lookeren Campagne, Michiel M. van; Driel, Roel van; Spek, Wouter; Haastert, Peter J.M. van; Pinas, Johan


    Cyclic AMP induces postaggregative differentiation in aggregation competent cells of Dictyostelium by interacting with cell surface cAMP receptors. We investigated the transduction pathway of this response and additional requirements for the induction of postaggregative differentiation. Optimal indu

  11. The alpha2-5'AMP-activated protein kinase is a site 2 glycogen synthase kinase in skeletal muscle and is responsive to glucose loading

    DEFF Research Database (Denmark)

    Jørgensen, Sebastian B; Nielsen, Jakob N.; Birk, Jesper Bratz


    The 5'AMP-activated protein kinase (AMPK) is a potential antidiabetic drug target. Here we show that the pharmacological activation of AMPK by 5-aminoimidazole-1-beta-4-carboxamide ribofuranoside (AICAR) leads to inactivation of glycogen synthase (GS) and phosphorylation of GS at Ser 7 (site 2). ...

  12. Experimental investigation of motion responses of tunnel element immerging by moored barge

    Institute of Scientific and Technical Information of China (English)

    左卫广; 王永学


    In this paper, the barge effect on the motion responses of the tunnel element immerging by the moored barge under waves is investigated experimentally. Both the motion responses of the tunnel element and the moored barge in the experiment are simultaneously acquired by the Untouched 6-D Measurement System. The results show that the sway motion responses of the tunnel element immerging by the moored barge are different from those without the barge. For the system of the tunnel element and the moored barge, the moored barge has two motion components in the sway direction. The high frequency motion of the moored barge has little effect on the high frequency motion of the tunnel element with moored barge. However, the low frequency motion of the moored barge has a significant effect on the sway motion of the tunnel element. The motion responses of the tunnel element and the barge in the heave and roll directions are mainly the high frequency motion.

  13. Induction of nuclear protein factors specific for hormone-responsive region during activation of thyroglobulin gene by thyrotropin in rat thyroid FRTL-5 cells. (United States)

    Lee, N T; Nayfeh, S N; Chae, C B


    We have investigated the mechanism of stimulation of thyroglobulin gene expression by thyrotropin (TSH) and cAMP in rat thyroid FRTL-5 cells. In contrast to the c-fos gene, induction of the thyroglobulin gene by TSH or cAMP is slow (10 h) and sensitive to cycloheximide treatment. We have identified a TSH and cAMP-responsive region of thyroglobulin gene between - 171 and - 140 base pairs from the transcription initiation site. The hormone-responsive region contains DNA sequence elements similar to the consensus cAMP-responsive element as well as the transcription factor AP-1-binding site but with opposite sequence polarity. Three DNA-protein complexes are formed when the hormone-responsive region is incubated with nuclear extracts of FRTL-5 cells. Formation of these complexes is dependent on TSH or cAMP stimulation, thus suggesting that the factors involved in binding to the hormone-responsive region may be induced by TSH. Although the identity of these factors is not yet known, they do not appear to be related to either cAMP-responsive element-binding protein or AP-1. These results suggest that thyroglobulin gene expression in FRTL-5 cells may be mediated by nuclear factors that are induced by cAMP in contrast to other genes (e.g. c-fos) whose activation involves post-translational modification of the pre-existing proteins specific for cAMP-responsive element.

  14. Lichens (Parmelia sulcata) time response model to environmental elemental availability

    NARCIS (Netherlands)

    Reis, M.A.; Alves, L.C.; Freitas, M.C.; Os, B. van; Wolterbeek, H.T.


    Parmelia sulcata transplants, collected in a non-polluted area, were exposed to new atmospheric conditions at six stations, of which five were located near power plants and one at an unpolluted area. Data were collected for a 1-year period, on rainfall, airborne particulates, elemental deposition an

  15. Protein kinase A and mitogen-activated protein kinase pathways mediate cAMP induction of alpha-epithelial Na+ channels (alpha-ENaC). (United States)

    Mustafa, Shamimunisa B; Castro, Robert; Falck, Alison J; Petershack, Jean A; Henson, Barbara M; Mendoza, Yvonne M; Choudary, Ahsan; Seidner, Steven R


    A major mechanism for Na+ transport across epithelia occurs through epithelial Na+ channels (ENaC). ENaC is a multimeric channel consisting of three subunits (alpha, beta, and gamma). The alpha-subunit is critical for ENaC function. In specific culture conditions, the rat submandibular gland epithelial cell line (SMG-C6) demonstrates minimal Na+ transport properties and exposure to dibutyryl cAMP (DbcAMP) for up to 48 h caused an elevation of alpha-ENaC mRNA and protein expression and amiloride-sensitive short-circuit current (I(SC)). Here we examined the early signaling pathways evoked by DbcAMP which contribute to the eventual increase in Na+ transport is present. Treatment with either of the protein kinase A (PKA) inhibitors KT5720 or H-89 followed by exposure to 1 mM DbcAMP for 24 h markedly attenuated DbcAMP-induced alpha-ENaC protein formation and I(SC). Exposure of SMG-C6 cells to 1 mM DbcAMP induced a rapid, transient phosphorylation of the cAMP response element binding protein (CREB). This response was attenuated in the presence of either KT5720 or H-89. Dominant-negative CREB decreased DbcAMP-induced alpha-ENaC expression. Suppression of the extracellular signal-regulated protein kinase (ERK 1,2) with PD98059 or the p38 mitogen-activated protein kinase (MAPK) pathway with SB203580 reduced DbcAMP-induced alpha-ENaC protein levels in SMG-C6 cells. DbcAMP-induced phosphorylation of CREB was markedly attenuated by PD98059 or SB203580. DbcAMP-induced activation of the either the p38 or the ERK 1,2 MAPK pathways was abolished by either of the PKA inhibitors, H-89 or KT5720. Cross talk between these signaling pathways induced by DbcAMP via the activation of CREB appears to contribute to increased levels of alpha-ENaC observed after 24 h of treatment in SMG-C6 epithelial cells.

  16. cAMP receptor protein (CRP) downregulates Klebsiella pneumoniae nif promoters in Escherichia coli

    Institute of Scientific and Technical Information of China (English)


    In enteric bacteria, in response to the PTS system, the cAMP receptor protein (CRP) mediates the glucose effect, via regulating s70-dependent catabolic genes at transcriptional level. In this study, it is observed that the nitrogen fixation capacity of Klebsiella pneumoniae varies strongly when cells are grown on different carbohydrates, and this carbon effect occurs at the level of nif gene expression. Here we show that CRP can repress s54-dependent nif promoters (nifB, nifE, nifF, nifH, nifJ, nifLA and nifU), in a cAMP dependent fashion, in closed related E. coli background. Sequence analysis of these nif promoters indicates that there is no direct correlation between the fold of CRP-cAMP-mediated inhibition and the upstream cis elements at the promoters. In addition, the crp gene of K. pneumoniae has been isolated and sequenced, which is structural and functional highly homologous to that of E. coli. This suggests that CRP-cAMP-mediated inhibition on the nif promoters could be the reason for carbon effect on nitrogen fixation and thus has its physiological significance. A novel regulatory linkage between carbon metabolism and nitrogen fixation is proposed.

  17. Finite element model calibration using frequency responses with damping equalization (United States)

    Abrahamsson, T. J. S.; Kammer, D. C.


    Model calibration is a cornerstone of the finite element verification and validation procedure, in which the credibility of the model is substantiated by positive comparison with test data. The calibration problem, in which the minimum deviation between finite element model data and experimental data is searched for, is normally characterized as being a large scale optimization problem with many model parameters to solve for and with deviation metrics that are nonlinear in these parameters. The calibrated parameters need to be found by iterative procedures, starting from initial estimates. Sometimes these procedures get trapped in local deviation function minima and do not converge to the globally optimal calibration solution that is searched for. The reason for such traps is often the multi-modality of the problem which causes eigenmode crossover problems in the iterative variation of parameter settings. This work presents a calibration formulation which gives a smooth deviation metric with a large radius of convergence to the global minimum. A damping equalization method is suggested to avoid the mode correlation and mode pairing problems that need to be solved in many other model updating procedures. By this method, the modal damping of a test data model and the finite element model is set to be the same fraction of critical modal damping. Mode pairing for mapping of experimentally found damping to the finite element model is thus not needed. The method is combined with model reduction for efficiency and employs the Levenberg-Marquardt minimizer with randomized starts to achieve the calibration solution. The performance of the calibration procedure, including a study of parameter bias and variance under noisy data conditions, is demonstrated by two numerical examples.

  18. Systems biology investigation of cAMP modulation to increase SMN levels for the treatment of spinal muscular atrophy.

    Directory of Open Access Journals (Sweden)

    Sean G Mack

    Full Text Available Spinal muscular atrophy (SMA, a leading genetic cause of infant death worldwide, is an autosomal recessive disorder caused by the loss of SMN1 (survival motor neuron 1, which encodes the protein SMN. The loss of SMN1 causes a deficiency in SMN protein levels leading to motor neuron cell death in the anterior horn of the spinal cord. SMN2, however, can also produce some functional SMN to partially compensate for loss of SMN1 in SMA suggesting increasing transcription of SMN2 as a potential therapy to treat patients with SMA. A cAMP response element was identified on the SMN2 promoter, implicating cAMP activation as a step in the transcription of SMN2. Therefore, we investigated the effects of modulating the cAMP signaling cascade on SMN production in vitro and in silico. SMA patient fibroblasts were treated with the cAMP signaling modulators rolipram, salbutamol, dbcAMP, epinephrine and forskolin. All of the modulators tested were able to increase gem formation, a marker for SMN protein in the nucleus, in a dose-dependent manner. We then derived two possible mathematical models simulating the regulation of SMN2 expression by cAMP signaling. Both models fit well with our experimental data. In silico treatment of SMA fibroblasts simultaneously with two different cAMP modulators resulted in an additive increase in gem formation. This study shows how a systems biology approach can be used to develop potential therapeutic targets for treating SMA.

  19. AMP N1-Oxide, a Unique Compound of Royal Jelly, Induces Neurite Outgrowth from PC12 Vells via Signaling by Protein Kinase A Independent of that by Mitogen-Activated Protein Kinase

    Directory of Open Access Journals (Sweden)

    Noriko Hattori


    Full Text Available Earlier we identified adenosine monophosphate (AMP N1-oxide as a unique compound of royal jelly (RJ that induces neurite outgrowth (neuritegenesis from cultured rat pheochromocytoma PC12 cells via the adenosine A2A receptor. Now, we found that AMP N1-oxide stimulated the phosphorylation of not only mitogen-activated protein kinase (MAPK but also that of cAMP/calcium-response element-binding protein (CREB in a dose-dependent manner. Inhibition of MAPK activation by a MEK inhibitor, PD98059, did not influence the AMP N1-oxide-induced neuritegenesis, whereas that of protein kinase A (PKA by a selective inhibitor, KT5720, significantly reduced neurite outgrowth. AMP N1-oxide also had the activity of suppressing the growth of PC12 cells, which correlated well with the neurite outgrowth-promoting activity. KT5720 restored the growth of AMP N1-oxide-treated PC12 cells. It is well known that nerve growth factor suppresses proliferation of PC12 cells before causing stimulation of neuronal differentiation. Thus, AMP N1-oxide elicited neuronal differentiation of PC12 cells, as evidenced by generation of neurites, and inhibited cell growth through adenosine A2A receptor-mediated PKA signaling, which may be responsible for characteristic actions of RJ.

  20. Modeling Reader's Emotional State Response on Document's Typographic Elements

    Directory of Open Access Journals (Sweden)

    Dimitrios Tsonos


    Full Text Available We present the results of an experimental study towards modeling the reader's emotional state variations induced by the typographic elements in electronic documents. Based on the dimensional theory of emotions we investigate how typographic elements, like font style (bold, italics, bold-italics and font (type, size, color and background color, affect the reader's emotional states, namely, Pleasure, Arousal, and Dominance (PAD. An experimental procedure was implemented conforming to International Affective Picture System guidelines and incorporating the Self-Assessment Manikin test. Thirty students participated in the experiment. The stimulus was a short paragraph of text for which any content, emotion, and/or domain dependent information was excluded. The Analysis of Variance revealed the dependency of (a all the three emotional dimensions on font size and font/background color combinations and (b the Pleasure dimension on font type and font style. We introduce a set of mapping rules showing how PAD vary on the discrete values of font style and font type elements. Moreover, we introduce a set of equations describing the PAD dimensions' dependency on font size. This novel model can contribute to the automated reader's emotional state extraction in order, for example, to enhance the acoustic rendition of the documents, utilizing text-to-speech synthesis.

  1. Finite-element impact response of debonded composite turbine blades (United States)

    Dey, Sudip; Karmakar, Amit


    This paper investigates on the transient behavior of debonded composite pretwisted rotating shallow conical shells which could be idealized as turbine blades subjected to low velocity normal impact using finite-element method. Lagrange's equation of motion is used to derive the dynamic equilibrium equation and the moderate rotational speeds are considered neglecting the Coriolis effect. An eight-noded isoparametric plate bending element is employed in the finite element formulation incorporating rotary inertia and effects of transverse shear deformation based on Mindlin's theory. The modified Hertzian contact law which accounts for permanent indentation is utilized to compute the impact parameters. The time-dependent equations are solved by using Newmark's time integration scheme. Parametric studies are performed to investigate the effects of triggering parameters like angle of twist, rotational speed, laminate configuration and location of debonding considering low velocity normal impact at the center of eight-layered graphite-epoxy composite cantilevered conical shells with bending stiff ([0o2/{±} 30o]s), torsion stiff ([45°/-45°/-45°/45°]s) and cross-ply ([0°/90°/0°/90°]s) laminate configurations.

  2. Increases in cAMP, MAPK activity, and CREB phosphorylation during REM sleep: implications for REM sleep and memory consolidation. (United States)

    Luo, Jie; Phan, Trongha X; Yang, Yimei; Garelick, Michael G; Storm, Daniel R


    The cyclic adenosine monophosphate (cAMP), mitogen-activated protein kinase (MAPK), and cAMP response element-binding protein (CREB) transcriptional pathway is required for consolidation of hippocampus-dependent memory. In mice, this pathway undergoes a circadian oscillation required for memory persistence that reaches a peak during the daytime. Because mice exhibit polyphasic sleep patterns during the day, this suggested the interesting possibility that cAMP, MAPK activity, and CREB phosphorylation may be elevated during sleep. Here, we report that cAMP, phospho-p44/42 MAPK, and phospho-CREB are higher in rapid eye movement (REM) sleep compared with awake mice but are not elevated in non-REM sleep. This peak of activity during REM sleep does not occur in mice lacking calmodulin-stimulated adenylyl cyclases, a mouse strain that learns but cannot consolidate hippocampus-dependent memory. We conclude that a preferential increase in cAMP, MAPK activity, and CREB phosphorylation during REM sleep may contribute to hippocampus-dependent memory consolidation.

  3. Inhibin alpha gene expression in human trophoblasts is regulated by interactions between TFAP2 and cAMP signaling pathways. (United States)

    Depoix, Christophe L; Debiève, Frédéric; Hubinont, Corinne


    Inhibin α (Inha) gene expression is regulated, in rat granulosa cells, via a cyclic 3',5'-adenosine monophosphate (AMP)-response element (CRE) found in a region of the promoter that is homologous to the human INHA promoter. We previously found that during in vitro cytotrophoblast differentiation, human INHA gene expression was regulated by TFAP2A via association with an AP-2 site located upstream of this CRE. The aim of this study was to evaluate if the human INHA gene was also regulated by cAMP in trophoblasts, and to investigate the possible crosstalk between TFAP2 and cAMP signaling pathways in the regulation of INHA gene expression. Treatment with cAMP or forskolin increased INHA mRNA expression by 7- and 2-fold in primary cytotrophoblasts and choriocarcinoma-derived BeWo cells, respectively. Treatment with the protein kinase A inhibitor H-89 reduced forskolin-induced luciferase activity by ∼40% in BeWo cells transfected with an INHA promoter-driven luciferase reporter vector. TFAP2 overexpression increased basal luciferase activity, whereas the dominant repressor KCREB abolished it. Surprisingly, mutation of the CRE also eliminated the TFAP2-induced transcription, although TFAP2 overexpression was still able to increase forskolin-induced luciferase activity when the AP-2 binding site, but not the CRE site, was mutated. Thus, INHA gene expression is upregulated by cAMP via CRE in human trophoblasts, and TFAP2 regulates this expression by interacting with CRE.

  4. Cis-element of the rice PDIL2-3 promoter is responsible for inducing the endoplasmic reticulum stress response. (United States)

    Takahashi, Hideyuki; Wang, Shuyi; Hayashi, Shimpei; Wakasa, Yuhya; Takaiwa, Fumio


    A protein disulfide isomerase (PDI) family oxidoreductase, PDIL2-3, is involved in endoplasmic reticulum (ER) stress responses in rice. We identified a critical cis-element required for induction of the ER stress response. The activation of PDIL2-3 in response to ER stress strongly depends on the IRE1-OsbZIP50 signaling pathway.

  5. YC-1 potentiates cAMP-induced CREB activation and nitric oxide production in alveolar macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, Tsong-Long, E-mail: [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China); Chinese Herbal Medicine Research Team, Healthy Aging Research Center, Chang Gung University, Kweishan, Taoyuan, Taiwan (China); Tang, Ming-Chi [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China); Kuo, Liang-Mou [Department of General Surgery, Chang Gung Memorial Hospital at Chia-Yi, Taiwan (China); Chang, Wen-De; Chung, Pei-Jen; Chang, Ya-Wen; Fang, Yao-Ching [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China)


    Alveolar macrophages play significant roles in the pathogenesis of several inflammatory lung diseases. Increases in exhaled nitric oxide (NO) are well documented to reflect disease severity in the airway. In this study, we investigated the effect of 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1), a known activator of soluble guanylyl cyclase, on prostaglandin (PG)E{sub 1} (a stable PGE{sub 2} analogue) and forskolin (a adenylate cyclase activator) induced NO production and inducible NO synthase (iNOS) expression in rat alveolar macrophages (NR8383). YC-1 did not directly cause NO production or iNOS expression, but drastically potentiated PGE{sub 1}- or forskolin-induced NO production and iNOS expression in NR8383 alveolar macrophages. Combination treatment with YC-1 and PGE{sub 1} significantly increased phosphorylation of the cAMP response element-binding protein (CREB), but not nuclear factor (NF)-κB activation. The combined effect on NO production, iNOS expression, and CREB phosphorylation was reversed by a protein kinase (PK)A inhibitor (H89), suggesting that the potentiating functions were mediated through a cAMP/PKA signaling pathway. Consistent with this, cAMP analogues, but not the cGMP analogue, caused NO release, iNOS expression, and CREB activation. YC-1 treatment induced an increase in PGE{sub 1}-induced cAMP formation, which occurred through the inhibition of cAMP-specific phosphodiesterase (PDE) activity. Furthermore, the combination of rolipram (an inhibitor of PDE4), but not milronone (an inhibitor of PDE3), and PGE{sub 1} also triggered NO production and iNOS expression. In summary, YC-1 potentiates PGE{sub 1}-induced NO production and iNOS expression in alveolar macrophages through inhibition of cAMP PDE activity and activation of the cAMP/PKA/CREB signaling pathway. Highlights: ► YC-1 potentiated PGE1-induced iNOS expression in alveolar macrophages. ► The combination of YC-1 and PGE1 increased CREB but not NFκB activation.

  6. Ecological responsibility of business as public-private partnership element

    Directory of Open Access Journals (Sweden)

    I.M. Potravniy


    Full Text Available In the article the questions of social and ecological responsibility of business are considered within the public-private partnership mechanism framework. Methods of the ecological significant project economic regulation are shown in wildlife management and environment protection sphere.

  7. Moral Responsibility: The Missing Element in Educational Leadership (United States)

    Vasillopulos, Christopher; Denney, Morgan


    We intend to deepen the understanding of leadership in general and educational leadership in particular by an analysis of Chester Barnard's (1938) concept of executive responsibility. By so doing we believe that we will reveal how an educational leader can foster the environment in which competent teachers can optimize their students' learning…

  8. Low-power laser irradiation suppresses inflammatory response of human adipose-derived stem cells by modulating intracellular cyclic AMP level and NF-κB activity. (United States)

    Wu, Jyun-Yi; Chen, Chia-Hsin; Wang, Chau-Zen; Ho, Mei-Ling; Yeh, Ming-Long; Wang, Yan-Hsiung


    Mesenchymal stem cell (MSC)-based tissue regeneration is a promising therapeutic strategy for treating damaged tissues. However, the inflammatory microenvironment that exists at a local injury site might restrict reconstruction. Low-power laser irradiation (LPLI) has been widely applied to retard the inflammatory reaction. The purpose of this study was to investigate the anti-inflammatory effect of LPLI on human adipose-derived stem cells (hADSCs) in an inflammatory environment. We showed that the hADSCs expressed Toll-like Receptors (TLR) 1, TLR2, TLR3, TLR4, and TLR6 and that lipopolysaccharide (LPS) significantly induced the production of pro-inflammatory cytokines (Cyclooxygenase-2 (Cox-2), Interleukin-1β (IL-1β), Interleukin-6 (IL-6), and Interleukin-8 (IL-8)). LPLI markedly inhibited LPS-induced, pro-inflammatory cytokine expression at an optimal dose of 8 J/cm². The inhibitory effect triggered by LPLI might occur through an increase in the intracellular level of cyclic AMP (cAMP), which acts to down-regulate nuclear factor kappa B (NF-κB) transcriptional activity. These data collectively provide insight for further investigations of the potential application of anti-inflammatory treatment followed by stem cell therapy.

  9. Preferential hydrophobic interactions are responsible for a preference of D-amino acids in the aminoacylation of 5'-AMP with hydrophobic amino acids (United States)

    Lacey, J. C. Jr; Wickramasinghe, N. S.; Sabatini, R. S.


    We have studied the chemistry of aminoacyl AMP to model reactions at the 3' terminus of aminoacyl tRNA for the purpose of understanding the origin of protein synthesis. The present studies relate to the D, L preference in the esterification of 5'-AMP. All N-acetyl amino acids we studied showed faster reaction of the D-isomer, with a generally decreasing preference for D-isomer as the hydrophobicity of the amino acid decreased. The beta-branched amino acids, Ile and Val, showed an extreme preference for D-isomer. Ac-Leu, the gamma-branched amino acid, showed a slightly low D/L ratio relative to its hydrophobicity. The molecular basis for these preferences for D-isomer is understandable in the light of our previous studies and seems to be due to preferential hydrophobic interaction of the D-isomer with adenine. The preference for hydrophobic D-amino acids can be decreased by addition of an organic solvent to the reaction medium. Conversely, peptidylation with Ac-PhePhe shows a preference for the LL isomer over the DD isomer.

  10. Low-power laser irradiation suppresses inflammatory response of human adipose-derived stem cells by modulating intracellular cyclic AMP level and NF-κB activity.

    Directory of Open Access Journals (Sweden)

    Jyun-Yi Wu

    Full Text Available Mesenchymal stem cell (MSC-based tissue regeneration is a promising therapeutic strategy for treating damaged tissues. However, the inflammatory microenvironment that exists at a local injury site might restrict reconstruction. Low-power laser irradiation (LPLI has been widely applied to retard the inflammatory reaction. The purpose of this study was to investigate the anti-inflammatory effect of LPLI on human adipose-derived stem cells (hADSCs in an inflammatory environment. We showed that the hADSCs expressed Toll-like Receptors (TLR 1, TLR2, TLR3, TLR4, and TLR6 and that lipopolysaccharide (LPS significantly induced the production of pro-inflammatory cytokines (Cyclooxygenase-2 (Cox-2, Interleukin-1β (IL-1β, Interleukin-6 (IL-6, and Interleukin-8 (IL-8. LPLI markedly inhibited LPS-induced, pro-inflammatory cytokine expression at an optimal dose of 8 J/cm². The inhibitory effect triggered by LPLI might occur through an increase in the intracellular level of cyclic AMP (cAMP, which acts to down-regulate nuclear factor kappa B (NF-κB transcriptional activity. These data collectively provide insight for further investigations of the potential application of anti-inflammatory treatment followed by stem cell therapy.

  11. Heterogeneity of Calcium Channel/cAMP-Dependent Transcriptional Activation. (United States)

    Kobrinsky, Evgeny


    The major function of the voltage-gated calcium channels is to provide the Ca(2+) flux into the cell. L-type voltage-gated calcium channels (Cav1) serve as voltage sensors that couple membrane depolarization to many intracellular processes. Electrical activity in excitable cells affects gene expression through signaling pathways involved in the excitation-transcription (E-T) coupling. E-T coupling starts with activation of the Cav1 channel and results in initiation of the cAMP-response element binding protein (CREB)-dependent transcription. In this review we discuss the new quantitative approaches to measuring E-T signaling events. We describe the use of wavelet transform to detect heterogeneity of transcriptional activation in nuclei. Furthermore, we discuss the properties of discovered microdomains of nuclear signaling associated with the E-T coupling and the basis of the frequency-dependent transcriptional regulation.

  12. The Popeye Domain Containing Genes and cAMP Signaling

    Directory of Open Access Journals (Sweden)

    Thomas Brand


    Full Text Available 3'-5'-cyclic adenosine monophosphate (cAMP is a second messenger, which plays an important role in the heart. It is generated in response to activation of G-protein-coupled receptors (GPCRs. Initially, it was thought that protein kinase A (PKA exclusively mediates cAMP-induced cellular responses such as an increase in cardiac contractility, relaxation, and heart rate. With the identification of the exchange factor directly activated by cAMP (EPAC and hyperpolarizing cyclic nucleotide-gated (HCN channels as cAMP effector proteins it became clear that a protein network is involved in cAMP signaling. The Popeye domain containing (Popdc genes encode yet another family of cAMP-binding proteins, which are prominently expressed in the heart. Loss-of-function mutations in mice are associated with cardiac arrhythmia and impaired skeletal muscle regeneration. Interestingly, the cardiac phenotype, which is present in both, Popdc1 and Popdc2 null mutants, is characterized by a stress-induced sinus bradycardia, suggesting that Popdc proteins participate in cAMP signaling in the sinuatrial node. The identification of the two-pore channel TREK-1 and Caveolin 3 as Popdc-interacting proteins represents a first step into understanding the mechanisms of heart rate modulation triggered by Popdc proteins.

  13. Brain-derived neurotrophic factor is produced by skeletal muscle cells in response to contraction and enhances fat oxidation via activation of AMP-activated protein kinase

    DEFF Research Database (Denmark)

    Matthews, V B; Åström, Maj-Brit; Chan, M H S


    C12 skeletal muscle cells were electrically stimulated to mimic contraction. L6 myotubes and isolated rat extensor digitorum longus muscles were treated with BDNF and phosphorylation of the proteins AMP-activated protein kinase (AMPK) (Thr(172)) and acetyl coenzyme A carboxylase beta (ACCbeta) (Ser...... kinase (p44/42 Thr(202)/Tyr(204)) phosphorylation in these muscles. In addition, phosphorylation of ACCbeta was markedly elevated in the Bdnf electroporated muscles. CONCLUSIONS/INTERPRETATION: These data identify BDNF as a contraction-inducible protein in skeletal muscle that is capable of enhancing......AIMS/HYPOTHESIS: Brain-derived neurotrophic factor (BDNF) is produced in skeletal muscle, but its functional significance is unknown. We aimed to determine the signalling processes and metabolic actions of BDNF. METHODS: We first examined whether exercise induced BDNF expression in humans. Next, C2...

  14. Prediction of transcriptional regulatory elements for plant hormone responses based on microarray data

    Directory of Open Access Journals (Sweden)

    Yamaguchi-Shinozaki Kazuko


    Full Text Available Abstract Background Phytohormones organize plant development and environmental adaptation through cell-to-cell signal transduction, and their action involves transcriptional activation. Recent international efforts to establish and maintain public databases of Arabidopsis microarray data have enabled the utilization of this data in the analysis of various phytohormone responses, providing genome-wide identification of promoters targeted by phytohormones. Results We utilized such microarray data for prediction of cis-regulatory elements with an octamer-based approach. Our test prediction of a drought-responsive RD29A promoter with the aid of microarray data for response to drought, ABA and overexpression of DREB1A, a key regulator of cold and drought response, provided reasonable results that fit with the experimentally identified regulatory elements. With this succession, we expanded the prediction to various phytohormone responses, including those for abscisic acid, auxin, cytokinin, ethylene, brassinosteroid, jasmonic acid, and salicylic acid, as well as for hydrogen peroxide, drought and DREB1A overexpression. Totally 622 promoters that are activated by phytohormones were subjected to the prediction. In addition, we have assigned putative functions to 53 octamers of the Regulatory Element Group (REG that have been extracted as position-dependent cis-regulatory elements with the aid of their feature of preferential appearance in the promoter region. Conclusions Our prediction of Arabidopsis cis-regulatory elements for phytohormone responses provides guidance for experimental analysis of promoters to reveal the basis of the transcriptional network of phytohormone responses.

  15. Effect of Large Negative Phase of Blast Loading on Structural Response of RC Elements

    Directory of Open Access Journals (Sweden)

    Syed Zubair Iman


    Full Text Available Structural response of reinforced concrete (RC elements for analysis and design are often obtained using the positive phase of the blast pressure curve disregarding the negative phase assuming insignificant contribution from the negative phase of the loading. Although, some insight on the effect of negative phase of blast pressure based on elastic single-degree-of-freedom (SDOF analysis was presented before, the influence of negative phase on different types of resistance functions of SDOF models and on realistic finite element analysis has not been explored. In this study, the effects of inclusion of pulse negative phase on structural response of RC elements from SDOF analysis and from more detailed finite element analysis have been investigated. Investigation of SDOF part has been conducted using MATLAB code that utilizes non-linear resistance functions of SDOF model. Detailed numerical investigation using finite element code DIANA was conducted on the significance of the negative phase on structural response. In the FE model, different support stiffness was used to explore the effect of support stiffness on the structural response due to blast negative phase. Results from SDOF and FE analyses present specific situations where the effect of large negative phase was found to be significant on the structural response of RC elements.

  16. Expression profiles of antimicrobial peptides (AMPs) and their regulation by Relish (United States)

    Wang, Dongdong; Li, Fuhua; Li, Shihao; Wen, Rong; Xiang, Jianhai


    Antimicrobial peptides (AMPs), as key immune effectors, play important roles in the innate immune system of invertebrates. Different types of AMPs, including Penaeidin, Crustin, ALF (antilipopolysaccharide factor) have been identified in different penaeid shrimp; however, systematic analyses on the function of different AMPs in shrimp responsive to different types of bacteria are very limited. In this study, we analyzed the expression profiles of AMPs in the Chinese shrimps, Fenneropenaeus chinensis, simultaneously by real-time RT-PCR (reverse transcription-polymerase chain reaction) when shrimp were challenged with Micrococcus lysodeikticus (Gram-positive, G+) or Vibrio anguillarium (Gram-negative, G-). Different AMPs showed different expression profiles when shrimp were injected with one type of bacterium, and one AMP also showed different expression profiles when shrimp were challenged with different bacteria. Furthermore, the expression of these AMPs showed temporal expression profiles, suggesting that different AMPs function coordinately in bacteria-infected shrimp. An RNA interference approach was used to study the function of the Relish transcription factor in regulating the transcription of different AMPs. The current study showed that Relish could regulate the transcription of different AMPs in shrimp. Differential expression profiles of AMPs in shrimp injected with different types of bacteria indicated that a complicated antimicrobial response network existed in shrimp. These data contribute to our understanding of immunity in shrimp and may provide a strategy for the control of disease in shrimp.

  17. Exchange factors directly activated by cAMP mediate melanocortin 4 receptor-induced gene expression (United States)

    Glas, Evi; Mückter, Harald; Gudermann, Thomas; Breit, Andreas


    Gs protein-coupled receptors regulate many vital body functions by activation of cAMP response elements (CRE) via cAMP-dependent kinase A (PKA)-mediated phosphorylation of the CRE binding protein (CREB). Melanocortin 4 receptors (MC4R) are prototypical Gs-coupled receptors that orchestrate the hypothalamic control of food-intake and metabolism. Remarkably, the significance of PKA for MC4R-induced CRE-dependent transcription in hypothalamic cells has not been rigorously interrogated yet. In two hypothalamic cell lines, we observed that blocking PKA activity had only weak or no effects on reporter gene expression. In contrast, inhibitors of exchange factors directly activated by cAMP-1/2 (EPAC-1/2) mitigated MC4R-induced CRE reporter activation and mRNA induction of the CREB-dependent genes c-fos and thyrotropin-releasing hormone. Furthermore, we provide first evidence that extracellular-regulated kinases-1/2 (ERK-1/2) activated by EPACs and not PKA are the elusive CREB kinases responsible for MC4R-induced CREB/CRE activation in hypothalamic cells. Overall, these data emphasize the pivotal role of EPACs rather than PKA in hypothalamic gene expression elicited by a prototypical Gs-coupled receptor. PMID:27612207

  18. Microgravity changes in heart structure and cyclic-AMP metabolism (United States)

    Philpott, D. E.; Fine, A.; Kato, K.; Egnor, R.; Cheng, L.


    The effects of microgravity on cardiac ultrastructure and cyclic AMP metabolism in tissues of rats flown on Spacelab 3 are reported. Light and electron microscope studies of cell structure, measurements of low and high Km phosphodiesterase activity, cyclic AMP-dependent protein kinase activity, and regulatory subunit compartmentation show significant deviations in flight animals when compared to ground controls. The results indicate that some changes have occurred in cellular responses associated with catecholamine receptor interactions and intracellular signal processing.

  19. State-of-the-art Review : Vol. 2A. Responsive Building Elements

    DEFF Research Database (Denmark)

    Blümel, Ernst; Haghighat, Fariborz; Li, Yuguo

    . The publication is an internal Annex report. With a focus on innovative building elements that dynamically respond to changes in climate and user demands, the report describes materials, components and systems that have been tested in laboratories and buildings around the world. This report is aimed......This report resumes and presents the activity done in Subtask A of IEA-ECBCS Annex 44 “Integrating Environmentally Responsive Elements in Buildings” concerning the state of the art review of Responsive Building Elements. It is based on the contributions from the participating countries...... at researchers in the field and gives an overview of how these elements work together with available performance data. It is hoped, that this report will be helpful for researchers in their search for new solutions to the problem of designing and constructing sustainable buildings....

  20. Genetic Environment and Transcription of ampC in an Acinetobacter baumannii Clinical Isolate


    Segal, Heidi; Nelson, E.C.; Elisha, B. Gay


    An ampC gene was cloned from a clinical isolate of Acinetobacter baumannii (strain RAN). DNA sequencing and primer extension studies showed that ampC is transcribed from a promoter contained within a putative insertion sequence element which has been found to abut several different genes in Acinetobacter spp.

  1. Insulin regulation of the glucagon gene is mediated by an insulin-responsive DNA element.



    Diabetes mellitus is characterized by insulin deficiency and high plasma glucagon levels, which can be normalized by insulin replacement. It has previously been reported that glucagon gene expression is negatively regulated by insulin at the transcriptional level. By transfection studies, I have now localized a DNA control element that mediates insulin effects on glucagon gene transcription. This element also confers insulin responsiveness to a heterologous promoter. DNA-binding proteins that...

  2. Response of removable epoxy foam exposed to fire using an element death model.

    Energy Technology Data Exchange (ETDEWEB)

    Hobbs, Michael L.


    Response of removable epoxy foam (REF) to high heat fluxes is described using a decomposition chemistry model [1] in conjunction with a finite element heat conduction code [2] that supports chemical kinetics and dynamic radiation enclosures. The chemistry model [1] describes the temporal transformation of virgin foam into carbonaceous residue by considering breakdown of the foam polymer structure, desorption of gases not associated with the foam polymer, mass transport of decomposition products from the reaction site to the bulk gas, and phase equilibrium. The finite element foam response model considers the spatial behavior of the foam by using measured and predicted thermophysical properties in combination with the decomposition chemistry model. Foam elements are removed from the computational domain when the condensed mass fractions of the foam elements are close to zero. Element removal, referred to as element death, creates a space within the metal confinement causing radiation to be the dominant mode of heat transfer between the surface of the remaining foam elements and the interior walls of the confining metal skin. Predictions were compared to front locations extrapolated from radiographs of foam cylinders enclosed in metal containers that were heated with quartz lamps [3,4]. The effects of the maximum temperature of the metal container, density of the foam, the foam orientation, venting of the decomposition products, pressurization of the metal container, and the presence or absence of embedded components are discussed.

  3. Finite element analysis of dynamic response and structure borne noise of gearbox

    Institute of Scientific and Technical Information of China (English)

    LIU Wen; LIN Teng-jiao; LI Run-fang; DU Xue-song


    A dynamic finite element method combined with finite element mixed formula for contact problem is used to analyze the dynamic characteristics of gear system. Considering the stiffness excitation, error excitation and meshing shock excitation, the dynamic finite element model is established for the entire gear system which includes gears, shafts, bearings and gearbox housing. By the software of I-DEAS, the natural frequency, normal mode, dynamic time-domain response, frequency-domain response and one-third octave velocity grade structure borne noise of gear system are studied by the method of theoretical modal analysis and dynamic response analysis. The maximum values of vibration and structure borne noise are occurred at the mesh frequency of output grade gearing.

  4. Multiple Facets of cAMP Signalling and Physiological Impact: cAMP Compartmentalization in the Lung

    Directory of Open Access Journals (Sweden)

    Martina Schmidt


    Full Text Available Therapies involving elevation of the endogenous suppressor cyclic AMP (cAMP are currently used in the treatment of several chronic inflammatory disorders, including chronic obstructive pulmonary disease (COPD. Characteristics of COPD are airway obstruction, airway inflammation and airway remodelling, processes encompassed by increased airway smooth muscle mass, epithelial changes, goblet cell and submucosal gland hyperplasia. In addition to inflammatory cells, airway smooth muscle cells and (myofibroblasts, epithelial cells underpin a variety of key responses in the airways such as inflammatory cytokine release, airway remodelling, mucus hypersecretion and airway barrier function. Cigarette smoke, being next to environmental pollution the main cause of COPD, is believed to cause epithelial hyperpermeability by disrupting the barrier function. Here we will focus on the most recent progress on compartmentalized signalling by cAMP. In addition to G protein-coupled receptors, adenylyl cyclases, cAMP-specific phospho-diesterases (PDEs maintain compartmentalized cAMP signalling. Intriguingly, spatially discrete cAMP-sensing signalling complexes seem also to involve distinct members of the A-kinase anchoring (AKAP superfamily and IQ motif containing GTPase activating protein (IQGAPs. In this review, we will highlight the interaction between cAMP and the epithelial barrier to retain proper lung function and to alleviate COPD symptoms and focus on the possible molecular mechanisms involved in this process. Future studies should include the development of cAMP-sensing multiprotein complex specific disruptors and/or stabilizers to orchestrate cellular functions. Compartmentalized cAMP signalling regulates important cellular processes in the lung and may serve as a therapeutic target.

  5. Effect of allergen-specific immunotherapy with purified Alt a1 on AMP responsiveness, exhaled nitric oxide and exhaled breath condensate pH: a randomized double blind study

    Directory of Open Access Journals (Sweden)

    Prieto Luis


    Full Text Available Abstract Background Little information is available on the effect of allergen-specific immunotherapy on airway responsiveness and markers in exhaled air. The aims of this study were to assess the safety of immunotherapy with purified natural Alt a1 and its effect on airway responsiveness to direct and indirect bronchoconstrictor agents and markers in exhaled air. Methods This was a randomized double-blind trial. Subjects with allergic rhinitis with or without mild/moderate asthma sensitized to A alternata and who also had a positive skin prick test to Alt a1 were randomized to treatment with placebo (n = 18 or purified natural Alt a1 (n = 22 subcutaneously for 12 months. Bronchial responsiveness to adenosine 5'-monophosphate (AMP and methacholine, exhaled nitric oxide (ENO, exhaled breath condensate (EBC pH, and serum Alt a1-specific IgG4 antibodies were measured at baseline and after 6 and 12 months of treatment. Local and systemic adverse events were also registered. Results The mean (95% CI allergen-specific IgG4 value for the active treatment group increased from 0.07 μg/mL (0.03-0.11 at baseline to 1.21 μg/mL (0.69-1.73, P 4 value increased nonsignificantly from 0.09 μg/mL (0.06-0.12 at baseline to 0.13 μg/mL (0.07-0.18 at 6 months and to 0.11 μg/mL (0.07-0.15 at 12 months of treatment. Changes in the active treatment group were significantly higher than in the placebo group both at 6 months (P Conclusion Although allergen-specific immunotherapy with purified natural Alt a1 is well tolerated and induces an allergen-specific IgG4 response, treatment is not associated with changes in AMP or methacholine responsiveness or with significant improvements in markers of inflammation in exhaled air. These findings suggest dissociation between the immunotherapy-induced increase in IgG4 levels and its effect on airway responsiveness and inflammation.

  6. Sleutelmag en amp

    Directory of Open Access Journals (Sweden)

    P. J. Rossouw


    Full Text Available As die Heidelbergse Kategismus (Sondag 31 handel oor die sleutels van die Koninkryk, dan word die betrokkenheid van die ampte slegs per implikasie veronderstel. Meer eksplisiet word die amp en sleutelmag in die Nederlandse Geloofsbelydenis gekoppel; “ons glo dat hierdie ware kerk ooreenkomstig die geestelike bestuurswyse wat ons Here ons in sy Woord geleer het, geregeer moet word... hulle (moet sorg dra dat die ware godsdiens onderhou (word, die ware leer orals versprei, die oortreders op geestelike wyse vermaan en in toom gehou (word... (N.G.B. art. 30 en: “Ons glo verder dat die regeerders van die kerk, al is dit nuttig en goed om onder mekaar ’n bepaalde orde tot instandhouding van die liggaam van die kerk in te stel en te handhaaf, tog noukeurig moet oppas om nie af te wyk van wat Christus, ons enigste Meester, vir ons ingestel het nie... Ons aanvaar derhalwe slegs wat kan dien om eendrag en eenheid te bewaar en te bevorder, en om alles in gehoorsaamheid van God te onderhou. Daarvoor is nodig die ban en alles wat daarmee saamhang, toegepas ooreenkomstig die Woord van God” (N.G.B. art. 32.

  7. Assisted Medical Procedures (AMP) Project (United States)

    National Aeronautics and Space Administration — Documentation and Development: The AMP was initially being developed as part the Advanced Integrated Clinical System (AICS)-Guided Medical Procedure System for the...

  8. A Computational Modeling and Simulation Approach to Investigate Mechanisms of Subcellular cAMP Compartmentation. (United States)

    Yang, Pei-Chi; Boras, Britton W; Jeng, Mao-Tsuen; Docken, Steffen S; Lewis, Timothy J; McCulloch, Andrew D; Harvey, Robert D; Clancy, Colleen E


    Subcellular compartmentation of the ubiquitous second messenger cAMP has been widely proposed as a mechanism to explain unique receptor-dependent functional responses. How exactly compartmentation is achieved, however, has remained a mystery for more than 40 years. In this study, we developed computational and mathematical models to represent a subcellular sarcomeric space in a cardiac myocyte with varying detail. We then used these models to predict the contributions of various mechanisms that establish subcellular cAMP microdomains. We used the models to test the hypothesis that phosphodiesterases act as functional barriers to diffusion, creating discrete cAMP signaling domains. We also used the models to predict the effect of a range of experimentally measured diffusion rates on cAMP compartmentation. Finally, we modeled the anatomical structures in a cardiac myocyte diad, to predict the effects of anatomical diffusion barriers on cAMP compartmentation. When we incorporated experimentally informed model parameters to reconstruct an in silico subcellular sarcomeric space with spatially distinct cAMP production sites linked to caveloar domains, the models predict that under realistic conditions phosphodiesterases alone were insufficient to generate significant cAMP gradients. This prediction persisted even when combined with slow cAMP diffusion. When we additionally considered the effects of anatomic barriers to diffusion that are expected in the cardiac myocyte dyadic space, cAMP compartmentation did occur, but only when diffusion was slow. Our model simulations suggest that additional mechanisms likely contribute to cAMP gradients occurring in submicroscopic domains. The difference between the physiological and pathological effects resulting from the production of cAMP may be a function of appropriate compartmentation of cAMP signaling. Therefore, understanding the contribution of factors that are responsible for coordinating the spatial and temporal

  9. Molecular characterization and expression of AMP-activated protein kinase in response to low-salinity stress in the Pacific white shrimp Litopenaeus vannamei. (United States)

    Xu, Chang; Li, Erchao; Xu, Zhixin; Wang, Shifeng; Chen, Ke; Wang, Xiaodan; Li, Tongyu; Qin, Jian G; Chen, Liqiao


    AMP-activated protein kinase (AMPK) serves as a major regulator of cellular energy metabolism by activating ATP production pathways and blocking ATP consumption. However, information on AMPK genes in aquatic animals is limited. In this study, three subunits of AMPK were cloned from the Pacific white shrimp Litopenaeus vannamei. The full-length cDNAs of the α, β and γ subunits were 1617, 1243 and 3467bp long, respectively, with open reading frames of 1566, 873 and 2988bp encoding for 521, 290 and 996 amino acids, respectively. Amino acid sequence alignments of the three subunits showed that the functional domains in the L. vannamei proteins retained the highest similarity with those of other animals, at 89%, 58%, and 75%, respectively. The expression levels of the three subunits were higher in the muscle and gills than in the eyestalk and hepatopancreas. The mRNA levels of AMPK-α and AMPK-β were up-regulated in the hepatopancreas and muscle after acute low-salinity stress at 3psu for 6h compared with control salinity at 20psu. After 8-week salinity stress at 3psu, AMPK-α and AMPK-β mRNA levels in the hepatopancreas were significantly higher than those of the control at 30psu. However, in the muscle only AMPK-γ mRNA was significantly up-regulated at low salinity relative to controls. Muscle and hepatopancreas showed increases in AMPK protein after 6h exposure to low salinity, but there were no differences seen after long term acclimation. The change patterns of protein were slightly differing from the mRNA patterns due to the distinguishing function of individual subunits of AMPK. These findings confirm that three AMPK subunits are present in L. vannamei and that all encode proteins with conserved functional domains. The three AMPK subunits are all regulated at the transcriptional and protein levels to manage excess energy expenditure during salinity stress.

  10. Expression profiles of antimicrobial peptides (AMPs) and their regulation by Relish

    Institute of Scientific and Technical Information of China (English)

    WANG Dongdong; LI Fuhua; LI Shihao; WEN Rong; XIANG Jianhai


    Antimicrobial peptides (AMPs),as key immune effectors,play important roles in the innate immune system of invertebrates.Different types of AMPs,including Penaeidin,Crustin,ALF (antilipopolysaccharide factor) have been identified in different penaeid shrimp; however,systematic analyses on the function of different AMPs in shrimp responsive to different types of bacteria are very limited.In this study,we analyzed the expression profiles of AMPs in the Chinese shrimps,Fenneropenaeus chinensis,simultaneously by real-time RT-PCR (reverse transcription-polymerase chain reaction) when shrimp were challenged with Micrococcus lysodeikticus (Gram-positive,G+) or Vibrio anguillarium (Gram-negative,G).Different AMPs showed different expression profiles when shrimp were injected with one type of bacterium,and one AMP also showed different expression profiles when shrimp were challenged with different bacteria.Furthermore,the expression of these AMPs showed temporal expression profiles,suggesting that different AMPs function coordinately in bacteria-infected shrimp.An RNA interference approach was used to study the function of the Relish transcription factor in regulating the transcription of different AM Ps.The current study showed that Relish could regulate the transcription of different AMPs in shrimp.Differential expression profiles of AMPs in shrimp injected with different types of bacteria indicated that a complicated antimicrobial response network existed in shrimp.These data contribute to our understanding of immunity in shrimp and may provide a strategy for the control of disease in shrimp.

  11. Three-dimensional measurement of cAMP gradients using hyperspectral confocal microscopy (United States)

    Rich, Thomas C.; Annamdevula, Naga; Britain, Andrea L.; Mayes, Samuel; Favreau, Peter F.; Leavesley, Silas J.


    Cyclic AMP (cAMP) is a ubiquitous second messenger known to differentially regulate many cellular functions over a wide range of timescales. Several lines of evidence have suggested that the distribution of cAMP within cells is not uniform, and that cAMP compartmentalization is largely responsible for signaling specificity within the cAMP signaling pathway. However, to date, no studies have experimentally measured three dimensional (3D) cAMP distributions within cells. Here we use both 2D and 3D hyperspectral microscopy to visualize cAMP gradients in endothelial cells from the pulmonary microvasculature (PMVECs). cAMP levels were measured using a FRETbased cAMP sensor comprised of a cAMP binding domain from EPAC sandwiched between FRET donors and acceptors -- Turquoise and Venus fluorescent proteins. Data were acquired using either a Nikon A1R spectral confocal microscope or custom spectral microscopy system. Analysis of hyperspectral image stacks from a single confocal slice or from summed images of all slices (2D analysis) indicated little or no cAMP gradients were formed within PMVECs under basal conditions or following agonist treatment. However, analysis of hyperspectral image stacks from 3D cellular geometries (z stacks) demonstrate marked cAMP gradients from the apical to basolateral membrane of PMVECs. These results strongly suggest that 2D imaging studies of cAMP compartmentalization -- whether epifluorescence or confocal microscopy -- may lead to erroneous conclusions about the existence of cAMP gradients, and that 3D studies are required to assess mechanisms of signaling specificity.

  12. Responses of trace elements to aerobic maximal exercise in elite sportsmen. (United States)

    Otag, Aynur; Hazar, Muhsin; Otag, Ilhan; Gürkan, Alper Cenk; Okan, Ilyas


    Trace elements are chemical elements needed in minute quantities for the proper growth, development, and physiology of the organism. In biochemistry, a trace element is also referred to as a micronutrient. Trace elements, such as nickel, cadmium, aluminum, silver, chromium, molybdenum, germanium, tin, titanium, tungsten, scandium, are found naturally in the environment and human exposure derives from a variety of sources, including air, drinking water and food. The Purpose of this study was investigated the effect of aerobic maximal intensity endurance exercise on serum trace elements as well-trained individuals of 28 wrestlers (age (year) 19.64±1.13, weight (Kg) 70.07 ± 15.69, height (cm) 176.97 ± 6.69) during and after a 2000 meter Ergometer test protocol was used to perform aerobic (75 %) maximal endurance exercise. Trace element serum levels were analyzed from blood samples taken before, immediately after and one hour after the exercise. While an increase was detected in Chromium (Cr), Nickel (Ni), Molybdenum (Mo) and Titanium (Ti) serum levels immediately after the exercise, a decrease was detected in Aluminum (Al), Scandium (Sc) and Tungsten (W) serum levels. Except for aluminum, the trace elements we worked on showed statistically meaningful responses (P sportsman performance but also in terms of future healthy life plans and clinically.

  13. Simulation of Electromagnetic Wave Logging Response in Deviated Wells Based on Vector Finite Element Method

    Institute of Scientific and Technical Information of China (English)

    LV Wei-Guo; CHU Zhao-Tan; ZHAO Xiao-Qing; FAN Yu-Xiu; SONG Ruo-Long; HAN Wei


    The vector finite element method of tetrahedral elements is used to model 3D electromagnetic wave logging response. The tangential component of the vector field at the mesh edges is used as a degree of freedom to overcome the shortcomings of node-based finite element methods. The algorithm can simulate inhomogeneous media with arbitrary distribution of conductivity and magnetic permeability. The electromagnetic response of well logging tools are studied in dipping bed layers with the borehole and invasion included. In order to simulate realistic logging tools, we take the transmitter antennas consisting of circular wire loops instead of magnetic dipoles. We also investigate the apparent resistivity of inhomogeneous formation for different dip angles.

  14. Are collapsars responsible for some r-process elements? How could we tell?

    Energy Technology Data Exchange (ETDEWEB)

    Pruet, J


    We consider the possibility that supernovae which form hyper-accreting black holes might be responsible for synthesis of r-process elements with mass A {approx}< 130. Calculations are presented which show that these elements are naturally synthesized in neutron-rich magnetically-dominated bubbles born in the inner regions of a black hole accretion disk. Simple considerations suggest that the total mass ejected in the form of these bubbles is about that needed to account for the entire galactic inventory of the 2nd-peak r-process elements. We also argue that if collapsars are responsible for, e.g., Ag synthesis, then Ag abundances should be correlated with Sc and/or Zn abundances in metal-poor stars.

  15. 33 CFR Appendix D to Part 154 - Training Elements for Oil Spill Response Plans (United States)


    .... Further, this section of the plan must work in harmony with those sections of the plan dealing with exercises, the spill management team, and the qualified individual. 1.3The material in this appendix D is... the elements that define the program as appropriate. 2.2An effective spill response training...

  16. Modulation of the cAMP signaling pathway after traumatic brain injury


    Atkins, Coleen M.; Oliva, Anthony A.; Alonso, Ofelia F.; Pearse, Damien D.; Bramlett, Helen M; Dietrich, W. Dalton


    Traumatic brain injury (TBI) results in both focal and diffuse brain pathologies that are exacerbated by the inflammatory response and progress from hours to days after the initial injury. Using a clinically relevant model of TBI, the parasagittal fluid-percussion brain injury (FPI) model, we found injury-induced impairments in the cyclic AMP (cAMP) signaling pathway. Levels of cAMP were depressed in the ipsilateral parietal cortex and hippocampus, as well as activation of its downstream targ...

  17. Dynamic Stationary Response of Reinforced Plates by the Boundary Element Method

    Directory of Open Access Journals (Sweden)

    Luiz Carlos Facundo Sanches


    Full Text Available A direct version of the boundary element method (BEM is developed to model the stationary dynamic response of reinforced plate structures, such as reinforced panels in buildings, automobiles, and airplanes. The dynamic stationary fundamental solutions of thin plates and plane stress state are used to transform the governing partial differential equations into boundary integral equations (BIEs. Two sets of uncoupled BIEs are formulated, respectively, for the in-plane state (membrane and for the out-of-plane state (bending. These uncoupled systems are joined to form a macro-element, in which membrane and bending effects are present. The association of these macro-elements is able to simulate thin-walled structures, including reinforced plate structures. In the present formulation, the BIE is discretized by continuous and/or discontinuous linear elements. Four displacement integral equations are written for every boundary node. Modal data, that is, natural frequencies and the corresponding mode shapes of reinforced plates, are obtained from information contained in the frequency response functions (FRFs. A specific example is presented to illustrate the versatility of the proposed methodology. Different configurations of the reinforcements are used to simulate simply supported and clamped boundary conditions for the plate structures. The procedure is validated by comparison with results determined by the finite element method (FEM.

  18. Endothelial responses of magnesium and other alloying elements in magnesium-based stent materials (United States)

    Zhao, Nan; Zhu, Donghui


    Biodegradable tailored magnesium (Mg) alloys are some of the most promising scaffolds for cardiovascular stents. During the course of degradation after implantation, all the alloying elements in the scaffold will be released to the surrounding vascular tissues. However, fundamental questions regarding the toxicity of alloying elements towards vascular cells, the maximum amount of each element that could be used in alloy design, or how each of the alloying elements affects vascular cellular activity and gene expression, are still not fully answered. This work systematically addressed these questions by revealing how application of different alloying elements commonly used in Mg stent materials influences several indices of human endothelial cell health, i.e., viability, proliferations, cytoskeletal reorganizations, migration, and the gene expression profile. The overall cell viability and proliferation showed a decreasing trend with increasing concentrations of the ions, and the half maximal effective concentrations (EC50) for each element were determined. When applied at a low concentration of around 10 mM, Mg had no adverse effects but improved cell proliferation and migration instead. Mg ions also altered endothelial gene expression significantly in a dose dependent manner. Most of the changed genes are related to angiogenesis and the cell adhesion signaling pathways. Findings from this work provide useful information on maximum safe doses of these ions for endothelial cells, endothelial responses towards these metal ions, and some guidance for future Mg stent design. PMID:25363018

  19. Dynamic Response of a Planetary Gear System Using a Finite Element/Contact Mechanics Model (United States)

    Parker, Robert G.; Agashe, Vinayak; Vijayakar, Sandeep M.


    The dynamic response of a helicopter planetary gear system is examined over a wide range of operating speeds and torques. The analysis tool is a unique, semianalytical finite element formulation that admits precise representation of the tooth geometry and contact forces that are crucial in gear dynamics. Importantly, no a priori specification of static transmission error excitation or mesh frequency variation is required; the dynamic contact forces are evaluated internally at each time step. The calculated response shows classical resonances when a harmonic of mesh frequency coincides with a natural frequency. However, peculiar behavior occurs where resonances expected to be excited at a given speed are absent. This absence of particular modes is explained by analytical relationships that depend on the planetary configuration and mesh frequency harmonic. The torque sensitivity of the dynamic response is examined and compared to static analyses. Rotation mode response is shown to be more sensitive to input torque than translational mode response.

  20. Creating Diversified Response Profiles from a Single Quenchometric Sensor Element by Using Phase-Resolved Luminescence

    Directory of Open Access Journals (Sweden)

    Elizabeth C. Tehan


    Full Text Available We report a new strategy for generating a continuum of response profiles from a single luminescence-based sensor element by using phase-resolved detection. This strategy yields reliable responses that depend in a predictable manner on changes in the luminescent reporter lifetime in the presence of the target analyte, the excitation modulation frequency, and the detector (lock-in amplifier phase angle. In the traditional steady-state mode, the sensor that we evaluate exhibits a linear, positive going response to changes in the target analyte concentration. Under phase-resolved conditions the analyte-dependent response profiles: (i can become highly non-linear; (ii yield negative going responses; (iii can be biphasic; and (iv can exhibit super sensitivity (e.g., sensitivities up to 300 fold greater in comparison to steady-state conditions.

  1. Creating Diversified Response Profiles from a Single Quenchometric Sensor Element by Using Phase-Resolved Luminescence (United States)

    Tehan, Elizabeth C.; Bukowski, Rachel M.; Chodavarapu, Vamsy P.; Titus, Albert H.; Cartwright, Alexander N.; Bright, Frank V.


    We report a new strategy for generating a continuum of response profiles from a single luminescence-based sensor element by using phase-resolved detection. This strategy yields reliable responses that depend in a predictable manner on changes in the luminescent reporter lifetime in the presence of the target analyte, the excitation modulation frequency, and the detector (lock-in amplifier) phase angle. In the traditional steady-state mode, the sensor that we evaluate exhibits a linear, positive going response to changes in the target analyte concentration. Under phase-resolved conditions the analyte-dependent response profiles: (i) can become highly non-linear; (ii) yield negative going responses; (iii) can be biphasic; and (iv) can exhibit super sensitivity (e.g., sensitivities up to 300 fold greater in comparison to steady-state conditions). PMID:25569752

  2. TSH-induced cyclic AMP production in an ovine thyroid cell line: OVNIS 5H. (United States)

    Fayet, G; Aouani, A; Hovsépian, S


    The TSH-induced cyclic AMP response was studied using a 3-year-old ovine thyroid cell line TSH-independent for growth: OVNIS 5H. The kinetics of cyclic AMP production was followed both in cell layers and in cell culture media, with or without phosphodiesterase inhibitor. It is noteworthy that following the first wave in cyclic AMP obtained within minutes, we observed later a sustained exponential increase in cyclic AMP during the 5 days following TSH stimulation. A bioassay of TSH was derived allowing measurement of 1 microU/ml TSH from a crude bTSH preparation.

  3. The cAMP Response Element Binding protein (CREB) is activated by Insulin-like Growth Factor-1 (IGF-1) and regulates myostatin gene expression in skeletal myoblast

    Energy Technology Data Exchange (ETDEWEB)

    Zuloaga, R. [Facultad de Ciencias Biológicas, Universidad Andres Bello, Santiago (Chile); Fuentes, E.N.; Molina, A. [Facultad de Ciencias Biológicas, Universidad Andres Bello, Santiago (Chile); Interdisciplinary Center for Aquaculture Research (INCAR), Víctor Lamas 1290, PO Box 160-C, Concepción (Chile); Valdés, J.A., E-mail: [Facultad de Ciencias Biológicas, Universidad Andres Bello, Santiago (Chile); Interdisciplinary Center for Aquaculture Research (INCAR), Víctor Lamas 1290, PO Box 160-C, Concepción (Chile)


    Highlights: •IGF-1 induces the activation of CREB via IGF-1R/PI3K/PLC signaling pathway. •Calcium dependent signaling pathways regulate myostatin gene expression. •IGF-1 regulates myostatin gene expression via CREB transcription in skeletal myoblast. -- Abstract: Myostatin, a member of the Transforming Growth Factor beta (TGF-β) superfamily, plays an important role as a negative regulator of skeletal muscle growth and differentiation. We have previously reported that IGF-1 induces a transient myostatin mRNA expression, through the activation of the Nuclear Factor of Activated T cells (NFAT) in an IP{sub 3}/calcium-dependent manner. Here we examined the activation of CREB transcription factor as downstream targets of IGF-1 during myoblast differentiation and its role as a regulator of myostatin gene expression. In cultured skeletal myoblast, IGF-1 induced the phosphorylation and transcriptional activation of CREB via IGF-1 Receptor/Phosphatidylinositol 3-Kinase (PI3K)/Phospholipase C gamma (PLC γ), signaling pathways. Also, IGF-1 induced calcium-dependent molecules such as Calmodulin Kinase II (CaMK II), Extracellular signal-regulated Kinases (ERK), Protein Kinase C (PKC). Additionally, we examined myostatin mRNA levels and myostatin promoter activity in differentiated myoblasts stimulated with IGF-1. We found a significant increase in mRNA contents of myostatin and its reporter activity after treatment with IGF-1. The expression of myostatin in differentiated myoblast was downregulated by the transfection of siRNA–CREB and by pharmacological inhibitors of the signaling pathways involved in CREB activation. By using pharmacological and genetic approaches together these data demonstrate that IGF-1 regulates the myostatin gene expression via CREB transcription factor during muscle cell differentiation.

  4. An Extended Finite Element Method Formulation for Modeling the Response of Polycrystalline Materials to Dynamic Loading (United States)

    Robbins, Joshua; Voth, Thomas E.


    The eXtended Finite Element Method (X-FEM) is a finite-element based discretization technique developed originally to model dynamic crack propagation [1]. Since that time the method has been used for modeling physics ranging from static meso-scale material failure to dendrite growth. Here we adapt the recent advances of Vitali and Benson [2] and Song et al. [3] to model dynamic loading of a polycrystalline material. We use demonstration problems to examine the method's efficacy for modeling the dynamic response of polycrystalline materials at the meso-scale. Specifically, we use the X-FEM to model grain boundaries. This approach allows us to i) eliminate ad-hoc mixture rules for multi-material elements and ii) avoid explicitly meshing grain boundaries.

  5. Response of hot element wall shear stress gages in laminar oscillating flows (United States)

    Cook, W. J.; Murphy, J. D.; Giddings, T. A.


    An experimental investigation of the time-dependent response of hot element wall shear stress gages in unsteady periodic air flows is reported. The study has focused on wall shear stress in laminar oscillating flows produced on a flat plate by a free stream velocity composed of a mean component and a superposed sinusoidal variation. Two types of hot element gages, platinum film and flush wire, were tested for values of reduced frequency ranging from 0.14 to 2.36. Values of the phase angle of the wall shear stress variation relative to the free stream velocity, as indicated by the hot element gages, are compared with numerical prediction. The comparisons show that the gages indicate a wall shear stress variation that lags the true variation, and that the gages will also not indicate the correct wall shear stress variation in periodic turbulent flows.

  6. The coordinated Response of the Physical and Antimicrobial Peptide Barriers of the Skin (United States)

    Borkowski, Andrew W.; Gallo, Richard L.


    Antimicrobial peptides (AMPs) are an essential and multifunctional element for immune defense of the skin during infection and injury. In this issue, Ahrens et al. characterize the response of β-defensins, a class of AMPs, following acute and chronic challenges to the permeability barrier of the skin. Their findings suggest that the antimicrobial and permeability barriers of the skin are closely linked. PMID:21228809

  7. Verification of Advective Bar Elements Implemented in the Aria Thermal Response Code.

    Energy Technology Data Exchange (ETDEWEB)

    Mills, Brantley [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)


    A verification effort was undertaken to evaluate the implementation of the new advective bar capability in the Aria thermal response code. Several approaches to the verification process were taken : a mesh refinement study to demonstrate solution convergence in the fluid and the solid, visually examining the mapping of the advective bar element nodes to the surrounding surfaces, and a comparison of solutions produced using the advective bars for simple geometries with solutions from commercial CFD software . The mesh refinement study has shown solution convergence for simple pipe flow in both temperature and velocity . Guidelines were provided to achieve appropriate meshes between the advective bar elements and the surrounding volume. Simulations of pipe flow using advective bars elements in Aria have been compared to simulations using the commercial CFD software ANSYS Fluent (r) and provided comparable solutions in temperature and velocity supporting proper implementation of the new capability. Verification of Advective Bar Elements iv Acknowledgements A special thanks goes to Dean Dobranich for his guidance and expertise through all stages of this effort . His advice and feedback was instrumental to its completion. Thanks also goes to Sam Subia and Tolu Okusanya for helping to plan many of the verification activities performed in this document. Thank you to Sam, Justin Lamb and Victor Brunini for their assistance in resolving issues encountered with running the advective bar element model. Finally, thanks goes to Dean, Sam, and Adam Hetzler for reviewing the document and providing very valuable comments.

  8. An extended finite element formulation for modeling the response of polycrystalline materials to shock loading (United States)

    Robbins, Joshua; Voth, Thomas


    The eXtended Finite Element Method (X-FEM) is a finite element based discretization technique developed originally to model dynamic crack propagation [1]. Since that time the method has been used for modeling physics ranging from static mesoscale material failure to dendrite growth. Here we adapt the recent advances of Benson et al. [2] and Belytchko et al. [3] to model shock loading of polycrystalline material. Through several demonstration problems we evaluate the method for modeling the shock response of polycrystalline materials at the mesoscale. Specifically, we use the X-FEM to model grain boundaries. This approach allows us to i) eliminate ad-hoc mixture rules for multi-material elements and ii) avoid explicitly meshing grain boundaries. ([1] N. Moes, J. Dolbow, J and T. Belytschko, 1999,``A finite element method for crack growth without remeshing,'' International Journal for Numerical Methods in Engineering, 46, 131-150. [2] E. Vitali, and D. J. Benson, 2006, ``An extended finite element formulation for contact in multi-material arbitrary Lagrangian-Eulerian calculations,'' International Journal for Numerical Methods in Engineering, 67, 1420-1444. [3] J-H Song, P. M. A. Areias and T. Belytschko, 2006, ``A method for dynamic crack and shear band propagation with phantom nodes,'' International Journal for Numerical Methods in Engineering, 67, 868-893.)

  9. Mechanical stress induces biotic and abiotic stress responses via a novel cis-element.

    Directory of Open Access Journals (Sweden)

    Justin W Walley


    Full Text Available Plants are continuously exposed to a myriad of abiotic and biotic stresses. However, the molecular mechanisms by which these stress signals are perceived and transduced are poorly understood. To begin to identify primary stress signal transduction components, we have focused on genes that respond rapidly (within 5 min to stress signals. Because it has been hypothesized that detection of physical stress is a mechanism common to mounting a response against a broad range of environmental stresses, we have utilized mechanical wounding as the stress stimulus and performed whole genome microarray analysis of Arabidopsis thaliana leaf tissue. This led to the identification of a number of rapid wound responsive (RWR genes. Comparison of RWR genes with published abiotic and biotic stress microarray datasets demonstrates a large overlap across a wide range of environmental stresses. Interestingly, RWR genes also exhibit a striking level and pattern of circadian regulation, with induced and repressed genes displaying antiphasic rhythms. Using bioinformatic analysis, we identified a novel motif overrepresented in the promoters of RWR genes, herein designated as the Rapid Stress Response Element (RSRE. We demonstrate in transgenic plants that multimerized RSREs are sufficient to confer a rapid response to both biotic and abiotic stresses in vivo, thereby establishing the functional involvement of this motif in primary transcriptional stress responses. Collectively, our data provide evidence for a novel cis-element that is distributed across the promoters of an array of diverse stress-responsive genes, poised to respond immediately and coordinately to stress signals. This structure suggests that plants may have a transcriptional network resembling the general stress signaling pathway in yeast and that the RSRE element may provide the key to this coordinate regulation.

  10. Chemotaxis to cyclic AMP and folic acid is mediated by different G proteins in Dictyostelium discoideum

    NARCIS (Netherlands)

    Kesbeke, Fanja; Haastert, Peter J.M. van; Wit, René J.W. de; Snaar-Jagalska, B. Ewa


    Mutant Frigid A (fgdA) of Dictyostelium discoideum is defective in a functional Gα2 subunit of a G protein and is characterized by a complete blockade of the cyclic AMP-mediated sensory transduction steps, including cyclic AMP relay, chemotaxis and the cyclic GMP response. Folic acid-mediated transm

  11. Simulation of the ultrasonic array response from real branched cracks using an efficient finite element method

    Energy Technology Data Exchange (ETDEWEB)

    Felice, Maria V. [Department of Mechanical Engineering, University of Bristol, Bristol BS8 1TR, United Kingdom and Rolls-Royce plc., Bristol BS34 7QE (United Kingdom); Velichko, Alexander; Wilcox, Paul D. [Department of Mechanical Engineering, University of Bristol, Bristol BS8 1TR (United Kingdom); Barden, Tim J.; Dunhill, Tony K. [Rolls-Royce plc., Bristol BS34 7QE (United Kingdom)


    A hybrid model to simulate the ultrasonic array response from stress corrosion cracks is presented. These cracks are branched and difficult to detect so the model is required to enable optimization of an array design. An efficient frequency-domain finite element method is described and selected to simulate the ultrasonic scattering. Experimental validation results are presented, followed by an example of the simulated ultrasonic array response from a real stress corrosion crack whose geometry is obtained from an X-ray Computed Tomography image. A simulation-assisted array design methodology, which includes the model and use of real crack geometries, is proposed.

  12. Finite element simulation for mechanical response of surface mounted solder joints under different temperature cycling

    Institute of Scientific and Technical Information of China (English)

    马鑫; 钱乙余


    Nonlinear finite element simulation for mechanical response of surface mounted solder joint under different temperature cycling was carried out. Seven sets of parameters were used in order to evaluate the influence of temperature cycling profile parameters. The results show that temperature cycling history has significant effect on the stress response of the solder joint. Based on the concept of relative damage stress proposed by the authors, it is found that enough high temperature holding time is necessary for designing the temperature cycling profile in accelerated thermal fatigue test.

  13. Isolation of transcription factors binding auxin response elements using a yeast one-hybrid system

    Institute of Scientific and Technical Information of China (English)

    齐眉; 黄美娟; 陈凡


    Plant hormones play an important role during higher plant embryogenesis. Auxin is central to the development of vascular tissues, formation of lateral and adventitious roots, control of apical dominance, and tropic responses. Auxin response element (AuxRE), present in the promoters of many auxin-induced genes, can confer auxin responsiveness. Using carrot somatic embryo under specific developmental phase, a cDNA expression library was constructed. Several plasmids were recombined containing the tetramer of AuxRE as a bait. After screening by a yeast one-hy- brid system, one positive clone was confirmed and characterized. Electrophoretic mobility shift assay showed that AxRF1 protein expressed in yeast cell could bind AuxRE in vitro. It suggests that AxRF1 participates in regulation of the expression of auxin responsive gene during carrot somatic embryogenesis.

  14. A Novel Peroxisome Proliferator Response Element Modulates Hepatic Low Density Lipoprotein Receptor Gene Transcription in Response to PPARδ Activation (United States)

    Shende, Vikram R.; Singh, Amar Bahadur; Liu, Jingwen


    The hepatic expression of LDLR gene is regulated primarily at the transcriptional level by a sterol-regulatory element (SRE) in its proximal promoter region which is the site of action of SRE-binding protein 2 (SREBP2). However whether additional cis-regulatory elements contribute to LDLR transcription has not been fully explored. We investigated the function of a putative PPAR-response element (PPRE) sequence motif located at −768 to −752 bases upstream of the transcription start site of human LDLR gene in response to PPARδ activation. Promoter luciferase reporter analyses showed that treating HepG2 cells with PPARδ agonist L165041 markedly increased the activity of a full-length LDLR promoter construct (pLDLR-1192) without any effects on the shorter promoter reporter pLDLR-234 that contains only the core regulatory elements SRE-1 and SP1 sites. Importantly, mutation of the PPRE sequence greatly attenuated the induction of the full-length LDLR promoter activity by L165041 without affecting rosuvastatin mediated transactivation. Electrophoretic mobility shift and chromatin immunoprecipitation assays further confirmed the binding of PPARδ to the LDLR-PPRE site. Treating HepG2 cells with L165041 elevated the mRNA and protein expressions of LDLR without affecting the LDLR mRNA decay rate. The induction of LDLR expression by PPARδ agonist was further observed in liver tissue of mice and hamsters treated with L165041. Altogether, our studies identify a novel PPRE-mediated regulatory mechanism for LDLR transcription and suggest that combined treatment of statin with PPARδ agonists may have advantageous effects on LDLR expression. PMID:26443862

  15. A novel peroxisome proliferator response element modulates hepatic low-density lipoprotein receptor gene transcription in response to PPARδ activation. (United States)

    Shende, Vikram R; Singh, Amar Bahadur; Liu, Jingwen


    The hepatic expression of low-density lipoprotein (LDL) receptor (LDLR) gene is regulated primarily at the transcriptional level by a sterol-regulatory element (SRE) in its proximal promoter region which is the site of action of SRE-binding protein 2 (SREBP2). However whether additional cis-regulatory elements contribute to LDLR transcription has not been fully explored. We investigated the function of a putative peroxisome proliferator-activated receptor (PPAR)-response element (PPRE) sequence motif located at -768 to -752 bases upstream of the transcription start site of human LDLR gene in response to PPARδ activation. Promoter luciferase reporter analyses showed that treating HepG2 cells with PPARδ agonist L165041 markedly increased the activity of a full-length LDLR promoter construct (pLDLR-1192) without any effects on the shorter promoter reporter pLDLR-234 that contains only the core regulatory elements SRE-1 and SP1 sites. Importantly, mutation of the PPRE sequence greatly attenuated the induction of the full-length LDLR promoter activity by L165041 without affecting rosuvastatin (RSV)-mediated transactivation. EMSA and ChIP assay further confirmed the binding of PPARδ to the LDLR-PPRE site. Treating HepG2 cells with L165041 elevated the mRNA and protein expressions of LDLR without affecting the LDLR mRNA decay rate. The induction of LDLR expression by PPARδ agonist was further observed in liver tissue of mice and hamsters treated with L165041. Altogether, our studies identify a novel PPRE-mediated regulatory mechanism for LDLR transcription and suggest that combined treatment of statin with PPARδ agonists may have advantageous effects on LDLR expression.

  16. Equivalent Dynamic Stiffness Mapping technique for identifying nonlinear structural elements from frequency response functions (United States)

    Wang, X.; Zheng, G. T.


    A simple and general Equivalent Dynamic Stiffness Mapping technique is proposed for identifying the parameters or the mathematical model of a nonlinear structural element with steady-state primary harmonic frequency response functions (FRFs). The Equivalent Dynamic Stiffness is defined as the complex ratio between the internal force and the displacement response of unknown element. Obtained with the test data of responses' frequencies and amplitudes, the real and imaginary part of Equivalent Dynamic Stiffness are plotted as discrete points in a three dimensional space over the displacement amplitude and the frequency, which are called the real and the imaginary Equivalent Dynamic Stiffness map, respectively. These points will form a repeatable surface as the Equivalent Dynamic stiffness is only a function of the corresponding data as derived in the paper. The mathematical model of the unknown element can then be obtained by surface-fitting these points with special functions selected by priori knowledge of the nonlinear type or with ordinary polynomials if the type of nonlinearity is not pre-known. An important merit of this technique is its capability of dealing with strong nonlinearities owning complicated frequency response behaviors such as jumps and breaks in resonance curves. In addition, this technique could also greatly simplify the test procedure. Besides there is no need to pre-identify the underlying linear parameters, the method uses the measured data of excitation forces and responses without requiring a strict control of the excitation force during the test. The proposed technique is demonstrated and validated with four classical single-degree-of-freedom (SDOF) numerical examples and one experimental example. An application of this technique for identification of nonlinearity from multiple-degree-of-freedom (MDOF) systems is also illustrated.

  17. ¬cAMP promotes the differentiation of neural progenitor cells in vitro via modulation of voltage-gated calcium channels

    Directory of Open Access Journals (Sweden)

    Guilherme eLepski


    Full Text Available The molecular mechanisms underlying the differentiation of neural progenitor cells (NPCs remain poorly understood. In this study we investigated the role of Ca2+ and cAMP (cyclic adenosine monophosphate in the differentiation of NPCs extracted from the subventricular zone of E14.5 rat embryos. Patch clamp recordings revealed that increasing cAMP-signaling with Forskolin or IBMX (3-isobutyl-1-methylxantine significantly facilitated neuronal functional maturation. A continuous application of IBMX to the differentiation medium substantially increased the functional expression of voltage-gated Na+ and K+ channels, as well as neuronal firing frequency. Furthermore, we observed an increase in the frequency of spontaneous synaptic currents and in the amplitude of evoked glutamatergic and GABAergic synaptic currents. The most prominent acute effect of applying IBMX was an increase in L-type Ca2+currents. Conversely, blocking L-type channels strongly inhibited dendritic outgrowth and synapse formation even in the presence of IBMX, indicating that voltage-gated Ca2+ influx plays a major role in neuronal differentiation. Finally, we found that nifedipine completely blocks IBMX-induced CREB phosphorylation (cAMP-response-element-binding protein, indicating that the activity of this important transcription factor equally depends on both enhanced cAMP and voltage-gated Ca2+-signaling. Taken together, these data indicate that the up-regulation of voltage-gated L-type Ca2+-channels and early electrical excitability are critical steps in the cAMP-dependent differentiation of SVZ-derived NPCs into functional neurons. To our knowledge, this is the first demonstration of the acute effects of cAMP on voltage-gated Ca+2channels in NPC-derived developing neurons.

  18. Dynamics of β-adrenergic/cAMP signaling and morphological changes in cultured astrocytes. (United States)

    Vardjan, Nina; Kreft, Marko; Zorec, Robert


    The morphology of astrocytes, likely regulated by cAMP, determines the structural association between astrocytes and the synapse, consequently modulating synaptic function. β-Adrenergic receptors (β-AR), which increase cytosolic cAMP concentration ([cAMP]i ), may affect cell morphology. However, the real-time dynamics of β-AR-mediated cAMP signaling in single live astrocytes and its effect on cell morphology have not been studied. We used the fluorescence resonance energy transfer (FRET)-based cAMP biosensor Epac1-camps to study time-dependent changes in [cAMP]i ; morphological changes in primary rat astrocytes were monitored by real-time confocal microscopy. Stimulation of β-AR by adrenaline, noradrenaline, and isoprenaline, a specific agonist of β-AR, rapidly increased [cAMP]i (∼15 s). The FRET signal response, mediated via β-AR, was faster than in the presence of forskolin (twofold) and dibutyryl-cAMP (>35-fold), which directly activate adenylyl cyclase and Epac1-camps, respectively, likely due to slow entry of these agents into the cytosol. Oscillations in [cAMP]i have not been recorded, indicating that cAMP-dependent processes operate in a slow time domain. Most Epac1-camps expressing astrocytes revealed a morphological change upon β-AR activation and attained a stellate morphology within 1 h. The morphological changes exhibited a bell-shaped dependency on [cAMP]i . The 5-10% decrease in cell cross-sectional area and the 30-50% increase in cell perimeter are likely due to withdrawal of the cytoplasm to the perinuclear region and the appearance of protrusions on the surface of astrocytes. Because astrocyte processes ensheath neurons, β-AR/cAMP-mediated morphological changes can modify the geometry of the extracellular space, affecting synaptic, neuronal, and astrocyte functions in health and disease.

  19. Validation of an interferon stimulatory response element reporter gene assay for quantifying type I interferons. (United States)

    McCoski, S R; Xie, M; Hall, E B; Mercadante, P M; Spencer, T E; Lonergan, P; Ealy, A D


    The goal of this work was to develop a virus-free, cell-based interferon (IFN) bioassay and determine the utility of this assay on biological samples that contained IFN-τ, the trophoblast-secreted maternal recognition of pregnancy factor in ruminants. Madin-Darby bovine kidney cells were transduced with lentiviral particles that contained a firefly luciferase reporter construct driven by an IFN stimulatory response element (ISRE). Stably transduced cells were selected with the use of puromycin resistance. A linear, dose-responsive response was detected with human IFN-α and ovine IFN-τ. Interferon activity was detected in conditioned media from bovine trophoblast cells and uterine flushes collected from sheep and cattle. Activity also was detected in media collected after individual or small group culture of in vitro-produced bovine blastocysts at day 8 to 10 after fertilization. In summary, this IFN stimulatory response element-reporter assay may be used as an alternative to virus-dependent, cytopathic assays. It contains a similar sensitivity to IFNs and can be completed in a shorter time than cytopathic assays and does not require heightened biosafety conditions after cell transduction.

  20. Elevated cAMP increases aquaporin-3 plasma membrane diffusion

    DEFF Research Database (Denmark)

    Marlar, Saw; Christensen, Eva Arnspang; Koffman, Jennifer Skaarup


    .05)]. Immunoelectron microscopy showed no obvious difference in AQP3-EGFP expression levels or localization in the plasma membrane upon forskolin stimulation. Thus AQP3-EGFP diffusion is altered upon increased cAMP, which may correspond to basolateral adaptations in response to the increased apical water readsorption...

  1. Analysis of Resonance Response Performance of C-Band Antenna Using Parasitic Element

    Directory of Open Access Journals (Sweden)

    M. R. Zaman


    Full Text Available Analysis of the resonance response improvement of a planar C-band (4–8 GHz antenna is proposed using parasitic element method. This parasitic element based method is validated for change in the active and parasitic antenna elements. A novel dual-band antenna for C-band application covering 5.7 GHz and 7.6 GHz is designed and fabricated. The antenna is composed of circular parasitic element with unequal microstrip lines at both sides and a rectangular partial ground plane. A fractional bandwidth of 13.5% has been achieved from 5.5 GHz to 6.3 GHz (WLAN band for the lower band. The upper band covers from 7.1 GHz to 8 GHz with a fractional bandwidth of 12%. A gain of 6.4 dBi is achieved at the lower frequency and 4 dBi is achieved at the upper frequency. The VSWR of the antenna is less than 2 at the resonance frequency.

  2. AMP-Conjugated Quantum Dots: Low Immunotoxicity Both In Vitro and In Vivo (United States)

    Dai, Tongcheng; Li, Na; Liu, Lu; Liu, Qin; Zhang, Yuanxing


    Quantum dots (QDs) are engineered nanoparticles that possess special optical and electronic properties and have shown great promise for future biomedical applications. In this work, adenosine 5'-monophosphate (AMP), a small biocompatible molecular, was conjugated to organic QDs to produce hydrophilic AMP-QDs. Using macrophage J774A.1 as the cell model, AMP-QDs exhibited both prior imaging property and low toxicity, and more importantly, triggered limited innate immune responses in macrophage, indicating low immunotoxicity in vitro. Using BALB/c mice as the animal model, AMP-QDs were found to be detained in immune organs but did not evoke robust inflammation responses or obvious histopathological abnormalities, which reveals low immunotoxicity in vivo. This work suggests that AMP is an excellent surface ligand with low immunotoxicity, and potentially used in surface modification for more extensive nanoparticles.

  3. Inhibition of thyrotropin-stimulated DNA synthesis by microinjection of inhibitors of cellular Ras and cyclic AMP-dependent protein kinase. (United States)

    Kupperman, E; Wen, W; Meinkoth, J L


    Microinjection of a dominant interfering mutant of Ras (N17 Ras) caused a significant reduction in thyrotropin (thyroid-stimulating hormone [TSH])-stimulated DNA synthesis in rat thyroid cells. A similar reduction was observed following injection of the heat-stable protein kinase inhibitor of the cyclic AMP-dependent protein kinase. Coinjection of both inhibitors almost completely abolished TSH-induced DNA synthesis. In contrast to TSH, overexpression of cellular Ras protein did not stimulate the expression of a cyclic AMP response element-regulated reporter gene. Similarly, injection of N17 Ras had no effect on TSH-stimulated reporter gene expression. Moreover, overexpression of cellular Ras protein stimulated similar levels of DNA synthesis in the presence or absence of the heat-stable protein kinase inhibitor. Together, these results suggest that in Wistar rat thyroid cells, a full mitogenic response to TSH requires both Ras and cyclic APK-dependent protein kinase.

  4. Dopamine receptors modulate cytotoxicity of natural killer cells via cAMP-PKA-CREB signaling pathway.

    Directory of Open Access Journals (Sweden)

    Wei Zhao

    Full Text Available Dopamine (DA, a neurotransmitter in the nervous system, has been shown to modulate immune function. We have previously reported that five subtypes of DA receptors, including D1R, D2R, D3R, D4R and D5R, are expressed in T lymphocytes and they are involved in regulation of T cells. However, roles of these DA receptor subtypes and their coupled signal-transduction pathway in modulation of natural killer (NK cells still remain to be clarified. The spleen of mice was harvested and NK cells were isolated and purified by negative selection using magnetic activated cell sorting. After NK cells were incubated with various drugs for 4 h, flow cytometry measured cytotoxicity of NK cells against YAC-1 lymphoma cells. NK cells expressed the five subtypes of DA receptors at mRNA and protein levels. Activation of D1-like receptors (including D1R and D5R with agonist SKF38393 enhanced NK cell cytotoxicity, but activation of D2-like receptors (including D2R, D3R and D4R with agonist quinpirole attenuated NK cells. Simultaneously, SKF38393 elevated D1R and D5R expression, cAMP content, and phosphorylated cAMP-response element-binding (CREB level in NK cells, while quinpirole reduced D3R and D4R expression, cAMP content, and phosphorylated CREB level in NK cells. These effects of SKF38393 were blocked by SCH23390, an antagonist of D1-like receptors, and quinpirole effects were abolished by haloperidol, an antagonist of D2-like receptors. In support these results, H89, an inhibitor of phosphokinase A (PKA, prevented the SKF38393-dependent enhancement of NK cells and forskolin, an activator of adenylyl cyclase (AC, counteracted the quinpirole-dependent suppression of NK cells. These findings show that DA receptor subtypes are involved in modulation of NK cells and suggest that D1-like receptors facilitate NK cells by stimulating D1R/D5R-cAMP-PKA-CREB signaling pathway and D2-like receptors suppress NK cells by inhibiting D3R/D4R-cAMP-PKA-CREB signaling pathway. The

  5. Finite element simulation of rate-dependent magneto-active polymer response (United States)

    Haldar, K.; Kiefer, B.; Menzel, A.


    This contribution is concerned with the embedding of constitutive relations for magneto-active polymers (MAP) into finite element simulations. To this end, a recently suggested, calibrated, and validated material model for magneto-mechanically coupled and rate-dependent MAP response is briefly summarized in its continuous and algorithmic settings. Moreover, the strongly coupled field equations of finite deformation magneto-mechanics are reviewed. For the purpose of numerical simulation, a finite element model is then established based on the usual steps of weak form representation, discretization and consistent linearization. Two verifying inhomogeneous numerical examples are presented in which a classical ‘plate with a hole’ geometry is equipped with MAP properties and subjected to different types of time-varying mechanical and magnetic loading.

  6. Identification of peroxisome-proliferator responsive element in the mouse HSL gene. (United States)

    Yajima, Hiroaki; Kobayashi, Yumie; Kanaya, Tomoka; Horino, Yoko


    Hormone-sensitive lipase (HSL) catalyzes the rate-limiting step of lipolysis in adipose tissue. Several studies suggest that protein phosphorylation regulates the HSL enzymatic activity. On the other hand, the precise mechanism of the transcriptional regulation of the HSL gene remains to be elucidated. Here, we identified a functional peroxisome-proliferator responsive element (PPRE) in the mouse HSL promoter by reporter assay in CV-1 cells using serial deletion and point mutants of the 5'-flanking region. Chromatin immunoprecipitation (ChIP) analysis revealed that both peroxisome-proliferator activated receptor (PPARgamma) and retinoid X receptor (RXRalpha) interacted with the region. Binding of the PPARgamma/RXRalpha heterodimer to the PPRE sequence was also confirmed by electrophoretic mobility shift assay. These results indicate that the HSL gene is transcriptionally regulated by PPARgamma/RXRalpha heterodimer, and suggest that a cis-acting element regulates the HSL gene expression.


    Institute of Scientific and Technical Information of China (English)

    HUA Lei-na; YU Xi-ping


    This study presents an effective numerical model for the dynamic response of poroelastic seabed under wave action with enhanced performance. The spatial discretization is based on the Element-Free Galerkin (EFG) method and the time integration based on the GN11 scheme. A stability strategy that adopts a smaller number of nodes for the pore water pressure compared with those for the displacements of the soil skeleton is suggested to resolve the similar difficulty as encountered in the finite element method for a problem with mixed formulation when the pore water is incompressible and the soil skeleton impervious. The accuracy of the numerical model is verified through applying it to a typical case with critical permeability. Good agreement between computational and analytical solutions is obtained.

  8. Effects of rare earth elements and REE-binding proteins on physiological responses in plants. (United States)

    Liu, Dongwu; Wang, Xue; Chen, Zhiwei


    Rare earth elements (REEs), which include 17 elements in the periodic table, share chemical properties related to a similar external electronic configuration. REEs enriched fertilizers have been used in China since the 1980s. REEs could enter the cell and cell organelles, influence plant growth, and mainly be bound with the biological macromolecules. REE-binding proteins have been found in some plants. In addition, the chlorophyll activities and photosynthetic rate can be regulated by REEs. REEs could promote the protective function of cell membrane and enhance the plant resistance capability to stress produced by environmental factors, and affect the plant physiological mechanism by regulating the Ca²⁺ level in the plant cells. The focus of present review is to describe how REEs and REE-binding proteins participate in the physiological responses in plants.

  9. Pseudomonas aeruginosa β-lactamase induction requires two permeases, AmpG and AmpP

    Directory of Open Access Journals (Sweden)

    Schneper Lisa


    Full Text Available Abstract Background In Enterobacteriaceae, β-lactam antibiotic resistance involves murein recycling intermediates. Murein recycling is a complex process with discrete steps taking place in the periplasm and the cytoplasm. The AmpG permease is critical to this process as it transports N-acetylglucosamine anhydrous N-acetylmuramyl peptides across the inner membrane. In Pseudomonadaceae, this intrinsic mechanism remains to be elucidated. Since the mechanism involves two cellular compartments, the characterization of transporters is crucial to establish the link. Results Pseudomonas aeruginosa PAO1 has two ampG paralogs, PA4218 (ampP and PA4393 (ampG. Topology analysis using β-galactosidase and alkaline phosphatase fusions indicates ampP and ampG encode proteins which possess 10 and 14 transmembrane helices, respectively, that could potentially transport substrates. Both ampP and ampG are required for maximum expression of β-lactamase, but complementation and kinetic experiments suggest they act independently to play different roles. Mutation of ampG affects resistance to a subset of β-lactam antibiotics. Low-levels of β-lactamase induction occur independently of either ampP or ampG. Both ampG and ampP are the second members of two independent two-gene operons. Analysis of the ampG and ampP operon expression using β-galactosidase transcriptional fusions showed that in PAO1, ampG operon expression is β-lactam and ampR-independent, while ampP operon expression is β-lactam and ampR-dependent. β-lactam-dependent expression of the ampP operon and independent expression of the ampG operon is also dependent upon ampP. Conclusions In P. aeruginosa, β-lactamase induction occurs in at least three ways, induction at low β-lactam concentrations by an as yet uncharacterized pathway, at intermediate concentrations by an ampP and ampG dependent pathway, and at high concentrations where although both ampP and ampG play a role, ampG may be of greater

  10. Differential interactions of promoter elements in stress responses of the Arabidopsis Adh gene. (United States)

    Dolferus, R; Jacobs, M; Peacock, W J; Dennis, E S


    The Adh (alcohol dehydrogenase, EC gene from Arabidopsis thaliana (L.) Heynh. can be induced by dehydration and cold, as well as by hypoxia. A 1-kb promoter fragment (CADH: -964 to +53) is sufficient to confer the stress induction and tissue-specific developmental expression characteristics of the Adh gene to a beta-glucuronidase reporter gene. Deletion mapping of the 5' end and site-specific mutagenesis identified four regions of the promoter essential for expression under the three stress conditions. Some sequence elements are important for response to all three stress treatments, whereas others are stress specific. The most critical region essential for expression of the Arabidopsis Adh promoter under all three environmental stresses (region IV: -172 to -141) contains sequences homologous to the GT motif (-160 to -152) and the GC motif (-147 to -144) of the maize Adh1 anaerobic responsive element. Region III (-235 to -172) contains two regions shown by R.J. Ferl and B.H. Laughner ([1989] Plant Mol Biol 12: 357-366) to bind regulatory proteins; mutation of the G-box-1 region (5'-CCACGTGG-3', -216 to -209) does not affect expression under uninduced or hypoxic conditions, but significantly reduces induction by cold stress and, to a lesser extent, by dehydration stress. Mutation of the other G-box-like sequence (G-box-2: 5'-CCAAGTGG-3', -193 to -182) does not change hypoxic response and affects cold and dehydration stress only slightly. G-box-2 mutations also promote high levels of expression under uninduced conditions. Deletion of region I (-964 to -510) results in increased expression under uninduced and all stress conditions, suggesting that this region contains a repressor binding site. Region II (-510 to -384) contains a positive regulatory element and is necessary for high expression levels under all treatments. PMID:7972489

  11. cAMP-independent signal pathways stimulate hyphal morphogenesis in Candida albicans. (United States)

    Parrino, Salvatore M; Si, Haoyu; Naseem, Shamoon; Groudan, Kevin; Gardin, Justin; Konopka, James B


    The fungal pathogen Candida albicans can transition from budding to hyphal growth, which promotes biofilm formation and invasive growth into tissues. Stimulation of adenylyl cyclase to form cAMP induces hyphal morphogenesis. The failure of cells lacking adenylyl cyclase (cyr1Δ) to form hyphae has suggested that cAMP signaling is essential for hyphal growth. However, cyr1Δ mutants also grow slowly and have defects in morphogenesis, making it unclear whether hyphal inducers must stimulate cAMP, or if normal basal levels of cAMP are required to maintain cellular health needed for hyphal growth. Interestingly, supplementation of cyr1Δ cells with low levels of cAMP enabled them to form hyphae in response to the inducer N-acetylglucosamine (GlcNAc), suggesting that a basal level of cAMP is sufficient for stimulation. Furthermore, we isolated faster-growing cyr1Δ pseudorevertant strains that can be induced to form hyphae even though they lack cAMP. The pseudorevertant strains were not induced by CO2 , consistent with reports that CO2 directly stimulates adenylyl cyclase. Mutational analysis showed that induction of hyphae in a pseudorevertant strain was independent of RAS1, but was dependent on the EFG1 transcription factor that acts downstream of protein kinase A. Thus, cAMP-independent signals contribute to the induction of hyphal responses.

  12. Piperine, a component of black pepper, decreases eugenol-induced cAMP and calcium levels in non-chemosensory 3T3-L1 cells. (United States)

    Yoon, Yeo Cho; Kim, Sung-Hee; Kim, Min Jung; Yang, Hye Jeong; Rhyu, Mee-Ra; Park, Jae-Ho


    This study investigated the effects of an ethanol extract of black pepper and its constituent, piperine, on odorant-induced signal transduction in non-chemosensory cells. An ethanol extract of black pepper decreased eugenol-induced cAMP and calcium levels in preadipocyte 3T3-L1 cells with no toxicity. Phosphorylation of CREB (cAMP response element-binding protein) was down-regulated by the black pepper extract. The concentration (133.8 mg/g) and retention time (5.5 min) of piperine in the ethanol extract were quantified using UPLC-MS/MS. Pretreatment with piperine decreased eugenol-induced cAMP and calcium levels in 3T3-L1 cells. Piperine also decreased the phosphorylation of CREB, which is up-regulated by eugenol. These results suggest that piperine inhibits the eugenol-induced signal transduction pathway through modulation of cAMP and calcium levels and phosphorylation of CREB in non-chemosensory cells.

  13. LAS0811: From Combinatorial Chemistry to Activation of Antioxidant Response Element


    Ming Zhu; Hyounggee Baek; Ruiwu Liu; Aimin Song; Kit Lam; Derick Lau


    The antioxidant response element (ARE) and its transcription factor, nuclear factor-erythroid 2 p45-related factor 2 (Nrf2), are potential targets for cancer chemoprevention. We sought to screen small molecules synthesized with combinatorial chemistry for activation of ARE. By high-throughput screening of 9400 small molecules from 10 combinatorial chemical libraries using HepG2 cells with an ARE-driven reporter, we have identified a novel small molecule, 1,2-dimethoxy-4,5-dinitrobenzene (LAS0...

  14. Identification of putative PPAR response elements in and around the murine UCP3 gene

    DEFF Research Database (Denmark)

    Siersbæk, Majken

    in thermogenesis in unerring, the physiological functions of UCP2 and UCP3 are at present not fully understood. Synthetic agonists for the peroxisome proliferator-activated receptors (PPARs) as well as fatty acids have been shown to increase murine UCP2 and UCP3 mRNA expression but response elements and mechanisms...... differentiation. Results from sequencing of chromatin immunoprecipitated (ChIP) material from 3T3-L1 adipocytes revealed three PPAR and retinoid X receptor (RXR) binding sites in and around the murine UCP3 gene. The recruitment of PPAR and RXR to the three potential PPREs was determined by ChIP combined...

  15. On the structure of AP-4 responsive element in the LTR of Jembrana disease virus

    Institute of Scientific and Technical Information of China (English)


    Previous studies with deletion and sequence analysis of JDV LTR showed that there is a putative AP-4 responsive element in LTR. By antisense transient assay and gel shifting assay, we for the first time demonstrated that AP-4 modulated JDV gene expression by binding DNA directly to bovine cells. The results, derived from site-directed mutagenesis experiments, suggest that the six base pairs of AP-4 binding site (CAGCTG) have different effects on JDV gene expression. When the first two base pairs changed to GC, JDV gene expression is severely decreased.

  16. Spatial Finite Element Analysis for Dynamic Response of Curved Thin-Walled Box Girder Bridges

    Directory of Open Access Journals (Sweden)

    Yinhui Wang


    Full Text Available According to the flexural and torsional characteristics of curved thin-walled box girder with the effect of initial curvature, 7 basic displacements of curved box girder are determined. And then the strain-displacement calculation correlations were established. Under the curvilinear coordinate system, a three-noded curved girder finite element which has 7 degrees of freedom per node for the vibration characteristic and dynamic response analysis of curved box girder is constructed. The shape functions are used as the interpolation functions of variable curvature and variable height to accommodate to the variation of curvature and section height. A MATLAB numerical analysis program has been implemented.

  17. The antioxidant responsive element (ARE) may explain the protective effects of cruciferous vegetables on cancer. (United States)

    Finley, John W


    Research supports the hypothesis that one's diet has a great impact on his or her risk of cancer. Many studies have found that increased fruit and vegetable intake decreases the risk of cancer. Cruciferous vegetables such as broccoli and cauliflower seem to be especially protective against cancer. Most studies show that phytochemicals in crucifers up-regulate many detoxification enzyme systems in the animal that consumes them. Recent reports of the molecular events involved in the activation of a gene promoter called the antioxidant responsive element have begun to provide clues as to how a single substance may induce a battery of many genes.

  18. Sesamin induces melanogenesis by microphthalmia-associated transcription factor and tyrosinase up-regulation via cAMP signaling pathway

    Institute of Scientific and Technical Information of China (English)

    Zequn Jiang; Shasha Li; Yunyi Liu; Pengyi Deng; Jianguo Huang; Guangyuan He


    In this study,we confirmed that sesamin,an active lignan isolated from sesame seed and oil,is a novel skin-tanning compound.The melanin content and tyrosinase activity were increased by sesamin in a dose-dependent manner in B16 melanoma cells.The mRNA and protein levels of tyrosinase were also enhanced after the treatment with sesamin.Western blot analysis revealed that sesamin induced and sustained up-regulation of microphthalmiaassociated transcription factor (MITF).Sesamin could activate cAMP response element (CRE) binding protein (CREB),but it had no effect on the phosphorylation of p38 mitogen-activated protein kinase (MAPK) or Akt.Moreover,sesamin activated protein kinase A (PKA) via a cAMP-dependent pathway.Consistent with these results,sesamin-mediated increase of melanin synthesis was reduced significantly by H-89,a PKA inhibitor,but not by SB203580,a p38 MAPK inhibitor or by LY294002,a phosphatidylinositol-3-kinase (PI3K) inhibitor.Sesamin-mediated phosphorylation of CREB and induction of MITF and tyrosinase expression were also inhibited by H-89.These findings indicated that sesamin could stimulate melanogenesis in B16 cells via the up-regulation of MITF and tyrosinase,which was,in turn,due to the activation of cAMP signaling.

  19. Sesamin induces melanogenesis by microphthalmia-associated transcription factor and tyrosinase up-regulation via cAMP signaling pathway. (United States)

    Jiang, Zequn; Li, Shasha; Liu, Yunyi; Deng, Pengyi; Huang, Jianguo; He, Guangyuan


    In this study, we confirmed that sesamin, an active lignan isolated from sesame seed and oil, is a novel skin-tanning compound. The melanin content and tyrosinase activity were increased by sesamin in a dose-dependent manner in B16 melanoma cells. The mRNA and protein levels of tyrosinase were also enhanced after the treatment with sesamin. Western blot analysis revealed that sesamin induced and sustained up-regulation of microphthalmia-associated transcription factor (MITF). Sesamin could activate cAMP response element (CRE) binding protein (CREB), but it had no effect on the phosphorylation of p38 mitogen-activated protein kinase (MAPK) or Akt. Moreover, sesamin activated protein kinase A (PKA) via a cAMP-dependent pathway. Consistent with these results, sesamin-mediated increase of melanin synthesis was reduced significantly by H-89, a PKA inhibitor, but not by SB203580, a p38 MAPK inhibitor or by LY294002, a phosphatidylinositol-3-kinase (PI3K) inhibitor. Sesamin-mediated phosphorylation of CREB and induction of MITF and tyrosinase expression were also inhibited by H-89. These findings indicated that sesamin could stimulate melanogenesis in B16 cells via the up-regulation of MITF and tyrosinase, which was, in turn, due to the activation of cAMP signaling.

  20. Analysis of nonexponential transient response due to a constant-phase element

    Energy Technology Data Exchange (ETDEWEB)

    Heuveln, F.H. van (Netherlands Energy Research Foundation, Petten (Netherlands))


    To characterize electrical losses of fuel cells or batteries, impedance spectroscopy (IS) or current interruption (CI) can be used. Analysis and parameter determination of impedance data is widely used. The system under study is usually represented by an equivalent circuit from which the system parameters can be determined. However, the analysis of current-interruption data is often carried out with too simple circuits, e.g., using pure exponential behavior, because analysis in the time domain (CI) is often much more awkward than analysis in the frequency domain (IS). A comparative study has been carried out on the analysis of a relatively ideal electrical circuit, containing a pure capacitor, and a more realistic circuit where the capacitor is replaced by a constant-phase element. Equations describing the response in the frequency and time domain are presented. Emphasis input upon the analysis of circuits containing a constant-phase element because impedance measurements clearly indicate the presence of such an element in many types of experiments, and because there is only limited literature available describing the behavior in the time domain.

  1. Analysis of non-exponential transient response due to a constant phase element

    Energy Technology Data Exchange (ETDEWEB)

    Van Heuveln, F.H.


    To characterize electrical losses of fuel cells or batteries, impedance spectroscopy (IS) or current interruption (CI) can be used. Analysis and parameter determination of impedance data is widely used. The system under study is usually represented by an equivalent circuit from which the system parameters can be determined. However, the analysis of current interruption data is often carried out with too simple circuits, e.g., using pure exponential behavior, because analysis in the time domain (CI) is often much more awkward then analysis in the frequency domain (IS). A comparative study has been carried out on the analysis of a relatively ideal electrical circuit, containing a pure capacitor, and a more realistic circuit where the capacitor is replaced by a constant phase element. Equations describing the response in the frequency and time domain are presented. Emphasis is put upon the analysis of circuits containing a constant phase element, because impedance measurements clearly indicate the presence of such an element in many types of experiments, and because there is only limited literature available describing the behavior in the time domain. 6 figs., 11 refs.

  2. Core influence on the frequency response analysis (FRA of power transformers through the finite element method

    Directory of Open Access Journals (Sweden)

    D. L. Alvarez


    Full Text Available In this paper the influence of core parameters in Frequency Response Analysis is analyzed through the equivalent circuit impedance matrix of the transformer winding; the parameters of the circuit have been computed using the Finite Element Method. In order to appreciate the behavior of the iron core in comparison to the air core, the frequency dependence of resonances is calculated to show how the air core only influences the results at low frequencies. The core is modeled using a complex permeability, and the parameters of conductivity and permeability are varied to show their influence in the resonances, which turned out to be negligible. In order to explain this behavior, the eigenvalues of the inverse impedance matrix are calculated showing that they are similar for different values of conductivity and permeability. Finally, the magnetic flux inside and outside the core and its influence in the frequency response is studied.

  3. Cyclic-AMP regulation of calcium-dependent K channels in an insect central neurone. (United States)

    David, J A; Pitman, R M


    In the cockroach fast coxal depressor motoneurone, either the muscarinic agonist McN-A-343 or dibutyryl cAMP (Db-cAMP) induced a reduction in voltage-dependent outward current. The response to McN is due to suppression of a calcium-dependent potassium current (IK,Ca) produced secondarily to a reduction in voltage-dependent calcium current (ICa). The response to Db-cAMP was investigated in order to establish whether cAMP might mediate the response to McN. ICa was suppressed by 3-isobutyl-1-methylxanthine (IBMX) but not by Db-cAMP. The effects of IBMX were therefore unlikely to be the result of phosphodiesterase inhibition. Since caffeine also suppressed ICa, the observed effect of IBMX is probably due to release of Ca2+ from intracellular stores. IK,Ca, evoked by injection of Ca2+, was reduced by Db-cAMP or forskolin but not by McN. These results indicate that the electrical response to McN in this neurone is not mediated by changes in cAMP.

  4. A common signaling pathway is activated in erythroid cells expressing high levels of fetal hemoglobin: a potential role for cAMP-elevating agents in β-globin disorders

    Directory of Open Access Journals (Sweden)

    Ikuta T


    Full Text Available Tohru Ikuta,1 Yuichi Kuroyanagi,1 Nadine Odo,1 Siyang Liu21Department of Anesthesiology and Perioperative Medicine, 2Department of Physiology, Medical College of Georgia, Georgia Regents University, Augusta, GA, USABackground: Although erythroid cells prepared from fetal liver, cord blood, or blood from β-thalassemia patients are known to express fetal hemoglobin at high levels, the underlying mechanisms remain elusive. We previously showed that cyclic nucleotides such as cAMP and cGMP induce fetal hemoglobin expression in primary erythroid cells. Here we report that cAMP signaling contributes to high-level fetal hemoglobin expression in erythroid cells prepared from cord blood and β-thalassemia.Methods: The status of the cAMP signaling pathway was investigated using primary erythroid cells prepared from cord blood and the mononuclear cells of patients with β-thalassemia; erythroid cells from adult bone marrow mononuclear cells served as the control.Results: We found that intracellular cAMP levels were higher in erythroid cells from cord blood and β-thalassemia than from adult bone marrow. Protein kinase A activity levels and cAMP-response element binding protein phosphorylation were higher in erythroid cells from cord blood or β-thalassemia than in adult bone marrow progenitors. Mitogen-activated protein kinase pathways, which play a role in fetal hemoglobin expression, were not consistently activated in cord blood or β-thalassemia erythroid cells. When cAMP signaling was activated in adult erythroid cells, fetal hemoglobin was induced at high levels and associated with reduced expression of BCL11A, a silencer of the β-globin gene.Conclusion: These results suggest that activated cAMP signaling may be a common mechanism among erythroid cells with high fetal hemoglobin levels, in part because of downregulation of BCL11A. Activation of the cAMP signaling pathway with cAMP-elevating agents may prove to be an important signaling mechanism to

  5. Molecular cloning and expression of chicken carbohydrate response element binding protein and Max-like protein X gene homologues (United States)

    Carbohydrate response element binding protein (ChREBP) and sterol regulatory element binding protein-1c (SREBP-1c) are transcription factors that are known to be key regulators of glucose metabolism and lipid synthesis in mammals. Since ChREBP and its co-activator Max-like protein X (Mlx) have not ...

  6. Cyclic 3', 5'-AMP relay in Dictyostelium discoideum: adaptation is independent of activation of adenylate cyclase



    In Dictyostelium discoideum, binding of cAMP to high affinity surface receptors leads to a rapid activation of adenylate cyclase followed by subsequent adaptation within several minutes. The rate of secretion of [ 3H ]cAMP, which reflects the state of activation of the enzyme, was measured. Caffeine noncompetitively inhibited the response to cAMP. Inhibition was rapidly reversible and pretreatment of cells with caffeine for up to 22 min had little effect on the subsequent responsiveness to cA...

  7. Cyclic AMP activates the mitogen-activated protein kinase cascade in PC12 cells

    DEFF Research Database (Denmark)

    Frödin, M; Peraldi, P; Van Obberghen, E


    Mitogen-activated protein (MAP) kinases are activated in response to a large variety of extracellular signals, including growth factors, hormones, and neurotransmitters, which activate distinct intracellular signaling pathways. Their activation by the cAMP-dependent pathway, however, has not been...... reported. In rat pheochromocytoma PC12 cells, we demonstrate here a stimulation of the MAP kinase isozyme extracellular signal-regulated kinase 1 (ERK1) following elevation of intracellular cAMP after exposure of the cells to isobutylmethylxanthine, cholera toxin, forskolin, or cAMP-analogues. cAMP acted...... synergistically with phorbol ester, an activator of protein kinase C, in the stimulation of ERK1. In accordance with this observation, the peptide neurotransmitter pituitary adenylate cyclase-activating polypeptide 38 (PACAP38), which stimulates cAMP production as well as phosphatidylinositol breakdown in PC12...

  8. Effects of Frequency and Acceleration Amplitude on Osteoblast Mechanical Vibration Responses: A Finite Element Study

    Directory of Open Access Journals (Sweden)

    Liping Wang


    Full Text Available Bone cells are deformed according to mechanical stimulation they receive and their mechanical characteristics. However, how osteoblasts are affected by mechanical vibration frequency and acceleration amplitude remains unclear. By developing 3D osteoblast finite element (FE models, this study investigated the effect of cell shapes on vibration characteristics and effect of acceleration (vibration intensity on vibrational responses of cultured osteoblasts. Firstly, the developed FE models predicted natural frequencies of osteoblasts within 6.85–48.69 Hz. Then, three different levels of acceleration of base excitation were selected (0.5, 1, and 2 g to simulate vibrational responses, and acceleration of base excitation was found to have no influence on natural frequencies of osteoblasts. However, vibration response values of displacement, stress, and strain increased with the increase of acceleration. Finally, stress and stress distributions of osteoblast models under 0.5 g acceleration in Z-direction were investigated further. It was revealed that resonance frequencies can be a monotonic function of cell height or bottom area when cell volumes and material properties were assumed as constants. These findings will be useful in understanding how forces are transferred and influence osteoblast mechanical responses during vibrations and in providing guidance for cell culture and external vibration loading in experimental and clinical osteogenesis studies.

  9. Targeting brain tumor cAMP: the case for sex-specific therapeutics

    Directory of Open Access Journals (Sweden)

    Nicole M Warrington


    Full Text Available A relationship between cyclic adenosine 3’, 5’-monophosphate (cAMP levels and brain tumor biology has been evident for nearly as long as cAMP and its synthetase, adenylate cyclase (ADCY have been known. The importance of the pathway in brain tumorigenesis has been demonstrated in vitro and in multiple animal models. Recently, we provided human validation for a cooperating oncogenic role for cAMP in brain tumorigenesis when we found that SNPs in ADCY8 were correlated with glioma (brain tumor risk in individuals with Neurofibromatosis type 1 (NF1. Together, these studies provide a strong rationale for targeting cAMP in brain tumor therapy. However, the cAMP pathway is well known to be sexually dimorphic, and SNPs in ADCY8 affected glioma risk in a sex-specific fashion, elevating the risk for females while protecting males. The cAMP pathway can be targeted at multiple levels in the regulation of its synthesis and degradation. Sex differences in response to drugs that target cAMP regulators indicate that successful targeting of the cAMP pathway for brain tumor patients is likely to require matching specific mechanisms of drug action with patient sex.

  10. Dibutyryl cAMP effects on thromboxane and leukotriene production in decompression-induced lung injury (United States)

    Little, T. M.; Butler, B. D.


    Decompression-induced venous bubble formation has been linked to increased neutrophil counts, endothelial cell injury, release of vasoactive eicosanoids, and increased vascular membrane permeability. These actions may account for inflammatory responses and edema formation. Increasing the intracellular cAMP has been shown to decrease eicosanoid production and edema formation in various models of lung injury. Reduction of decompression-induced inflammatory responses was evaluated in decompressed rats pretreated with saline (controls) or dibutyryl cAMP (DBcAMP, an analog of cAMP). After pretreatment, rats were exposed to either 616 kPa for 120 min or 683 kPa for 60 min. The observed increases in extravascular lung water ratios (pulmonary edema), bronchoalveolar lavage, and pleural protein in the saline control group (683 kPa) were not evident with DBcAMP treatment. DBcAMP pretreatment effects were also seen with the white blood cell counts and the percent of neutrophils in the bronchoalveolar lavage. Urinary levels of thromboxane B2, 11-dehydrothromboxane B2, and leukotriene E4 were significantly increased with the 683 kPa saline control decompression exposure. DBcAMP reduced the decompression-induced leukotriene E4 production in the urine. Plasma levels of thromboxane B2, 11-dehydrothromboxane B2, and leukotriene E4 were increased with the 683-kPa exposure groups. DBcAMP treatment did not affect these changes. The 11-dehydrothromboxane B2 and leukotriene E4 levels in the bronchoalveolar lavage were increased with the 683 kPa exposure and were reduced with the DBcAMP treatment. Our results indicate that DBcAMP has the capability to reduce eicosanoid production and limit membrane permeability and subsequent edema formation in rats experiencing decompression sickness.

  11. Prognostic value of coexistence of abnormal expression of micro-RNA-200b and cyclic adenosine monophosphate-responsive element-binding protein 1 in human astrocytoma. (United States)

    Zhang, Jun-qing; Yao, Qing-he; Kuang, Yong-qin; Ma, Yuan; Yang, Li-bin; Huang, Hai-dong; Cheng, Jing-ming; Yang, Tao; Liu, En-yu; Liang, Liang; Fan, Ke-xia; Zhao, Kai; Xia, Xun; Gu, Jian-wen


    Our aim was to investigate the expression of micro-RNA-200b (miR-200b) and cAMP-responsive element-binding protein 1 (CREB-1) in astrocytoma and its efficacy for predicting outcome. Both miR-200b and CREB-1 messenger RNA expression was measured in 122 astrocytomas and 30 nonneoplastic brain specimens by quantitative real-time polymerase chain reaction. Expression of miR-200b was significantly lower in astrocytoma than in nonneoplastic brain (P RNA expression was significantly elevated in the tumors (P < .001). Both miR-200b down-regulation and CREB-1 up-regulation were significantly associated with advanced pathologic grade (P = .002 and P = .006, respectively). Low miR-200b expression correlated negatively with Karnofsky performance score (P = .03), and high CREB-1 expression correlated positively with mean tumor diameter (P = .03). By Kaplan-Meier analysis, low miR-200b, high CREB-1, and coexistence of abnormal miR-200b and CREB-1 expression (low miR-200b/high CREB-1) were predictive of shorter progression-free survival and overall survival in both grade III and grade IV astrocytoma. By multivariate analysis, only low miR-200b/high CREB-1 expression was an independent prognostic factor for poor prognosis in astrocytoma of advanced grade. Both miR-200b and CREB-1 may play important cooperative roles in the progression of human astrocytoma. The efficacy of miR-200b and CREB-1 together as a predictor of prognosis in astrocytoma patients is shown for the first time.

  12. A Space-Time Finite Element Model for Design and Control Optimization of Nonlinear Dynamic Response

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    P.P. Moita


    Full Text Available A design and control sensitivity analysis and multicriteria optimization formulation is derived for flexible mechanical systems. This formulation is implemented in an optimum design code and it is applied to the nonlinear dynamic response. By extending the spatial domain to the space-time domain and treating the design variables as control variables that do not change with time, the design space is included in the control space. Thus, one can unify in one single formulation the problems of optimum design and optimal control. Structural dimensions as well as lumped damping and stiffness parameters plus control driven forces, are considered as decision variables. The dynamic response and its sensitivity with respect to the design and control variables are discretized via space-time finite elements, and are integrated at-once, as it is traditionally used for static response. The adjoint system approach is used to determine the design sensitivities. Design optimization numerical examples are performed. Nonlinear programming and optimality criteria may be used for the optimization process. A normalized weighted bound formulation is used to handle multicriteria problems.

  13. Identification and Validation of a Putative Polycomb Responsive Element in the Human Genome.

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    Hemant Bengani

    Full Text Available Epigenetic cellular memory mechanisms that involve polycomb and trithorax group of proteins are well conserved across metazoans. The cis-acting elements interacting with these proteins, however, are poorly understood in mammals. In a directed search we identified a potential polycomb responsive element with 25 repeats of YY1 binding motifthatwe designate PRE-PIK3C2B as it occurs in the first intron of human PIK3C2B gene. It down regulates reporter gene expression in HEK cells and the repression is dependent on polycomb group of proteins (PcG. We demonstrate that PRE-PIK3C2B interacts directly with YY1 in vitro and recruits PRC2 complex in vivo. The localization of PcG proteins including YY1 to PRE-PIK3C2B in HEK cells is decreased on knock-down of either YY1 or SUZ12. Endogenous PRE-PIK3C2B shows bivalent marking having H3K27me3 and H3K4me3 for repressed and active state respectively. In transgenic Drosophila, PRE-PIK3C2B down regulates mini-white expression, exhibits variegation and pairing sensitive silencing (PSS, which has not been previously demonstrated for mammalian PRE. Taken together, our results strongly suggest that PRE-PIK3C2B functions as a site of interaction for polycomb proteins.

  14. HIV Rev Assembly on the Rev Response Element (RRE: A Structural Perspective

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    Jason W. Rausch


    Full Text Available HIV-1 Rev is an ~13 kD accessory protein expressed during the early stage of virus replication. After translation, Rev enters the nucleus and binds the Rev response element (RRE, a ~350 nucleotide, highly structured element embedded in the env gene in unspliced and singly spliced viral RNA transcripts. Rev-RNA assemblies subsequently recruit Crm1 and other cellular proteins to form larger complexes that are exported from the nucleus. Once in the cytoplasm, the complexes dissociate and unspliced and singly-spliced viral RNAs are packaged into nascent virions or translated into viral structural proteins and enzymes, respectively. Rev binding to the RRE is a complex process, as multiple copies of the protein assemble on the RNA in a coordinated fashion via a series of Rev-Rev and Rev-RNA interactions. Our understanding of the nature of these interactions has been greatly advanced by recent studies using X-ray crystallography, small angle X-ray scattering (SAXS and single particle electron microscopy as well as biochemical and genetic methodologies. These advances are discussed in detail in this review, along with perspectives on development of antiviral therapies targeting the HIV-1 RRE.

  15. Gold-nanoparticle-based assay for instantaneous detection of nuclear hormone receptor-response elements interactions. (United States)

    Tan, Yen Nee; Su, Xiaodi; Liu, Edison T; Thomsen, Jane S


    Gold nanoparticles (AuNPs) are widely used as colorimetric probes for biosensing, relying on their unique particle size-dependent and/or interparticle distance-dependent extinction spectrum and solution color. Herein, we describe an AuNP-based colorimetric assay to detect binding interactions between nuclear hormone receptors and their corresponding DNA-binding elements, particularly the human estrogen receptors (ERalpha and ERbeta) and their cognate estrogen response elements (EREs). We found that the protein-DNA (ER-ERE) complexes can stabilize citrate anion-capped AuNPs against salt-induced aggregation to a larger extent than the protein (ER) or the DNA (ERE) alone, due to their unique molecular size and charge properties that provide a strong electrosteric protection. Moreover, our results show that the extent of stabilization is sequence-dependent and can distinguish a single base variation in the ERE associated with minor changes in protein-DNA binding affinity. With this assay, many important parameters of protein-DNA binding events (e.g., sequence selectivity, distinct DNA binding properties of protein subtypes, binding stoichiometry, and sequence-independent transient binding) can be determined instantly without using labels, tedious sample preparations, and sophisticated instrumentation. These benefits, in particular the high-throughput potential, could enable this assay to become the assay of choice to complement conventional techniques for large scale characterization of protein-DNA interactions, a key aspect in biological research.

  16. Application of finite element, global polynomial, and kriging response surfaces in Progressive Lattice Sampling designs

    Energy Technology Data Exchange (ETDEWEB)



    This paper examines the modeling accuracy of finite element interpolation, kriging, and polynomial regression used in conjunction with the Progressive Lattice Sampling (PLS) incremental design-of-experiments approach. PLS is a paradigm for sampling a deterministic hypercubic parameter space by placing and incrementally adding samples in a manner intended to maximally reduce lack of knowledge in the parameter space. When combined with suitable interpolation methods, PLS is a formulation for progressive construction of response surface approximations (RSA) in which the RSA are efficiently upgradable, and upon upgrading, offer convergence information essential in estimating error introduced by the use of RSA in the problem. The three interpolation methods tried here are examined for performance in replicating an analytic test function as measured by several different indicators. The process described here provides a framework for future studies using other interpolation schemes, test functions, and measures of approximation quality.

  17. A novel human polycomb binding site acts as a functional polycomb response element in Drosophila.

    Directory of Open Access Journals (Sweden)

    Suresh Cuddapah

    Full Text Available Polycomb group (PcG proteins are key chromatin regulators implicated in multiple processes including embryonic development, tissue homeostasis, genomic imprinting, X-chromosome inactivation, and germ cell differentiation. The PcG proteins recognize target genomic loci through cis DNA sequences known as Polycomb Response Elements (PREs, which are well characterized in Drosophila. However, mammalian PREs have been elusive until two groups reported putative mammalian PREs recently. Consistent with the existence of mammalian PREs, here we report the identification and characterization of a potential PRE from human T cells. The putative human PRE has enriched binding of PcG proteins, and such binding is dependent on a key PcG component SUZ12. We demonstrate that the putative human PRE carries both genetic and molecular features of Drosophila PRE in transgenic flies, implying that not only the trans PcG proteins but also certain features of the cis PREs are conserved between mammals and Drosophila.

  18. Linking cellular actin status with cAMP signaling in Candida albicans. (United States)

    Wang, Yue; Zou, Hao; Fang, Hao-Ming; Zhu, Yong


    The fungal pathogen Candida albicans has a remarkable ability to switch growth forms. Particularly, the yeast-to-hyphae switch is closely linked with its virulence. A range of chemicals and conditions can promote hyphal growth including serum, peptidoglycan, CO2, neutral pH, and elevated temperature. All these signals act essentially through the adenylyl cyclase Cyr1 that synthesizes cAMP. Cells lacking Cyr1 are completely defective in hyphal growth. Recently, cellular actin status is found to influence cAMP synthesis. However, how Cyr1 senses and processes multiple external and internal signals to produce a contextually proper level of cAMP remains unclear. We hypothesized that Cyr1 itself possesses multiple sensors for different signals and achieves signal integration through a combined allosteric effect on the catalytic center. To test this hypothesis, we affinity-purified a Cyr1-containing complex and found that it could enhance cAMP synthesis upon treatment with serum, peptidoglycan or CO2 in vitro. The data indicate that the complex is an essentially intact sensor/effector apparatus for cAMP synthesis. The complex contains two more subunits, the cyclase-associated protein Cap1 and G-actin. We discovered that G-actin plays a regulatory role, rendering cAMP synthesis responsive to actin dynamics. These findings shed new lights on the mechanisms that regulate cAMP-mediated responses in fungi.

  19. Activation of a Non-cAMP/PKA Signaling Pathway Downstream of the PTH/PTHrP Receptor Is Essential for a Sustained Hypophosphatemic Response to PTH Infusion in Male Mice (United States)

    Song, Lige; Liu, Minlin; Segawa, Hiroko; Miyamoto, Ken-Ichi; Bringhurst, F. Richard; Kronenberg, Henry M.; Jüppner, Harald


    PTH increases urinary Pi excretion by reducing expression of two renal cotransporters [NaPi-IIa (Npt2a) and NaPi-IIc (Npt2c)]. In contrast to acute transporter regulation that is cAMP/protein kinase A dependent, long-term effects require phospholipase C (PLC) signaling by the PTH/PTHrP receptor (PPR). To determine whether the latter pathway regulates Pi through Npt2a and/or Npt2c, wild-type mice (Wt) and animals expressing a mutant PPR incapable of PLC activation (DD) were tested in the absence of one (Npt2a−/− or Npt2c−/−) or both phosphate transporters (2a/2c-dko). PTH infusion for 8 days caused a rapid and persistent decrease in serum Pi in Wt mice, whereas serum Pi in DD mice fell only transiently for the first 2 days. Consistent with these findings, fractional Pi excretion index was increased initially in both animals, but this increase persisted only when the PPR Wt was present. The hypophosphatemic response to PTH infusion was impaired only slightly in PPR Wt/Npt2c−/− or DD/Npt2c−/− mice. Despite lower baselines, PTH infusion in PPR Wt/Npt2a−/− mice decreased serum Pi further, an effect that was attenuated in DD/Npt2a−/− mice. Continuous PTH had no effect on serum Pi in 2a/2c-dko mice. PTH administration increased serum 1,25 dihydroxyvitamin D3 levels in Wt and DD mice and increased levels above the elevated baseline with ablation of either but not of both transporters. Continuous PTH elevated serum fibroblast growth factor 23 and blood Ca2+ equivalently in all groups of mice. Our data indicate that PLC signaling at the PPR contributes to the long-term effect of PTH on Pi homeostasis but not to the regulation of 1,25 dihydroxyvitamin D3, fibroblast growth factor 23, or blood Ca2+. PMID:23515284

  20. cAMP-binding proteins in medullary tubules from rat kidney: effect of ADH

    Energy Technology Data Exchange (ETDEWEB)

    Gapstur, S.M.; Homma, S.; Dousa, T.P.


    Little is known of the regulatory steps in the cellular action of vasopressin (AVP) on the renal epithelium, subsequent to the cAMP generation. We studied cAMP-binding proteins in the medullary collecting tubule (MCT) and the thick ascending limb of Henle's loop (MTAL) microdissected from the rat kidney by use of photoaffinity labeling. Microdissected tubules were homogenized and photoaffinity labeled by incubation with 1 microM 32P-labeled 8-azido-adenosine 3',5'-cyclic monophosphate (N3-8-(32P)-cAMP); the incorporated 32P was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Both in MCT and MTAL preparations, the analyses showed incorporation of N3-8-(32P)cAMP into two bands (Mr = 49,000 and Mr = 55,000) that comigrated with standards of the cAMP-dependent protein kinase regulatory subunits RI and RII. In MCT, most of the 32P (80%) was incorporated into RI, whereas in MTAL the 32P incorporated into RI and RII was equivalent. When freshly dissected MCT segments were incubated with 10(-12)-10(-6) M AVP, the subsequent photoaffinity labeling of RI with N3-8-(32P)cAMP was markedly diminished in a dose-dependent manner compared with controls. Our results suggest that cAMP binds in MCT and MTAL to regulatory subunits RI and RII of cAMP-dependent protein kinase. However, in MCT the dominant type of cAMP-dependent protein kinase appears to be type I. The outlined procedure is suitable to indirectly measure the occupancy of RI by endogenous cAMP generated in MCT cells in response to physiological levels (10(-12) M) of AVP.

  1. Intrasteric control of AMPK via the gamma1 subunit AMP allosteric regulatory site. (United States)

    Adams, Julian; Chen, Zhi-Ping; Van Denderen, Bryce J W; Morton, Craig J; Parker, Michael W; Witters, Lee A; Stapleton, David; Kemp, Bruce E


    AMP-activated protein kinase (AMPK) is a alphabetagamma heterotrimer that is activated in response to both hormones and intracellular metabolic stress signals. AMPK is regulated by phosphorylation on the alpha subunit and by AMP allosteric control previously thought to be mediated by both alpha and gamma subunits. Here we present evidence that adjacent gamma subunit pairs of CBS repeat sequences (after Cystathionine Beta Synthase) form an AMP binding site related to, but distinct from the classical AMP binding site in phosphorylase, that can also bind ATP. The AMP binding site of the gamma(1) CBS1/CBS2 pair, modeled on the structures of the CBS sequences present in the inosine monophosphate dehydrogenase crystal structure, contains three arginine residues 70, 152, and 171 and His151. The yeast gamma homolog, snf4 contains a His151Gly substitution, and when this is introduced into gamma(1), AMP allosteric control is substantially lost and explains why the yeast snf1p/snf4p complex is insensitive to AMP. Arg70 in gamma(1) corresponds to the site of mutation in human gamma(2) and pig gamma(3) genes previously identified to cause an unusual cardiac phenotype and glycogen storage disease, respectively. Mutation of any of AMP binding site Arg residues to Gln substantially abolishes AMP allosteric control in expressed AMPK holoenzyme. The Arg/Gln mutations also suppress the previously described inhibitory properties of ATP and render the enzyme constitutively active. We propose that ATP acts as an intrasteric inhibitor by bridging the alpha and gamma subunits and that AMP functions to derepress AMPK activity.

  2. Opposing needling promotes behavior recovery and exerts neuroprotection via the cAMP/PKA/CREB signal transduction pathway in transient MCAO rats (United States)



    The aim of the present study was to investigate whether the cyclic adenosine 3′,5′-monophosphate (cAMP)/protein kinase A(PKA)/cAMP-responsive element binding protein (CREB) signal transduction pathway triggered by γ-aminobutyric acid class B (GABAB) receptor activation is involved in neuroprotection against ischemia and behavioral recovery induced by opposing needling (ON). A total of 80 rats were randomly divided into four groups: A sham operation group, an ischemia group, an ON group and an ON group effectively inhibited by the GABAB receptor antagonist, CGP35384 (n=20/group). The behavior of the rats was assessed by their neurological deficit score, whereas the impairment of gait was examined using the CatWalk system. The volume of cerebral infarction was examined upon treatment with 2,3,5-triphenyltetrazolium chloride. The expression levels of CREB, GABAB1 and GABAB2 were examined by western blotting and reverse transcription-quantitative polymerase chain reaction, and the activity of adenylyl cyclase (AC), cAMP and PKA in the serum was detected using an enzyme-linked immunosorbent assay. In the present study, in comparison with other groups, the ON group exhibited a reduced score for the neurological deficit, the stride length and swing speed were improved, and the volume of infarction was reduced. However, these effects were reversed upon administration of CGP35384. Additionally, the expression levels of CREB, GABAB1 and GABAB2 were increased in the ON group. The levels of AC, cAMP and PKA in the serum were also increased in the ON group, whereas the addition of CGP35384 reversed these effects. The results of the present study demonstrated that ON markedly protected the brain against transient cerebral ischemic injury, and this effect was possibly mediated by the activation of the GABAB/cAMP/PKA/CREB signal transduction pathway. These findings implied that ON may be a potential therapeutic method for treating stroke. PMID:26780954

  3. Responses of plant calmodulin to endocytosis induced by rare earth elements. (United States)

    Wang, Lihong; Cheng, Mengzhu; Chu, Yunxia; Li, Xiaodong; Chen, David D Y; Huang, Xiaohua; Zhou, Qing


    The wide application of rare earth elements (REEs) have led to their diffusion and accumulation in the environment. The activation of endocytosis is the primary response of plant cells to REEs. Calmodulin (CaM), as an important substance in calcium (Ca) signaling systems, regulating almost all of the physiological activities in plants, such as cellular metabolism, cell growth and division. However, the response of CaM to endocytosis activated by REEs remains unknown. By using immunofluorescence labeling and a confocal laser scanning microscope, we found that trivalent lanthanum [La(III)], an REE ion, affected the expression of CaM in endocytosis. Using circular dichroism, X-ray photoelectron spectroscopy and computer simulations, we demonstrated that a low concentration of La(III) could interact with extracellular CaM by electrostatic attraction and was then bound to two Ca-binding sites of CaM, making the molecular structure more compact and orderly, whereas a high concentration of La(III) could be coordinated with cytoplasmic CaM or bound to other Ca-binding sites, making the molecular structure more loose and disorderly. Our results provide a reference for revealing the action mechanisms of REEs in plant cells.

  4. Characterizing response to elemental unit of acoustic imaging noise: an FMRI study. (United States)

    Tamer, Gregory G; Luh, Wen-Ming; Talavage, Thomas M


    Acoustic imaging noise produced during functional magnetic resonance imaging (fMRI) studies can hinder auditory fMRI research analysis by altering the properties of the acquired time-series data. Acoustic imaging noise can be especially confounding when estimating the time course of the hemodynamic response (HDR) in auditory event-related fMRI (fMRI) experiments. This study is motivated by the desire to establish a baseline function that can serve not only as a comparison to other quantities of acoustic imaging noise for determining how detrimental is one's experimental noise, but also as a foundation for a model that compensates for the response to acoustic imaging noise. Therefore, the amplitude and spatial extent of the HDR to the elemental unit of acoustic imaging noise (i.e., a single ping) associated with echoplanar acquisition were characterized and modeled. Results from this fMRI study at 1.5 T indicate that the group-averaged HDR in left and right auditory cortex to acoustic imaging noise (duration of 46 ms) has an estimated peak magnitude of 0.29% (right) to 0.48% (left) signal change from baseline, peaks between 3 and 5 s after stimulus presentation, and returns to baseline and remains within the noise range approximately 8 s after stimulus presentation.

  5. Seismic response of continuous span bridges through fiber-based finite element analysis

    Institute of Scientific and Technical Information of China (English)

    Chiara Casarotti; Rui Pinho


    It is widely recognized that nonlinear time-history analysis constitutes the most accurate way to simulate the response of structures subjected to strong levels of seismic excitation. This analytical method is based on sound underlying principles and has the capability to reproduce the intrinsic inelastic dynamic behavior of structures. Nonetheless,comparisons with experimental results from large-scale testing of structures are still needed, in order to ensure adequate levels of confidence in this numerical methodology. The fiber modelling approach employed in the current endeavor inherently accounts for geometric nonlinearities and material inelasticity, without a need for calibration of plastic hinges mechanisms,typical in concentrated plasticity models. The resulting combination of analysis accuracy and modelling simplicity, allows thus to overcome the perhaps not fully justifiable sense of complexity associated to nonlinear dynamic analysis. The fiberbased modelling approach is employed in the framework of a finite element program downloaded from the Internet for seismic response analysis of framed structures. The reliability and accuracy of the program are demonstrated by numerically reproducing pseudo-dynamic tests on a four span continuous deck concrete bridge. Modelling assumptions are discussed,together with their implications on numerical results of the nonlinear time-history analyses, which were found to be in good agreement with experimental results.

  6. Quantifying crustal response to deep active intrusions with geodesy-based finite element modeling (United States)

    Henderson, S. T.; Pritchard, M. E.; Elliott, J.


    The Altiplano-Puna Volcanic Complex (APVC, 21-24 S, 66-69 W) is a first order feature of the Central Andes Volcanic Arc. The APVC consists of over 10,000 km^3 of dacitic ignimbrites deposited in the late Miocene, making it one of the largest concentrations of silicic volcanism in the world. The persistent and intense magmatic flux in this region has likely contributed to the thickened crust (50-70 km), elevated geotherm (>50 C/km) and extensive partial melt (mass balance between source and sink reservoirs, and require physically realistic rheological parameters of the crust. Modeling is performed with Pylith finite element software on a cylindrical three dimensional domain with a radius of 300 km and depth of 200 km. Our models assume pressurization in a viscoelastic crust, and therefore describe crustal response rather than dynamic processes of fluid creation and motion. Surface deformation timescales are highly dependent on geothermal gradient and source pressurization history. While still inconclusive, the potential for an abrupt end to surface deformation has important implications for the characterization of creep versus relaxation processes occurring in response to deep intrusions. Ultimately, crustal scale numerical models provide a means to check petrologically-derived depths of fluid accumulation, in addition to theoretical estimates of softening that occurs with distributed partial melt. The specific models presented are tuned to the subsurface conditions of the APVC, but may be relevant to arc intrusions below the brittle-ductile transition elsewhere.

  7. Finite element modeling of human brain response to football helmet impacts. (United States)

    Darling, T; Muthuswamy, J; Rajan, S D


    The football helmet is used to help mitigate the occurrence of impact-related traumatic (TBI) and minor traumatic brain injuries (mTBI) in the game of American football. While the current helmet design methodology may be adequate for reducing linear acceleration of the head and minimizing TBI, it however has had less effect in minimizing mTBI. The objectives of this study are (a) to develop and validate a coupled finite element (FE) model of a football helmet and the human body, and (b) to assess responses of different regions of the brain to two different impact conditions - frontal oblique and crown impact conditions. The FE helmet model was validated using experimental results of drop tests. Subsequently, the integrated helmet-human body FE model was used to assess the responses of different regions of the brain to impact loads. Strain-rate, strain, and stress measures in the corpus callosum, midbrain, and brain stem were assessed. Results show that maximum strain-rates of 27 and 19 s(-1) are observed in the brain-stem and mid-brain, respectively. This could potentially lead to axonal injuries and neuronal cell death during crown impact conditions. The developed experimental-numerical framework can be used in the study of other helmet-related impact conditions.

  8. Genetically-encoded yellow fluorescent cAMP indicator with an expanded dynamic range for dual-color imaging.

    Directory of Open Access Journals (Sweden)

    Haruki Odaka

    Full Text Available Cyclic AMP is a ubiquitous second messenger, which mediates many cellular responses mainly initiated by activation of cell surface receptors. Various Förster resonance energy transfer-based ratiometric cAMP indicators have been created for monitoring the spatial and temporal dynamics of cAMP at the single-cell level. However, single fluorescent protein-based cAMP indicators have been poorly developed, with improvement required for dynamic range and brightness. Based on our previous yellow fluorescent protein-based cAMP indicator, Flamindo, we developed an improved yellow fluorescent cAMP indicator named Flamindo2. Flamindo2 has a 2-fold expanded dynamic range and 8-fold increased brightness compared with Flamindo by optimization of linker peptides in the vicinity of the chromophore. We found that fluorescence intensity of Flamindo2 was decreased to 25% in response to cAMP. Live-cell cAMP imaging of the cytosol and nucleus in COS7 cells using Flamindo2 and nlsFlamindo2, respectively, showed that forskolin elevated cAMP levels in each compartment with different kinetics. Furthermore, dual-color imaging of cAMP and Ca2+ with Flamindo2 and a red fluorescent Ca2+ indicator, R-GECO, showed that cAMP and Ca2+ elevation were induced by noradrenaline in single HeLa cells. Our study shows that Flamindo2, which is feasible for multi-color imaging with other intracellular signaling molecules, is useful and is an alternative tool for live-cell imaging of intracellular cAMP dynamics.

  9. Genetically-Encoded Yellow Fluorescent cAMP Indicator with an Expanded Dynamic Range for Dual-Color Imaging (United States)

    Odaka, Haruki; Arai, Satoshi; Inoue, Takafumi; Kitaguchi, Tetsuya


    Cyclic AMP is a ubiquitous second messenger, which mediates many cellular responses mainly initiated by activation of cell surface receptors. Various Förster resonance energy transfer-based ratiometric cAMP indicators have been created for monitoring the spatial and temporal dynamics of cAMP at the single-cell level. However, single fluorescent protein-based cAMP indicators have been poorly developed, with improvement required for dynamic range and brightness. Based on our previous yellow fluorescent protein-based cAMP indicator, Flamindo, we developed an improved yellow fluorescent cAMP indicator named Flamindo2. Flamindo2 has a 2-fold expanded dynamic range and 8-fold increased brightness compared with Flamindo by optimization of linker peptides in the vicinity of the chromophore. We found that fluorescence intensity of Flamindo2 was decreased to 25% in response to cAMP. Live-cell cAMP imaging of the cytosol and nucleus in COS7 cells using Flamindo2 and nlsFlamindo2, respectively, showed that forskolin elevated cAMP levels in each compartment with different kinetics. Furthermore, dual-color imaging of cAMP and Ca2+ with Flamindo2 and a red fluorescent Ca2+ indicator, R-GECO, showed that cAMP and Ca2+ elevation were induced by noradrenaline in single HeLa cells. Our study shows that Flamindo2, which is feasible for multi-color imaging with other intracellular signaling molecules, is useful and is an alternative tool for live-cell imaging of intracellular cAMP dynamics. PMID:24959857

  10. Identification of the interleukin-6/oncostatin M response element in the rat tissue inhibitor of metalloproteinases-1 (TIMP-1) promoter. (United States)

    Bugno, M; Graeve, L; Gatsios, P; Koj, A; Heinrich, P C; Travis, J; Kordula, T


    The rat tissue inhibitor of metalloproteinase 1 (TIMP-1) gene is expressed in rat hepatocytes, and this expression is up-regulated by interleukin 6 (IL-6). We report here the cloning of the 5' flanking region of the rat TIMP-1 gene and identification of an IL-6/oncostatin M (OSM) response element at -64 to -36 which functions in hepatic cells. Within this element we have identified two functional binding sites for transcription factors AP-1 (activatory protein-1) and STAT (signal transducer and activator of transcription). IL-6/OSM stimulation induces binding of a protein, identified as STAT3, to the IL-6/OSM response element, while binding of the AP-1 protein was constitutive. Binding sites for both AP-1 and STAT3 are necessary for full responsiveness of the TIMP-1 promoter to IL-6/OSM, as shown by deletion and mutation analysis. Furthermore, the entire IL-6/OSM response element conferred responsiveness onto a heterologous promoter, whereas this has not been observed when AP-1 and STAT elements were separately tested. Images PMID:8559663


    Institute of Scientific and Technical Information of China (English)



    It had been observed that there was a close relationship between cyclic AMP and the motility, energy metabolism, capaeitation and acrosome reaction of spermatozoa. In this study a radio-immunoassay procedure for measuring cAMP concentration in sperm and

  12. Use of phenylthiocarbamide for assessing cAMP-dependent resistance to anoxia in animals. (United States)

    Bolekhan, E A; Semenov, D G; Gerasimova, I A; Samoilov, M O


    The responses of cats with different levels of taste sensitivity to phenylthiocarbamide (PTC) bitters to five-minute hypoxia were studied; PTC sensitivity is a genetic marker of the activity of the cAMP system. Animals able to perceive PTC showed a number of functional differences, with higher levels of resistance to anoxia, than those which could not perceive PTC. The groups showed significant differences in the basal cAMP content in the cerebral cortex, and in the time course of changes in the cAMP level during anoxia and subsequent reoxygenation. It is suggested that these differences result from genetically determined features of the cAMP system, which is involved in forming adaptive responses.

  13. Liver X receptor regulates hepatic nuclear O-GlcNAc signaling and carbohydrate responsive element-binding protein activity

    DEFF Research Database (Denmark)

    Bindesbøll, Christian; Fan, Qiong; Nørgaard, Rikke C;


    Liver X receptor (LXR)α and LXRβ play key roles in hepatic de novo lipogenesis through their regulation of lipogenic genes, including sterol regulatory element-binding protein (SREBP)-1c and carbohydrate responsive element-binding protein (ChREBP). LXRs activate lipogenic gene transcription...... metabolic sensors upstream of ChREBP by modulating GK expression, nuclear O-GlcNAc signaling, and ChREBP expression and activity....

  14. Waterborne Epoxy Resin Modified by AMPS

    Institute of Scientific and Technical Information of China (English)


    A stable epoxy emulsion was prepared with epoxy resin (EP) as raw material, 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) as modifier and benzoyl peroxide as initiator. By criterion of yield of the copolymer AMPS-EP, water-solubility, change of the acid value and intrinsic viscosity [η] along with reaction time, the copolymerization course was deduced. It is found that during the process, AMPS takes part in both the grafting copolymerization with epoxy principal chain and the ring-opening polyaddition with epoxy group. It is also discovered that the yield of AMPS-EP and water dispersing varies with reaction time. When it reaches 1.5 h,AMPS-EP can obtain good water-solubility; but the water-solubility will go bad gradually ifit exceeds 3.5 h.( )R spectrum analysis indicates that partial epoxy group partially remains and the others create sulfonic ester.

  15. The critical roles of cyclic AMP/cyclic AMP-dependent protein kinase in platelet physiology

    Institute of Scientific and Technical Information of China (English)

    Rong YAN; Suping LI; Kesheng DAI


    Platelets are the primary players in both thrombosis and hemostasis.Cyclic AMP (cAMP) and cAMP-dependent protein kinase (PKA) are important signaling molecules in the regulation of platelet function,such as adhesion,aggregation,and secretion.Elevation of intracellular cAMP,which induces the activation of PKA,results in the inhibition of platelet function.Thus,tight control of the intracellular cAMP/PKA signaling pathway has great implications for platelet-dependent hemostasis and effective cardiovascular therapy.In this review,we summarize the PKA substrates and their contributions to platelet function,especially the advancing understanding of the cAMP/PKA-dependent signaling pathway in platelet physiology.In addition,we suggest the possibility that cAMP/PKA is involved in the platelet procoagulant process and receptor ectodomain shedding.

  16. Effects of segregation of primary alloying elements on the creep response in magnesium alloys

    DEFF Research Database (Denmark)

    Huang, Y.D.; Dieringa, H.; Hort, N.


    The segregation of primary alloying elements deteriorates the high temperature creep resistance of magnesium alloys. Annealing at high temperatures alleviating their segregations can improve the creep resistance. Present investigation on the effect of segregation of primary alloying elements...

  17. Three Yersinia enterocolitica AmpD Homologs Participate in the Multi-Step Regulation of Chromosomal Cephalosporinase, AmpC. (United States)

    Liu, Chang; Wang, Xin; Chen, Yuhuang; Hao, Huijing; Li, Xu; Liang, Junrong; Duan, Ran; Li, Chuchu; Zhang, Jing; Shao, Shihe; Jing, Huaiqi


    In many gram negative bacilli, AmpD plays a key role in both cell well-recycling pathway and β-lactamase regulation, inactivation of the ampD causes the accumulation of 1,6-anhydromuropeptides, and results in the ampC overproduction. In Yersinia enterocolitica, the regulation of ampC expression may also rely on the ampR-ampC system, the role of AmpD in this species is still unknown. In this study, three AmpD homologs (AmpD1, AmpD2, and AmpD3) have been identified in complete sequence of strain Y. enterocolitica subsp. palearctica 105.5R(r). To understand the role of three AmpD homologs, several mutant strains were constructed and analyzed where a rare ampC regulation mechanism was observed: low-effective ampD2 and ampD3 cooperate with the high-effective ampD1 in the three levels regulation of ampC expression. Enterobacteriaceae was used to be supposed to regulate ampC expression by two steps, three steps regulation was only observed in Pseudomonas aeruginosa. In this study, we first reported that Enterobacteriaceae Y. enterocolitica can also possess a three steps stepwise regulation mechanism, regulating the ampC expression precisely.

  18. A chromatin insulator driving three-dimensional Polycomb response element (PRE) contacts and Polycomb association with the chromatin fiber

    DEFF Research Database (Denmark)

    Comet, Itys; Schuettengruber, Bernd; Sexton, Tom;


    to insulate genes from regulatory elements or to take part in long-distance interactions. Using a high-resolution chromatin conformation capture (H3C) method, we show that the Drosophila gypsy insulator behaves as a conformational chromatin border that is able to prohibit contacts between a Polycomb response...... element (PRE) and a distal promoter. On the other hand, two spaced gypsy elements form a chromatin loop that is able to bring an upstream PRE in contact with a downstream gene to mediate its repression. Chromatin immunoprecipitation (ChIP) profiles of the Polycomb protein and its associated H3K27me3...

  19. Evidence for cAMP as a mediator of gonadotropin secretion from male pituitaries

    Energy Technology Data Exchange (ETDEWEB)

    Bourne, G.A.; Baldwin, D.M.


    The purpose of this study was to use sodium flufenamate, a compound that inhibits gonadotropin-releasing hormone (GnRH)-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) production in the pituitary, to evaluate the potential role of cAMP as a mediator of GnRH-stimulated gonadotropin secretion from male pituitaries. Quartered male pituitaries were perifused at 37/sup 0/C and sequential effluent fractions collected every 10 min. Infusions of GnRH resulted in a twofold increase in luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion. Cycloheximide, 5, completely inhibited the GnRH-stimulated LH and FSH secretion. Infusions of 0.1 mM flufenamate had similar effects on gonadotropin secretion as cycloheximide, whereas the administration of 5 mM dibutyryl cAMP in combination with GnRH and flufenamate restored the secretory responses of both hormones. The flufenamate-inhibited GnRH stimulated LH and FSH release, which was restored by DBcAMP and appeared to be protein synthesis dependent and specific for cAMP.These results suggest an indirect role for cAMP as a mediator of gonadotropin secretion from male pituitaries. However, in contrast to female pituitaries, the secretion of these hormones form male pituitaries is completely dependent on cAMP and de novo protein synthesis.

  20. Looking downstream: the role of cyclic AMP-regulated genes in axonal regeneration

    Directory of Open Access Journals (Sweden)

    Mustafa M Siddiq


    Full Text Available Elevation of intracellular cyclic AMP (cAMP levels has proven to be one of the most effective means of overcoming inhibition of axonal regeneration by myelin-associated inhibitors such as myelin-associated glycoprotein, Nogo, and oligodendrocyte myelin glycoprotein. Pharmacological manipulation of cAMP through the administration of dibutyryl cAMP or rolipram leads to enhanced axonal growth both in vivo and in vitro, and importantly, upregulation of cAMP within dorsal root ganglion neurons is responsible for the conditioning lesion effect, which indicates that cAMP plays a significant role in the endogenous mechanisms that promote axonal regeneration. The effects of cAMP are transcription-dependent and are mediated through the activation of protein kinase A and the transcription factor CREB. This leads to the induction of a variety of genes, several of which have been shown to overcome myelin-mediated inhibition in their own right. In this review, we will highlight the pro-regenerative effects of arginase I, interleukin-6, secretory leukocyte protease inhibitor, and metallothionein-I/II, and discuss their potential for therapeutic use in spinal cord injury.

  1. Icariin upregulates phosphorylated cyclic adenosine monophosphate response element binding protein levels in the hippocampus of the senescence- accelerated mouse

    Institute of Scientific and Technical Information of China (English)

    Zhanwei Zhang; Ting Zhang; Keli Dong


    At 8 weeks after intragastric administration of icariin to senescence-accelerated mice (P8 strain), Morris water maze results showed that escape latency was shortened, and the number of platform crossings was increased. Immunohistochemical staining and western blot assay detected signifi-cantly increased levels of cyclic adenosine monophosphate response element binding protein. These results suggest that icariin upregulates phosphorylated cyclic adenosine monophosphate response element binding protein levels and improves learning and memory functions in hippo-campus of the senescence-accelerated mouse.

  2. Software Design Document for the AMP Nuclear Fuel Performance Code

    Energy Technology Data Exchange (ETDEWEB)

    Philip, Bobby [ORNL; Clarno, Kevin T [ORNL; Cochran, Bill [ORNL


    The purpose of this document is to describe the design of the AMP nuclear fuel performance code. It provides an overview of the decomposition into separable components, an overview of what those components will do, and the strategic basis for the design. The primary components of a computational physics code include a user interface, physics packages, material properties, mathematics solvers, and computational infrastructure. Some capability from established off-the-shelf (OTS) packages will be leveraged in the development of AMP, but the primary physics components will be entirely new. The material properties required by these physics operators include many highly non-linear properties, which will be replicated from FRAPCON and LIFE where applicable, as well as some computationally-intensive operations, such as gap conductance, which depends upon the plenum pressure. Because there is extensive capability in off-the-shelf leadership class computational solvers, AMP will leverage the Trilinos, PETSc, and SUNDIALS packages. The computational infrastructure includes a build system, mesh database, and other building blocks of a computational physics package. The user interface will be developed through a collaborative effort with the Nuclear Energy Advanced Modeling and Simulation (NEAMS) Capability Transfer program element as much as possible and will be discussed in detail in a future document.

  3. Isoniazid suppresses antioxidant response element activities and impairs adipogenesis in mouse and human preadipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Yanyan [Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC 27709 (United States); The First Affiliated Hospital, China Medical University, Shenyang 110001 (China); Xue, Peng [Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC 27709 (United States); Key Laboratory of the Public Health Safety, Ministry of Education, School of Public Health, Fudan University, Shanghai (China); Hou, Yongyong [Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC 27709 (United States); School of Public Health, China Medical University, Shenyang 110001 (China); Zhang, Hao [Key Laboratory of the Public Health Safety, Ministry of Education, School of Public Health, Fudan University, Shanghai (China); Zheng, Hongzhi [Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC 27709 (United States); The First Affiliated Hospital, China Medical University, Shenyang 110001 (China); Zhou, Tong [Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC 27709 (United States); Qu, Weidong [Key Laboratory of the Public Health Safety, Ministry of Education, School of Public Health, Fudan University, Shanghai (China); Teng, Weiping [The First Affiliated Hospital, China Medical University, Shenyang 110001 (China); Zhang, Qiang; Andersen, Melvin E. [Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC 27709 (United States); Pi, Jingbo, E-mail: [Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC 27709 (United States); School of Public Health, China Medical University, Shenyang 110001 (China)


    Transcriptional signaling through the antioxidant response element (ARE), orchestrated by the Nuclear factor E2-related factor 2 (Nrf2), is a major cellular defense mechanism against oxidative or electrophilic stress. Here, we reported that isoniazid (INH), a widely used antitubercular drug, displays a substantial inhibitory property against ARE activities in diverse mouse and human cells. In 3T3-L1 preadipocytes, INH concentration-dependently suppressed the ARE-luciferase reporter activity and mRNA expression of various ARE-dependent antioxidant genes under basal and oxidative stressed conditions. In keeping with our previous findings that Nrf2-ARE plays a critical role in adipogenesis by regulating expression of CCAAT/enhancer-binding protein β (C/EBPβ) and peroxisome proliferator-activated receptor γ (PPARγ), suppression of ARE signaling by INH hampered adipogenic differentiation of 3T3-L1 cells and human adipose-derived stem cells (ADSCs). Following adipogenesis induced by hormonal cocktails, INH-treated 3T3-L1 cells and ADSCs displayed significantly reduced levels of lipid accumulation and attenuated expression of C/EBPα and PPARγ. Time-course studies in 3T3-L1 cells revealed that inhibition of adipogenesis by INH occurred in the early stage of terminal adipogenic differentiation, where reduced expression of C/EBPβ and C/EBPδ was observed. To our knowledge, the present study is the first to demonstrate that INH suppresses ARE signaling and interrupts with the transcriptional network of adipogenesis, leading to impaired adipogenic differentiation. The inhibition of ARE signaling may be a potential underlying mechanism by which INH attenuates cellular antioxidant response contributing to various complications. - Highlights: • Isoniazid suppresses ARE-mediated transcriptional activity. • Isoniazid inhibits adipogenesis in preadipocytes. • Isoniazid suppresses adipogenic gene expression during adipogenesis.

  4. A peroxisome proliferator response elements regulatory system in xenopus oocytes and its application

    Institute of Scientific and Technical Information of China (English)

    YAN Jin; FAN Chun-lei; WO Xing-de; GAO Li-ping


    Background Peroxisome proliferator-activated receptor-gamma (PPARγ) is a kind of ligand-activated transcription factors binding to peroxisome proliferator response element (PPRE), a specific recognition site. It is thought to play a critical role in glucose and lipid metabolism and in inflammation control. The aim of this study was to establish a new cellular model for the quick screening of lipid-lowering drugs, which may be effective as PPAR-γ ligands on the PPRE-mediated pathway regulatory system. Methods Two plasmids were constructed: pXOE-PPARγ, in which the human PPARγ gene was in the downstream of TFⅢA gene promoter, and pLXRN-PPRE-d2EGFP, in which the enhanced green fluorescent protein (EGFP) gene was subcloned into PPRE. The xenopus oocytes were injected with these two plamids, and consequently treated with prostaglandin E1, pioglitazone, and different kinds of lipid-lowering drugs. After 3 days, the oocytes were observed under a fluorescence microscope. To confirm the drug action,we injected pXOE-PPARγ plasmid into the oocytes, which then treated with prostaglandin E1and Hawthorn flavonoids. The mass of expressed lipoprotein lipase (LPL) in the cells was determined by enzyme labeling linked immunosorbent assay (ELISA).Conclusions It is possible to establish a PPRE regulatory EGFP reporter system in xenopus oocytes to monitor the activity of PPARγ ligand. Hawthorn flavonoids can increase the expression of gene downsteam of PPRE by effect on the PPRE pathway regulatory system.

  5. Farnesoid X Receptor Inhibits the Transcriptional Activity of Carbohydrate Response Element Binding Protein in Human Hepatocytes (United States)

    Caron, Sandrine; Huaman Samanez, Carolina; Dehondt, Hélène; Ploton, Maheul; Briand, Olivier; Lien, Fleur; Dorchies, Emilie; Dumont, Julie; Postic, Catherine; Cariou, Bertrand; Lefebvre, Philippe


    The glucose-activated transcription factor carbohydrate response element binding protein (ChREBP) induces the expression of hepatic glycolytic and lipogenic genes. The farnesoid X receptor (FXR) is a nuclear bile acid receptor controlling bile acid, lipid, and glucose homeostasis. FXR negatively regulates hepatic glycolysis and lipogenesis in mouse liver. The aim of this study was to determine whether FXR regulates the transcriptional activity of ChREBP in human hepatocytes and to unravel the underlying molecular mechanisms. Agonist-activated FXR inhibits glucose-induced transcription of several glycolytic genes, including the liver-type pyruvate kinase gene (L-PK), in the immortalized human hepatocyte (IHH) and HepaRG cell lines. This inhibition requires the L4L3 region of the L-PK promoter, known to bind the transcription factors ChREBP and hepatocyte nuclear factor 4α (HNF4α). FXR interacts directly with ChREBP and HNF4α proteins. Analysis of the protein complex bound to the L4L3 region reveals the presence of ChREBP, HNF4α, FXR, and the transcriptional coactivators p300 and CBP at high glucose concentrations. FXR activation does not affect either FXR or HNF4α binding to the L4L3 region but does result in the concomitant release of ChREBP, p300, and CBP and in the recruitment of the transcriptional corepressor SMRT. Thus, FXR transrepresses the expression of genes involved in glycolysis in human hepatocytes. PMID:23530060

  6. LAS0811: From Combinatorial Chemistry to Activation of Antioxidant Response Element

    Directory of Open Access Journals (Sweden)

    Ming Zhu


    Full Text Available The antioxidant response element (ARE and its transcription factor, nuclear factor-erythroid 2 p45-related factor 2 (Nrf2, are potential targets for cancer chemoprevention. We sought to screen small molecules synthesized with combinatorial chemistry for activation of ARE. By high-throughput screening of 9400 small molecules from 10 combinatorial chemical libraries using HepG2 cells with an ARE-driven reporter, we have identified a novel small molecule, 1,2-dimethoxy-4,5-dinitrobenzene (LAS0811, as an activator of the ARE. LAS0811 upregulated the activity of NAD(PH:quinone oxidoreductase 1 (NQO1, a representative antioxidative enzyme regulated by ARE. It enhanced production of an endogenous reducing agent, glutathione (GSH. In addition, LAS0811 induced expression of heme oxygenase 1 (HO1, which is an ARE-regulated enzyme with anti-inflammatory activity. Furthermore, LAS0811 reduced cell death due to the cytotoxic stress of a strong oxidant, t-butyl hydroperoxide (t-BOOH. Mechanistically, LAS0811 upregulated the expression of Nrf2 and promoted its translocation into the nuclei leading to subsequent ARE activation. Taken together, LAS0811 is a novel activator of the ARE and its associated detoxifying genes and, thus, a potential agent for cancer chemoprevention.

  7. Killing of Brain Tumor Cells by Hypoxia-Responsive Element Mediated Expression of BAX

    Directory of Open Access Journals (Sweden)

    Hangjun Ruan


    Full Text Available The presence of radioresistant hypoxic cells in human brain tumors limits the overall effectiveness of conventional fractionated radiation therapy. Tumor-specific therapies that target hypoxic cells are clearly needed. We have investigated the expression of suicide genes under hypoxia by a hypoxia-responsive element (HRE, which can be activated through hypoxia-inducible factor-1 (HIF-1. We transfected plasmids containing multiple copies of HIRE into U-87 MG and U-251 MG-NCI human brain tumor cells and tested their ability to induce LacZ gene expression under anoxia. Gene expression under anoxia versus oxia was increased about 12-fold for U-87 MG cells and about fourfold for U-251 MG-NCI cells. At intermediate hypoxic conditions, increased LacZ gene expression in U-87 MG cells was induced by the plasmid that contained three HREs, but not by the plasmid with two HREs. Lastly, when we placed a suicide gene BAX under the control of HREs, cells transfected with the BAX plasmids were preferentially killed through apoptosis under anoxia. Our studies demonstrate that HRE-regulated gene expression is active in brain tumor cells, and that the amount of increased gene expression obtained is dependent on the cell line, the HIRE copy number, and the degree of hypoxia.

  8. [Response of a finite element model of the pelvis to different side impact loads]. (United States)

    Ruan, Shijie; Zheng, Huijing; Li, Haiyan; Zhao, Wei


    The pelvis is one of the most likely affected areas of the human body in case of side impact, especially while people suffer from motor vehicle crashes. With the investigation of pelvis injury on side impact, the injury biomechanical behavior of pelvis can be found, and the data can help design the vehicle security devices to keep the safety of the occupants. In this study, a finite element (FE) model of an isolated human pelvis was used to study the pelvic dynamic response under different side impact conditions. Fracture threshold was established by applying lateral loads of 1000, 2000, 3000, 4000 and 5000 N, respectively, to the articular surface of the right acetabulum. It was observed that the smaller the lateral loads were, the smaller the von Mises stress and the displacement in the direction of impact were. It was also found that the failure threshold load was near 3000 N, based on the fact that the peak stress would not exceed the average compressive strength of the cortical bone. It could well be concluded that with better design of car-door and hip-pad so that the side impact force was brought down to 3000 N or lower, the pelvis would not be injured.

  9. The Role of Carbohydrate Response Element Binding Protein in Intestinal and Hepatic Fructose Metabolism

    Directory of Open Access Journals (Sweden)

    Katsumi Iizuka


    Full Text Available Many articles have discussed the relationship between fructose consumption and the incidence of obesity and related diseases. Fructose is absorbed in the intestine and metabolized in the liver to glucose, lactate, glycogen, and, to a lesser extent, lipids. Unabsorbed fructose causes bacterial fermentation, resulting in irritable bowl syndrome. Therefore, understanding the mechanisms underlying intestinal and hepatic fructose metabolism is important for the treatment of metabolic syndrome and fructose malabsorption. Carbohydrate response element binding protein (ChREBP is a glucose-activated transcription factor that controls approximately 50% of de novo lipogenesis in the liver. ChREBP target genes are involved in glycolysis (Glut2, liver pyruvate kinase, fructolysis (Glut5, ketohexokinase, and lipogenesis (acetyl CoA carboxylase, fatty acid synthase. ChREBP gene deletion protects against high sucrose diet-induced and leptin-deficient obesity, because Chrebp−/− mice cannot consume fructose or sucrose. Moreover, ChREBP contributes to some of the physiological effects of fructose on sweet taste preference and glucose production through regulation of ChREBP target genes, such as fibroblast growth factor-21 and glucose-6-phosphatase catalytic subunits. Thus, ChREBP might play roles in fructose metabolism. Restriction of excess fructose intake will be beneficial for preventing not only metabolic syndrome but also irritable bowl syndrome.

  10. Finite element prediction of seismic response modification of monumental structures utilizing base isolation (United States)

    Spanos, Konstantinos; Anifantis, Nikolaos; Kakavas, Panayiotis


    The analysis of the mechanical behavior of ancient structures is an essential engineering task concerning the preservation of architectural heritage. As many monuments of classical antiquity are located in regions of earthquake activity, the safety assessment of these structures, as well as the selection of possible restoration interventions, requires numerical models capable of correctly representing their seismic response. The work presented herein was part of a research project in which a better understanding of the dynamics of classical column-architrave structures was sought by means of numerical techniques. In this paper, the seismic behavior of ancient monumental structures with multi-drum classical columns is investigated. In particular, the column-architrave classical structure under strong ground excitations was represented by a finite element method. This approach simulates the individual rock blocks as distinct rigid blocks interconnected with slidelines and incorporates seismic isolation dampers under the basement of the structure. Sliding and rocking motions of individual stone blocks and drums are modeled utilizing non-linear frictional contact conditions. The seismic isolation is modeled through the application of pad bearings under the basement of the structure. These pads are interpreted by appropriate rubber and steel layers. Time domain analyses were performed, considering the geometric and material non-linear behavior at the joints and the characteristics of pad bearings. The deformation and failure modes of drum columns subject to seismic excitations of various types and intensities were analyzed. The adverse influence of drum imperfections on structural safety was also examined.

  11. Copper toxicity in expanding leaves of Phaseolus vulgaris L.: antioxidant enzyme response and nutrient element uptake. (United States)

    Bouazizi, Houda; Jouili, Hager; Geitmann, Anja; El Ferjani, Ezzeddine


    Bioaccumulation and toxicity of copper (CuSO4) were assessed in expanding leaves of 14-day-old bean seedlings. CuSO4 was administrated in the growth medium for three days and changes in the activities of the antioxidant enzymes guaiacol peroxidase (GPX) and catalase (CAT), and in the H2O2 production and mineral element contents were measured. Copper accumulated in exposed plants caused severe symptoms such as chlorosis and necrosis as well as a dramatic reduction in dry weight production. Simultaneously, concentrations of iron, zinc and potassium were reduced significantly suggesting that a change in nutrient homeostasis may be responsible for the observed symptoms. Contrary to mature tissues, the expanding leaves did not display significant oxidative stress, since malondialdehyde (MDA) content was unchanged, the activities of GPX and CAT were lowered or unaltered, and endogenous H2O2 only increased at high copper concentrations. Our results suggest that while excess copper slightly alters the activity of the antioxidative enzyme system in young expanding leaves of bean plants, it exerts its toxicity primarily through causing a disturbance in the nutrient balance.

  12. Domain- and nucleotide-specific Rev response element regulation of feline immunodeficiency virus production. (United States)

    Na, Hong; Huisman, Willem; Ellestad, Kristofor K; Phillips, Tom R; Power, Christopher


    Computational analysis of feline immunodeficiency virus (FIV) RNA sequences indicated that common FIV strains contain a rev response element (RRE) defined by a long unbranched hairpin with 6 stem-loop sub-domains, termed stem-loop A (SLA). To examine the role of the RNA secondary structure of the RRE, mutational analyses were performed in both an infectious FIV molecular clone and a FIV CAT-RRE reporter system. These studies disclosed that the stems within SLA (SA1, 2, 3, 4, and 5) of the RRE were critical but SA6 was not essential for FIV replication and CAT expression. These studies also revealed that the secondary structure rather than an antisense protein (ASP) mediates virus expression and replication in vitro. In addition, a single synonymous mutation within the FIV-RRE, SA3/45, reduced viral reverse transcriptase activity and p24 expression after transfection but in addition also showed a marked reduction in viral expression and production following infection.

  13. Characterization of cyclic AMP accumulation in cultured human corpus cavernosum smooth muscle cells. (United States)

    Palmer, L S; Valcic, M; Melman, A; Giraldi, A; Wagner, G; Christ, G J


    Intracavernous pharmacotherapy relies heavily on the use of vasoactive agents which act by increasing intracellular cAMP levels in human corpus cavernosum smooth muscle. Yet little is known about the cAMP generating system in this tissue, and how it may affect observed patient variability. Thus, the goal of these studies was to better characterize the biochemistry of cAMP formation in human corpus cavernosum smooth muscle, and thus provide more insight into the mechanisms of corporal smooth muscle relaxation in vivo. We studied both receptor and nonreceptor mediated increases in cAMP formation in short-term cultures of human corpus cavernosum smooth muscle cells. Both isoproterenol (ISO) and prostaglandin E1 (PGE1) produced concentration-dependent increases in cAMP, but histamine, serotonin and vasoactive intestinal polypeptide did not. Forskolin, a relatively specific activator of adenylate cyclase, was also a potent stimulant of cAMP formation in these cells. Moreover, there was a direct correlation between the degree of forskolin-induced cAMP accumulation in cultured corporal smooth muscle cells and the magnitude of the forskolin-induced relaxation response of precontracted isolated corporal smooth muscle strips. Prostaglandin E1 and ISO concentration response curves (CRCs) were then assayed in the absence and presence of subthreshold forskolin (0.1 microM.). In the presence of forskolin, the calculated maximal PGE1-induced cAMP accumulation (Emax) was significantly greater than that elicited by PGE1 alone, ISO alone, or ISO + forskolin (p protocol was used to demonstrate that both 80:20 and 70:30 FMRs (but not 95:5 or 90:10), were associated with significantly greater cAMP Emax values than that observed for PGE1 alone (p < or = 0.01). These data provide direct evidence that the degree of cAMP formation in cultured corporal smooth muscle cells is strongly correlated with the magnitude of relaxation of isolated corporal smooth muscle strips. In addition, since

  14. Finite Element Analysis of the Random Response Suppression of Composite Panels at Elevated Temperatures using Shape Memory Alloy Fibers (United States)

    Turner, Travis L.; Zhong, Z. W.; Mei, Chuh


    A feasibility study on the use of shape memory alloys (SMA) for suppression of the random response of composite panels due to acoustic loads at elevated temperatures is presented. The constitutive relations for a composite lamina with embedded SMA fibers are developed. The finite element governing equations and the solution procedures for a composite plate subjected to combined acoustic and thermal loads are presented. Solutions include: 1) Critical buckling temperature; 2) Flat panel random response; 3) Thermal postbuckling deflection; 4) Random response of a thermally buckled panel. The preliminary results demonstrate that the SMA fibers can completely eliminate the thermal postbuckling deflection and significantly reduce the random response at elevated temperatures.

  15. Discovery of a cAMP deaminase that quenches cyclic AMP-dependent regulation. (United States)

    Goble, Alissa M; Feng, Youjun; Raushel, Frank M; Cronan, John E


    An enzyme of unknown function within the amidohydrolase superfamily was discovered to catalyze the hydrolysis of the universal second messenger, cyclic-3',5'-adenosine monophosphate (cAMP). The enzyme, which we have named CadD, is encoded by the human pathogenic bacterium Leptospira interrogans. Although CadD is annotated as an adenosine deaminase, the protein specifically deaminates cAMP to cyclic-3',5'-inosine monophosphate (cIMP) with a kcat/Km of 2.7 ± 0.4 × 10(5) M(-1) s(-1) and has no activity on adenosine, adenine, or 5'-adenosine monophosphate (AMP). This is the first identification of a deaminase specific for cAMP. Expression of CadD in Escherichia coli mimics the loss of adenylate cyclase in that it blocks growth on carbon sources that require the cAMP-CRP transcriptional activator complex for expression of the cognate genes. The cIMP reaction product cannot replace cAMP as the ligand for CRP binding to DNA in vitro and cIMP is a very poor competitor of cAMP activation of CRP for DNA binding. Transcriptional analyses indicate that CadD expression represses expression of several cAMP-CRP dependent genes. CadD adds a new activity to the cAMP metabolic network and may be a useful tool in intracellular study of cAMP-dependent processes.

  16. Extended-spectrum β-lactamase/AmpC- and carbapenemase-producing Enterobacteriaceae in animals: a threat for humans? (United States)

    Madec, J-Y; Haenni, M; Nordmann, P; Poirel, L


    There has been a great and long-term concern that extended-spectrum β-lactamase (ESBL)/AmpC- and carbapenemase-producing Enterobacteriaceae occurring in animals may constitute a public-health issue. A large number of factors with complex interrelations contribute to the spread of those bacteria among animals and humans. ESBL/AmpC- or carbapenemase-encoding genes are most often located on mobile genetic elements favouring their dissemination. Some shared reservoirs of ESBL/AmpC or carbapenemase genes, plasmids or clones have been identified and suggest cross-transmissions. Even though exposure to animals is regarded as a risk factor, evidence for a direct transfer of ESBL/AmpC-producing bacteria from animals to humans through close contacts is limited. Nonetheless, the size of the commensal ESBL/AmpC reservoir in non-human sources is dramatically rising. This may constitute an indirect risk to public health by increasing the gene pool from which pathogenic bacteria can pick up ESBL/AmpC/carbapenemase genes. The extent to which food contributes to potential transmission of ESBL/AmpC producers to humans is also not well established. Overall, events leading to the occurrence of ESBL/AmpC- and carbapenemase-encoding genes in animals seem very much multifactorial. The impact of animal reservoirs on human health still remains debatable and unclear; nonetheless, there are some examples of direct links that have been identified.

  17. CREB activity in the nucleus accumbens shell controls gating of behavioral responses to emotional stimuli

    NARCIS (Netherlands)

    Barrot, Michel; Olivier, Jocelien D A; Perrotti, Linda I; DiLeone, Ralph J; Berton, Olivier; Eisch, Amelia J; Impey, Soren; Storm, Daniel R; Neve, Rachael L; Yin, Jerry C; Zachariou, Venetia; Nestler, Eric J


    The transcription factor cAMP response element (CRE)-binding protein (CREB) has been shown to regulate neural plasticity. Drugs of abuse activate CREB in the nucleus accumbens, an important part of the brain's reward pathways, and local manipulations of CREB activity have been shown to affect cocain

  18. Involvement of Phosphorylated "Apis Mellifera" CREB in Gating a Honeybee's Behavioral Response to an External Stimulus (United States)

    Gehring, Katrin B.; Heufelder, Karin; Feige, Janina; Bauer, Paul; Dyck, Yan; Ehrhardt, Lea; Kühnemund, Johannes; Bergmann, Anja; Göbel, Josefine; Isecke, Marlene; Eisenhardt, Dorothea


    The transcription factor cAMP-response element-binding protein (CREB) is involved in neuronal plasticity. Phosphorylation activates CREB and an increased level of phosphorylated CREB is regarded as an indicator of CREB-dependent transcriptional activation. In honeybees ("Apis mellifera") we recently demonstrated a particular high…

  19. An HD-domain phosphodiesterase mediates cooperative hydrolysis of c-di-AMP to affect bacterial growth and virulence (United States)

    Huynh, TuAnh Ngoc; Luo, Shukun; Pensinger, Daniel; Sauer, John-Demian; Tong, Liang; Woodward, Joshua J.


    The nucleotide cyclic di-3′,5′- adenosine monophosphate (c-di-AMP) was recently identified as an essential and widespread second messenger in bacterial signaling. Among c-di-AMP–producing bacteria, altered nucleotide levels result in several physiological defects and attenuated virulence. Thus, a detailed molecular understanding of c-di-AMP metabolism is of both fundamental and practical interest. Currently, c-di-AMP degradation is recognized solely among DHH-DHHA1 domain-containing phosphodiesterases. Using chemical proteomics, we identified the Listeria monocytogenes protein PgpH as a molecular target of c-di-AMP. Biochemical and structural studies revealed that the PgpH His-Asp (HD) domain bound c-di-AMP with high affinity and specifically hydrolyzed this nucleotide to 5′-pApA. PgpH hydrolysis activity was inhibited by ppGpp, indicating a cross-talk between c-di-AMP signaling and the stringent response. Genetic analyses supported coordinated regulation of c-di-AMP levels in and out of the host. Intriguingly, a L. monocytogenes mutant that lacks c-di-AMP phosphodiesterases exhibited elevated c-di-AMP levels, hyperinduced a host type-I IFN response, and was significantly attenuated for infection. Furthermore, PgpH homologs, which belong to the 7TMR-HD family, are widespread among hundreds of c-di-AMP synthesizing microorganisms. Thus, PgpH represents a broadly conserved class of c-di-AMP phosphodiesterase with possibly other physiological functions in this crucial signaling network. PMID:25583510

  20. Opposing roles of PKA and EPAC in the cAMP-dependent regulation of schwann cell proliferation and differentiation [corrected].

    Directory of Open Access Journals (Sweden)

    Ketty Bacallao

    Full Text Available In Schwann cells (SCs, cyclic adenosine monophosphate (cAMP not only induces differentiation into a myelinating SC-related phenotype, but also synergistically enhances the mitogenic action of growth factors such as neuregulin. To better understand the molecular mechanism by which cAMP exerts these apparently contradictory functions, we investigated the role of the two main effectors of cAMP, protein kinase A (PKA and the exchange protein activated by cAMP (EPAC, on the proliferation and differentiation of both isolated and axon-related SCs. For these studies, a variety of PKA and EPAC agonists and antagonists were used, including pathway-selective analogs of cAMP and pharmacological inhibitors. Our studies indicated that the activity of PKA rather than EPAC was required for the adjuvant effect of cAMP on S-phase entry, whereas the activity of EPAC rather than PKA was required for SC differentiation and myelin formation. Even though selective EPAC activation had an overall anti-proliferative effect in SCs, it failed to drive the expression of Krox-20, a master regulator of myelination, and that of myelin-specific proteins and lipids, suggesting that EPAC activation was insufficient to drive a full differentiating response. Interestingly, inhibition of EPAC activity resulted in a drastic impairment of SC differentiation and myelin formation but not Krox-20 expression, which indicates an independent mechanism of Krox-20 regulation in response to cAMP. In conclusion, our data supports the idea that the outcome of cAMP signaling in SCs depends on the particular set of effectors activated. Whereas the mitogenic action of cAMP relies exclusively on PKA activity, the differentiating action of cAMP requires a PKA-independent (non-canonical cAMP-specific pathway that is partially transduced by EPAC.

  1. Opposing roles of PKA and EPAC in the cAMP-dependent regulation of schwann cell proliferation and differentiation [corrected]. (United States)

    Bacallao, Ketty; Monje, Paula V


    In Schwann cells (SCs), cyclic adenosine monophosphate (cAMP) not only induces differentiation into a myelinating SC-related phenotype, but also synergistically enhances the mitogenic action of growth factors such as neuregulin. To better understand the molecular mechanism by which cAMP exerts these apparently contradictory functions, we investigated the role of the two main effectors of cAMP, protein kinase A (PKA) and the exchange protein activated by cAMP (EPAC), on the proliferation and differentiation of both isolated and axon-related SCs. For these studies, a variety of PKA and EPAC agonists and antagonists were used, including pathway-selective analogs of cAMP and pharmacological inhibitors. Our studies indicated that the activity of PKA rather than EPAC was required for the adjuvant effect of cAMP on S-phase entry, whereas the activity of EPAC rather than PKA was required for SC differentiation and myelin formation. Even though selective EPAC activation had an overall anti-proliferative effect in SCs, it failed to drive the expression of Krox-20, a master regulator of myelination, and that of myelin-specific proteins and lipids, suggesting that EPAC activation was insufficient to drive a full differentiating response. Interestingly, inhibition of EPAC activity resulted in a drastic impairment of SC differentiation and myelin formation but not Krox-20 expression, which indicates an independent mechanism of Krox-20 regulation in response to cAMP. In conclusion, our data supports the idea that the outcome of cAMP signaling in SCs depends on the particular set of effectors activated. Whereas the mitogenic action of cAMP relies exclusively on PKA activity, the differentiating action of cAMP requires a PKA-independent (non-canonical) cAMP-specific pathway that is partially transduced by EPAC.

  2. The Indirect Boundary Element Method (IBEM) for Seismic Response of Topographical Irregularities in Layered Media (United States)

    Contreras Zazueta, M. A.; Perton, M.; Sanchez-Sesma, F. J.; Sánchez-Alvaro, E.


    The seismic hazard assessment of extended developments, such as a dam, a bridge or a pipeline, needs the strong ground motion simulation taking into account the effects of surface geology. In many cases the incoming wave field can be obtained from attenuation relations or simulations for layered media using Discrete Wave Number (DWN). Sometimes there is a need to include in simulations the seismic source as well. A number of methods to solve these problems have been developed. Among them the Finite Element and Finite Difference Methods (FEM and FDM) are generally preferred because of the facility of use. Nevertheless, the analysis of realistic dynamic loading induced by earthquakes requires a thinner mesh of the entire domain to consider high frequencies. Consequently this may imply a high computational cost. The Indirect Boundary Element Method (IBEM) can also be employed. Here it is used to study the response of a site to historical seismic activity. This method is particularly suited to model wave propagation through wide areas as it requires only the meshing of boundaries. Moreover, it is well suited to represent finely the diffraction that can occur on a fault. However, the IBEM has been applied mainly to simple geometrical configurations. In this communication significant refinements of the formulation are presented. Using IBEM we can simulate wave propagation in complex geometrical configurations such as a stratified medium crossed by thin faults or having a complex topography. Two main developments are here described; one integrates the DWN method inside the IBEM in order to represent the Green's functions of stratified media with relatively low computational cost but assuming unbounded parallel flat layers, and the other is the extension of IBEM to deal with multi-regions in contact which allows more versatility with a higher computational cost compared to the first one but still minor to an equivalent FEM formulation. The two approaches are fully

  3. A study of the diffusional response of refractory and other elements in superalloy systems during diffusion coating

    Energy Technology Data Exchange (ETDEWEB)

    Purvis, A.L.; Warnes, B.M. [Howmet Thermatech Coatings, Whitehall, MI (United States)


    Samples of commercially pure nickel and two common superalloys were prepared by electroplating a thin layer of platinum on the surface, then exposing the samples to temperatures of 950 C and 1080 C for periods of two and six hours. Using electron probe micro analysis (EMPA), elemental composition profiles were obtained from the samples following the diffusion steps. The relative diffusion coefficients for a number of elements were determined using a classical Boltzmann-Matano method. As expected, it was discovered that elements such as cobalt, chromium, titanium and tantalum displayed a significant diffusional response in this relatively short time, while tungsten, molybdenum and rhenium diffused to a lesser degree under these conditions. It was discovered that there is significant interaction between many of the alloying elements in these systems during the diffusion anneal. The limitations of the analytical technique are summarized. (orig.)

  4. The role of a retinoic acid response element in establishing the anterior neural expression border of Hoxd4 transgenes. (United States)

    Nolte, Christof; Amores, Angel; Nagy Kovács, Erzsébet; Postlethwait, John; Featherstone, Mark


    The zebrafish hoxd4a locus was compared to its murine ortholog, Hoxd4. The sequence of regulatory elements, including a DR5 type retinoic acid response element (RARE) required for Hoxd4 neural enhancer activity, are highly conserved. Additionally, zebrafish and mouse neural enhancers function identically in transgenic mouse embryos. We tested whether sequence conservation reflects functional importance by altering the spacing and sequence of the RARE in the Hoxd4 neural enhancer. Stabilizing receptor-DNA interactions did not anteriorize transgene expression. By contrast, conversion of the RARE from a DR5 to a DR2 type element decreased receptor-DNA stability and posteriorized expression. Hence, the setting of the Hox anterior expression border is not a simple function of the affinity of retinoid receptors for their cognate element.

  5. Widespread Alu repeat-driven expansion of consensus DR2 retinoic acid response elements during primate evolution

    Directory of Open Access Journals (Sweden)

    Wang Tian-Tian


    Full Text Available Abstract Background Nuclear receptors are hormone-regulated transcription factors whose signaling controls numerous aspects of development and physiology. Many receptors recognize DNA hormone response elements formed by direct repeats of RGKTCA motifs separated by 1 to 5 bp (DR1-DR5. Although many known such response elements are conserved in the mouse and human genomes, it is unclear to which extent transcriptional regulation by nuclear receptors has evolved specifically in primates. Results We have mapped the positions of all consensus DR-type hormone response elements in the human genome, and found that DR2 motifs, recognized by retinoic acid receptors (RARs, are heavily overrepresented (108,582 elements. 90% of these are present in Alu repeats, which also contain lesser numbers of other consensus DRs, including 50% of consensus DR4 motifs. Few DR2s are in potentially mobile AluY elements and the vast majority are also present in chimp and macaque. 95.5% of Alu-DR2s are distributed throughout subclasses of AluS repeats, and arose largely through deamination of a methylated CpG dinucleotide in a non-consensus motif present in AluS sequences. We find that Alu-DR2 motifs are located adjacent to numerous known retinoic acid target genes, and show by chromatin immunoprecipitation assays in squamous carcinoma cells that several of these elements recruit RARs in vivo. These findings are supported by ChIP-on-chip data from retinoic acid-treated HL60 cells revealing RAR binding to several Alu-DR2 motifs. Conclusion These data provide strong support for the notion that Alu-mediated expansion of DR elements contributed to the evolution of gene regulation by RARs and other nuclear receptors in primates and humans.

  6. cAMP/CREB-regulated LINC00473 marks LKB1-inactivated lung cancer and mediates tumor growth. (United States)

    Chen, Zirong; Li, Jian-Liang; Lin, Shuibin; Cao, Chunxia; Gimbrone, Nicholas T; Yang, Rongqiang; Fu, Dongtao A; Carper, Miranda B; Haura, Eric B; Schabath, Matthew B; Lu, Jianrong; Amelio, Antonio L; Cress, W Douglas; Kaye, Frederic J; Wu, Lizi


    The LKB1 tumor suppressor gene is frequently mutated and inactivated in non-small cell lung cancer (NSCLC). Loss of LKB1 promotes cancer progression and influences therapeutic responses in preclinical studies; however, specific targeted therapies for lung cancer with LKB1 inactivation are currently unavailable. Here, we have identified a long noncoding RNA (lncRNA) signature that is associated with the loss of LKB1 function. We discovered that LINC00473 is consistently the most highly induced gene in LKB1-inactivated human primary NSCLC samples and derived cell lines. Elevated LINC00473 expression correlated with poor prognosis, and sustained LINC00473 expression was required for the growth and survival of LKB1-inactivated NSCLC cells. Mechanistically, LINC00473 was induced by LKB1 inactivation and subsequent cyclic AMP-responsive element-binding protein (CREB)/CREB-regulated transcription coactivator (CRTC) activation. We determined that LINC00473 is a nuclear lncRNA and interacts with NONO, a component of the cAMP signaling pathway, thereby facilitating CRTC/CREB-mediated transcription. Collectively, our study demonstrates that LINC00473 expression potentially serves as a robust biomarker for tumor LKB1 functional status that can be integrated into clinical trials for patient selection and treatment evaluation, and implicates LINC00473 as a therapeutic target for LKB1-inactivated NSCLC.

  7. Profiling Environmental Chemicals for Activity in the Antioxidant Response Element Signaling Pathway Using a High-Throughput Screening Approach (United States)

    1 ABSTRACT 2 3 BACKGROUND: Oxidative stress has been implicated in the pathogenesis of a variety 4 of diseases ranging from cancer to neurodegeneration, the need to identify 5 chemicals that can induce this effect. The antioxidant response element (ARE)...

  8. Cyclic AMP (cAMP)-mediated stimulation of adipocyte differentiation requires the synergistic action of Epac- and cAMP-dependent protein kinase-dependent processes

    DEFF Research Database (Denmark)

    Petersen, Rasmus Koefoed; Madsen, Lise; Pedersen, Lone Møller;


    Cyclic AMP (cAMP)-dependent processes are pivotal during the early stages of adipocyte differentiation. We show that exchange protein directly activated by cAMP (Epac), which functions as a guanine nucleotide exchange factor for the Ras-like GTPases Rap1 and Rap2, was required for cAMP-dependent ......Cyclic AMP (cAMP)-dependent processes are pivotal during the early stages of adipocyte differentiation. We show that exchange protein directly activated by cAMP (Epac), which functions as a guanine nucleotide exchange factor for the Ras-like GTPases Rap1 and Rap2, was required for c......AMP-dependent stimulation of adipocyte differentiation. Epac, working via Rap, acted synergistically with cAMP-dependent protein kinase (protein kinase A [PKA]) to promote adipogenesis. The major role of PKA was to down-regulate Rho and Rho-kinase activity, rather than to enhance CREB phosphorylation. Suppression of Rho...

  9. Signal transduction in Dictyostelium fgd A mutants with a defective interaction between surface cAMP receptors and a GTP-binding regulatory protein [published erratum appears in J Cell Biol 1988 Dec;107(6 Pt 1):following 2463



    Transmembrane signal transduction was investigated in four Dictyostelium discoideum mutants that belong to the fgd A complementation group. The results show the following. (a) Cell surface cAMP receptors are present in fgd A mutants, but cAMP does not induce any of the intracellular responses, including the activation of adenylate or guanylate cyclase and chemotaxis. (b) cAMP induces down- regulation and the covalent modification (presumably phosphorylation) of the cAMP receptor. (c) The inhi...

  10. Characterization and localization of metal-responsive-element-binding transcription factors from tilapia

    Energy Technology Data Exchange (ETDEWEB)

    Cheung, Andrew Pok-Lap; Au, Candy Yee-Man; Chan, William Wai-Lun [Department of Biochemistry, Chinese University of Hong Kong, Sha Tin, N.T., Hong Kong (Hong Kong); Chan, King Ming, E-mail: [Department of Biochemistry, Chinese University of Hong Kong, Sha Tin, N.T., Hong Kong (Hong Kong)


    Two isoforms of MTF-1, MTF-1L (long form) and MTF-1S (short form), were cloned in tilapia (Ti) and characterized in a tilapia liver cell line, Hepa-T1. The cloned tiMTF-1L has the characteristics of all of the tiMTF-1S identified so far with the zinc finger domain having six fingers, the acidic-rich, proline-rich, and serine/threonine-rich domains; however, the short form encodes for the zinc finger domain with five zinc fingers only and no other domains. The transient transfection of tiMTF-1L into human HepG2 cells showed both constitutive and zinc-induced metal-responsive-element (MRE)-driven reporter gene expression. However, the transfection of tiMTF-1S (which lacks all three transactivation domains) into a human cell line showed reduced transcriptional activities compared with an endogenous control in both basal- and Zn{sup 2+}-induced conditions. The tiMTF-1 isoforms were tagged with GFP and transfected into Hepa-T1 cells (tilapia hepatocytes). The nuclear translocation of tiMTF-1L was observed when the cells were exposed to a sufficient concentration of metals for 6 h. However, tiMTF-1S, was localized in the nucleus with or without metal treatment. Electrophoretic mobility shift assay (EMSA) confirmed that both of the isoforms were able to bind to the MRE specifically in vitro. Tissue distribution studies showed that tiMTF-1L was more abundant than tiMTF-1S in all of the tissues tested.

  11. Long-term cilostazol administration ameliorates memory decline in senescence-accelerated mouse prone 8 (SAMP8) through a dual effect on cAMP and blood-brain barrier. (United States)

    Yanai, Shuichi; Toyohara, Jun; Ishiwata, Kiichi; Ito, Hideki; Endo, Shogo


    Phosphodiesterases (PDEs), which hydrolyze and inactivate 3', 5'-cyclic adenosine monophosphate (cAMP) and 3', 5'-cyclic guanosine monophosphate (cGMP), play an important role in synaptic plasticity that underlies memory. Recently, several PDE inhibitors were assessed for their possible therapeutic efficacy in treating cognitive disorders. Here, we examined how cilostazol, a selective PDE3 inhibitor, affects brain functions in senescence-accelerated mouse prone 8 (SAMP8), an animal model of age-related cognitive impairment. Long-term administration of cilostazol restored the impaired context-dependent conditioned fear memory of SAMP8 to match that in normal aging control substrain SAMR1. Cilostazol also increased the number of cells containing phosphorylated cAMP-responsive element binding protein (CREB), a downstream component of the cAMP pathway. Finally, cilostazol improves blood-brain barrier (BBB) integrity, demonstrated by reduced extravasation of 2-deoxy-2-(18)F-fluoro-d-glucose and Evans Blue dye in the brains of SAMP8. This improvement in BBB integrity was associated with an increased amount of zona occludens protein 1 (ZO-1) and occludin proteins, components of tight junctions integral to the BBB. The results suggest that long-term administration of cilostazol exerts its beneficial effects on age-related cognitive impairment through a dual mechanism: by enhancing the cAMP system in the brain and by maintaining or improving BBB integrity.

  12. What is the primary beam response of an interferometer with unequal elements?

    NARCIS (Netherlands)

    Strom, R.G.; Bachiller, R.; Colomer, F.; Desmurs, J.F.; de Vicente, P.


    The EVN stations encompass elements with a range of diameters, even including an interferometer (the Westerbork Telescope, with up to 14 elements used together as a tied array). In combination, the various station pairs will each produce their own primary beam envelopes, with which the interferomete

  13. Plant-soil distribution of potentially toxic elements in response to elevated atmospheric CO2. (United States)

    Duval, Benjamin D; Dijkstra, Paul; Natali, Susan M; Megonigal, J Patrick; Ketterer, Michael E; Drake, Bert G; Lerdau, Manuel T; Gordon, Gwyneth; Anbar, Ariel D; Hungate, Bruce A


    The distribution of contaminant elements within ecosystems is an environmental concern because of these elements' potential toxicity to animals and plants and their ability to hinder microbial ecosystem services. As with nutrients, contaminants are cycled within and through ecosystems. Elevated atmospheric CO2 generally increases plant productivity and alters nutrient element cycling, but whether CO2 causes similar effects on the cycling of contaminant elements is unknown. Here we show that 11 years of experimental CO2 enrichment in a sandy soil with low organic matter content causes plants to accumulate contaminants in plant biomass, with declines in the extractable contaminant element pools in surface soils. These results indicate that CO2 alters the distribution of contaminant elements in ecosystems, with plant element accumulation and declining soil availability both likely explained by the CO2 stimulation of plant biomass. Our results highlight the interdependence of element cycles and the importance of taking a broad view of the periodic table when the effects of global environmental change on ecosystem biogeochemistry are considered.

  14. Warmer amps for the LHC

    CERN Multimedia

    Anaïs Schaeffer


    CERN is working together with an Italian company to develop superconducting cables that can function at temperatures of up to 25 K (-248°C). This will make it possible to move LHC magnet power supplies out of the tunnel, protecting them from exposure to the showers of very high-energy particles produced by the accelerator.   Figure 1: devices of this type, which measure approximately 10 metres in length, are inserted between the accelerating magnets at different points along the LHC. When it comes to consuming electricity, the magnets that steer particles through large accelerators can be characterised with just one word: greedy. For the LHC, the total current can reach 1.5 million amps. At the present time, this current is brought in via copper cables of up to 10 cm in diameter. In the tunnel, these cables connect the current leads - which provide the transition between the ambient-temperature cables and the magnets in their bath of superfluid helium - to the power supply. In the a...

  15. Cyclic AMP functions as a primary sexual signal in gametes of Chlamydomonas reinhardtii. (United States)

    Pasquale, S M; Goodenough, U W


    When Chlamydomonas reinhardtii gametes of opposite mating type are mixed together, they adhere by a flagella-mediated agglutination that triggers three rapid mating responses: flagellar tip activation, cell wall loss, and mating structure activation accompanied by actin polymerization. Here we show that a transient 10-fold elevation of intracellular cAMP levels is also triggered by sexual agglutination. We further show that gametes of a single mating type can be induced to undergo all three mating responses when presented with exogenous dibutyryl-cAMP (db-cAMP). These events are also induced by cyclic nucleotide phosphodiesterase inhibitors, which elevate endogenous cAMP levels and act synergistically with db-cAMP. Non-agglutinating mutants of opposite mating type will fuse efficiently in the presence of db-cAMP. No activation of mating events is induced by calcium plus ionophores, 8-bromo-cGMP, dibutyryl-cGMP, nigericin at alkaline pH, phorbol esters, or forskolin. H-8, an inhibitor of cyclic nucleotide-dependent protein kinase, inhibits mating events in agglutinating cells and antagonizes the effects of cAMP on non-agglutinating cells. Adenylate cyclase activity was detected in both the gamete cell body and flagella, with the highest specific activity displayed in flagellar membrane fractions. The flagellar membrane adenylate cyclase is preferentially stimulated by Mn++, unresponsive to NaF, GTP, GTP gamma S, AlF4-, and forskolin, and is inhibited by trifluoperazine. Cyclic nucleotide phosphodiesterase activity is also present in flagella. Our observations indicate that cAMP is a sufficient initial signal for all of the known mating reaction events in C. reinhardtii, and suggest that the flagellar cyclase and/or phosphodiesterase may be important loci of control for the agglutination-stimulated production of this signal.

  16. 21 CFR 862.1230 - Cyclic AMP test system. (United States)


    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cyclic AMP test system. 862.1230 Section 862.1230....1230 Cyclic AMP test system. (a) Identification. A cyclic AMP test system is a device intended to measure the level of adenosine 3′, 5′-monophosphate (cyclic AMP) in plasma, urine, and other body...

  17. Diatom acclimation to elevated CO2 via cAMP signalling and coordinated gene expression (United States)

    Hennon, Gwenn M. M.; Ashworth, Justin; Groussman, Ryan D.; Berthiaume, Chris; Morales, Rhonda L.; Baliga, Nitin S.; Orellana, Mónica V.; Armbrust, E. V.


    Diatoms are responsible for ~40% of marine primary productivity, fuelling the oceanic carbon cycle and contributing to natural carbon sequestration in the deep ocean. Diatoms rely on energetically expensive carbon concentrating mechanisms (CCMs) to fix carbon efficiently at modern levels of CO2 (refs , , ). How diatoms may respond over the short and long term to rising atmospheric CO2 remains an open question. Here we use nitrate-limited chemostats to show that the model diatom Thalassiosira pseudonana rapidly responds to increasing CO2 by differentially expressing gene clusters that regulate transcription and chromosome folding, and subsequently reduces transcription of photosynthesis and respiration gene clusters under steady-state elevated CO2. These results suggest that exposure to elevated CO2 first causes a shift in regulation, and then a metabolic rearrangement. Genes in one CO2-responsive cluster included CCM and photorespiration genes that share a putative cAMP-responsive cis-regulatory sequence, implying these genes are co-regulated in response to CO2, with cAMP as an intermediate messenger. We verified cAMP-induced downregulation of CCM gene δ-CA3 in nutrient-replete diatom cultures by inhibiting the hydrolysis of cAMP. These results indicate an important role for cAMP in downregulating CCM and photorespiration genes under elevated CO2 and provide insights into mechanisms of diatom acclimation in response to climate change.

  18. Compartmentalisation of cAMP-dependent signalling in blood platelets: The role of lipid rafts and actin polymerisation. (United States)

    Raslan, Zaher; Naseem, Khalid M


    Prostacyclin (PGI2) inhibits blood platelets through the activation of membrane adenylyl cyclases (ACs) and cyclic adenosine 3',5'-monophosphate (cAMP)-mediated signalling. However, the molecular mechanism controlling cAMP signalling in blood platelet remains unclear, and in particular how individual isoforms of AC and protein kinase A (PKA) are coordinated to target distinct substrates in order to modulate platelet activation. In this study, we demonstrate that lipid rafts and the actin cytoskeleton may play a key role in regulating platelet responses to cAMP downstream of PGI2. Disruption of lipid rafts with methyl-beta-cyclodextrin (MβCD) increased platelet sensitivity to PGI2 and forskolin, a direct AC cyclase activator, resulting in greater inhibition of collagen-stimulated platelet aggregation. In contrast, platelet inhibition by the direct activator of PKA, 8-CPT-6-Phe-cAMP was unaffected by MβCD treatment. Consistent with the functional data, lipid raft disruption increased PGI2-stimulated cAMP formation and proximal PKA-mediated signalling events. Platelet inhibition, cAMP formation and phosphorylation of PKA substrates in response to PGI2 were also increased in the presence of cytochalasin D, indicating a role for actin cytoskeleton in signalling in response to PGI2. A potential role for lipid rafts in cAMP signalling is strengthened by our finding that a pool of ACV/VI and PKA was partitioned into lipid rafts. Our data demonstrate partial compartmentalisation of cAMP signalling machinery in platelets, where lipid rafts and the actin cytoskeleton regulate the inhibitory effects induced by PGI2. The increased platelet sensitivity to cAMP-elevating agents signalling upon raft and cytoskeleton disruption suggests that these compartments act to restrain basal cAMP signalling.

  19. Preserved cardiac function despite marked impairment of cAMP generation.

    Directory of Open Access Journals (Sweden)

    Mei Hua Gao

    Full Text Available OBJECTIVES: So many clinical trials of positive inotropes have failed, that it is now axiomatic that agents that increase cAMP are deleterious to the failing heart. An alternative strategy is to alter myocardial Ca(2+ handling or myofilament response to Ca(2+ using agents that do not affect cAMP. Although left ventricular (LV function is tightly linked to adenylyl cyclase (AC activity, the beneficial effects of AC may be independent of cAMP and instead stem from effects on Ca(2+ handling. Here we ask whether an AC mutant molecule that reduces LV cAMP production would have favorable effects on LV function through its effects on Ca(2+ handling alone. METHODS AND RESULTS: We generated transgenic mice with cardiac-directed expression of an AC6 mutant (AC6mut. Cardiac myocytes showed impaired cAMP production in response to isoproterenol (74% reduction; p<0.001, but LV size and function were normal. Isolated hearts showed preserved LV function in response to isoproterenol stimulation. AC6mut expression was associated with increased sarcoplasmic reticulum Ca(2+ uptake and the EC50 for SERCA2a activation was reduced. Cardiac myocytes isolated from AC6mut mice showed increased amplitude of Ca(2+ transients in response to isoproterenol (p = 0.0001. AC6mut expression also was associated with increased expression of LV S100A1 (p = 0.03 and reduced expression of phospholamban protein (p = 0.01. CONCLUSION: LV AC mutant expression is associated with normal cardiac function despite impaired cAMP generation. The mechanism appears to be through effects on Ca(2+ handling - effects that occur despite diminished cAMP.

  20. The chitin-binding capability of Cy-AMP1 from cycad is essential to antifungal activity. (United States)

    Yokoyama, Seiya; Iida, Yuto; Kawasaki, Yousuke; Minami, Yuji; Watanabe, Keiichi; Yagi, Fumio


    Antimicrobial peptides are important components of the host innate immune responses by exerting broad-spectrum microbicidal activity against pathogenic microbes. Cy-AMP1 found in the cycad (Cycas revoluta) seeds has chitin-binding ability, and the chitin-binding domain was conserved in knottin-type and hevein-type antimicrobial peptides. The recombinant Cy-AMP1 was expressed in Escherichia coli and purified to study the role of chitin-binding domain. The mutants of Cy-AMP1 lost chitin-binding ability completely, and its antifungal activity was markedly decreased in comparison with native Cy-AMP1. However, the antimicrobial activities of the mutant peptides are nearly identical to that of native one. It was suggested that the chitin-binding domain plays an essential role in antifungal, but not antimicrobial, activity of Cy-AMP1.

  1. Regulation of cyclic GMP, cyclic amp and lactate dehydrogenase by putative neutrotransmitters in the C6 rat glioma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Bottenstein, J.E.; de Vellis, J.


    In C6 cells norepinephrine and dopamine caused transient increases in cyclic GMP and cyclic AMP, as well as an induction of lactate dehydrogenase. All of these responses were blocked by 1-propranolol, suggesting mediation by a ..beta..-receptor. Phentolamine potentiated the NE-increased cAMP levels by 5-fold when NE was used at suboptimal doses, suggesting the presence of ..cap alpha..-adrenergic receptors in C6 cells. Carbamylcholine decreased the levels of both cyclic nucleotides, with hexamethonium partially reversing the effect on cyclic GMP. Dibutyryl-cyclic GMP or carbamylcholine reduced catecholamine-induced cyclic AMP levels. Serotonin increased cyclic GMP levels 60% and decreased cyclic AMP levels 36%. Calcium- and magnesium-free media inhibited the norepinephrine-induced levels of cyclic GMP and cyclic AMP respectively.

  2. Regulation of cyclic GMP, cyclic AMP and lactate dehydrogenase by putative neurotransmitters in the C6 rat glioma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Bottenstein, J.E.; de Vellis, J.


    In C6 cells norepinephrine and dopamine caused transient increases in cyclic GMP and cyclic AMP, as well as an induction of lactate dehydrogenase. All of these responses were blocked by l-propranolol, suggesting mediation by a ..beta..-receptor. Phentolamine potentiated the NE-increased cAMP levels by 5-fold when NE was used at suboptimal doses, suggesting the presence of ..cap alpha..-adrenergic receptors in C6 cells. Carbamylcholine decreased the levels of both cyclic nucleotides, with hexamethonium partially reversing the effect on cyclic GMP. Dibutyryl-cyclic GMP or carbamylcholine reduced catecholamine-induced cyclic AMP levels. Serotonin increased cyclic GMP levels 60% and decreased cyclic AMP levels 36%. Calcium- and magnesium-free media inhibited the norepinephrine-induced levels of cyclic GMP and cyclic AMP respectively.

  3. Quality assessment of structure and language elements of written responses given by seven Scandinavian drug information centres

    DEFF Research Database (Denmark)

    Reppe, Linda Amundstuen; Spigset, Olav; Kampmann, Jens Peter


    for which queries were part of the study. The responses were assessed qualitatively by six clinical pharmacologists (internal experts) and six general practitioners (GPs, external experts). In addition, linguistic aspects of the responses were evaluated by a plain language expert. RESULTS: The quality......PURPOSE: The aim of this study was to identify structure and language elements affecting the quality of responses from Scandinavian drug information centres (DICs). METHODS: Six different fictitious drug-related queries were sent to each of seven Scandinavian DICs. The centres were blinded...... of responses was generally judged as satisfactory to good. Presenting specific advice and conclusions were considered to improve the quality of the responses. However, small nuances in language formulations could affect the individual judgments of the experts, e.g. on whether or not advice was given. Some...

  4. Cyclic AMP control measured in two compartments in HEK293 cells: phosphodiesterase K(M is more important than phosphodiesterase localization.

    Directory of Open Access Journals (Sweden)

    Karina Matthiesen

    Full Text Available The intracellular second messenger cyclic AMP (cAMP is degraded by phosphodiesterases (PDE. The knowledge of individual families and subtypes of PDEs is considerable, but how the different PDEs collaborate in the cell to control a cAMP signal is still not fully understood. In order to investigate compartmentalized cAMP signaling, we have generated a membrane-targeted variant of the cAMP Bioluminiscence Resonance Energy Transfer (BRET sensor CAMYEL and have compared intracellular cAMP measurements with it to measurements with the cytosolic BRET sensor CAMYEL in HEK293 cells. With these sensors we observed a slightly higher cAMP response to adenylyl cyclase activation at the plasma membrane compared to the cytosol, which is in accordance with earlier results from Fluorescence Resonance Energy Transfer (FRET sensors. We have analyzed PDE activity in fractionated lysates from HEK293 cells using selective PDE inhibitors and have identified PDE3 and PDE10A as the major membrane-bound PDEs and PDE4 as the major cytosolic PDE. Inhibition of membrane-bound or cytosolic PDEs can potentiate the cAMP response to adenylyl cyclase activation, but we see no significant difference between the potentiation of the cAMP response at the plasma membrane and in cytosol when membrane-bound and cytosolic PDEs are inhibited. When different levels of stimulation were tested, we found that PDEs 3 and 10 are mainly responsible for cAMP degradation at low intracellular cAMP concentrations, whereas PDE4 is more important for control of cAMP at higher concentrations.

  5. The effect of hypoxia on PGE2-stimulated cAMP generation in HMEC-1. (United States)

    Wiktorowska-Owczarek, Anna; Owczarek, Jacek


    Prostaglandin E2 (PGE2) is generated in various cells, including endothelial cells, and is responsible for various functions, such as vascular relaxation and angiogenesis. Effects of PGE2 are mediated via receptors EP1-EP4, among which EP2 and EP4 are coupled to Gs protein which activates adenylate cyclase (AC) and cAMP synthesis. The aim of this work was to study the ability of human microvascular endothelial cells (HMEC-1) to synthesize cAMP in the presence of PGE2, and to determine the effect of hypoxia on the PGE2- stimulated cAMP level. It was decided to evaluate the effect of PGE2 on the secretion of VEGF, an inducer of angiogenesis. In summary, our findings show that PGE2 induces cAMP production, but hypoxia may impair PGE2-stimulated activity of the AC-cAMP signaling pathway. These results suggest that the cardioprotective effect of PGE2/EP4/cAMP may be attenuated during ischemia. Furthermore, this study indicates that the pro-angiogenic effect of PGE2 is not associated with VEGF secretion in HMEC-1 cells.

  6. SANTOS - a two-dimensional finite element program for the quasistatic, large deformation, inelastic response of solids

    Energy Technology Data Exchange (ETDEWEB)

    Stone, C.M.


    SANTOS is a finite element program designed to compute the quasistatic, large deformation, inelastic response of two-dimensional planar or axisymmetric solids. The code is derived from the transient dynamic code PRONTO 2D. The solution strategy used to compute the equilibrium states is based on a self-adaptive dynamic relaxation solution scheme, which is based on explicit central difference pseudo-time integration and artificial mass proportional damping. The element used in SANTOS is a uniform strain 4-node quadrilateral element with an hourglass control scheme to control the spurious deformation modes. Finite strain constitutive models for many common engineering materials are included. A robust master-slave contact algorithm for modeling sliding contact is implemented. An interface for coupling to an external code is also provided. 43 refs., 22 figs.

  7. Separation of effects of adenosine on energy metabolism from those on cyclic AMP in rat thymic lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Nordeen, S.K.; Young, D.A.


    In rat thymic lymphocytes incubated for 2 h without exogenous energy-providing substrate, adenosine may be substituted for glucose as a means of maximally restoring energy metabolism and those cellular functions whose rates are sensitive to small changes in the energy balance, such as protein synthesis and uridine utilization for RNA synthesis. Since effects of adenosine in thymocytes and other cells have frequently been attributed to changes in cyclic AMP, this report investigates its possible involvement in these glucose-like restorative actions of adenosine. Although the same range of doses of adenosine effective at raising cyclic AMP also elicit roughly parallel stimulations of protein synthesis and uridine utilization, further results dissociate the restorative actions from those on cyclic AMP. (a) Other purine nucleosides mimic the glucose-like actions of adenosine without increasing cyclic AMP; (b) conversely, prostaglandin E/sub 1/ mimics the cyclic AMP response without restoring energy metabolism or energy-dependent functions; and (c) potentiation of the cyclic AMP response, either by inhibiting phosphodiesterase or adenosine deaminase, does not enhance the restorative response to a range of doses of adenosine. Finally, cyclic AMP-mediated glycogenolysis cannot account for the glucose-like effects since addition of adenosine increases, not decreases, levels of glycogen.

  8. cAMP stimulation of StAR expression and cholesterol metabolism is modulated by co-expression of labile suppressors of transcription and mRNA turnover. (United States)

    Jefcoate, Colin R; Lee, Jinwoo; Cherradi, Nadia; Takemori, Hiroshi; Duan, Haichuan


    The steroidogenic acute regulatory (StAR) protein is generated in rodents from 1.6 kb and 3.5 kb mRNA formed by alternative polyadenylation. The zinc finger protein, TIS11B (also Znf36L1), is elevated by cAMP in adrenal cells in parallel with StAR mRNA. TIS11b selectively destabilizes the 3.5 kb mRNA through AU-rich sequences at the end of the 3'UTR. siRNA suppression shows that TIS11b surprisingly increases StAR protein and cholesterol metabolism. StAR transcription is directly activated by PKA phosphorylation. cAMP responsive element binding (CREB) protein 1 phosphorylation is a key step leading to recruitment of the co-activator, CREB binding protein (CBP). A second protein, CREB regulated transcription coactivator (TORC/CRTC), enhances this recruitment, but is inhibited by salt inducible kinase (SIK). Basal StAR transcription is constrained through this phosphorylation of TORC. PKA provides an alternative stimulation by phosphorylating SIK, which prevents TORC inactivation. PKA stimulation of StAR nuclear transcripts substantially precedes TORC recruitment to the StAR promoter, which may, therefore, mediate a later step in mRNA production. Inhibition of SIK by staurosporine elevates StAR transcription and TORC recruitment to maximum levels, but without CREB phosphorylation. TORC suppression by SIK evidently limits basal StAR transcription. Staurosporine and cAMP stimulate synergistically. SIK targets the phosphatase, PP2a (activation), and Type 2 histone de-acetylases (inhibition), which may each contribute to suppression. Staurosporine stimulation through SIK inhibition is repeated in cAMP stimulation of many steroidogenic genes regulated by steroidogenic factor 1 (SF-1) and CREB. TIS11b and SIK may combine to attenuate StAR expression when hormonal stimuli decline.

  9. Anxiety and depression with neurogenesis defects in exchange protein directly activated by cAMP 2-deficient mice are ameliorated by a selective serotonin reuptake inhibitor, Prozac (United States)

    Zhou, L; Ma, S L; Yeung, P K K; Wong, Y H; Tsim, K W K; So, K F; Lam, L C W; Chung, S K


    Intracellular cAMP and serotonin are important modulators of anxiety and depression. Fluoxetine, a selective serotonin reuptake inhibitor (SSRI) also known as Prozac, is widely used against depression, potentially by activating cAMP response element-binding protein (CREB) and increasing brain-derived neurotrophic factor (BDNF) through protein kinase A (PKA). However, the role of Epac1 and Epac2 (Rap guanine nucleotide exchange factors, RAPGEF3 and RAPGEF4, respectively) as potential downstream targets of SSRI/cAMP in mood regulations is not yet clear. Here, we investigated the phenotypes of Epac1 (Epac1−/−) or Epac2 (Epac2−/−) knockout mice by comparing them with their wild-type counterparts. Surprisingly, Epac2−/− mice exhibited a wide range of mood disorders, including anxiety and depression with learning and memory deficits in contextual and cued fear-conditioning tests without affecting Epac1 expression or PKA activity. Interestingly, rs17746510, one of the three single-nucleotide polymorphisms (SNPs) in RAPGEF4 associated with cognitive decline in Chinese Alzheimer's disease (AD) patients, was significantly correlated with apathy and mood disturbance, whereas no significant association was observed between RAPGEF3 SNPs and the risk of AD or neuropsychiatric inventory scores. To further determine the detailed role of Epac2 in SSRI/serotonin/cAMP-involved mood disorders, we treated Epac2−/− mice with a SSRI, Prozac. The alteration in open field behavior and impaired hippocampal cell proliferation in Epac2−/− mice were alleviated by Prozac. Taken together, Epac2 gene polymorphism is a putative risk factor for mood disorders in AD patients in part by affecting the hippocampal neurogenesis. PMID:27598965


    Miki, Toshihiro; Mizusawa, Tomisaku; Yamada, Osamu; Toda, Tomoki

    This paper studies the earthquake response of steel portal frames when the shear collapse occurs at the centre of the beam. The pseudodynamic simulation technique for the earthquake response analysis of the frames is developed in correspondence to the pseudodynamic substructure testing method. For the thin-walled box element under shear force in the middle of beam, the numerical process is utilized by a general-purpose finite element analysis program. The numerical results show the shear collapse behaviour in stiffened box beams and corresponding restoring force - displacement relationship of frames. The advantages of shear collapse of beams for the use in frames during earthquakes are discussed from the point of view of the hysteretic energy dissipated by the column base.

  11. CFTR: a hub for kinases and crosstalk of cAMP and Ca2+. (United States)

    Kunzelmann, Karl; Mehta, Anil


    Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR). The resulting disease is pleiotropic consistent with the idea that CFTR acts as a node within a network of signalling proteins. CFTR is not only a regulator of multiple transport proteins and controlled by numerous kinases but also participates in many signalling pathways that are disrupted after expression of its commonest mutant (F508del-CFTR). It operates in membrane compartments creating a scaffold for cytoskeletal elements, surface receptors, kinases and phosphodiesterases. CFTR is exposed to membrane-local second messengers such that a CFTR-interacting, low cellular energy sensor kinase (AMP- and ADP-activated kinase, AMPK) signals through a high energy phosphohistidine protein kinase (nucleoside diphosphate kinase, NDPK). CFTR also translocates a Ca(2+)-dependent adenylate cyclase to its proximity so that a rigid separation between cAMP-dependent and Ca(2+)-dependent regulation of Cl(-) transport becomes obsolete. In the presence of wild-type CFTR, parallel activation of CFTR and outwardly rectifying anoctamin 6 Cl(-) channels is observed, while the Ca(2+)-activated anoctamin 1 Cl(-) channel is inhibited. In contrast, in CF cells, CFTR is missing/mislocalized and the outwardly rectifying chloride channel is attenuated while Ca(2+)-dependent Cl(-) secretion (anoctamin 1) appears upregulated. Additionally, we consider the idea that F508del-CFTR when trapped in the endoplasmic reticulum augments IP3-mediated Ca(2+) release by providing a shunt pathway for Cl(-). CFTR and the IP3 receptor share the characteristic that they both assemble their partner proteins to increase the plasticity of their hub responses. In CF, the CFTR hub fails to form at the plasma membrane, with widespread detrimental consequences for cell signalling.

  12. Enhancement of DNA vaccine-induced immune responses by a 72-bp element from SV40 enhancer

    Institute of Scientific and Technical Information of China (English)

    LI Hai-shan; XU Jian-qing; HONG Kun-xue; SHAO Yi-ming; LIU Yong; LI Ding-feng; ZHANG Ran-ran; TANG Hai-li; ZHANG Yu-wei; HUANG Wei; LIU Ying; PENG Hong


    Background Although DNA vaccine is considered as the next generation of vaccine, most DNA vaccine candidates are still suffering from the relatively weak immunogenicity despite the increased dosage of plasmid DNA administered. In order to enhance the immune responses elicited by a codon-optimized HIV gag DNA vaccine, a modified plasmid vector pDRVI1.0 and a booster immunization with replicating Tiantan vaccinia (RTV) strain expressing the same gene were employed.Methods Vector pDRVI1.0 was constructed through inserting the 72-bp element from the SV40 enhancer, which was reported promoting nuclear transport of plasmid DNA, to the upstream of cytomegalovirus enhancer/promoter region of the plasmid vector pVR1012. Gene expression levels from expression plasmids based on pDRVI1.0 and pVR1012 were tested. Humoral and cellular immune responses induced by DNA vaccine alone or DNA prime-RTV boost regimen were determined in mice.Results It was shown that the 72-bp element significantly enhanced the gene expression level in non-dividing cells.gag-specific humoral and cellular immune responses induced by DNA vaccination were both significantly improved, while the Th1/Th2 balance was not obviously affected by the 72-bp element. RTV boosting further significantly enhanced DNA vaccine-primed antibody and T cell responses in a Th1-biased manner.Conclusions The 72-bp SV40 enhancer element should be included in the DNA vaccine vector and RTV strain is a very efficient live vector for boosting immunization.

  13. Dis3- and exosome subunit-responsive 3 Prime mRNA instability elements

    Energy Technology Data Exchange (ETDEWEB)

    Kiss, Daniel L.; Hou, Dezhi [Case Western Reserve University School of Medicine, Department of Molecular Biology and Microbiology, Cleveland, OH 44106 (United States); Gross, Robert H. [Dartmouth College, Department of Biological Sciences, Life Sciences Center 343, Hanover, NH 03755 (United States); Andrulis, Erik D., E-mail: [Case Western Reserve University School of Medicine, Department of Molecular Biology and Microbiology, Cleveland, OH 44106 (United States)


    Highlights: Black-Right-Pointing-Pointer Successful use of a novel RNA-specific bioinformatic tool, RNA SCOPE. Black-Right-Pointing-Pointer Identified novel 3 Prime UTR cis-acting element that destabilizes a reporter mRNA. Black-Right-Pointing-Pointer Show exosome subunits are required for cis-acting element-mediated mRNA instability. Black-Right-Pointing-Pointer Define precise sequence requirements of novel cis-acting element. Black-Right-Pointing-Pointer Show that microarray-defined exosome subunit-regulated mRNAs have novel element. -- Abstract: Eukaryotic RNA turnover is regulated in part by the exosome, a nuclear and cytoplasmic complex of ribonucleases (RNases) and RNA-binding proteins. The major RNase of the complex is thought to be Dis3, a multi-functional 3 Prime -5 Prime exoribonuclease and endoribonuclease. Although it is known that Dis3 and core exosome subunits are recruited to transcriptionally active genes and to messenger RNA (mRNA) substrates, this recruitment is thought to occur indirectly. We sought to discover cis-acting elements that recruit Dis3 or other exosome subunits. Using a bioinformatic tool called RNA SCOPE to screen the 3 Prime untranslated regions of up-regulated transcripts from our published Dis3 depletion-derived transcriptomic data set, we identified several motifs as candidate instability elements. Secondary screening using a luciferase reporter system revealed that one cassette-harboring four elements-destabilized the reporter transcript. RNAi-based depletion of Dis3, Rrp6, Rrp4, Rrp40, or Rrp46 diminished the efficacy of cassette-mediated destabilization. Truncation analysis of the cassette showed that two exosome subunit-sensitive elements (ESSEs) destabilized the reporter. Point-directed mutagenesis of ESSE abrogated the destabilization effect. An examination of the transcriptomic data from exosome subunit depletion-based microarrays revealed that mRNAs with ESSEs are found in every up-regulated mRNA data set but are

  14. Evidence for cAMP as a mediator of gonadotropin secretion from female pituitaries

    Energy Technology Data Exchange (ETDEWEB)

    Bourne, G.A.; Baldwin, D.M.


    Sodium flufenamate, which inhibited gonadotropin-releasing hormone (GnRH)-stimulated increases in adenosine 3',5'-cyclic monophosphate (cAMP), was used to evaluate the potential role of cAMP as a mediator of GnRH-stimulated gonadotropin secretion. Quartered pituitaries from diestrous II female rats were perifused at 37/sup 0/C, and sequential effluent fractions were collected every 10 min. Administration of GnRH resulted in a characteristic biphasic response for both luteinizing hormone (LH) and follicle-stimulating hormone (FSH), whereas 5 cycloheximide inhibited the secondary augmented responses (phase II) of both hormones. Infusions of 0.1 mM flufenamate inhibited GnRH-stimulated gonadotropin secretion in a manner similar to that of cycloheximide, whereas the administration of 5 mM dibutyryl cAMP in combination with GnRH and flufenamate resulted in the restoration of LH and FSH secretion. The dibutyryl cAMP-restored response appeared to be protein synthesis dependent and specific for cAMP. These results suggest that although the cyclic nucleotide is not involved in the acute release of LH and FSH, it does appear to play a pivotal but indirect role in phase II release of the hormones, by effects involving the stimulation of de novo protein synthesis.

  15. Purification, characterization, and sequencing of antimicrobial peptides, Cy-AMP1, Cy-AMP2, and Cy-AMP3, from the Cycad (Cycas revoluta) seeds. (United States)

    Yokoyama, Seiya; Kato, Kouji; Koba, Atsuko; Minami, Yuji; Watanabe, Keiichi; Yagi, Fumio


    Novel antimicrobial peptides (AMP), designated Cy-AMP1, Cy-AMP2, and Cy-AMP3, were purified from seeds of the cycad (Cycas revoluta) by a CM cellulofine column, ion-exchange HPLC on SP COSMOGEL, and reverse-phase HPLC. They had molecular masses of 4583.2 Da, 4568.9 Da and 9275.8 Da, respectively, by MALDI-TOF MS analysis. Half of the amino acid residues of Cy-AMP1 and Cy-AMP2 were cysteine, glycine and proline, and their sequences were similar. The sequence of Cy-AMP3 showed high homology to various lipid transfer proteins. For Cy-AMP1 and Cy-AMP2, the concentrations of peptides required for 50% inhibition (IC(50)) of the growth of plant pathogenic fungi, Gram-positive and Gram-negative bacteria were 7.0-8.9 microg/ml. The Cy-AMP3 had weak antimicrobial activity. The structural and antimicrobial characteristics of Cy-AMP1 and Cy-AMP2 indicated that they are a novel type of antimicrobial peptide belonging to a plant defensin family.

  16. cAMP target sequences enhCRE and CNRE sense low-salt intake to increase human renin gene expression in vivo. (United States)

    Desch, Michael; Harlander, Sabine; Neubauer, Björn; Gerl, Melanie; Germain, Stephane; Castrop, Hayo; Todorov, Vladimir T


    This study aimed to assess the role of cAMP target sequences enhancer cAMP response element (enhCRE) and cAMP and overlapping negative response element (CNRE) in the control of human renin gene (REN) in vivo. enhCRE and CNRE were silenced by mutations in a 12.2-kb human renin promoter fused to LacZ reporter gene. This construct was used to generate transgenic mice (RENMut-LacZ). The expression of the transgene was correctly targeted to the juxtaglomerular portions of renal afferent arterioles which express endogenous mouse renin. Therefore, enhCRE and CNRE do not seem to be relevant for the control of the cell-specific expression of the human renin gene. The β-adrenoreceptor agonist isoproterenol (10 mg/kg/day, for 2 days) stimulated the endogenous renin, but not the LacZ mRNA expression. Treatment of RENMut-LacZ mice with the angiotensin converting enzyme inhibitor (enalapril 10 mg/kg/day, for 7 days) or their crossing to angiotensin receptor type 1a knockout mice led to increased renin and LacZ mRNA levels. Renin expression was upregulated by low-salt diet (0.03% NaCl, for 10 days) and downregulated by high-salt diet (4% NaCl, for 10 days). In contrast, low-salt diet did not influence, while high-salt diet inhibited the expression of LacZ. In summary, enhCRE and CNRE appear to be necessary for the transactivation of the human renin gene through β-adrenoreceptors and by low-salt diet. Our data also suggest that different intracellular mechanisms mediate the effect of low- and high-salt intake on renin expression in vivo.

  17. Androgen regulation of the TMPRSS2 gene and the effect of a SNP in an androgen response element. (United States)

    Clinckemalie, Liesbeth; Spans, Lien; Dubois, Vanessa; Laurent, Michaël; Helsen, Christine; Joniau, Steven; Claessens, Frank


    More than 50% of prostate cancers have undergone a genomic reorganization that juxtaposes the androgen-regulated promoter of TMPRSS2 and the protein coding parts of several ETS oncogenes. These gene fusions lead to prostate-specific and androgen-induced ETS expression and are associated with aggressive lesions, poor prognosis, and early-onset prostate cancer. In this study, we showed that an enhancer at 13 kb upstream of the TMPRSS2 transcription start site is crucial for the androgen regulation of the TMPRSS2 gene when tested in bacterial artificial chromosomal vectors. Within this enhancer, we identified the exact androgen receptor binding sequence. This newly identified androgen response element is situated next to two binding sites for the pioneer factor GATA2, which were identified by DNase I footprinting. Both the androgen response element and the GATA-2 binding sites are involved in the enhancer activity. Importantly, a single nucleotide polymorphism (rs8134378) within this androgen response element reduces binding and transactivation by the androgen receptor. The presence of this SNP might have implications on the expression and/or formation levels of TMPRSS2 fusions, because both have been shown to be influenced by androgens.

  18. Identification of a functional antioxidant responsive element in the promoter of the Chinese hamster carbonyl reductase 3 (Chcr3) gene. (United States)

    Miura, Takeshi; Taketomi, Ayako; Nakabayashi, Toshikatsu; Nishinaka, Toru; Terada, Tomoyuki


    CHCR3, a member of the short-chain dehydrogenase/reductase superfamily, is a carbonyl reductase 3 enzyme in Chinese hamsters. Carbonyl reductase 3 in humans has been believed to involve the metabolism and/or pharmacokinetics of anthracycline drugs, and the mechanism underlying the gene regulation has been investigated. In this study, the nucleotide sequence of the Chcr3 promoter was originally determined, and its promoter activity was characterised. The proximal promoter region is TATA-less and GC-rich, similar to the promoter region of human carbonyl reductase 3. Cobalt stimulated the transcriptional activity of the Chcr3 gene. The results of a luciferase gene reporter assay demonstrated that cobalt-induced stimulation required an antioxidant responsive element. Forced expression of Nrf2, the transcription factor that binds to antioxidant responsive elements, enhanced the transcriptional activity of the Chcr3 gene. These results suggest that cobalt induces the expression of the Chcr3 gene via the Nrf2-antioxidant responsive element pathway.

  19. 33 CFR Appendix C to Part 155 - Training Elements for Oil Spill Response Plans (United States)


    ... delivered to the various personnel. Further, this section of the plan must work in harmony with those sections of the plan dealing with exercises, the spill management team, and the qualified individual. 1... plan, it is necessary for the plan to convey the elements that define the program as appropriate....

  20. cAMP-dependent protein kinase and c-Jun N-terminal kinase mediate stathmin phosphorylation for the maintenance of interphase microtubules during osmotic stress. (United States)

    Yip, Yan Y; Yeap, Yvonne Y C; Bogoyevitch, Marie A; Ng, Dominic C H


    Dynamic microtubule changes after a cell stress challenge are required for cell survival and adaptation. Stathmin (STMN), a cytoplasmic microtubule-destabilizing phosphoprotein, regulates interphase microtubules during cell stress, but the signaling mechanisms involved are poorly defined. In this study ectopic expression of single alanine-substituted phospho-resistant mutants demonstrated that STMN Ser-38 and Ser-63 phosphorylation were specifically required to maintain interphase microtubules during hyperosmotic stress. STMN was phosphorylated on Ser-38 and Ser-63 in response to hyperosmolarity, heat shock, and arsenite treatment but rapidly dephosphorylated after oxidative stress treatment. Two-dimensional PAGE and Phos-tag gel analysis of stress-stimulated STMN phospho-isoforms revealed rapid STMN Ser-38 phosphorylation followed by subsequent Ser-25 and Ser-63 phosphorylation. Previously, we delineated stress-stimulated JNK targeting of STMN. Here, we identified cAMP-dependent protein kinase (PKA) signaling as responsible for stress-induced STMN Ser-63 phosphorylation. Increased cAMP levels induced by cholera toxin triggered potent STMN Ser-63 phosphorylation. Osmotic stress stimulated an increase in PKA activity and elevated STMN Ser-63 and CREB (cAMP-response element-binding protein) Ser-133 phosphorylation that was substantially attenuated by pretreatment with H-89, a PKA inhibitor. Interestingly, PKA activity and subsequent phosphorylation of STMN were augmented in the absence of JNK activation, indicating JNK and PKA pathway cross-talk during stress regulation of STMN. Taken together our study indicates that JNK- and PKA-mediated STMN Ser-38 and Ser-63 phosphorylation are required to preserve interphase microtubules in response to hyperosmotic stress.

  1. The central role of cAMP in regulating Plasmodium falciparum merozoite invasion of human erythrocytes.

    Directory of Open Access Journals (Sweden)

    Amrita Dawn


    Full Text Available All pathogenesis and death associated with Plasmodium falciparum malaria is due to parasite-infected erythrocytes. Invasion of erythrocytes by P. falciparum merozoites requires specific interactions between host receptors and parasite ligands that are localized in apical organelles called micronemes. Here, we identify cAMP as a key regulator that triggers the timely secretion of microneme proteins enabling receptor-engagement and invasion. We demonstrate that exposure of merozoites to a low K+ environment, typical of blood plasma, activates a bicarbonate-sensitive cytoplasmic adenylyl cyclase to raise cytosolic cAMP levels and activate protein kinase A, which regulates microneme secretion. We also show that cAMP regulates merozoite cytosolic Ca2+ levels via induction of an Epac pathway and demonstrate that increases in both cAMP and Ca2+ are essential to trigger microneme secretion. Our identification of the different elements in cAMP-dependent signaling pathways that regulate microneme secretion during invasion provides novel targets to inhibit blood stage parasite growth and prevent malaria.

  2. Dual role of cAMP and involvement of both G-proteins and ras in regulation of ERK2 in Dictyostelium discoideum. (United States)

    Knetsch, M L; Epskamp, S J; Schenk, P W; Wang, Y; Segall, J E; Snaar-Jagalska, B E


    Dictyostelium discoideum expresses two Extracellular signal Regulated Kinases, ERK1 and ERK2, which are involved in growth, multicellular development and regulation of adenylyl cyclase. Binding of extracellular cAMP to cAMP receptor 1, a G-protein coupled cell surface receptor, transiently stimulates phosphorylation, activation and nuclear translocation of ERK2. Activation of ERK2 by cAMP is dependent on heterotrimeric G-proteins, since activation of ERK2 is absent in cells lacking the Galpha4 subunit. The small G-protein rasD also activates ERK2. In cells overexpressing a mutated, constitutively active rasD, ERK2 activity is elevated prior to cAMP stimulation. Intracellular cAMP and cAMP-dependent protein kinase (PKA) are essential for adaptation of the ERK2 response. This report shows that multiple signalling pathways are involved in regulation of ERK2 activity in D.discoideum.

  3. Activation of extracellular signal-regulated kinases, NF-kappa B, and cyclic adenosine 5'-monophosphate response element-binding protein in lung neutrophils occurs by differing mechanisms after hemorrhage or endotoxemia. (United States)

    Abraham, E; Arcaroli, J; Shenkar, R


    Acute lung injury is frequently associated with sepsis or blood loss and is characterized by a proinflammatory response and infiltration of activated neutrophils into the lungs. Hemorrhage or endotoxemia result in activation of cAMP response element-binding protein (CREB) and NF-kappa B in lung neutrophils as well as increased expression of proinflammatory cytokines, such as TNF-alpha and macrophage-inflammatory peptide-2, by these cells. Activation of the extracellular regulated kinase (ERK) pathway occurs in stress responses and is involved in CREB activation. In the present experiments, hemorrhage or endotoxemia produced increased activation of mitogen-activated protein kinase kinase (MEK)1/2 and ERK2 (p42), but not of ERK1 (p44), in lung neutrophils. ERK1, ERK2, and MEK1/2 were not activated in peripheral blood neutrophils after hemorrhage or endotoxemia. Inhibition of xanthine oxidase led to further increase in the activation of MEK1/2 and ERK2 in lung neutrophils after hemorrhage, but not after endotoxemia. Alpha-adrenergic blockade before hemorrhage resulted in increased activation in lung neutrophils of MEK1/2, ERK1, ERK2, and CREB, but decreased activation of NF-kappa B. In contrast, alpha-adrenergic blockade before endotoxemia was associated with decreased activation of MEK1/2, ERK2, and CREB, but increased activation of NF-kappa B. Beta-adrenergic blockade before hemorrhage did not alter MEK1/2 or ERK1 activation in lung neutrophils, but decreased activation of ERK2 and CREB, while increasing activation of NF-kappa B. Beta-adrenergic inhibition before endotoxemia did not affect activation of MEK1/2, ERK1, ERK2, CREB, or NF-kappa B. These data indicate that the pathways leading to lung neutrophil activation after hemorrhage are different from those induced by endotoxemia.

  4. Modeling and assessment of the response of super-light elements to fire

    DEFF Research Database (Denmark)

    Hertz, Kristian Dahl; Campeanu, B.M.; Giraudo, M.


    in those cases, where a safe evacuation of the building is ensured, the high costs associated with the downtime and reparation of the building can be very high and not acceptable in the view of a safe and sustainable design of structures. In this respect, the newly patented building technology....... The implementation of a three-dimensional Finite Element Model (FEM) of the SL-deck is described and modelling aspects are discussed with particular reference to the sensitivity of the model to the thermal properties of the strong and light concrete. The results of the investigation are compared with the outcomes...... of the fire performances of complex concrete elements and in the focus on modelling issues related to the handling of the uncertainties. © 2013 Taylor & Francis Group....

  5. Modeling and assessment of the response of super-light elements to fire

    DEFF Research Database (Denmark)

    Hertz, Kristian Dahl; Campeanu, B.M.; Giraudo, M.

    in those cases, where a safe evacuation of the building is ensured, the high costs associated with the downtime and reparation of the building can be very high and not acceptable in the view of a safe and sustainable design of structures. In this respect, the newly patented building technology....... The implementation of a three-dimensional finite element model (FEM) of the SL-deck is described and modelling aspects are discussed with particular reference to the sensitivity of the model to the thermal properties of the strong and light concrete. The results of the investigation are compared with the outcomes...... of the fire performances of complex concrete elements and in the focus on modelling issues related to the handling of the uncertainties....

  6. A topology-motivated mixed finite element method for dynamic response of porous media

    CERN Document Server

    Lotfian, Zahrasadat


    In this paper, we propose a numerical method for computing solutions to Biot's fully dynamic model of incompressible saturated porous media [Biot;1956]. Our spatial discretization scheme is based on the three-field formulation (u-w-p) and the coupling of a lowest order Raviart-Thomas mixed element [Raviart,Thomas;1977] for fluid variable fields (w, p ) and a nodal Galerkin finite element for skeleton variable field (u). These mixed spaces are constructed based on the natural topology of the variables; hence, are physically compatible and able to exactly model the kind of continuity which is expected. The method automatically satisfies the well known LBB (inf-sup) stability condition and avoids locking that usually occurs in the numerical computations in the incompressible limit and very low hydraulic conductivity. In contrast to the majority of approaches, our three-field formulation can fully capture dynamic behavior of porous media even in high frequency loading phenomena with considerable fluid acceleratio...

  7. Study of the Internal Mechanical response of an asphalt mixture by 3-D Discrete Element Modeling

    DEFF Research Database (Denmark)

    Feng, Huan; Pettinari, Matteo; Hofko, Bernhard


    for all the distinct elements. The dynamic modulus and phase angle from uniaxial complex modulus tests of the asphalt mixtures in the laboratory have been collected. A macro-scale Burger’s model was first established and the input parameters of Burger’s contact model were calibrated by fitting......In this paper the viscoelastic behavior of asphalt mixture was investigated by employing a three-dimensional Discrete Element Method (DEM). The cylinder model was filled with cubic array of spheres with a specified radius, and was considered as a whole mixture with uniform contact properties...... with the lab test data of the complex modulus of the asphalt mixture. The Burger’s contact model parameters are usually calibrated for each frequency. While in this research a constant set of Burger’s parameters has been calibrated and used for all the test frequencies, the calibration procedure...

  8. Cyclic GMP-AMP displays mucosal adjuvant activity in mice.

    Directory of Open Access Journals (Sweden)

    Ivana Škrnjug

    Full Text Available The recently discovered mammalian enzyme cyclic GMP-AMP synthase produces cyclic GMP-AMP (cGAMP after being activated by pathogen-derived cytosolic double stranded DNA. The product can stimulate STING-dependent interferon type I signaling. Here, we explore the efficacy of cGAMP as a mucosal adjuvant in mice. We show that cGAMP can enhance the adaptive immune response to the model antigen ovalbumin. It promotes antigen specific IgG and a balanced Th1/Th2 lymphocyte response in immunized mice. A characteristic of the cGAMP-induced immune response is the slightly reduced induction of interleukin-17 as a hallmark of Th17 activity--a distinct feature that is not observed with other cyclic di-nucleotide adjuvants. We further characterize the innate immune stimulation activity in vitro on murine bone marrow-derived dendritic cells and human dendritic cells. The observed results suggest the consideration of cGAMP as a candidate mucosal adjuvant for human vaccines.

  9. Impact of Derepressed AmpC β-Lactamase ACT-9 on the Clinical Efficacy of Ertapenem▿


    Lee, Yi-Tzu; Chen, Te-Li; Siu, Leung-Kei; Chen, Chien-Pei; Fung, Chang-Phone


    An in vivo development of Pantoea agglomerans mutants (isolates PA2 to PA4) with reduced ertapenem susceptibility from that of isolate PA1 was associated with an inadequate clinical response to ertapenem therapy. All four isolates harbored the blaACT-9 AmpC β-lactamase gene. However, a loss-of-function mutation in the ampD gene in PA2 to PA4, but not PA1, led to derepressed ACT-9. The reduced ertapenem susceptibility caused by derepressed ACT-9 was confirmed with an ampD knockout mutant of PA1.

  10. Geological occurrence response to trace elemental migration in coal liquefaction based on SPSS: take no. 11 coalbed in Antaibao mine for example (United States)

    Xia, Xiaohong; Qin, Yong; Yang, Weifeng


    Coal liquefaction is an adoptable method to transfer the solid fossil energy into liquid oil in large scale, but the dirty material in which will migrate to different step of liquefaction. The migration rule of some trace elements is response to the react activity of macerals in coal and the geological occurrence of the element nature of itself. In this paper, from the SPSS data correlation analysis and hierarchical clustering dendrogram about the trace elements with macerals respond to coal liquefaction yield, it shows the trace elements in No.11 Antaibao coal seam originated from some of lithophile and sulphophle elements. Correlation coefficient between liquefaction yield of three organic macerals and migration of the elements in liquefaction residue indicated that the lithophile are easy to transfer to residue, while sulphophle are apt to in the liquid products. The activated macerals are response to sulphophle trace elements. The conclusion is useful to the coal blending and environmental effects on coal direct liquefaction.

  11. Escherichia coli exports cyclic AMP via TolC. (United States)

    Hantke, Klaus; Winkler, Karin; Schultz, Joachim E


    In Escherichia coli more than 180 genes are regulated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex. However, more than 90% of cAMP that is made by intracellular adenylyl cyclases is found in the culture medium. How is cAMP exported from E. coli? In a tolC mutant, 0.03 mM IPTG (isopropyl-β-d-thiogalactopyranoside) was sufficient to induce β-galactosidase compared to 0.1 mM IPTG in the parent strain. In a cya mutant unable to produce cAMP about 1 mM extracellular cAMP was required to induce β-galactosidase, whereas in a cya tolC mutant 0.1 mM cAMP was sufficient. When cAMP in E. coli cya was generated intracellularly by a recombinant, weakly active adenylyl cyclase from Corynebacterium glutamicum, the critical level of cAMP necessary for induction of maltose degradation was only achieved in a tolC mutant and not in the parent strain. Deletion of a putative cAMP phosphodiesterase of E. coli, CpdA, resulted in a slightly similar, yet more diffuse phenotype. The data demonstrate that export of cAMP via TolC is a most efficient way of E. coli to lower high concentrations of cAMP in the cell and maintain its sensitivity in changing metabolic environments.

  12. Low-Velocity Impact Response and Finite Element Analysis of Four-Step 3-D Braided Composites (United States)

    Sun, Baozhong; Zhang, Yan; Gu, Bohong


    The low-velocity impact characters of 3-D braided carbon/epoxy composites were investigated from experimental and finite element simulation approaches. The quasi-static tests were carried out at a constant velocity of 2 mm/min on MTS 810.23 material tester system to obtain the indentation load-displacement curves and indentation damages. The low-velocity tests were conducted at the velocities from 1 m/s to 6 m/s (corresponding to the impact energy from 3.22 J to 116 J) on Instron Dynatup 9250 impact tester. The peak force, energy for peak force, time to peak force, and total energy absorption were obtained to determine the impact responses of 3-D braided composites. A unit cell model was established according to the microstructure of 3-D braided composites to derive the constitutive equation. Based on the model, a user-defined material subroutine (VUMAT) has been compiled by FORTRAN and connected with commercial finite element code ABAQUS/Explicit to calculate the impact damage. The unit cell model successfully predicted the impact response of 3-D braided composites. Furthermore, the stress wave propagation and failure mechanisms have been revealed from the finite element simulation results and ultimate damage morphologies of specimens.

  13. MYC cis-Elements in PsMPT Promoter Is Involved in Chilling Response of Paeonia suffruticosa.

    Directory of Open Access Journals (Sweden)

    Yuxi Zhang

    Full Text Available The MPT transports Pi to synthesize ATP. PsMPT, a chilling-induced gene, was previously reported to promote energy metabolism during bud dormancy release in tree peony. In this study, the regulatory elements of PsMPT promoter involved in chilling response were further analyzed. The PsMPT transcript was detected in different tree peony tissues and was highly expressed in the flower organs, including petal, stigma and stamen. An 1174 bp of the PsMPT promoter was isolated by TAIL-PCR, and the PsMPT promoter::GUS transgenic Arabidopsis was generated and analyzed. GUS staining and qPCR showed that the promoter was active in mainly the flower stigma and stamen. Moreover, it was found that the promoter activity was enhanced by chilling, NaCl, GA, ACC and NAA, but inhibited by ABA, mannitol and PEG. In transgenic plants harboring 421 bp of the PsMPT promoter, the GUS gene expression and the activity were significantly increased by chilling treatment. When the fragment from -421 to -408 containing a MYC cis-element was deleted, the chilling response could not be observed. Further mutation analysis confirmed that the MYC element was one of the key motifs responding to chilling in the PsMPT promoter. The present study provides useful information for further investigation of the regulatory mechanism of PsMPT during the endo-dormancy release.

  14. Requirement of cAMP signaling for Schwann cell differentiation restricts the onset of myelination. (United States)

    Bacallao, Ketty; Monje, Paula V


    Isolated Schwann cells (SCs) respond to cAMP elevation by adopting a differentiated post-mitotic state that exhibits high levels of Krox-20, a transcriptional enhancer of myelination, and mature SC markers such as the myelin lipid galactocerebroside (O1). To address how cAMP controls myelination, we performed a series of cell culture experiments which compared the differentiating responses of isolated and axon-related SCs to cAMP analogs and ascorbate, a known inducer of axon ensheathment, basal lamina formation and myelination. In axon-related SCs, cAMP induced the expression of Krox-20 and O1 without a concomitant increase in the expression of myelin basic protein (MBP) and without promoting axon ensheathment, collagen synthesis or basal lamina assembly. When cAMP was provided together with ascorbate, a dramatic enhancement of MBP expression occurred, indicating that cAMP primes SCs to form myelin only under conditions supportive of basal lamina formation. Experiments using a combination of cell permeable cAMP analogs and type-selective adenylyl cyclase (AC) agonists and antagonists revealed that selective transmembrane AC (tmAC) activation with forskolin was not sufficient for full SC differentiation and that the attainment of an O1 positive state also relied on the activity of the soluble AC (sAC), a bicarbonate sensor that is insensitive to forskolin and GPCR activation. Pharmacological and immunological evidence indicated that SCs expressed sAC and that sAC activity was required for morphological differentiation and the expression of myelin markers such as O1 and protein zero. To conclude, our data indicates that cAMP did not directly drive myelination but rather the transition into an O1 positive state, which is perhaps the most critical cAMP-dependent rate limiting step for the onset of myelination. The temporally restricted role of cAMP in inducing differentiation independently of basal lamina formation provides a clear example of the uncoupling of signals

  15. Requirement of cAMP signaling for Schwann cell differentiation restricts the onset of myelination.

    Directory of Open Access Journals (Sweden)

    Ketty Bacallao

    Full Text Available Isolated Schwann cells (SCs respond to cAMP elevation by adopting a differentiated post-mitotic state that exhibits high levels of Krox-20, a transcriptional enhancer of myelination, and mature SC markers such as the myelin lipid galactocerebroside (O1. To address how cAMP controls myelination, we performed a series of cell culture experiments which compared the differentiating responses of isolated and axon-related SCs to cAMP analogs and ascorbate, a known inducer of axon ensheathment, basal lamina formation and myelination. In axon-related SCs, cAMP induced the expression of Krox-20 and O1 without a concomitant increase in the expression of myelin basic protein (MBP and without promoting axon ensheathment, collagen synthesis or basal lamina assembly. When cAMP was provided together with ascorbate, a dramatic enhancement of MBP expression occurred, indicating that cAMP primes SCs to form myelin only under conditions supportive of basal lamina formation. Experiments using a combination of cell permeable cAMP analogs and type-selective adenylyl cyclase (AC agonists and antagonists revealed that selective transmembrane AC (tmAC activation with forskolin was not sufficient for full SC differentiation and that the attainment of an O1 positive state also relied on the activity of the soluble AC (sAC, a bicarbonate sensor that is insensitive to forskolin and GPCR activation. Pharmacological and immunological evidence indicated that SCs expressed sAC and that sAC activity was required for morphological differentiation and the expression of myelin markers such as O1 and protein zero. To conclude, our data indicates that cAMP did not directly drive myelination but rather the transition into an O1 positive state, which is perhaps the most critical cAMP-dependent rate limiting step for the onset of myelination. The temporally restricted role of cAMP in inducing differentiation independently of basal lamina formation provides a clear example of the

  16. GmDREB2A;2, a canonical DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN2-type transcription factor in soybean, is posttranslationally regulated and mediates dehydration-responsive element-dependent gene expression. (United States)

    Mizoi, Junya; Ohori, Teppei; Moriwaki, Takashi; Kidokoro, Satoshi; Todaka, Daisuke; Maruyama, Kyonoshin; Kusakabe, Kazuya; Osakabe, Yuriko; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko


    Soybean (Glycine max) is an important crop around the world. Abiotic stress conditions, such as drought and heat, adversely affect its survival, growth, and production. The DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN2 (DREB2) group includes transcription factors that contribute to drought and heat stress tolerance by activating transcription through the cis-element dehydration-responsive element (DRE) in response to these stress stimuli. Two modes of regulation, transcriptional and posttranslational, are important for the activation of gene expression by DREB2A in Arabidopsis (Arabidopsis thaliana). However, the regulatory system of DREB2 in soybean is not clear. We identified a new soybean DREB2 gene, GmDREB2A;2, that was highly induced not only by dehydration and heat but also by low temperature. GmDREB2A;2 exhibited a high transactivation activity via DRE and has a serine/threonine-rich region, which corresponds to a negative regulatory domain of DREB2A that is involved in its posttranslational regulation, including destabilization. Despite the partial similarity between these sequences, the activity and stability of the GmDREB2A;2 protein were enhanced by removal of the serine/threonine-rich region in both Arabidopsis and soybean protoplasts, suggestive of a conserved regulatory mechanism that involves the recognition of serine/threonine-rich sequences with a specific pattern. The heterologous expression of GmDREB2A;2 in Arabidopsis induced DRE-regulated stress-inducible genes and improved stress tolerance. However, there were variations in the growth phenotypes of the transgenic Arabidopsis, the induced genes, and their induction ratios between GmDREB2A;2 and DREB2A. Therefore, the basic function and regulatory machinery of DREB2 have been maintained between Arabidopsis and soybean, although differentiation has also occurred.

  17. Melanocortin 1 receptor mutations impact differentially on signalling to the cAMP and the ERK mitogen-activated protein kinase pathways. (United States)

    Herraiz, Cecilia; Jiménez-Cervantes, Celia; Zanna, Paola; García-Borrón, José C


    Melanocortin 1 receptor (MC1R), a Gs protein-coupled receptor expressed in melanocytes, is a major determinant of skin pigmentation, phototype and cancer risk. MC1R activates cAMP and mitogen-activated protein kinase ERK1/ERK2 signalling. When expressed in rat pheochromocytoma cell line cells, the R151C, R160W and D294H MC1R variants associated with melanoma and impaired cAMP signalling mediated ERK activation and ERK-dependent, agonist-induced neurite outgrowth comparable with wild-type. Dose-response curves for ERK activation and cAMP production indicated higher sensitivity of the ERK response. Thus, the melanoma-associated MC1R mutations impact differently on cAMP and ERK signalling, suggesting that cAMP is not responsible for functional coupling of MC1R to the ERK cascade.

  18. Thyroid hormone-regulated gene expression in juvenile mouse liver: identification of thyroid response elements using microarray profiling and in silico analyses

    Directory of Open Access Journals (Sweden)

    Paquette Martin A


    Full Text Available Abstract Background Disruption of thyroid hormone signalling can alter growth, development and energy metabolism. Thyroid hormones exert their effects through interactions with thyroid receptors that directly bind thyroid response elements and can alter transcriptional activity of target genes. The effects of short-term thyroid hormone perturbation on hepatic mRNA transcription in juvenile mice were evaluated, with the goal of identifying genes containing active thyroid response elements. Thyroid hormone disruption was induced from postnatal day 12 to 15 by adding goitrogens to dams' drinking water (hypothyroid. A subgroup of thyroid hormone-disrupted pups received intraperitoneal injections of replacement thyroid hormones four hours prior to sacrifice (replacement. An additional group received only thyroid hormones four hours prior to sacrifice (hyperthyroid. Hepatic mRNA was extracted and hybridized to Agilent mouse microarrays. Results Transcriptional profiling enabled the identification of 28 genes that appeared to be under direct thyroid hormone-regulation. The regulatory regions of the genome adjacent to these genes were examined for half-site sequences that resemble known thyroid response elements. A bioinformatics search identified 33 thyroid response elements in the promoter regions of 13 different genes thought to be directly regulated by thyroid hormones. Thyroid response elements found in the promoter regions of Tor1a, 2310003H01Rik, Hect3d and Slc25a45 were further validated by confirming that the thyroid receptor is associated with these sequences in vivo and that it can bind directly to these sequences in vitro. Three different arrangements of thyroid response elements were identified. Some of these thyroid response elements were located far up-stream (> 7 kb of the transcription start site of the regulated gene. Conclusions Transcriptional profiling of thyroid hormone disrupted animals coupled with a novel bioinformatics search

  19. Differential selection of single-step AmpC or efflux mutants of Pseudomonas aeruginosa by using cefepime, ceftazidime, or ceftobiprole. (United States)

    Queenan, Anne Marie; Shang, Wenchi; Bush, Karen; Flamm, Robert K


    Single-step Pseudomonas aeruginosa mutants, selected with ceftobiprole, ceftazidime, or cefepime, were generated at frequencies of 10(-6) to ceftobiprole did not overexpress AmpC; 90% of these produced elevated levels of mexXY RNA, indicating that increased efflux, not AmpC derepression, is the predominant response to ceftobiprole during first-step mutations in P. aeruginosa.

  20. Signal Transduction in Dictyostelium fgd A Mutants with a Defective Interaction between Surface cAMP Receptors and a GTP-binding Regulatory Protein

    NARCIS (Netherlands)

    Kesbeke, Fanja; Snaar-Jagalska, B. Ewa; Haastert, Peter J.M. van


    Transmembrane signal transduction was investigated in four Dictyostelium discoideum mutants that belong to the fgd A complementation group. The results show the following. (a) Cell surface cAMP receptors are present in fgd A mutants, but cAMP does not induce any of the intracellular responses, inclu

  1. Sequence elements in the human osteocalcin gene confer basal activation and inducible response to hormonal vitamin D sub 3

    Energy Technology Data Exchange (ETDEWEB)

    Kerner, S.A.; Scott, R.A.; Pike, J.W. (Baylor College of Medicine, Houston, TX (USA))


    Osteoblast-specific expression of the bone protein osteocalcin is controlled at the transcriptional level by the steroid hormone 1{alpha},25-dihydroxyvitamin D{sub 3}. As this protein may represent a marker for bone activity in human disease, the authors examined the regulation of its expression at the molecular level by evaluating human osteocalcin gene promoter function. They describe regions within the promoter that contribute to basal expression of the gene in osteoblast-like cells in culture. Further, they define a 21-base-pair DNA element with the sequence 5{prime}-GTGACTCACCGGGTGAACGGG-3{prime}, which acts in cis to mediate 1{alpha},25-dihydroxyvitamin D{sub 3} inducibility of the osteocalcin gene. This response element bears sequence similarity with other short DNA segments, particularly those for estrogen and thyroid hormone, which act together with their respective trans-acting receptors to modulate gene transcription.

  2. A highly specific pathogen-responsive promoter element from the immediate-early activated CMPG1 gene in Petroselinum crispum. (United States)

    Kirsch, C; Logemann, E; Lippok, B; Schmelzer, E; Hahlbrock, K


    Within the complex signalling network from pathogen-derived elicitor perception to defense-related gene activation, some immediate-early responding genes may have pivotal roles in downstream transcriptional regulation. We have identified the parsley (Petroselinum crispum) ELI17 gene as a particularly fast-responding gene possessing a new type of W box-containing, elicitor-responsive promoter element, E17. Highly selective E17-mediated reporter gene expression at pathogen infection sites in transgenic Arabidopsis thaliana plants demonstrated the potential of this promoter element for designing new strategies in resistance breeding as well as for further analysis of the early components of defense-related gene activation mechanisms. The protein encoded by the ELI17 gene exhibits various structural characteristics of established transcription factors and is designated as a CMPG protein according to the first four strictly conserved amino acids defining a newly emerging class of plant-specific proteins.

  3. Enrichment of Conserved Synaptic Activity-Responsive Element in Neuronal Genes Predicts a Coordinated Response of MEF2, CREB and SRF (United States)

    Rodríguez-Tornos, Fernanda M.; San Aniceto, Iñigo; Cubelos, Beatriz; Nieto, Marta


    A unique synaptic activity-responsive element (SARE) sequence, composed of the consensus binding sites for SRF, MEF2 and CREB, is necessary for control of transcriptional upregulation of the Arc gene in response to synaptic activity. We hypothesize that this sequence is a broad mechanism that regulates gene expression in response to synaptic activation and during plasticity; and that analysis of SARE-containing genes could identify molecular mechanisms involved in brain disorders. To search for conserved SARE sequences in the mammalian genome, we used the SynoR in silico tool, and found the SARE cluster predominantly in the regulatory regions of genes expressed specifically in the nervous system; most were related to neural development and homeostatic maintenance. Two of these SARE sequences were tested in luciferase assays and proved to promote transcription in response to neuronal activation. Supporting the predictive capacity of our candidate list, up-regulation of several SARE containing genes in response to neuronal activity was validated using external data and also experimentally using primary cortical neurons and quantitative real time RT-PCR. The list of SARE-containing genes includes several linked to mental retardation and cognitive disorders, and is significantly enriched in genes that encode mRNA targeted by FMRP (fragile X mental retardation protein). Our study thus supports the idea that SARE sequences are relevant transcriptional regulatory elements that participate in plasticity. In addition, it offers a comprehensive view of how activity-responsive transcription factors coordinate their actions and increase the selectivity of their targets. Our data suggest that analysis of SARE-containing genes will reveal yet-undescribed pathways of synaptic plasticity and additional candidate genes disrupted in mental disease. PMID:23382855

  4. Enrichment of conserved synaptic activity-responsive element in neuronal genes predicts a coordinated response of MEF2, CREB and SRF.

    Directory of Open Access Journals (Sweden)

    Fernanda M Rodríguez-Tornos

    Full Text Available A unique synaptic activity-responsive element (SARE sequence, composed of the consensus binding sites for SRF, MEF2 and CREB, is necessary for control of transcriptional upregulation of the Arc gene in response to synaptic activity. We hypothesize that this sequence is a broad mechanism that regulates gene expression in response to synaptic activation and during plasticity; and that analysis of SARE-containing genes could identify molecular mechanisms involved in brain disorders. To search for conserved SARE sequences in the mammalian genome, we used the SynoR in silico tool, and found the SARE cluster predominantly in the regulatory regions of genes expressed specifically in the nervous system; most were related to neural development and homeostatic maintenance. Two of these SARE sequences were tested in luciferase assays and proved to promote transcription in response to neuronal activation. Supporting the predictive capacity of our candidate list, up-regulation of several SARE containing genes in response to neuronal activity was validated using external data and also experimentally using primary cortical neurons and quantitative real time RT-PCR. The list of SARE-containing genes includes several linked to mental retardation and cognitive disorders, and is significantly enriched in genes that encode mRNA targeted by FMRP (fragile X mental retardation protein. Our study thus supports the idea that SARE sequences are relevant transcriptional regulatory elements that participate in plasticity. In addition, it offers a comprehensive view of how activity-responsive transcription factors coordinate their actions and increase the selectivity of their targets. Our data suggest that analysis of SARE-containing genes will reveal yet-undescribed pathways of synaptic plasticity and additional candidate genes disrupted in mental disease.

  5. Experimental Validation of Two-dimensional Finite Element Method for Simulating Constitutive Response of Polycrystals During High Temperature Plastic Deformation (United States)

    Agarwal, Sumit; Briant, Clyde L.; Krajewski, Paul E.; Bower, Allan F.; Taleff, Eric M.


    A finite element method was recently designed to model the mechanisms that cause superplastic deformation (A.F. Bower and E. Wininger, A Two-Dimensional Finite Element Method for Simulating the Constitutive Response and Microstructure of Polycrystals during High-Temperature Plastic Deformation, J. Mech. Phys. Solids, 2004, 52, p 1289-1317). The computations idealize the solid as a collection of two-dimensional grains, separated by sharp grain boundaries. The grains may deform plastically by thermally activated dislocation motion, which is modeled using a conventional crystal plasticity law. The solid may also deform by sliding on the grain boundaries, or by stress-driven diffusion of atoms along grain boundaries. The governing equations are solved using a finite element method, which includes a front-tracking procedure to monitor the evolution of the grain boundaries and surfaces in the solid. The goal of this article is to validate these computations by systematically comparing numerical predictions to experimental measurements of the elevated-temperature response of aluminum alloy AA5083 (M.-A. Kulas, W.P. Green, E.M. Taleff, P.E. Krajewski, and T.R. McNelley, Deformation Mechanisms in Superplastic AA5083 materials. Metall. Mater. Trans. A, 2005, 36(5), p 1249-1261). The experimental work revealed that a transition occurs from grain-boundary sliding to dislocation (solute-drag) creep at approximately 0.001/s for temperatures between 425 and 500 °C. In addition, increasing the grain size from 7 to 10 μm decreased the transition to significantly lower strain rates. Predictions from the finite element method accurately predict the effect of grain size on the transition in deformation mechanisms.

  6. Quantitative analysis of polycomb response elements (PREs at identical genomic locations distinguishes contributions of PRE sequence and genomic environment

    Directory of Open Access Journals (Sweden)

    Okulski Helena


    Full Text Available Abstract Background Polycomb/Trithorax response elements (PREs are cis-regulatory elements essential for the regulation of several hundred developmentally important genes. However, the precise sequence requirements for PRE function are not fully understood, and it is also unclear whether these elements all function in a similar manner. Drosophila PRE reporter assays typically rely on random integration by P-element insertion, but PREs are extremely sensitive to genomic position. Results We adapted the ΦC31 site-specific integration tool to enable systematic quantitative comparison of PREs and sequence variants at identical genomic locations. In this adaptation, a miniwhite (mw reporter in combination with eye-pigment analysis gives a quantitative readout of PRE function. We compared the Hox PRE Frontabdominal-7 (Fab-7 with a PRE from the vestigial (vg gene at four landing sites. The analysis revealed that the Fab-7 and vg PREs have fundamentally different properties, both in terms of their interaction with the genomic environment at each site and their inherent silencing abilities. Furthermore, we used the ΦC31 tool to examine the effect of deletions and mutations in the vg PRE, identifying a 106 bp region containing a previously predicted motif (GTGT that is essential for silencing. Conclusions This analysis showed that different PREs have quantifiably different properties, and that changes in as few as four base pairs have profound effects on PRE function, thus illustrating the power and sensitivity of ΦC31 site-specific integration as a tool for the rapid and quantitative dissection of elements of PRE design.

  7. Expression and purification of full length mouse metal response element binding transcription factor-1 using Pichia pastoris. (United States)

    Huyck, Ryan W; Keightley, Andrew; Laity, John H


    The metal response element binding transcription factor-1 (MTF-1) is an important stress response, heavy metal detoxification, and zinc homeostasis factor in eukaryotic organisms from Drosophila to humans. MTF-1 transcriptional regulation is primarily mediated by elevated levels of labile zinc, which direct MTF-1 to bind the metal response element (MRE). This process involves direct zinc binding to the MTF-1 zinc fingers, and zinc dependent interaction of the MTF-1 acidic region with the p300 coactivator protein. Here, the first recombinant expression system for mutant and wild type (WT) mouse MTF-1 (mMTF-1) suitable for biochemical and biophysical studies in vitro is reported. Using the methyltropic yeast Pichia pastoris, nearly half-milligram recombinant WT and mutant mMTF-1 were produced per liter of P. pastoris cell culture, and purified by a FLAG-tag epitope. Using a first pass ammonium sulfate purification, followed by anti-FLAG affinity resin, mMTF-1 was purified to >95% purity. This recombinant mMTF-1 was then assayed for direct protein-protein interactions with p300 by co-immunoprecipitation. Surface plasmon resonance studies on mMTF-1 provided the first quantitative DNA binding affinity measurements to the MRE promotor element (K(d)=5±3 nM). Both assays demonstrated the functional activity of the recombinant mMTF-1, while elucidating the molecular basis for mMTF-1-p300 functional synergy, and provided new insights into the mMTF-1 domain specific roles in DNA binding. Overall, this production system provides accessibility for the first time to a multitude of in vitro studies using recombinant mutant and WT mMTF-1, which greatly facilitates new approaches to understanding the complex and varied functions of this protein.

  8. Regulation of CYP3A4 by pregnane X receptor: The role of nuclear receptors competing for response element binding

    Energy Technology Data Exchange (ETDEWEB)

    Istrate, Monica A., E-mail: [Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany, and University of Tuebingen, Auerbachstr. 112, D-70376 Stuttgart (Germany); Nussler, Andreas K., E-mail: [Department of Traumatology, Technical University Munich, Ismaningerstr. 22, 81675 Munich (Germany); Eichelbaum, Michel, E-mail: [Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany, and University of Tuebingen, Auerbachstr. 112, D-70376 Stuttgart (Germany); Burk, Oliver, E-mail: [Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany, and University of Tuebingen, Auerbachstr. 112, D-70376 Stuttgart (Germany)


    Induction of the major drug metabolizing enzyme CYP3A4 by xenobiotics contributes to the pronounced interindividual variability of its expression and often results in clinically relevant drug-drug interactions. It is mainly mediated by PXR, which regulates CYP3A4 expression by binding to several specific elements in the 5' upstream regulatory region of the gene. Induction itself shows a marked interindividual variability, whose underlying determinants are only partly understood. In this study, we investigated the role of nuclear receptor binding to PXR response elements in CYP3A4, as a potential non-genetic mechanism contributing to interindividual variability of induction. By in vitro DNA binding experiments, we showed that several nuclear receptors bind efficiently to the proximal promoter ER6 and distal xenobiotic-responsive enhancer module DR3 motifs. TR{alpha}1, TR{beta}1, COUP-TFI, and COUP-TFII further demonstrated dose-dependent repression of PXR-mediated CYP3A4 enhancer/promoter reporter activity in transient transfection in the presence and absence of the PXR inducer rifampin, while VDR showed this effect only in the absence of treatment. By combining functional in vitro characterization with hepatic expression analysis, we predict that TR{alpha}1, TR{beta}1, COUP-TFI, and COUP-TFII show a strong potential for the repression of PXR-mediated activation of CYP3A4 in vivo. In summary, our results demonstrate that nuclear receptor binding to PXR response elements interferes with PXR-mediated expression and induction of CYP3A4 and thereby contributes to the interindividual variability of induction.

  9. Non-linear finite element analysis for prediction of seismic response of buildings considering soil-structure interaction

    Directory of Open Access Journals (Sweden)

    E. Çelebi


    Full Text Available The objective of this paper focuses primarily on the numerical approach based on two-dimensional (2-D finite element method for analysis of the seismic response of infinite soil-structure interaction (SSI system. This study is performed by a series of different scenarios that involved comprehensive parametric analyses including the effects of realistic material properties of the underlying soil on the structural response quantities. Viscous artificial boundaries, simulating the process of wave transmission along the truncated interface of the semi-infinite space, are adopted in the non-linear finite element formulation in the time domain along with Newmark's integration. The slenderness ratio of the superstructure and the local soil conditions as well as the characteristics of input excitations are important parameters for the numerical simulation in this research. The mechanical behavior of the underlying soil medium considered in this prediction model is simulated by an undrained elasto-plastic Mohr-Coulomb model under plane-strain conditions. To emphasize the important findings of this type of problems to civil engineers, systematic calculations with different controlling parameters are accomplished to evaluate directly the structural response of the vibrating soil-structure system. When the underlying soil becomes stiffer, the frequency content of the seismic motion has a major role in altering the seismic response. The sudden increase of the dynamic response is more pronounced for resonance case, when the frequency content of the seismic ground motion is close to that of the SSI system. The SSI effects under different seismic inputs are different for all considered soil conditions and structural types.

  10. A Bioinformatics Method for the Design of Live Attenuated Virus Vaccine Utilizing Host MicroRNA Response Elements. (United States)

    Wichadakul, Duangdao


    The host microRNA machinery has been employed to control viral replication. To improve safety for live attenuated virus vaccines, the binding sites of the host microRNAs, so-called microRNA response elements (MREs), were incorporated into the virus sequences. These MREs were typically designed for a specific host microRNA and virus sequence with the effectiveness evaluated by experimental trials. Here, we describe a computational flow that can be used to simultaneously design and prioritize the effective MREs in large-scale.

  11. 7 CFR 772.14 - Reamortization of AMP loans. (United States)


    ... 7 Agriculture 7 2010-01-01 2010-01-01 false Reamortization of AMP loans. 772.14 Section 772.14... AGRICULTURE SPECIAL PROGRAMS SERVICING MINOR PROGRAM LOANS § 772.14 Reamortization of AMP loans. The Agency may approve reamortization of AMP loans provided: (a) There is no extension of the final maturity...

  12. S-AMP: Approximate Message Passing for General Matrix Ensembles

    DEFF Research Database (Denmark)

    Cakmak, Burak; Winther, Ole; Fleury, Bernard H.


    We propose a novel iterative estimation algorithm for linear observation models called S-AMP. The fixed points of S-AMP are the stationary points of the exact Gibbs free energy under a set of (first- and second-) moment consistency constraints in the large system limit. S-AMP extends...

  13. Multiple facets of cAMP signalling and physiological impact : cAMP compartmentalization in the lung

    NARCIS (Netherlands)

    Oldenburger, Anouk; Maarsingh, Harm; Schmidt, Martina


    Therapies involving elevation of the endogenous suppressor cyclic AMP (cAMP) are currently used in the treatment of several chronic inflammatory disorders, including chronic obstructive pulmonary disease (COPD). Characteristics of COPD are airway obstruction, airway inflammation and airway remodelli

  14. Detection of cyclic di-AMP using a competitive ELISA with a unique pneumococcal cyclic di-AMP binding protein (United States)

    Underwood, Adam J.; Zhang, Yang; Metzger, Dennis W.; Bai, Guangchun


    Cyclic di-AMP (c-di-AMP) is a signaling molecule that has been shown to play important roles in bacterial physiology and infections. Currently, c-di-AMP detection and quantification relies mostly on the use of high-performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS). In this study, a competitive enzyme-linked immunosorbent assay (ELISA) for the quantification of c-di-AMP was developed, which utilizes a novel pneumococcal c-di-AMP binding protein (CabP) and a newly commercialized c-di-AMP derivative. With this new method, c-di-AMP concentrations in biological samples can be quickly and accurately quantified. Furthermore, this assay is much more efficient than current methods as it requires less overall cost and training while processing many samples at once. Therefore, this assay can be extensively used in research into c-di-AMP signaling. PMID:25239824

  15. cAMP dependent and independent regulation of thyroglobulin synthesis by two clones of the OVNIS 6H thyroid cell line. (United States)

    Aouani, A; Hovsépian, S; Fayet, G


    The hormonal regulation of thyroglobulin synthesis has been studied using two independent clones of the OVNIS 6H cell line. Insulin, hydrocortisone and TSH were able to stimulate thyroglobulin synthesis, whereas transferrin, somatostatin and glycyl-histidyl-lysine were without effect. Insulin stimulated thyroglobulin synthesis without affecting cAMP production. Hydrocortisone, when combined with insulin was a stimulator too; this stimulation was not accompanied by an increase in cAMP. TSH alone was unable to stimulate either cAMP or thyroglobulin synthesis. The stimulatory effect of TSH on thyroglobulin synthesis took place only when combined with insulin or insulin plus hydrocortisone, and was mediated by cAMP. Consequently, insulin and hydrocortisone stimulated thyroglobulin synthesis by cAMP-independent mechanisms, whereas TSH acted via the cAMP system. Forskolin mimicked TSH effects on cAMP and thyroglobulin synthesis. Calf serum inhibited cAMP and thyroglobulin production. Optimal cAMP and thyroglobulin synthesis as well as TSH responsiveness were obtained in serum-free medium supplemented with 5 micrograms/ml insulin, 100 nM hydrocortisone and 1 mU/ml TSH.

  16. Microbial Response to the Application of Amendments in a Contaminated Soil with Trace Elements

    Directory of Open Access Journals (Sweden)

    A. Branzini


    Full Text Available Problem Statement: The anthropogenic activities can cause adverse effects in soils, increasing in some situations trace elements contents, impacting negatively both the microbial biomass and activity. Among the practices used for the recovery of soil quality we can find the application of organic amendments or the product of their composting. These can adsorb trace elements decreasing their availability and increasing the soil microbial biomass. The microorganisms of the soil use to be considered as sensitive biological indicators of the changes produced in the soil quality. Approach: One processes to quantify soil biological activity is the respiration. The aim was to evaluate the effects of two organic amendments application on soil microbial activity, in a soil contaminated with copper (Cu, zinc (Zn and chromium (Cr. To prove the raised aim we quantified CO2-C release. Results: The results showed that at the end of the incubation period, as much in contaminated soils as in soils without contamination, the total activity of microorganisms was significantly increased by the application of organic amendments (p = 0.0062 and p = 0.0005, respectively. The application of both composts to slightly acid soils increased the initial and final values of pH. There was no evidence of modification in Electrical Conductivity (EC because of compost application. At the end of the incubation period a negative relationship was observed between EC and CO2-C (R2 = 0.74, p = 0.0028. Conclusions: The obtained results in this study suggested that it was possible to increase the total activity of soil microorganisms and to reduce the bioavailability of Cu, Cr and Zn in a contaminated soil. As a result, CO2-C release is a sensitive index of the soil quality, at least in the experimental conditions of this essay.

  17. Characterization of a proximal Sp1 response element in the mouse Dlk2 gene promoter

    Directory of Open Access Journals (Sweden)

    Rivero Samuel


    Full Text Available Abstract Background DLK2 is an EGF-like membrane protein, closely related to DLK1, which is involved in adipogenesis. Both proteins interact with the NOTCH1 receptor and are able to modulate its activation. The expression of the gene Dlk2 is coordinated with that of Dlk1 in several tissues and cell lines. Unlike Dlk1, the mouse Dlk2 gene and its locus at chromosome 17 are not fully characterized. Results The goal of this work was the characterization of Dlk2 mRNA, as well as the analysis of the mechanisms that control its basal transcription. First, we analyzed the Dlk2 transcripts expressed by several mouse cells lines and tissues, and mapped the transcription start site by 5' Rapid Amplification of cDNA Ends. In silico analysis revealed that Dlk2 possesses a TATA-less promoter containing minimal promoter elements associated with a CpG island, and sequences for Inr and DPE elements. Besides, it possesses six GC-boxes, considered as consensus sites for the transcription factor Sp1. Indeed, we report that Sp1 directly binds to the Dlk2 promoter, activates its transcription, and regulates its level of expression. Conclusions Our results provide the first characterization of Dlk2 transcripts, map the location of the Dlk2 core promoter, and show the role of Sp1 as a key regulator of Dlk2 transcription, providing new insights into the molecular mechanisms that contribute to the expression of the Dlk2 gene.

  18. Opposing actions of dibutyryl cyclic AMP and GMP on temperature in conscious guinea-pigs (United States)

    Kandasamy, S. B.; Williaes, B. A.


    It is shown that the intracerebroventricular administration of dibutyryl cyclic AMP (Db-cAMP) induced hyperthermia in guinea pigs which was not mediated through prostaglandins or norepinephrine since a prostaglandin synthesis inhibitor and an alpha-adrenergic receptor blocking agent did not antagonize the hyperthermia. However, the hyperthermic response to Db-cAMP was attenuated by the central administration of a beta-adrenergic receptor antagonist, which indicates that cAMP may be involved, through beta-adrenergic receptors, in the central regulation of heat production and conservation. The central administration of Db-cGMP produced hypothermia which was not mediated via histamine H1 or H2 receptors and serotonin. The antagonism of hypothermia induced by Db-cGMP and acetylcholine + physostigmine by central administration of a cholinergic muscarine receptor antagonist and not by a cholinergic nicotinic receptor antagonist suggests that cholinoceptive neurons and endogenous cGMP may regulate heat loss through cholinergic muscarine receptors. It is concluded that these results indicate a regulatory role in thermoregulation provided by a balance between opposing actions of cAMP and cGMP in guinea pigs.

  19. Effects of diazepam on orthodontic tooth movement and alveolar bone cAMP levels in cats. (United States)

    Burrow, S J; Sammon, P J; Tuncay, O C


    Cyclic AMP has been suggested as a possible intracellular mediator in bone remodeling during tooth movement. Accordingly, an increase in the level of this nucleotide should result in faster tooth movement. Breakdown of cAMP was inhibited by administration of diazepam in eight cats undergoing orthodontic tooth movement; another matched group of eight animals served as controls. Orthodontic appliances consisted of coil springs stretching between the right side maxillary and mandibular canines and third premolars. The data for tooth movement and cAMP concentrations were analyzed by repeated measures factorial analyses of variance. The results indicated that administration of diazepam increased the rate of tooth movement at P less than 0.0005 and, interestingly, although diazepam had no effect on undisturbed tissues, it lowered the cAMP levels in the periodontal tissues of orthodontically moved teeth at P less than 0.01. On the basis of these results, it was concluded that the concentration of cAMP did not correlate with bone remodeling in this model and perhaps should not be used as an index of periodontal-tissue response during orthodontic tooth movement.

  20. cAMP/PKA Signaling Inhibits Osteogenic Differentiation and Bone Formation in Rodent Models

    NARCIS (Netherlands)

    Siddappa, Ramakrishnaiah; Mulder, Winfried; Steeghs, Ilse; Klundert, van de Christine; Fernandes, Hugo; Liu, Jun; Arends, Roel; Blitterswijk, van Clemens; Boer, de Jan


    We previously demonstrated that cAMP-mediated protein kinase A (PKA) activation induces in vitro osteogenesis and in vivo bone formation by human mesenchymal stem cells (hMSCs). To analyze the species-specific response of this phenomenon and to translate our findings into a clinical trial, suitable

  1. Investigation of mechanical responses to the tactile perception of surfaces with different textures using the finite element method

    Directory of Open Access Journals (Sweden)

    Wei Tang


    Full Text Available Tactile perception is essential for humans to perceive the world, and it usually results in mechanical responses from the finger. In this study, a nonlinear, viscoelastic, and multilayered finite element model of the finger was developed. The relationship between the mechanical responses within the finger and tactile perception while the finger scanned different surface textures was studied. The results showed that the sensitivity of tactile perception is affected by the peak value of von Mises stress, which is itself determined by the shape and density of a given texture. The von Mises stress varies periodically with time, and this variation depends on the periodicity of the texture. Displacement signals around Pacinian corpuscles have periodic variation. The period of displacement decreases as the density of the texture increases. The spectral centroid increases as the spacing of the texture decreases. The related mechanisms are discussed in this article.

  2. A Method for Studying cAMP-relay in Dictyostelium discoideum : the Effect of Temperature on cAMP-relay

    NARCIS (Netherlands)

    Haastert, Peter J.M. van


    A simple assay has been developed to quantify the cAMP-relay in Dictyostelium discoideum. The assay is based on the stimulation of cells, in the presence of a phosphodiesterase inhibitor, with 2'-deoxyadenosine 3',5'-monophosphate (dcAMP) at a concentration which saturates cell surface cAMP receptor

  3. Elements That Regulate the DNA Damage Response of Proteins Defective in Cockayne Syndrome. (United States)

    Iyama, Teruaki; Wilson, David M


    Cockayne syndrome (CS) is a premature aging disorder characterized by developmental defects, multisystem progressive degeneration and sensitivity to ultraviolet light. CS is divided into two primary complementation groups, A and B, with the CSA and CSB proteins presumably functioning in DNA repair and transcription. Using laser microirradiation and confocal microscopy, we characterized the nature and regulation of the CS protein response to oxidative DNA damage, double-strand breaks (DSBs), angelicin monoadducts and trioxsalen interstrand crosslinks (ICLs). Our data indicate that CSB recruitment is influenced by the type of DNA damage and is most rapid and robust as follows: ICLs>DSBs>monoadducts>oxidative lesions. Transcription inhibition reduced accumulation of CSB at sites of monoadducts and ICLs, but it did not affect recruitment to (although slightly affected retention at) oxidative damage. Inhibition of histone deacetylation altered the dynamics of CSB assembly, suggesting a role for chromatin status in the response to DNA damage, whereas the proteasome inhibitor MG132 had no effect. The C-terminus of CSB and, in particular, its ubiquitin-binding domain were critical to recruitment, while the N-terminus and a functional ATPase domain played a minor role at best in facilitating protein accumulation. Although the absence of CSA had no effect on CSB recruitment, CSA itself localized at sites of ICLs, DSBs and monoadducts but not at oxidative lesions. Our results reveal molecular components of the CS protein response and point to a major involvement of complex lesions in the pathology of CS.

  4. PPARgamma-dependent regulation of adenylate cyclase 6 amplifies the stimulatory effect of cAMP on renin gene expression. (United States)

    Desch, Michael; Schubert, Thomas; Schreiber, Andrea; Mayer, Sandra; Friedrich, Björn; Artunc, Ferruh; Todorov, Vladimir T


    The second messenger cAMP plays an important role in the regulation of renin gene expression. Nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) is known to stimulate renin gene transcription acting through PPARγ-binding sequences in renin promoter. We show now that activation of PPARγ by unsaturated fatty acids or thiazolidinediones drastically augments the cAMP-dependent increase of renin mRNA in the human renin-producing cell line Calu-6. The underlying mechanism involves potentiation of agonist-induced cAMP increase and up-regulation of adenylate cyclase 6 (AC6) gene expression. We identified a palindromic element with a 3-bp spacer (Pal3) in AC6 intron 1 (AC6Pal3). AC6Pal3 bound PPARγ and mediated trans-activation by PPARγ agonist. AC6 knockdown decreased basal renin mRNA level and attenuated the maximal PPARγ-dependent stimulation of the cAMP-induced renin gene expression. AC6Pal3 decoy oligonucleotide abrogated the PPARγ-dependent potentiation of cAMP-induced renin gene expression. Treatment of mice with PPARγ agonist increased AC6 mRNA kidney levels. Our data suggest that in addition to its direct effect on renin gene transcription, PPARγ "sensitizes" renin gene to cAMP via trans-activation of AC6 gene. AC6 has been identified as PPARγ target gene with a functional Pal3 sequence.

  5. Structural Responses and Finite Element Modeling of Hakka Tulou Rammed Earth Structures (United States)

    Sranislawski, Daniel

    Hakka Tulous are rammed earth structures that have survived the effects of aging and natural elements upwards of even over a thousand years. These structures have housed the Hakka people of the Fujian Province, China in natural yet modern housing that has provided benefits over newer building materials. The key building material, rammed earth, which is used for the walls of the Hakka Tulou structures, has provided structural stability along with thermal comfort to the respective inhabitants of the Hakka Tulous. Through material testing and analysis this study has examined how the Tulou structures have maintained their structural stability while also providing thermal comfort. Reports of self healing cracks in the rammed earth walls were also analyzed for their validity in this study. The study has found that although the story of the self healing crack cannot be validated, there is reason to believe that with the existence of lime, some type of autogenous healing could occur on a small scale. The study has also found, through the use of nondestructive testing, that both the internal wooden systems (flooring, roof, and column support) and the rammed earth walls, are still structurally sound. Also, rammed earth's high thermal mass along with the use of sufficient shading has allowed for a delay release of heat energy from the walls of the Tulous, thus providing thermal comfort that can be felt during both night and day temperatures. The Hakka Tulou structures have been found to resist destruction from natural disasters such as strong earthquakes even when more modern construction has not. Through finite element modeling, this study has shown that the high volume of rammed earth used in the construction of the Hakka Tulous helps dissipate lateral force energy into much lower stresses for the rammed earth wall. This absorption of lateral force energy allows the rammed earth structures to survive even the strongest of earthquakes experienced in the region. The Hakka

  6. Mitral valve finite element modeling: implications of tissues' nonlinear response and annular motion. (United States)

    Stevanella, Marco; Votta, Emiliano; Redaelli, Alberto


    Finite element modeling represents an established method for the comprehension of the mitral function and for the simulation of interesting clinical scenarios. However, current models still do not include all the key aspects of the real system. We implemented a new structural finite element model that considers (i) an accurate morphological description of the valve, (ii) a description of the tissues' mechanical properties that accounts for anisotropy and nonlinearity, and (iii) dynamic boundary conditions that mimic annulus and papillary muscles' contraction. The influence of such contraction on valve biomechanics was assessed by comparing the computed results with the ones obtained through an auxiliary model with fixed annulus and papillary muscles. At the systolic peak, the leaflets' maximum principal stress contour showed peak values in the anterior leaflet at the strut chordae insertion zone (300 kPa) and near the annulus (200-250 kPa), while much lower values were detected in the posterior leaflet. Both leaflets underwent larger tensile strains in the longitudinal direction, while in the circumferential one the anterior leaflet experienced nominal tensile strains up to 18% and the posterior one experienced compressive strains up to 23% associated with the folding of commissures and paracommissures, consistently with tissue redundancy. The force exerted by papillary muscles at the systolic peak was equal to 4.11 N, mainly borne by marginal chordae (76% of the force). Local reaction forces up to 45 mN were calculated on the annulus, leading to tensions of 89 N/m and 54 N/m for its anterior and posterior tracts, respectively. The comparison with the results of the auxiliary model showed that annular contraction mainly affects the leaflets' circumferential strains. When it was suppressed, no more compressive strains could be observed and peak strain values were located in the belly of the anterior leaflet. Computational results agree to a great extent with

  7. Micoses superficiais e os elementos da resposta imune Superficial mycosis and the immune response elements

    Directory of Open Access Journals (Sweden)

    Paulo Ricardo Criado


    Full Text Available As micoses superficiais são prevalentes em todo o mundo, geralmente ocasionadas por dermatófitos e restritas à camada córnea. A resposta imunológica do hospedeiro às infecções dos fungos dermatófitos depende basicamente das defesas do hospedeiro a metabólitos do fungo, da virulência da cepa ou da espécie infectante e da localização anatômica da infecção. Serão revistos alguns dos fatores da defesa imunológica do hospedeiro que influenciam na eficácia da resposta imune. Em especial, a participação dos receptores de padrão de reconhecimento (PRRs, tais como os receptores toll-like ou os da família lectina (DC-SIGN e dectin-2, que participam da resposta imune inata, conferindo-lhe especificidade e definindo o padrão da resposta imune como um todo. O predomínio celular ou humoral da resposta imune definirá o quadro clínico e o prognóstico da infecção, levando à cura ou cronicidadeSuperficial mycoses are prevalent worldwide. They are often caused by dermatophytes and restricted to the stratum corneum. The host's immune response against infections caused by dermatophytes basically depends on the host's defense against metabolites of the fungi, virulence of the infecting strain or species and anatomical site of the infection. We will review some of the factors of the host's immune defense that influence the efficacy of the immune response. We will particularly review the role of pattern recognition receptors (PRRs, such as toll-like receptors or lectin receptors (DCSIGN and Dectin 2, which participate in the innate immune response, bringing specificity to the immune response and setting its pattern. The predominance of a cellular or humoral immune response determines the clinical manifestations and the prognosis of the infection, leading to healing or chronicity

  8. Element Cycling and Energy Flux Responses in Ecosystem Simulations Conducted at the Chinese Lunar Palace-1 (United States)

    Dong, Chen; Fu, Yuming; Xie, Beizhen; Wang, Minjuan; Liu, Hong


    Bioregenerative life-support systems (BLSS) address interactions between organisms and their environment as an integrated system through the study of factors that regulate the pools and fluxes of materials and energy through ecological systems. As a simple model, using BLSS is very important in the investigation of element cycling and energy flux for sustainable development on Earth. A 105-day experiment with a high degree of closure was carried out in this system from February to May, 2014, with three volunteers. The results indicate that 247 g·d-1 carbon was imported into the system from stored food. Most hydrogen is circulated as water, and more than 99% H2O can be lost through leaf transpiration into the atmosphere. A total of 1.8 g·d-1 "unknown oxygen" emerged between the input and output of the plant growth module. For the urine processing module, 20.5% nitrogen was reused and 5.35 g·d-1 was put into the nutrient solution.

  9. Explicit incremental-update algorithm for modeling crystal elasto-viscoplastic response in finite element simulation

    Institute of Scientific and Technical Information of China (English)

    LI Hong-wei; YANG He; SUN Zhi-chao


    Computational stability and efficiency are the key problems for numerical modeling of crystal plasticity,which will limit its development and application in finite element (FE) simulation evidently. Since implicit iterative algorithms are inefficient and have difficulty to determine initial values,an explicit incremental-update algorithm for the elasto-viscoplastic constitutive relation was developed in the intermediate frame by using the second Piola-Kirchoff (P-K) stress and Green stain. The increment of stress and slip resistance were solved by a calculation loop of linear equations sets. The reorientation of the crystal as well as the elastic strain can be obtained from a polar decomposition of the elastic deformation gradient. User material subroutine VUMAT was developed to combine crystal elasto-viscoplastic constitutive model with ABAQUS/Explicit. Numerical studies were performed on a cubic upset model with OFHC material (FCC crystal). The comparison of the numerical results with those obtained by implicit iterative algorithm and those from experiments demonstrates that the explicit algorithm is reliable. Furthermore,the effect rules of material anisotropy,rate sensitivity coefficient (RSC) and loading speeds on the deformation were studied. The numerical studies indicate that the explicit algorithm is suitable and efficient for large deformation analyses where anisotropy due to texture is important.

  10. Identification of Regulatory Mutations in SERPINC1 Affecting Vitamin D Response Elements Associated with Antithrombin Deficiency (United States)

    Toderici, Mara; de la Morena-Barrio, María Eugenia; Padilla, José; Miñano, Antonia; Antón, Ana Isabel; Iniesta, Juan Antonio; Herranz, María Teresa; Fernández, Nuria; Vicente, Vicente; Corral, Javier


    Antithrombin is a crucial anticoagulant serpin whose even moderate deficiency significantly increases the risk of thrombosis. Most cases with antithrombin deficiency carried genetic defects affecting exons or flanking regions of SERPINC1.We aimed to identify regulatory mutations inSERPINC1 through sequencing the promoter, intron 1 and 2 of this gene in 23 patients with antithrombin deficiency but without known genetic defects. Three cases with moderate antithrombin deficiency (63–78%) carried potential regulatory mutations. One located 200 bp before the initiation ATG and two in intron 1. These mutations disrupted two out of five potential vitamin D receptor elements (VDRE) identified in SERPINC1 with different software. One genetic defect, c.42-1060_-1057dupTTGA, was a new low prevalent polymorphism (MAF: 0.01) with functional consequences on plasma antithrombin levels. The relevance of the vitamin D pathway on the regulation of SERPINC1 was confirmed in a cell model. Incubation of HepG2 with paricalcitol, a vitamin D analog, increased dose-dependently the levels of SERPINC1transcripts and antithrombin released to the conditioned medium. This study shows further evidence of the transcriptional regulation of SERPINC1 by vitamin D and first describes the functional and pathological relevance of mutations affecting VDRE of this gene. Our study opens new perspectives in the search of new genetic defects involved in antithrombin deficiency and the risk of thrombosis as well as in the design of new antithrombotic treatments. PMID:27003919

  11. Evaluation of Head Response to Blast Using Sagittal and Transverse Finite Element Head Models (United States)


    scenario nce in results s and the air layer of nod ration caused b ssue Material priate constitu al parameters yperelastic ma curves at diff s were...viscous material model with bulk properties similar to those of water (Table 2). The viscosity was set to that of water , providing minimal resistance...expected odel predicte ere not include ssue by the nu response in th he brain in th ive parameter imum effectiv in rates did no odel predicte el. at 4 m

  12. High adenylyl cyclase activity and in vivo cAMP fluctuations in corals suggest central physiological role. (United States)

    Barott, K L; Helman, Y; Haramaty, L; Barron, M E; Hess, K C; Buck, J; Levin, L R; Tresguerres, M


    Corals are an ecologically and evolutionarily significant group, providing the framework for coral reef biodiversity while representing one of the most basal of metazoan phyla. However, little is known about fundamental signaling pathways in corals. Here we investigate the dynamics of cAMP, a conserved signaling molecule that can regulate virtually every physiological process. Bioinformatics revealed corals have both transmembrane and soluble adenylyl cyclases (AC). Endogenous cAMP levels in live corals followed a potential diel cycle, as they were higher during the day compared to the middle of the night. Coral homogenates exhibited some of the highest cAMP production rates ever to be recorded in any organism; this activity was inhibited by calcium ions and stimulated by bicarbonate. In contrast, zooxanthellae or mucus had >1000-fold lower AC activity. These results suggest that cAMP is an important regulator of coral physiology, especially in response to light, acid/base disturbances and inorganic carbon levels.

  13. Cyclic AMP in female mouse brain is altered by the adrenocorticotropic hormone(4-9) analogue organon 2766. (United States)

    Schneider, D R; Felt, B T; Murphy, S; Goldman, H


    Cyclic AMP content was determined in 12 brain regions of young adult female mice at 30 min and at 24 h following an intraperitoneal injection of the tri-substituted adrenocorticotropic hormone(4-9) [ACTH(4-9)] analogue Organon 2766 [ORG 2766]. Animals were killed by focused 3.5 kW microwave radiation applied for 350 ms. Unlike previously reported responses in male mice, at 30 min post-injection there were no detectable differences in cyclic AMP content between the placebo and ORG 2766-treated animals. By contrast, 24 h after injection, the content of cyclic AMP was changed significantly in 8 of the 12 brain regions examined: medulla-pons, septal area, thalamus, hypothalamus, hippocampus, olfactory bulb, and parietal and occipital cortices. In most of the regions examined, differences consisted of 50% or greater reductions of tissue cyclic AMP content. The changes were unrelated to the estrus cycle of these animals.

  14. Two estrogen response element sequences near the PCNA gene are not responsible for its estrogen-enhanced expression in MCF7 cells.

    Directory of Open Access Journals (Sweden)

    Cheng Wang

    Full Text Available BACKGROUND: The proliferating cell nuclear antigen (PCNA is an essential component of DNA replication, cell cycle regulation, and epigenetic inheritance. High expression of PCNA is associated with poor prognosis in patients with breast cancer. The 5'-region of the PCNA gene contains two computationally-detected estrogen response element (ERE sequences, one of which is evolutionarily conserved. Both of these sequences are of undocumented cis-regulatory function. We recently demonstrated that estradiol (E2 enhances PCNA mRNA expression in MCF7 breast cancer cells. MCF7 cells proliferate in response to E2. METHODOLOGY/PRINCIPAL FINDINGS: Here, we demonstrate that E2 rapidly enhanced PCNA mRNA and protein expression in a process that requires ERalpha as well as de novo protein synthesis. One of the two upstream ERE sequences was specifically bound by ERalpha-containing protein complexes, in vitro, in gel shift analysis. Yet, each ERE sequence, when cloned as a single copy, or when engineered as two tandem copies of the ERE-containing sequence, was not capable of activating a luciferase reporter construct in response to E2. In MCF7 cells, neither ERE-containing genomic region demonstrated E2-dependent recruitment of ERalpha by sensitive ChIP-PCR assays. CONCLUSION/SIGNIFICANCE: We conclude that E2 enhances PCNA gene expression by an indirect process and that computational detection of EREs, even when evolutionarily conserved and when near E2-responsive genes, requires biochemical validation.

  15. Cyclic AMP (cAMP) confers drug resistance against DNA damaging agents via PKAIA in CML cells. (United States)

    Xiao, Ling-Yi; Kan, Wai-Ming


    Cyclic adenosine monophosphate (cAMP) regulates many vital functions such as metabolism, proliferation, differentiation and death. Depending on cell types and stimulators, cAMP could either promote or attenuate cell death. cAMP signal can be transduced by protein kinase A (PKA) and/or exchange protein directly activated by cAMP (EPAC). In CML cells, cAMP may suppress their proliferation and enhance their differentiation. However, the role of cAMP on DNA damaging agent toxicity and the mechanism involved has not been studied. In this study, we studied the effect of cAMP on the sensitivity of CML cells to DNA damaging agents. We observed that forskolin (FSK) and dibutyryl-cAMP (DBcAMP) decreased cisplatin and etoposide-induced cell death in K562 cells. Moreover, PKA activator prevented K562 cells from DNA damaging agent-induced cell death while EPAC activator had no effect. Furthermore, we found that the PKA subtype, PKAIA, was involved in cAMP-attenuated resistance in K562 cells. Taken together, our results suggest that increased cAMP level confers CML cells to acquire a novel mechanism against DNA damaging agent toxicity via PKAIA. Thus, PKAIA inhibitor may be helpful in overcoming the resistance to DNA damaging agents in CML cells.

  16. The Monge-Ampère equation

    CERN Document Server

    Gutiérrez, Cristian E


    Now in its second edition, this monograph explores the Monge-Ampère equation and the latest advances in its study and applications. It provides an essentially self-contained systematic exposition of the theory of weak solutions, including regularity results by L. A. Caffarelli. The geometric aspects of this theory are stressed using techniques from harmonic analysis, such as covering lemmas and set decompositions. An effort is made to present complete proofs of all theorems, and examples and exercises are offered to further illustrate important concepts. Some of the topics considered include generalized solutions, non-divergence equations, cross sections, and convex solutions. New to this edition is a chapter on the linearized Monge-Ampère equation and a chapter on interior Hölder estimates for second derivatives. Bibliographic notes, updated and expanded from the first edition, are included at the end of every chapter for further reading on Monge-Ampère-type equations and their diverse applications in th...

  17. The Applied Mathematics for Power Systems (AMPS)

    Energy Technology Data Exchange (ETDEWEB)

    Chertkov, Michael [Los Alamos National Laboratory


    Increased deployment of new technologies, e.g., renewable generation and electric vehicles, is rapidly transforming electrical power networks by crossing previously distinct spatiotemporal scales and invalidating many traditional approaches for designing, analyzing, and operating power grids. This trend is expected to accelerate over the coming years, bringing the disruptive challenge of complexity, but also opportunities to deliver unprecedented efficiency and reliability. Our Applied Mathematics for Power Systems (AMPS) Center will discover, enable, and solve emerging mathematics challenges arising in power systems and, more generally, in complex engineered networks. We will develop foundational applied mathematics resulting in rigorous algorithms and simulation toolboxes for modern and future engineered networks. The AMPS Center deconstruction/reconstruction approach 'deconstructs' complex networks into sub-problems within non-separable spatiotemporal scales, a missing step in 20th century modeling of engineered networks. These sub-problems are addressed within the appropriate AMPS foundational pillar - complex systems, control theory, and optimization theory - and merged or 'reconstructed' at their boundaries into more general mathematical descriptions of complex engineered networks where important new questions are formulated and attacked. These two steps, iterated multiple times, will bridge the growing chasm between the legacy power grid and its future as a complex engineered network.

  18. Effects of pyridoxine on a high-fat diet-induced reduction of cell proliferation and neuroblast differentiation depend on cyclic adenosine monophosphate response element binding protein in the mouse dentate gyrus. (United States)

    Yoo, Dae Young; Kim, Woosuk; Yoo, Ki-Yeon; Nam, Sung Min; Chung, Jin Young; Yoon, Yeo Sung; Won, Moo-Ho; Hwang, In Koo


    In this study, we challenged pyridoxine to mice fed a high-fat diet (HFD) and investigated the effects of pyridoxine on HFD-induced phenotypes such as blood glucose, reduction of cell proliferation and neuroblast differentiation in the dentate gyrus using Ki67 and doublecortin (DCX), respectively. Mice were fed a commercially available low-fat diet (LFD) as control diet or HFD (60% fat) for 8 weeks. After 5 weeks of LFD or HFD treatment, 350 mg/kg pyridoxine was administered for 3 weeks. The administration of pyridoxine significantly decreased body weight in the HFD-treated group. In addition, there were no significant differences in hepatic histology and pancreatic insulin-immunoreactive (-ir) and glucagon-ir cells of the HFD-treated group after pyridoxine treatment. In the HFD-fed group, Ki67-positive nuclei and DCX-ir neuroblasts were significantly decreased in the dentate gyrus compared with those in the LFD-fed mice. However, the administration of pyridoxine significantly increased Ki67-positive nuclei and DCX-ir neuroblasts in the dentate gyrus in both LFD- and HFD-fed mice. In addition, the administration of pyridoxine significantly increased the protein levels of glutamic acid decarboxylase 67 (GAD67) and brain-derived neurotrophic factor (BDNF) and the immunoreactivity of phosphorylated cyclic AMP response element binding protein (pCREB) compared with the vehicle-treated LFD- and HFD-fed mice. In contrast, the administration of pyridoxine significantly decreased HFD-induced malondialdehyde (MDA) levels in the hippocampus. These results showed that pyridoxine supplement reduced the HFD-induced reduction of cell proliferation and neuroblast differentiation in the dentate gyrus via controlling the levels of GAD67, pCREB, BDNF, and MDA.

  19. The Dynamic Response of an Euler-Bernoulli Beam on an Elastic Foundation by Finite Element Analysis using the Exact Stiffness Matrix (United States)

    Kim, Jeong Soo; Kyum Kim, Moon


    In this study, finite element analysis of beam on elastic foundation, which received great attention of researchers due to its wide applications in engineering, is performed for estimating dynamic responses of shallow foundation using exact stiffness matrix. First, element stiffness matrix based on the closed solution of beam on elastic foundation is derived. Then, we performed static finite element analysis included exact stiffness matrix numerically, comparing results from the analysis with some exact analysis solutions well known for verification. Finally, dynamic finite element analysis is performed for a shallow foundation structure under rectangular pulse loading using trapezoidal method. The dynamic analysis results exist in the reasonable range comparing solution of single degree of freedom problem under a similar condition. The results show that finite element analysis using exact stiffness matrix is evaluated as a good tool of estimating the dynamic response of structures on elastic foundation.

  20. Genomic Regulation of the Response of an Agroecosystem to Elements of Global Change

    Energy Technology Data Exchange (ETDEWEB)

    DeLucia, Evan, H.


    This document outlines some of the major accomplishments from this project: (1) New tools for analyzing and visualizing microarray data from soybean gene expression experiments; (2) Physiological, biochemical, and gene array evidence that acclimation of carbon metabolism to elevated CO{sub 2} is governed in significant part by changes in gene expression associated with respiratory metabolism; (3) Increased carbon assimilation in soybeans grown at elevated CO{sub 2} altered pools of carbohydrates and transcripts that control growth and expansion of young leaves; (4) Growth at elevated CO{sub 2} increases the abundance of transcripts controlling cell wall polysaccharide synthesis but not transcripts controlling lignin synthesis; (5) The total antioxidant capacity of soybeans varies among cultivars and in response to atmospheric change; (6) Accelerated leaf senescence at elevated O{sub 3} coincides with reduced abundance of transcripts controlling protein synthesis; (7) Growth under elevated CO{sub 2} increases the susceptibility of soybean to insect herbivores by increasing insect lifespan and fecundity through altered leaf chemistry and by defeating molecular induction of plant defenses; (8) Exposure to elevated CO{sub 2} and O{sub 3} alters flavonoid metabolism in soybean; (9) Exposure to elevated CO{sub 2} or O{sub 3} conferred resistance to soybean mosaic virus by cross inducing defense- and stress-related signaling pathways; and (10) Exposure to elevated CO{sub 2} accelerates decomposition by changing chemical and biotic properties of the soil.

  1. Validation of Shoulder Response of Human Body Finite-Element Model (GHBMC) Under Whole Body Lateral Impact Condition. (United States)

    Park, Gwansik; Kim, Taewung; Panzer, Matthew B; Crandall, Jeff R


    In previous shoulder impact studies, the 50th-percentile male GHBMC human body finite-element model was shown to have good biofidelity regarding impact force, but under-predicted shoulder deflection by 80% compared to those observed in the experiment. The goal of this study was to validate the response of the GHBMC M50 model by focusing on three-dimensional shoulder kinematics under a whole-body lateral impact condition. Five modifications, focused on material properties and modeling techniques, were introduced into the model and a supplementary sensitivity analysis was done to determine the influence of each modification to the biomechanical response of the body. The modified model predicted substantially improved shoulder response and peak shoulder deflection within 10% of the observed experimental data, and showed good correlation in the scapula kinematics on sagittal and transverse planes. The improvement in the biofidelity of the shoulder region was mainly due to the modifications of material properties of muscle, the acromioclavicular joint, and the attachment region between the pectoralis major and ribs. Predictions of rib fracture and chest deflection were also improved because of these modifications.

  2. CAP1, an adenylate cyclase-associated protein gene, regulates bud-hypha transitions, filamentous growth, and cyclic AMP levels and is required for virulence of Candida albicans. (United States)

    Bahn, Y S; Sundstrom, P


    In response to a wide variety of environmental stimuli, the opportunistic fungal pathogen Candida albicans exits the budding cycle, producing germ tubes and hyphae concomitant with expression of virulence genes, such as that encoding hyphal wall protein 1 (HWP1). Biochemical studies implicate cyclic AMP (cAMP) increases in promoting bud-hypha transitions, but genetic evidence relating genes that control cAMP levels to bud-hypha transitions has not been reported. Adenylate cyclase-associated proteins (CAPs) of nonpathogenic fungi interact with Ras and adenylate cyclase to increase cAMP levels under specific environmental conditions. To initiate studies on the relationship between cAMP signaling and bud-hypha transitions in C. albicans, we identified, cloned, characterized, and disrupted the C. albicans CAP1 gene. C. albicans strains with inactivated CAP1 budded in conditions that led to germ tube formation in isogenic strains with CAP1. The addition of 10 mM cAMP and dibutyryl cAMP promoted bud-hypha transitions and filamentous growth in the cap1/cap1 mutant in liquid and solid media, respectively, showing clearly that cAMP promotes hypha formation in C. albicans. Increases in cytoplasmic cAMP preceding germ tube emergence in strains having CAP1 were markedly diminished in the budding cap1/cap1 mutant. C. albicans strains with deletions of both alleles of CAP1 were avirulent in a mouse model of systemic candidiasis. The avirulence of a germ tube-deficient cap1/cap1 mutant coupled with the role of Cap1 in regulating cAMP levels shows that the Cap1-mediated cAMP signaling pathway is required for bud-hypha transitions, filamentous growth, and the pathogenesis of candidiasis.

  3. Geogenic and Anthropogenic Moss Responsiveness to Element Distribution Around a Pb-Zn Mine, Toranica, Republic of Macedonia. (United States)

    Angelovska, Svetlana; Stafilov, Trajče; Šajn, Robert; Balabanova, Biljana


    Moss species (Homalothecium lutescens, Hypnum cupressiforme, Brachythecium glareosum, and Campthotecium lutescens) were used as suitable sampling media for biomonitoring the origin of heavy-metal pollution in the lead-zinc (Pb-Zn) mine "Toranica" near the Kriva Palanka town, Eastern Macedonia. The contents of 20 elements-silver (Ag), aluminum (Al), arsenic (As), barium (Ba), calcium (Ca), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), potassium (K), lithium (Li), magnesium (Mg), manganese (Mn), sodium (Na), nickel (Ni), Pb, strontium (Sr), vanadium (V), and (Zn) were determined by atomic emission spectrometry with inductively coupled plasma. Data processing was applied with combinations of multivariate statistical methods: factor analysis, principal component analysis, and cluster analysis. Moss' responsiveness to the atmospheric distribution of the selected elements was investigated in correlation to the specific geology of the region (soil dusting). Lithogenic distribution was characterized with the distribution of three dominant geochemical associations: F1: Al-Li-V-Cr-Ni-Co, F2: Ba-Ca-Sr, and F3: Cd-Zn-Pb-Cu. Spatial distribution was constructed for visualization of the factor deposition. Furthermore, air distribution (passive biomonitoring) versus soil geochemistry of the analyzed elements was examined. Significant correlations were singled out for Pb, Zn, and Cd and for Mg(moss)/Na(soil). Characteristic lithological anomaly characterized the presence of the oldest geological volcanic rocks. Zone 1 (Pb-Zn mine surrounding) presents a unique area with hydrothermal action of Pb-Zn mineralization leading to polymetallic enrichments in soil. This phenomenon strongly affects the environment, which is a natural geochemical imprint in this unique area (described with the strong dominance of the geochemical association Cd-Zn-Pb-Cu).

  4. Growth responses of selected freshwater algae to trace elements and scrubber ash slurry generated by coal-fired power plants

    Energy Technology Data Exchange (ETDEWEB)

    Vocke, R.W.


    The development and implementation of standard toxicity tests is a necessity if consistent and reliable data are to be obtained for water quality criteria. The adapted EPA AAPBT is an ideal static algal toxicity test system. The algal test medium has a chemical composition similar to natural unpolluted waters of low ionic strength. It is appropriate to use MATC water quality criteria when assessing the potential impact of pollutants generated by coal-fired power stations because these energy-generated pollutants typically enter aquatic systems in small quantities over long periods. The MATC water quality criteria are estimates of trace element and SASE levels, based on the most sensitive alga investigated, that will not cause significant changes in naturally-functioning algal populations. These levels are 0.016f mg L/sup -1/ As(V), 0.001 mg L/sup -1/ Cd(II), 0.004 mg L/sup -1/ Hg(II), 0.006 mg L/sup -1/ Se(VI), and 0.344% SASE. To provide viable working water quality criteria, an extrapolation from the laboratory to the natural environment must be made. Therefore, those oxidation states of the trace elements were selected which are the dominant states occurring in natural, unpolluted, slightly alkaline freshwaters. It must be pointed out that these MATC values are based on algal responses to single toxicants and no allowance is made for synergistic, additive, or antagonistic relationships which could occur in natural aquatic systems. Additionally, natural chelation may influence toxicity. The highly toxic nature of potential pollutants from coal-fired generating plants emphasizes the need for minimizing stack effluent pollutants and retaining scrubber ash slurry for proper disposal in an effort to maintain trace elements in concentration ranges compatible with naturally-functioning ecosystems.

  5. Cyclic AMP synergizes with butyrate in promoting β-defensin 9 expression in chickens. (United States)

    Sunkara, Lakshmi T; Zeng, Xiangfang; Curtis, Amanda R; Zhang, Guolong


    Host defense peptides (HDP) have both microbicidal and immunomodulatory properties. Specific induction of endogenous HDP synthesis has emerged as a novel approach to antimicrobial therapy. Cyclic adenosine monophosphate (cAMP) and butyrate have been implicated in HDP induction in humans. However, the role of cAMP signaling and the possible interactions between cAMP and butyrate in regulating HDP expression in other species remain unknown. Here we report that activation of cAMP signaling induces HDP gene expression in chickens as exemplified by β-defensin 9 (AvBD9). We further showed that, albeit being weak inducers, cAMP agonists synergize strongly with butyrate or butyrate analogs in AvBD9 induction in macrophages and primary jejunal explants. Additionally, oral supplementation of forskolin, an adenylyl cyclase agonist in the form of a Coleus forskohlii extract, was found to induce AvBD9 expression in the crop of chickens. Furthermore, feeding with both forskolin and butyrate showed an obvious synergy in triggering AvBD9 expression in the crop and jejunum of chickens. Surprisingly, inhibition of the MEK-ERK mitogen-activated protein kinase (MAPK) pathway augmented the butyrate-FSK synergy, whereas blocking JNK or p38 MAPK pathway significantly diminished AvBD9 induction in chicken macrophages and jejunal explants in response to butyrate and FSK individually or in combination. Collectively, these results suggest the potential for concomitant use of butyrate and cAMP signaling activators in enhancing HDP expression, innate immunity, and disease resistance in both animals and humans.

  6. Refinement of the androgen response element based on ChIP-Seq in androgen-insensitive and androgen-responsive prostate cancer cell lines. (United States)

    Wilson, Stephen; Qi, Jianfei; Filipp, Fabian V


    Sequence motifs are short, recurring patterns in DNA that can mediate sequence-specific binding for proteins such as transcription factors or DNA modifying enzymes. The androgen response element (ARE) is a palindromic, dihexameric motif present in promoters or enhancers of genes targeted by the androgen receptor (AR). Using chromatin immunoprecipitation sequencing (ChIP-Seq) we refined AR-binding and AREs at a genome-scale in androgen-insensitive and androgen-responsive prostate cancer cell lines. Model-based searches identified more than 120,000 ChIP-Seq motifs allowing for expansion and refinement of the ARE. We classified AREs according to their degeneracy and their transcriptional involvement. Additionally, we quantified ARE utilization in response to somatic copy number amplifications, AR splice-variants, and steroid treatment. Although imperfect AREs make up 99.9% of the motifs, the degree of degeneracy correlates negatively with validated transcriptional outcome. Weaker AREs, particularly ARE half sites, benefit from neighboring motifs or cooperating transcription factors in regulating gene expression. Taken together, ARE full sites generate a reliable transcriptional outcome in AR positive cells, despite their low genome-wide abundance. In contrast, the transcriptional influence of ARE half sites can be modulated by cooperating factors.

  7. Investigation of Micro-mechanical Response of Asphalt Mixtures by a Three-dimensional Discrete Element Model

    Institute of Scientific and Technical Information of China (English)

    HOU Shuguang; ZHANG Dong; HUANG Xiaoming; ZHAO Yongli


    The micro-mechanical response of asphalt mixtures was studied using the discrete element method. The discrete element sample of stone mastic asphalt was generated first and the vehicle load was applied to the sample. A user-written program was coded with the FISH language in PFC3D to extract the contact forces within the sample and the displacements of the particles. Then, the contact forces within the whole sample, in asphalt mastic, in coarse aggregates and between asphalt mastic and coarse aggregates were investigated. Finally, the movement of the particles in the sample was analyzed. The sample was divided into 15 areas and a figure was drawn to show how the balls move in each area according to the displacements of the balls in each area. The displacements of asphalt mastic balls and coarse aggregates were also analyzed. The experimental results explain how the asphalt mixture bears vehicle load and the potential reasons why the rutting forms from a micro-mechanical view.


    Institute of Scientific and Technical Information of China (English)

    谢久永; 刘国仰; 王梅; 黄尚志; 罗会元


    All mammalian metallothionaln genes studied to dare have several metal responsive elements (MRE) with consensus sequences of TGCRCNC (R, purlne) in their regulatory region. MRE-11ke sequeaees were also found in many other metal-related genes. To see whether there is also such a sequence at the genetic locus (13q14. 3) d Wilstm disease, which is a genetic disorder d copper metabolisa''n, junction-trapping method baaed on the MRE sequence was used. A fragment containing MRE and MRE-like sequences from YAC 27D8 at the WND locus was successfully cloned and mapped back to the YAC by PC, R, Presence of such a sequence in the copper transporter gene at the W''D locus might imply that it has a possible interesting role in the regulation of WD gene expression.

  9. Activation of estrogen response elements is mediated both via estrogen and muscle contractions in rat skeletal muscle myotubes

    DEFF Research Database (Denmark)

    Wiik, A.; Hellsten, Ylva; Berthelson, P.


    The aim of the present study was to investigate the activation of estrogen response elements (EREs) by estrogen and muscle contractions in rat myotubes in culture and to assess whether the activation is dependent on the estrogen receptors (ERs). In addition, the effect of estrogen and contraction...... then differentiated into myotubes and subjected to either estrogen or electrical stimulation. Activation of the ERE sequence was determined by measurement of luciferase activity. The results show that both ERalpha and ERbeta are expressed in myotubes from rats. Both estrogen stimulation and muscle contraction...... increased (P muscle contraction. Use of ER antagonists showed that, whereas the estrogen-induced transactivation is mediated via ERs, the effect of muscle contraction...

  10. Development of a Finite Element Model of the Human Shoulder to Investigate the Mechanical Responses and Injuries in Side Impact (United States)

    Iwamoto, Masami; Miki, Kazuo; Yang, King H.

    Previous studies in both fields of automotive safety and orthopedic surgery have hypothesized that immobilization of the shoulder caused by the shoulder injury could be related to multiple rib fractures, which are frequently life threatening. Therefore, for more effective occupant protection, it is important to understand the relationship between shoulder injury and multiple rib fractures in side impact. The purpose of this study is to develop a finite element model of the human shoulder in order to understand this relationship. The shoulder model included three bones (the humerus, scapula and clavicle) and major ligaments and muscles around the shoulder. The model also included approaches to represent bone fractures and joint dislocations. The relationships between shoulder injury and immobilization of the shoulder are discussed using model responses for lateral shoulder impact. It is also discussed how the injury can be related to multiple rib fractures.

  11. Fuzzy tandem repeats containing p53 response elements may define species-specific p53 target genes.

    Directory of Open Access Journals (Sweden)

    Iva Simeonova


    Full Text Available Evolutionary forces that shape regulatory networks remain poorly understood. In mammals, the Rb pathway is a classic example of species-specific gene regulation, as a germline mutation in one Rb allele promotes retinoblastoma in humans, but not in mice. Here we show that p53 transactivates the Retinoblastoma-like 2 (Rbl2 gene to produce p130 in murine, but not human, cells. We found intronic fuzzy tandem repeats containing perfect p53 response elements to be important for this regulation. We next identified two other murine genes regulated by p53 via fuzzy tandem repeats: Ncoa1 and Klhl26. The repeats are poorly conserved in evolution, and the p53-dependent regulation of the murine genes is lost in humans. Our results indicate a role for the rapid evolution of tandem repeats in shaping differences in p53 regulatory networks between mammalian species.

  12. Regulation of brain-derived neurotrophic factor exon IV transcription through calcium responsive elements in cortical neurons.

    Directory of Open Access Journals (Sweden)

    Fei Zheng

    Full Text Available Activity-dependent transcription of brain-derived neurotrophic factor (BDNF has been studied as an important model to elucidate the mechanisms underlying numerous aspects of neuroplasticity. It has been extensively emphasized that Ca(2+ influx through different routes may have significantly different effects on BDNF transcription. Here, we examined the regulatory property of the major calcium responsive elements (CaRE in BDNF promoter IV in cultured rat cortical neurons. BDNF promoter IV, as well as CaRE1 and CaRE3, was significantly activated by Ca(2+ influx through L-type voltage-gated calcium channel (L-VGCC or NMDA receptor (NMDAR. However, the L-VGCC- and NMDAR-mediated activation of CaRE was differentially regulated by different Ca(2+-stimulated protein kinases. Specifically, PKA, CaMKI, and CaMKIV activity were required for L-VGCC-, but not NMDAR-mediated CaRE1 activation. CaMKI activity was required for NMDAR- but not L-VGCC-mediated CaRE3 activation. Surprisingly, the activation of CaRF, a previously identified transcription factor for CaRE1, was stimulated via L-VGCC but not NMDAR, and required MEK, PI3K and CaMKII activity. These results suggest a new working model that activity-dependent BDNF IV up-regulation may be coordinately mediated by CaRE1 and CaRE3 activity, which show different responses to Ca(2+-stimulated kinases. Our data also explain how the individual cis-element in BDNF promoter is distinctively coupled to different Ca(2+ routes.

  13. P53 family members modulate the expression of PRODH, but not PRODH2, via intronic p53 response elements.

    Directory of Open Access Journals (Sweden)

    Ivan Raimondi

    Full Text Available The tumor suppressor p53 was previously shown to markedly up-regulate the expression of the PRODH gene, encoding the proline dehydrogenase (PRODH enzyme, which catalyzes the first step in proline degradation. Also PRODH2, which degrades 4-hydroxy-L-proline, a product of protein (e.g. collagen catabolism, was recently described as a p53 target. Here, we confirmed p53-dependent induction of endogenous PRODH in response to genotoxic damage in cell lines of different histological origin. We established that over-expression of TAp73β or TAp63β is sufficient to induce PRODH expression in p53-null cells and that PRODH expression parallels the modulation of endogenous p73 by genotoxic drugs in several cell lines. The p53, p63, and p73-dependent transcriptional activation was linked to specific intronic response elements (REs, among those predicted by bioinformatics tools and experimentally validated by a yeast-based transactivation assay. p53 occupancy measurements were validated in HCT116 and MCF7 human cell lines. Conversely, PRODH2 was not responsive to p63 nor p73 and, at best, could be considered a weak p53 target. In fact, minimal levels of PRODH2 transcript induction by genotoxic stress was observed exclusively in one of four p53 wild-type cell lines tested. Consistently, all predicted p53 REs in PRODH2 were poor matches to the p53 RE consensus and showed very weak responsiveness, only to p53, in the functional assay. Taken together, our results highlight that PRODH, but not PRODH2, expression is under the control of p53 family members, specifically p53 and p73. This supports a deeper link between proteins of the p53-family and metabolic pathways, as PRODH modulates the balance of proline and glutamate levels and those of their derivative alpha-keto-glutarate (α-KG under normal and pathological (tumor conditions.

  14. Liver AMP/ATP ratio and fructokinase expression are related to gender differences in AMPK activity and glucose intolerance in rats ingesting liquid fructose. (United States)

    Vilà, Laia; Roglans, Núria; Perna, Victoria; Sánchez, Rosa M; Vázquez-Carrera, Manuel; Alegret, Marta; Laguna, Juan C


    Women, but not men, show an association between fructose consumption and an increased risk of Type 2 diabetes mellitus. As rats are considered a model for human fructose metabolism, we sought to determine whether such a gender-related difference is present in Sprague-Dawley rats and to analyze the molecular mechanism behind. Male and female Sprague-Dawley rats had free access to water or to a 10% w/v fructose solution for 14 days. Plasma analytes, liver triglycerides and enzyme activities and the expression of enzymes and transcription factors related to fatty acid metabolism, insulin signaling and glucose tolerance were determined. Fructose-fed rats had hypertriglyceridemia, steatosis and reduced fatty acid oxidation activity, although the metabolic pattern of fructose-fed female rats was different to that observed for male rats. Fructose-fed female, but not male rats, showed no change in plasma leptin; they had hyperinsulinemia, an altered glucose tolerance test and less liver insulin receptor substrate-2. Further, only fructose-fed female rats had increased adenosine 5'-monophosphate (AMP)-activated protein kinase activity, resulting in a decreased expression of hepatic nuclear factor 4 and sterol response element binding protein 1. These differences were related to the fact that liver expression of the enzyme fructokinase, controlling fructose metabolism, was markedly induced by fructose ingestion in female, but not in male rats, resulting in a significant increase in the AMP/adenosine 5'-triphosphate (ATP) ratio and, thus, AMP-activated protein kinase activation, in female rats only. The difference in fructokinase induction could explain the higher metabolic burden produced by fructose ingestion in the livers of female Sprague-Dawley rats.

  15. Motor Skill Learning Is Associated with Phase-Dependent Modifications in the Striatal cAMP/PKA/DARPP-32 Signaling Pathway in Rodents.

    Directory of Open Access Journals (Sweden)

    Yu Qian

    Full Text Available Abundant evidence points to a key role of dopamine in motor skill learning, although the underlying cellular and molecular mechanisms are still poorly understood. Here, we used a skilled-reaching paradigm to first examine changes in the expression of the plasticity-related gene Arc to map activity in cortico-striatal circuitry during different phases of motor skill learning in young animals. In the early phase, Arc mRNA was significantly induced in the medial prefrontal cortex (mPFC, cingulate cortex, primary motor cortex, and striatum. In the late phase, expression of Arc did not change in most regions, except in the mPFC and dorsal striatum. In the second series of experiments, we studied the learning-induced changes in the phosphorylation state of dopamine and cAMP-regulated phosphoprotein, 32k Da (DARPP-32. Western blot analysis of the phosphorylation state of DARPP-32 and its downstream target cAMP response element-binding protein (CREB in the striatum revealed that the early, but not late, phase of motor skill learning was associated with increased levels of phospho-Thr34-DARPP-32 and phospho-Ser133-CREB. Finally, we used the DARPP-32 knock-in mice with a point mutation in the Thr34 regulatory site (i.e., protein kinase A site to test the significance of this pathway in motor skill learning. In accordance with our hypothesis, inhibition of DARPP-32 activity at the Thr34 regulatory site strongly attenuated the motor learning rate and skilled reaching performance of mice. These findings suggest that the cAMP/PKA/DARPP-32 signaling pathway is critically involved in the acquisition of novel motor skills, and also demonstrate a dynamic shift in the contribution of cortico-striatal circuitry during different phases of motor skill learning.

  16. Altered beta-adrenergic receptor-stimulated cAMP formation in cultured skin fibroblasts from Alzheimer donors. (United States)

    Huang, H M; Gibson, G E


    An alteration in signal transduction systems in Alzheimer's disease would likely be of pathophysiological significance, because these steps are critical to normal brain function. Since dynamic processes are difficult to study in autopsied brain, the current studies utilized cultured skin fibroblasts. The beta-adrenergic-stimulated increase in cAMP was reduced approximately 80% in fibroblasts from Alzheimer's disease compared with age-matched controls. The deficit in Alzheimer fibroblasts in response to various adrenergic agonists paralleled their beta-adrenergic potency, and enhancement of cAMP accumulation by a non-adrenergic agonist, such as prostaglandin E1, was similar in Alzheimer and control fibroblasts. Diminished adenylate cyclase activity did not underlie these abnormalities, since direct stimulation of adenylate cyclase by forskolin elevated cAMP production equally in Alzheimer and control fibroblasts. Cholera toxin equally stimulated cAMP formation in Alzheimer and control fibroblasts. Moreover, cholera toxin partially reduced isoproterenol-induced cAMP deficit in Alzheimer fibroblasts. Pertussis toxin, on the other hand, did not alter the Alzheimer deficits. The results suggest either that the coupling of the GTP-binding protein(s) to the beta-adrenergic receptor is abnormal or that the sensitivity of receptor is altered with Alzheimer's disease. Further, any hypothesis about Alzheimer's disease must explain why a reduced beta-adrenergic-stimulated cAMP formation persists in tissue culture.

  17. Exposure to residual concentrations of elements from a remediated coal fly ash spill does not adversely influence stress and immune responses of nestling tree swallows. (United States)

    Beck, Michelle L; Hopkins, William A; Hallagan, John J; Jackson, Brian P; Hawley, Dana M


    Anthropogenic activities often produce pollutants that can affect the physiology, growth and reproductive success of wildlife. Many metals and trace elements play important roles in physiological processes, and exposure to even moderately elevated concentrations of essential and non-essential elements could have subtle effects on physiology, particularly during development. We examined the effects of exposure to a number of elements from a coal fly ash spill that occurred in December 2008 and has since been remediated on the stress and immune responses of nestling tree swallows. We found that nestlings at the site of the spill had significantly greater blood concentrations of Cu, Hg, Se and Zn in 2011, but greater concentrations only of Se in 2012, in comparison to reference colonies. The concentrations of elements were below levels of significant toxicological concern in both years. In 2011, we found no relationship between exposure to elements associated with the spill and basal or stress-induced corticosterone concentrations in nestlings. In 2012, we found that Se exposure was not associated with cell-mediated immunity based on the response to phytohaemagglutinin injection. However, the bactericidal capacity of nestling plasma had a positive but weak association with blood Se concentrations, and this association was stronger at the spill site. Our results indicate that exposure to these low concentrations of elements had few effects on nestling endocrine and immune physiology. The long-term health consequences of low-level exposure to elements and of exposure to greater element concentrations in avian species require additional study.

  18. Cyclic di-AMP Is Critical for Listeria monocytogenes Growth, Cell Wall Homeostasis, and Establishment of Infection (United States)

    Witte, Chelsea E.; Whiteley, Aaron T.; Burke, Thomas P.; Sauer, John-Demian; Portnoy, Daniel A.; Woodward, Joshua J.


    ABSTRACT Listeria monocytogenes infection leads to robust induction of an innate immune signaling pathway referred to as the cytosolic surveillance pathway (CSP), characterized by expression of beta interferon (IFN-β) and coregulated genes. We previously identified the IFN-β stimulatory ligand as secreted cyclic di-AMP. Synthesis of c-di-AMP in L. monocytogenes is catalyzed by the diadenylate cyclase DacA, and multidrug resistance transporters are necessary for secretion. To identify additional bacterial factors involved in L. monocytogenes detection by the CSP, we performed a forward genetic screen for mutants that induced altered levels of IFN-β. One mutant that stimulated elevated levels of IFN-β harbored a transposon insertion in the gene lmo0052. Lmo0052, renamed here PdeA, has homology to a cyclic di-AMP phosphodiesterase, GdpP (formerly YybT), of Bacillus subtilis and is able to degrade c-di-AMP to the linear dinucleotide pApA. Reduction of c-di-AMP levels by conditional depletion of the di-adenylate cyclase DacA or overexpression of PdeA led to marked decreases in growth rates, both in vitro and in macrophages. Additionally, mutants with altered levels of c-di-AMP had different susceptibilities to peptidoglycan-targeting antibiotics, suggesting that the molecule may be involved in regulating cell wall homeostasis. During intracellular infection, increases in c-di-AMP production led to hyperactivation of the CSP. Conditional depletion of dacA also led to increased IFN-β expression and a concomitant increase in host cell pyroptosis, a result of increased bacteriolysis and subsequent bacterial DNA release. These data suggest that c-di-AMP coordinates bacterial growth, cell wall stability, and responses to stress and plays a crucial role in the establishment of bacterial infection. PMID:23716572

  19. Expression of MUC17 is regulated by HIF1α-mediated hypoxic responses and requires a methylation-free hypoxia responsible element in pancreatic cancer.

    Directory of Open Access Journals (Sweden)

    Sho Kitamoto

    Full Text Available MUC17 is a type 1 membrane-bound glycoprotein that is mainly expressed in the digestive tract. Recent studies have demonstrated that the aberrant overexpression of MUC17 is correlated with the malignant potential of pancreatic ductal adenocarcinomas (PDACs; however, the exact regulatory mechanism of MUC17 expression has yet to be identified. Here, we provide the first report of the MUC17 regulatory mechanism under hypoxia, an essential feature of the tumor microenvironment and a driving force of cancer progression. Our data revealed that MUC17 was significantly induced by hypoxic stimulation through a hypoxia-inducible factor 1α (HIF1α-dependent pathway in some pancreatic cancer cells (e.g., AsPC1, whereas other pancreatic cancer cells (e.g., BxPC3 exhibited little response to hypoxia. Interestingly, these low-responsive cells have highly methylated CpG motifs within the hypoxia responsive element (HRE, 5'-RCGTG-3', a binding site for HIF1α. Thus, we investigated the demethylation effects of CpG at HRE on the hypoxic induction of MUC17. Treatment of low-responsive cells with 5-aza-2'-deoxycytidine followed by additional hypoxic incubation resulted in the restoration of hypoxic MUC17 induction. Furthermore, DNA methylation of HRE in pancreatic tissues from patients with PDACs showed higher hypomethylation status as compared to those from non-cancerous tissues, and hypomethylation was also correlated with MUC17 mRNA expression. Taken together, these findings suggested that the HIF1α-mediated hypoxic signal pathway contributes to MUC17 expression, and DNA methylation of HRE could be a determinant of the hypoxic inducibility of MUC17 in pancreatic cancer cells.

  20. Roles of Salicylic Acid-responsive Cis-acting Elements and W-boxes in Salicylic Acid Induction of VCH3 Promoter in Transgenic Tobaccos

    Institute of Scientific and Technical Information of China (English)

    Hai-Yan LI; Wei WEI; Yu LI


    A salicylic acid (SA)-inducible VCH3 promoter was recently identified from grapevine (Vitis amurensis) that contains two inverse SA-responsive cis-acting elements and four W-boxes. To further demonstrate the roles of these elements, four fragments with lengths from -1187, -892, -589, -276 to +7 bp were fused with the β-glucuronidase (GUS) reporter gene and transferred to Nicotiana tobacum,together with another four VCH3 promoter fragments with mutation in the two inverse SA-responsive elements. The functions of each promoter fragment were examined by analysis of GUS activity in the transgenic tobacco root treated with SA. Enhanced GUS activity was shown in the roots of transgenic tobaccos with the VCH3 (-1187)-GUS construct containing two SA-responsive cis-acting elements and four W-boxes. However, GUS activity directed by the VCH3 (-892)-GUS construct, containing one SA cisacting element and four W-boxes, was reduced by up to 35% compared with that in tobaccos transformed with the VCH3 (-1187)-GUS construct, indicating that the SA cis-acting element plays an important role in SA induction of the VCH3 promoter. Neither the m2VCH3 (-1187)-GUS nor the m VCH3 (-892)-GUSconstruct, with mutation on the SA-responsive elements, abolished the expression of GUS activity, demonstrating that the W-boxes in the VCH3 promoter are also involved in SA induction. Histochemical analysis of GUS activity directed by each of the eight VCH3 promoter fragments showed that GUS was expressed specifically in vascular tissue. It was concluded that both the SA-responsive cis-acting elements and the Wboxes are important for the SA induction of the VCH3 promoter. This promoter might have a potential use in plant genetic engineering.

  1. Hydroponics: A Versatile System to Study Nutrient Allocation and Plant Responses to Nutrient Availability and Exposure to Toxic Elements. (United States)

    Nguyen, Nga T; McInturf, Samuel A; Mendoza-Cózatl, David G


    Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several crops, including lettuce and tomato. Within the plant research community, numerous hydroponic systems have been designed to study plant responses to biotic and abiotic stresses. Here we present a hydroponic protocol that can be easily implemented in laboratories interested in pursuing studies on plant mineral nutrition. This protocol describes the hydroponic system set up in detail and the preparation of plant material for successful experiments. Most of the materials described in this protocol can be found outside scientific supply companies, making the set up for hydroponic experiments less expensive and convenient. The use of a hydroponic growth system is most advantageous in situations where the nutrient media need to be well controlled and when intact roots need to be harvested for downstream applications. We also demonstrate how nutrient concentrations can be modified to induce plant responses to both essential nutrients and toxic non-essential elements.

  2. Finite Element Modeling and Analysis of Nonlinear Impact and Frictional Motion Responses Including Fluid—Structure Coupling Effects

    Directory of Open Access Journals (Sweden)

    Yong Zhao


    Full Text Available A nonlinear three dimensional (3D single rack model and a nonlinear 3D whole pool multi-rack model are developed for the spent fuel storage racks of a nuclear power plant (NPP to determine impacts and frictional motion responses when subjected to 3D excitations from the supporting building floor. The submerged free standing rack system and surrounding water are coupled due to hydrodynamic fluid-structure interaction (FSI using potential theory. The models developed have features that allow consideration of geometric and material nonlinearities including (1 the impacts of fuel assemblies to rack cells, a rack to adjacent racks or pool walls, and rack support legs to the pool floor; (2 the hydrodynamic coupling of fuel assemblies with their storing racks, and of a rack with adjacent racks, pool walls, and the pool floor; and (3 the dynamic motion behavior of rocking, twisting, and frictional sliding of rack modules. Using these models 3D nonlinear time history dynamic analyses are performed per the U.S. Nuclear Regulatory Commission (USNRC criteria. Since few such modeling, analyses, and results using both the 3D single and whole pool multiple rack models are available in the literature, this paper emphasizes description of modeling and analysis techniques using the SOLVIA general purpose nonlinear finite element code. Typical response results with different Coulomb friction coefficients are presented and discussed.

  3. Discrete element modeling of Martian pit crater formation in response to extensional fracturing and dilational normal faulting (United States)

    Smart, Kevin J.; Wyrick, Danielle Y.; Ferrill, David A.


    Pit craters, circular to elliptical depressions that lack a raised rim or ejecta deposits, are common on the surface of Mars. Similar structures are also found on Earth, Venus, the Moon, and smaller planetary bodies, including some asteroids. While it is generally accepted that these pits form in response to material drainage into a subsurface void space, the primary mechanism(s) responsible for creating the void is a subject of debate. Previously proposed mechanisms include collapse into lave tubes, dike injection, extensional fracturing, and dilational normal faulting. In this study, we employ two-dimensional discrete element models to assess both extensional fracturing and dilational normal faulting as mechanisms for forming pit craters. We also examine the effect of mechanical stratigraphy (alternating strong and weak layers) and variation in regolith thickness on pit morphology. Our simulations indicate that both extensional fracturing and dilational normal faulting are viable mechanisms. Both mechanisms lead to generally convex (steepening downward) slope profiles; extensional fracturing results in generally symmetric pits, whereas dilational normal faulting produces strongly asymmetric geometries. Pit width is established early, whereas pit depth increases later in the deformation history. Inclusion of mechanical stratigraphy results in wider and deeper pits, particularly for the dilational normal faulting, and the presence of strong near-surface layers leads to pits with distinct edges as observed on Mars. The modeling results suggest that a thicker regolith leads to wider but shallower pits that are less distinct and may be more difficult to detect in areas of thick regolith.

  4. Minocycline upregulates cyclic AMP response element binding protein and brain-derived neurotrophic factor in the hippocampus of cerebral ischemia rats and improves behavioral deficits


    Zhao Y.; Xiao M; He W; Cai Z.


    Yu Zhao,1 Ming Xiao,2 Wenbo He,3 Zhiyou Cai3 1Department of Neurology, the Fourth Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province, People’s Republic of China; 2Department of Anatomy, Nanjing Medical University, Nanjing, Jiangsu, People’s Republic of China; 3Department of Neurology, Renmin Hospital, Hubei University of Medicine, Shiyan Renmin Hospital, Shiyan, Hubei Province, People’s Republic o...

  5. Radioprotection of the rat parotid gland by cAMP

    Energy Technology Data Exchange (ETDEWEB)

    Sodicoff, M.; Conger, A.D.


    Most earlier studies showing a radioprotective effect by cAMP show only slight degrees of protection. The present study demonstrates a substantial protective effect (DMF, 1.63) of exogenously administered cAMP on the rat parotid gland and supports the mechanism suggested previously for protection afforded the parotid glands by the ..beta..-adrenergic agonist isoproterenol, which is known to elevate endogenous intracellular cAMP.

  6. Elevated dopamine D2 receptor in prefrontal cortex of CUMS rats is associated with downregulated cAMP-independent signaling pathway. (United States)

    Chen, Cheng; Yang, Jing-mo; Hu, Ting-ting; Xu, Ting-juan; Xu, Wei-ping; Wei, Wei


    Because depression is associated with significant morbidity and functional disability, it is important to reveal the mechanism of action. A variety of studies have suggested the involvement of dopaminergic receptors in the pathophysiological mechanism of non-stress-associated depression-like behavior in rodents. Nevertheless, controversy exists about whether chronic stress acts on dopaminergic receptors in the prefrontal cortex. Thus, we investigated the level of dopamine D2 receptors (DRD2) and the possible mechanisms involved in a chronic unpredictable mild stress (CUMS) rat model of depression. The results showed CUMS-induced, depression-like symptoms in the rat, characterized by reduced sucrose consumption and body mass, and increased duration of immobility in a forced swimming test. Moreover, chronic stress upregulated the expression of DRD2 but downregulated protein kinase A (PKA), transcription factor cAMP response element binding protein (CREB), and phospho-CREB (p-CREB) in the prefrontal cortex, as demonstrated by Western blot. Notably, in the rat model of depression, decreased cyclic adenine monophosphate (cAMP) levels and PKA activity were present at the same time, which is consistent with clinical findings in depressed patients. Our findings suggested that dopaminergic system dysfunction could play a central role in stress-related disorders such as depression.

  7. Amp C酶及其耐药性

    Institute of Scientific and Technical Information of China (English)

    赵虎; 孔宪涛


    本文叙述了Amp C酶的定义、Amp C酶的基因结构与功能、Amp C酶的合成、Amp C酶的耐药性及其耐药机制,以及Amp C酶的检测方法。Amp C酶是由肠杆菌科细菌和铜绿假单胞菌等产生的一类头孢菌素酶,可水解头孢菌素类抗生素,导致细菌对这类抗生素产生耐药性。编码产生Amp C酶的基因包括结构基因-amp C和四种调控基因-ampR、ampD、ampE以及ampG,但其具体的转录、调控机制目前尚未完全明了。Amp C酶的合成具有明显的诱导性,其诱导有菌种依赖性、抗生素依赖性和生长条件依赖性。Amp C酶的耐药机制主要是作用于头胞菌素的β-内酰胺环上的羰基,形成酰化酶中间体,然后在水分子的作用下导致β-内酰胺环开环而失活。大部分Amp C酶都是由细菌染色体所介导,但近年来陆续在质粒上发现这些基因,并在肺炎克雷伯菌、大肠埃希菌、产气肠杆菌和沙门菌中持续高水平的表达,还可以通过质粒在细菌间相互传播,导致耐药菌的广泛传播。产Amp C酶菌株的检测以改良的酶提取物三维试验法较佳。

  8. S-AMP for non-linear observation models

    DEFF Research Database (Denmark)

    Cakmak, Burak; Winther, Ole; Fleury, Bernard H.


    Recently we presented the S-AMP approach, an extension of approximate message passing (AMP), to be able to handle general invariant matrix ensembles. In this contribution we extend S-AMP to non-linear observation models. We obtain generalized AMP (GAMP) as the special case when the measurement...... matrix has zero-mean iid Gaussian entries. Our derivation is based upon 1) deriving expectation-propagation-(EP)-like equations from the stationary-points equations of the Gibbs free energy under first- and second-moment constraints and 2) applying additive free convolution in free probability theory...

  9. AmpC酶的研究进展

    Institute of Scientific and Technical Information of China (English)




  10. Qushi Huayu Decoction Inhibits Hepatic Lipid Accumulation by Activating AMP-Activated Protein Kinase In Vivo and In Vitro

    Directory of Open Access Journals (Sweden)

    Qin Feng


    Full Text Available Qushi Huayu Decoction (QHD, a Chinese herbal formula, has been proven effective on alleviating nonalcoholic fatty liver disease (NAFLD in human and rats. The present study was conducted to investigate whether QHD could inhibit hepatic lipid accumulation by activating AMP-activated protein kinase (AMPK in vivo and in vitro. Nonalcoholic fatty liver (NAFL model was duplicated with high-fat diet in rats and with free fatty acid (FFA in L02 cells. In in vivo experimental condition, QHD significantly decreased the accumulation of fatty droplets in livers, lowered low-density lipoprotein cholesterol (LDL-c, alanine aminotransferase (ALT, and aspartate aminotransferase (AST levels in serum. Moreover, QHD supplementation reversed the HFD-induced decrease in the phosphorylation levels of AMPK and acetyl-CoA carboxylase (ACC and decreased hepatic nuclear protein expression of sterol regulatory element-binding protein-1 (SREBP-1 and carbohydrate-responsive element-binding protein (ChREBP in the liver. In in vitro, QHD-containing serum decreased the cellular TG content and alleviated the accumulation of fatty droplets in L02 cells. QHD supplementation reversed the FFA-induced decrease in the phosphorylation levels of AMPK and ACC and decreased the hepatic nuclear protein expression of SREBP-1 and ChREBP. Overall results suggest that QHD has significant effect on inhibiting hepatic lipid accumulation via AMPK pathway in vivo and in vitro.

  11. Antagonists of chemoattractants reveal separate receptors for cAMP, folic acid and pterin in Dictyostelium

    NARCIS (Netherlands)

    Haastert, Peter J.M. van; Wit, René J.W. de; Konijn, Theo M.


    Adenosine 3’,5’-monophosphate (cAMP), folic acid and pterin are chemoattractants in the cellular slime molds. The cAMP analog, 3’-amino-cAMP, inhibits a chemotactic reaction to cAMP at a concentration at which the analog is chemotactically inactive. The antagonistic effect of 3’-amino-cAMP on the ch

  12. Identification of fungus-responsive cis-acting element in the promoter of Brassica juncea chitinase gene, BjCHI1. (United States)

    Gao, Ying; Zan, Xin-Li; Wu, Xue-Feng; Yao, Lei; Chen, Yu-Ling; Jia, Shuang-Wei; Zhao, Kai-Jun


    Chitinases are a group of pathogenesis-related proteins. The Brassica juncea chitinase gene BjCHI1 is highly inducible by pathogenic fungal infection, suggesting that the promoter of BjCHI1 might contain specific cis-acting element responsive to fungal attack. To identify the fungus-responsive element in BjCHI1 promoter (BjC-P), a series of binary plant transformation vectors were constructed by fusing the BjC-P or its deletion-derivatives to β-glucuronidase (GUS) reporter gene. Expression of the GUS reporter gene was systematically assayed by a transient gene expression system in Nicotiana benthamiana leaves treated with fungal elicitor Hexa-N-Acetyl-Chitohexaose, as well as in transgenic Arabidopsis plants inoculated with fungus Botrytis cinerea. The histochemical and quantitative GUS assays showed that the W-box-like element (GTAGTGACTCAT) in the region (-668 to -657) was necessary for the fungus-response, although there were another five W-box-like elements in BjC-P. In addition, gain-of-function analysis demonstrated that the fragment (-409 to -337) coupled to the W-box-like element was needed for full magnitude of the fungal induction. These results revealed the existence of a novel regulation mechanism of W-box-like element involved in plant pathogenic resistance, and will benefit the potential application of BjC-P in engineering crops.

  13. Engineering of a red-light-activated human cAMP/cGMP-specific phosphodiesterase. (United States)

    Gasser, Carlos; Taiber, Sandra; Yeh, Chen-Min; Wittig, Charlotte Helene; Hegemann, Peter; Ryu, Soojin; Wunder, Frank; Möglich, Andreas


    Sensory photoreceptors elicit vital physiological adaptations in response to incident light. As light-regulated actuators, photoreceptors underpin optogenetics, which denotes the noninvasive, reversible, and spatiotemporally precise perturbation by light of living cells and organisms. Of particular versatility, naturally occurring photoactivated adenylate cyclases promote the synthesis of the second messenger cAMP under blue light. Here, we have engineered a light-activated phosphodiesterase (LAPD) with complementary light sensitivity and catalytic activity by recombining the photosensor module of Deinococcus radiodurans bacterial phytochrome with the effector module of Homo sapiens phosphodiesterase 2A. Upon red-light absorption, LAPD up-regulates hydrolysis of cAMP and cGMP by up to sixfold, whereas far-red light can be used to down-regulate activity. LAPD also mediates light-activated cAMP and cGMP hydrolysis in eukaryotic cell cultures and in zebrafish embryos; crucially, the biliverdin chromophore of LAPD is available endogenously and does not need to be provided exogenously. LAPD thus establishes a new optogenetic modality that permits light control over diverse cAMP/cGMP-mediated physiological processes. Because red light penetrates tissue more deeply than light of shorter wavelengths, LAPD appears particularly attractive for studies in living organisms.

  14. Synapse number and synaptic efficacy are regulated by presynaptic cAMP and protein kinase A. (United States)

    Munno, David W; Prince, David J; Syed, Naweed I


    The mechanisms by which neurons regulate the number and strength of synapses during development and synaptic plasticity have not yet been defined fully. This lack of fundamental knowledge in the fields of neurodevelopment and synaptic plasticity can be attributed, in part, to compensatory mechanisms by which neurons accommodate for the loss of function in their synaptic partners. This is generally achieved either by scaling up neuronal transmitter release capabilities or by enhancing the postsynaptic responsiveness. Here, we demonstrate that regulation of synaptic strength and number between identified Lymnaea neurons visceral dorsal 4 (VD4, the presynaptic cell) and left pedal dorsal 1 (LPeD1, the postsynaptic cell) requires presynaptic activation of a cAMP-PKA-dependent signal. Experimental activation of the cAMP-PKA pathway resulted in reduced synaptic efficacy, whereas inhibition of the cAMP-PKA cascade permitted hyperinnervation and an overall enhancement of synaptic strength. Because synaptic transmission between VD4 and LPeD1 does not require a cAMP-PKA pathway, our data show that these messengers may play a novel role in regulating the synaptic efficacy during early synaptogenesis and plasticity.

  15. Coordinated induction of GST and MRP2 by cAMP in Caco-2 cells: Role of protein kinase A signaling pathway and toxicological relevance

    Energy Technology Data Exchange (ETDEWEB)

    Arana, Maite Rocío, E-mail: [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Tocchetti, Guillermo Nicolás, E-mail: [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Domizi, Pablo, E-mail: [Instituto de Biología Molecular y Celular de Rosario (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Arias, Agostina, E-mail: [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Rigalli, Juan Pablo, E-mail: [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Ruiz, María Laura, E-mail: [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); and others


    The cAMP pathway is a universal signaling pathway regulating many cellular processes including metabolic routes, growth and differentiation. However, its effects on xenobiotic biotransformation and transport systems are poorly characterized. The effect of cAMP on expression and activity of GST and MRP2 was evaluated in Caco-2 cells, a model of intestinal epithelium. Cells incubated with the cAMP permeable analog dibutyryl cyclic AMP (db-cAMP: 1,10,100 μM) for 48 h exhibited a dose–response increase in GST class α and MRP2 protein expression. Incubation with forskolin, an activator of adenylyl cyclase, confirmed the association between intracellular cAMP and upregulation of MRP2. Consistent with increased expression of GSTα and MRP2, db-cAMP enhanced their activities, as well as cytoprotection against the common substrate 1-chloro-2,4-dinitrobenzene. Pretreatment with protein kinase A (PKA) inhibitors totally abolished upregulation of MRP2 and GSTα induced by db-cAMP. In silico analysis together with experiments consisting of treatment with db-cAMP of Caco-2 cells transfected with a reporter construct containing CRE and AP-1 sites evidenced participation of these sites in MRP2 upregulation. Further studies involving the transcription factors CREB and AP-1 (c-JUN, c-FOS and ATF2) demonstrated increased levels of total c-JUN and phosphorylation of c-JUN and ATF2 by db-cAMP, which were suppressed by a PKA inhibitor. Co-immunoprecipitation and ChIP assay studies demonstrated that db-cAMP increased c-JUN/ATF2 interaction, with further recruitment to the region of the MRP2 promoter containing CRE and AP-1 sites. We conclude that cAMP induces GSTα and MRP2 expression and activity in Caco-2 cells via the PKA pathway, thus regulating detoxification of specific xenobiotics. - Highlights: • cAMP positively modulates the expression and activity of GST and MRP2 in Caco-2 cells. • Such induction resulted in increased cytoprotection against chemical injury. • PKA

  16. p85 regulatory subunit of PI3K mediates cAMP-PKA and estrogens biological effects on growth and survival. (United States)

    Cosentino, C; Di Domenico, M; Porcellini, A; Cuozzo, C; De Gregorio, G; Santillo, M R; Agnese, S; Di Stasio, R; Feliciello, A; Migliaccio, A; Avvedimento, E V


    Cyclic adenosine 3'5' monophosphate (cAMP) and protein kinase A (PKA) cooperate with phosphatidylinositol 3' kinase (PI3K) signals in the control of growth and survival. To determine the molecular mechanism(s) involved, we identified and mutagenized a specific serine (residue 83) in p85alpha(PI3K), which is phosphorylated in vivo and in vitro by PKA. Expression of p85alpha(PI3K) mutants (alanine or aspartic substitutions) significantly altered the biological responses of the cells to cAMP. cAMP protection from anoikis was reduced in cells expressing the alanine version p85alpha(PI3K). These cells did not arrest in G1 in the presence of cAMP, whereas cells expressing the aspartic mutant p85D accumulated in G1 even in the absence of cAMP. S phase was still efficiently inhibited by cAMP in cells expressing both mutants. The binding of PI3K to Ras p21 was greatly reduced in cells expressing p85A in the presence or absence of cAMP. Conversely, expression of the aspartic mutant stimulated robustly the binding of PI3K to p21 Ras in the presence of cAMP. Mutation in the Ser 83 inhibited cAMP, but not PDGF stimulation of PI3K. Conversely, the p85D aspartic mutant amplified cAMP stimulation of PI3K activity. Phosphorylation of Ser 83 by cAMP-PKA in p85alpha(PI3K) was also necessary for estrogen signaling as expression of p85A or p85D mutants inhibited or amplified, respectively, the binding of estrogen receptor to p85alpha and AKT phosphorylation induced by estrogens. The data presented indicate that: (1) phosphorylation of Ser 83 in p85alpha(PI3K) is critical for cAMP-PKA induced G1 arrest and survival in mouse 3T3 fibroblasts; (2) this site is necessary for amplification of estrogen signals by cAMP-PKA and related receptors. Finally, these data suggest a general mechanism of PI3K regulation by cAMP, operating in various cell types and under different conditions.

  17. Berberine attenuates cAMP-induced lipolysis via reducing the inhibition of phosphodiesterase in 3T3-L1 adipocytes. (United States)

    Zhou, Libin; Wang, Xiao; Yang, Ying; Wu, Ling; Li, Fengying; Zhang, Rong; Yuan, Guoyue; Wang, Ning; Chen, Mingdao; Ning, Guang


    Berberine, a hypoglycemic agent, has been shown to decrease plasma free fatty acids (FFAs) level in insulin-resistant rats. In the present study, we explored the mechanism responsible for the antilipolytic effect of berberine in 3T3-L1 adipocytes. It was shown that berberine attenuated lipolysis induced by catecholamines, cAMP-raising agents, and a hydrolyzable cAMP analog, but not by tumor necrosis factor α and a nonhydrolyzable cAMP analog. Unlike insulin, the inhibitory effect of berberine on lipolysis in response to isoproterenol was not abrogated by wortmannin, an inhibitor of phosphatidylinositol 3-kinase, but additive to that of PD98059, an extracellular signal-regulated kinase kinase inhibitor. Prior exposure of adipocytes to berberine decreased the intracellular cAMP production induced by isoproterenol, forskolin, and 3-isobutyl-1-methylxanthine (IBMX), along with hormone-sensitive lipase (HSL) Ser-563 and Ser-660 dephosphorylation, but had no effect on perilipin phosphorylation. Berberine stimulated HSL Ser-565 as well as adenosine monophosphate-activated protein kinase (AMPK) phosphorylation. However, compound C, an AMPK inhibitor, did not reverse the regulatory effect of berberine on HSL Ser-563, Ser-660, and Ser-565 phosphorylation, nor the antilipolytic effect of berberine. Knockdown of AMPK using RNA interference also failed to restore berberine-suppressed lipolysis. cAMP-raising agents increased AMPK activity, which was not additive to that of berberine. Stimulation of adipocytes with berberine increased phosphodiesterase (PDE) 3B and PDE4 activity measured by hydrolysis of (3)[H]cAMP. These results suggest that berberine exerts an antilipolytic effect mainly by reducing the inhibition of PDE, leading to a decrease in cAMP and HSL phosphorylation independent of AMPK pathway.

  18. cAMP elevators inhibit LPS-induced IL-12 p40 expression by interfering with phosphorylation of p38 MAPK in Murine Peritoneal Macrophages

    Institute of Scientific and Technical Information of China (English)



    cAMP mediated signaling may play a suppressive role in immune response. We previously found thatthe cAMP-elevators (CTx and 8-Br-cAMP) inhibited IL-12, IL-la, IL-6 gene expression, but increasedthe transcriptional levels of IL-10 and IL-1Ra in LPS-treated murine peritoneal macrophages. The presentstudy examined a possible molecular mechanism involved in cAMP elevators-induced inhibition of IL-12 p40expression in response to LPS. Our data demonstrated that cAMP elevators downregulated IL-12 p40 mRNAexpression and IL-12 p70 production in murine peritoneal macrophages. Subsequent studies revealed thatcAMP-elevators blocked phosphorylation of p38 MAPK, but did not affect the activity of NF-κB bindingto IL-12 promoter (-136/-112). This is the first report that cAMP elevators inhibit LPS-induced IL-12production by a mechanism that is associated, at least in part, with p38-dependent inhibition by cAMPsignaling pathways.

  19. Cyclic AMP effectors in African trypanosomes revealed by genome-scale RNA interference library screening for resistance to the phosphodiesterase inhibitor CpdA. (United States)

    Gould, Matthew K; Bachmaier, Sabine; Ali, Juma A M; Alsford, Sam; Tagoe, Daniel N A; Munday, Jane C; Schnaufer, Achim C; Horn, David; Boshart, Michael; de Koning, Harry P


    One of the most promising new targets for trypanocidal drugs to emerge in recent years is the cyclic AMP (cAMP) phosphodiesterase (PDE) activity encoded by TbrPDEB1 and TbrPDEB2. These genes were genetically confirmed as essential, and a high-affinity inhibitor, CpdA, displays potent antitrypanosomal activity. To identify effectors of the elevated cAMP levels resulting from CpdA action and, consequently, potential sites for adaptations giving resistance to PDE inhibitors, resistance to the drug was induced. Selection of mutagenized trypanosomes resulted in resistance to CpdA as well as cross-resistance to membrane-permeable cAMP analogues but not to currently used trypanocidal drugs. Resistance was not due to changes in cAMP levels or in PDEB genes. A second approach, a genome-wide RNA interference (RNAi) library screen, returned four genes giving resistance to CpdA upon knockdown. Validation by independent RNAi strategies confirmed resistance to CpdA and suggested a role for the identified cAMP Response Proteins (CARPs) in cAMP action. CARP1 is unique to kinetoplastid parasites and has predicted cyclic nucleotide binding-like domains, and RNAi repression resulted in >100-fold resistance. CARP2 and CARP4 are hypothetical conserved proteins associated with the eukaryotic flagellar proteome or with flagellar function, with an orthologue of CARP4 implicated in human disease. CARP3 is a hypothetical protein, unique to Trypanosoma. CARP1 to CARP4 likely represent components of a novel cAMP signaling pathway in the parasite. As cAMP metabolism is validated as a drug target in Trypanosoma brucei, cAMP effectors highly divergent from the mammalian host, such as CARP1, lend themselves to further pharmacological development.

  20. Inhibition of hormone-sensitive lipase gene expression by cAMP and phorbol esters in 3T3-F442A and BFC-1 adipocytes. (United States)

    Plée-Gautier, E; Grober, J; Duplus, E; Langin, D; Forest, C


    Hormone-sensitive lipase (HSL) catalyses the rate-limiting step in adipocyte lipolysis. Short-term hormonal regulation of HSL activity is well characterized, whereas little is known about the control of HSL gene expression. We have measured HSL mRNA content of 3T3-F442A and BFC-1 adipocytes in response to the cAMP analogue 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP) and to the phorbol ester phorbol 12-myristate 13-acetate (PMA) by Northern blot, using a specific mouse cDNA fragment. Treatment of the cells for 12 or 6 h with, respectively, 0.5 mM 8-CPT-cAMP or 1 microM PMA produced a maximal decrease of about 60% in HSL mRNA. These effects were unaffected by the protein-synthesis inhibitor anisomycin, suggesting that cAMP and PMA actions were direct. The reduction in HSL mRNA was accompanied by a reduction in HSL total activity. The intracellular routes that cAMP and PMA follow for inducing such an effect seemed clearly independent. (i) After desensitization of the protein kinase C regulation pathway by a 24 h treatment of the cells with 1 microM PMA, PMA action was abolished whereas cAMP was still fully active. (ii) Treatment with saturating concentrations of both agents produced an additive effect. (iii) The synthetic glucocorticoid dexamethasone had no proper effect on HSL gene expression but potentiated cAMP action without affecting PMA action. cAMP inhibitory action on HSL is unexpected. Indeed, the second messenger of catecholamines is the main activator of HSL by phosphorylation. We envision that a long-term cAMP treatment of adipocytes induces a counter-regulatory process that reduces HSL content and, ultimately, limits fatty acid depletion from stored triacylglycerols.

  1. Revisiting cAMP signaling in the carotid body

    Directory of Open Access Journals (Sweden)

    Ana Rita eNunes


    Full Text Available Chronic carotid body (CB activation is now recognized as being essential in the development of hypertension and promoting insulin resistance; thus, it is imperative to characterize the chemotransduction mechanisms of this organ in order to modulate its activity and improve patient outcomes. For several years, and although controversial, cyclic adenosine monophosphate (cAMP was considered an important player in initiating the activation of the CB. However, its relevance was partially displaced in the 90s by the emerging role of the mitochondria and molecules such as AMP-activated protein kinase (AMPK and O2-sensitive K+ channels. Neurotransmitters/neuromodulators binding to metabotropic receptors are essential to chemotransmission in the CB, and cAMP is central to this process. cAMP also contributes to raise intracellular Ca2+ levels, and is intimately related to the cellular energetic status (AMP/ATP ratio. Furthermore, cAMP signaling is a target of multiple current pharmacological agents used in clinical practice. This review provides an outline on 1 the classical view of the cAMP-signaling pathway in the CB that originally supported its role in the O2/CO2 sensing mechanism, 2 present recent evidence on CB cAMP neuromodulation and 3 discuss how CB activity is affected by current clinical therapies that modify cAMP-signaling, namely dopaminergic drugs, caffeine (modulation of A2A/A2B receptors and roflumilast (PDE4 inhibitors. cAMP is key to any process that involves metabotropic receptors and the intracellular pathways involved in CB disease states are likely to involve this classical second messenger. Research examining the potential modification of cAMP levels and/or interactions with molecules associated with CB hyperactivity is currently in its beginning and this review will open doors for future explorations.

  2. Induction of the mammalian stress response gene GADD153 by oxidative stress: role of AP-1 element. (United States)

    Guyton, K Z; Xu, Q; Holbrook, N J


    GADD153 is a CCAAT/enhancer-binding-protein-related gene that may function to control cellular growth in response to stress signals. In this study, a variety of oxidant treatments were shown to stimulate endogenous GADD153 mRNA expression and to transcriptionally activate a GADD153 promoter-reporter gene construct in transfected HeLa cells. Both commonalities and distinctions in the induction of GADD153 by H2O2 and the thiol-reactive compound arsenite were demonstrated. GADD153 mRNA induction by both H2O2 and arsenite was potentiated by GSH depletion, and completely inhibited by N-acetyl-cysteine. o-Phenanthroline and mannitol blocked GADD153 induction by H2O2, indicating that iron-generated hydroxyl radical mediates this induction. Concordantly, GSH peroxidase overexpression in WI38 cells attenuated GADD153 mRNA induction by H2O2. However, GADD153 induction by arsenite was only modestly reduced in the same cells, suggesting a lesser contribution of peroxides to gene activation by arsenite. We also demonstrated that oxidative stress participates in the induction of GADD153 by UVC (254 nm) irradiation. Finally, both promoter-deletion analysis and point mutation of the AP-1 site in an otherwise intact promoter support a significant role for AP-1 in transcriptional activation of GADD153 by UVC or oxidant treatment. Indeed, exposure of cells to oxidants or UVC stimulated binding of Fos and Jun to the GADD153 AP-1 element. Together, these results demonstrate that both free-radical generation and thiol modification can transcriptionally activate GADD153, and that AP-1 is critical to oxidative regulation of this gene. This study further supports a role for the GADD153 gene product in the cellular response to oxidant injury. PMID:8670069

  3. Intrauterine growth restriction perturbs nucleosome depletion at a growth hormone-responsive element in the mouse IGF-1 gene. (United States)

    McKnight, Robert A; Yost, Christian C; Yu, Xing; Wiedmeier, Julia E; Callaway, Christopher W; Brown, Ashley S; Lane, Robert H; Fung, Camille M


    Intrauterine growth restriction (IUGR) is a common human pregnancy complication. IUGR offspring carry significant postnatal risk for early-onset metabolic syndrome, which is associated with persistent reduction in IGF-1 protein expression. We have previously shown that preadolescent IUGR male mice have decreased hepatic IGF-1 mRNA and circulating IGF-1 protein at postnatal day 21, the age when growth hormone (GH) normally upregulates hepatic IGF-1 expression. Here we studied nucleosome occupancy and CpG methylation at a putative growth hormone-responsive element in intron 2 (in2GHRE) of the hepatic IGF-1 gene in normal, sham-operated, and IUGR mice. Nucleosome occupancy and CpG methylation were determined in embryonic stem cells (ESCs) and in liver at postnatal days 14, 21, and 42. For CpG methylation, additional time points out to 2 yr were analyzed. We confirmed the putative mouse in2GHRE was GH-responsive, and in normal mice, a single nucleosome was displaced from the hepatic in2GHRE by postnatal day 21, which exposed two STAT5b DNA binding sites. Nucleosome displacement correlated with developmentally programmed CpG demethylation. Finally, IUGR significantly altered the nucleosome-depleted region (NDR) at the in2GHRE of IGF-1 on postnatal day 21, with either complete absence of the NDR or with a shifted NDR exposing only one of two STAT5b DNA binding sites. An NDR shift was also seen in offspring of sham-operated mothers. We conclude that prenatal insult such as IUGR or anesthesia/surgery could perturb the proper formation of a well-positioned NDR at the mouse hepatic IGF-1 in2GHRE necessary for transitioning to an open chromatin state.

  4. Cloning and characterization of the dehydration-responsive element-binding protein 2A gene in Eruca vesicaria subsp sativa. (United States)

    Huang, B L; Zhang, X K; Li, Y Y; Li, D Y; Ma, M Y; Cai, D T; Wu, W H; Huang, B Q


    Eruca vesicaria subsp sativa is one of the most tolerant Cruciferae species to drought, and dehydration-responsive element-binding protein 2A (DREB2A) is involved in responses to salinity, heat, and particularly drought. In this study, a gene encoding EvDREB2A was cloned and characterized in E. vesicaria subsp sativa. The full-length EvDREB2A cDNA sequence contained a 388-bp 5'-untranslated region (UTR), a 348-bp 3'-UTR, and a 1002-bp open reading frame that encoded 334 amino acid residues. The theoretical isoelectric point of the EvDREB2A protein was 4.80 and the molecular weight was 37.64 kDa. The genomic sequence of EvDREB2A contained no introns. Analysis using SMART indicated that EvDREB2A contains a conserved AP2 domain, similar to other plant DREBs. Phylogenetic analysis revealed that EvDREB2A and DREB2As from Brassica rapa, Eutrema salsugineum, Arabidopsis thaliana, Arabidopsis lyrata, and Arachis hypogaea formed a small subgroup, which clustered with DREB2Bs from A. lyrata, A. thaliana, Camelina sativa, and B. rapa to form a larger subgroup. EvDREB2A is most closely related to B. rapa DREB2A, followed by DREB2As from E. salsugineum, A. thaliana, A. hypogaea, and A. lyrata. A quantitative real-time polymerase chain reaction indicated that EvDREB2A expression was highest in the leaves, followed by the roots and hypocotyls, and was lowest in the flower buds. EvDREB2A could be used to improve drought tolerance in crops.

  5. Three-dimensional integral imaging displays using a quick-response encoded elemental image array: an overview (United States)

    Markman, A.; Javidi, B.


    Quick-response (QR) codes are barcodes that can store information such as numeric data and hyperlinks. The QR code can be scanned using a QR code reader, such as those built into smartphone devices, revealing the information stored in the code. Moreover, the QR code is robust to noise, rotation, and illumination when scanning due to error correction built in the QR code design. Integral imaging is an imaging technique used to generate a three-dimensional (3D) scene by combining the information from two-dimensional (2D) elemental images (EIs) each with a different perspective of a scene. Transferring these 2D images in a secure manner can be difficult. In this work, we overview two methods to store and encrypt EIs in multiple QR codes. The first method uses run-length encoding with Huffman coding and the double-random-phase encryption (DRPE) to compress and encrypt an EI. This information is then stored in a QR code. An alternative compression scheme is to perform photon-counting on the EI prior to compression. Photon-counting is a non-linear transformation of data that creates redundant information thus improving image compression. The compressed data is encrypted using the DRPE. Once information is stored in the QR codes, it is scanned using a smartphone device. The information scanned is decompressed and decrypted and an EI is recovered. Once all EIs have been recovered, a 3D optical reconstruction is generated.

  6. The expression of cyclic adenosine monophosphate responsive element modulator in rat sertoli cells following seminal extract administration (United States)

    Akmal, Muslim; Siregar, Tongku Nizwan; Wahyuni, Sri; Hamny; Nasution, Mustafa Kamal; Indriati, Wiwik; Panjaitan, Budianto; Aliza, Dwinna


    Aim: This study aims to determine the effect of seminal vesicle extract on cyclic adenosine monophosphate responsive element modulator (CREM) expression in rat Sertoli cells. Materials and Methods: This study examined the expression of CREM on 20 male rats (Rattus norvegicus) at 4 months of age, weighing 250-300 g. The rats were divided into four groups: K0, KP1, KP2, and KP3. K0 group was injected with 0.2 ml normal saline; KP1 was injected with 25 mg cloprostenol (Prostavet C, Virbac S. A); KP2 and KP3 were injected with 0.2 and 0.4 ml seminal vesicle extract, respectively. The treatments were conducted 5 times within 12-day interval. At the end of the study, the rats were euthanized by cervical dislocation; then, the testicles were necropsied and processed for histology observation using immunohistochemistry staining. Results: CREM expression in rat Sertoli cells was not altered by the administration of either 0.2 or 0.4 ml seminal vesicle extract. Conclusion: The administration of seminal vesicle extract is unable to increase CREM expression in rat Sertoli cells. PMID:27733803

  7. Immunohistochemical Study of Nrf2-Antioxidant Response Element as Indicator of Oxidative Stress Induced by Cadmium in Developing Rats

    Directory of Open Access Journals (Sweden)

    Sergio Montes


    Full Text Available In developing animals, Cadmium (Cd induces toxicity to many organs including brain. Reactive oxygen species (ROS are often implicated in Cd-inducedtoxicity and it has been clearly demonstrated that oxidative stress interferes with the expression of genes as well as transcriptional factors such as Nrf2-dependent Antioxidant Response Element (Nrf2-ARE. Cd-generated oxidative stress and elevated Nrf2 activity have been reported in vitro and in situ cells. In this study we evaluated the morphological changes and the expression pattern of Nrf2 and correlated them with the Cd concentrations in different ages of developing rats in heart, lung, kidney, liver, and brain. The Cd content in different organs of rats treated with the metal was increased in all ages assayed. Comparatively, lower Cd brain levels were found in rats intoxicated at the age of 12 days, then pups treated at 5, 10, or 15 days old, at the same metal dose. No evident changes, as a consequence of cadmium exposure, were evident in the morphological analysis in any of the ages assayed. However, Nrf2-ARE immunoreactivity was observed in 15-day-old rats exposed to Cd. Our results support that fully developed blood-brain barrier is an important protector against Cd entrance to brain and that Nrf2 increased expression is a part of protective mechanism against cadmium-induced toxicity.

  8. Bile acid-activated nuclear receptor FXR suppresses apolipoprotein A-I transcription via a negative FXR response element (United States)

    Claudel, Thierry; Sturm, Ekkehard; Duez, Hélène; Torra, Inés Pineda; Sirvent, Audrey; Kosykh, Vladimir; Fruchart, Jean-Charles; Dallongeville, Jean; Hum, Dean W.; Kuipers, Folkert; Staels, Bart


    Serum levels of HDL are inversely correlated with the risk of coronary heart disease. The anti-atherogenic effect of HDL is partially mediated by its major protein constituent apoA-I. In this study, we identify bile acids that are activators of the nuclear receptor farnesoid X receptor (FXR) as negative regulators of human apoA-I expression. Intrahepatocellular accumulation of bile acids, as seen in patients with progressive familial intrahepatic cholestasis and biliary atresia, was associated with diminished apoA-I serum levels. In human apoA-I transgenic mice, treatment with the FXR agonist taurocholic acid strongly decreased serum concentrations and liver mRNA levels of human apoA-I, which was associated with reduced serum HDL levels. Incubation of human primary hepatocytes and hepatoblastoma HepG2 cells with bile acids resulted in a dose-dependent downregulation of apoA-I expression. Promoter mutation analysis and gel-shift experiments in HepG2 cells demonstrated that bile acid–activated FXR decreases human apoA-I promoter activity by a negative FXR response element mapped to the C site. FXR bound this site and repressed transcription in a manner independent of retinoid X receptor. The nonsteroidal synthetic FXR agonist GW4064 likewise decreased apoA-I mRNA levels and promoter activity in HepG2 cells. PMID:11927623

  9. The structure formed by inverted repeats in p53 response elements determines the transactivation activity of p53 protein. (United States)

    Brázda, Václav; Čechová, Jana; Battistin, Michele; Coufal, Jan; Jagelská, Eva B; Raimondi, Ivan; Inga, Alberto


    The TP53 gene is the most frequently mutated gene in human cancer and p53 protein plays a crucial role in gene expression and cancer protection. Its role is manifested by interactions with other proteins and DNA. p53 is a transcription factor that binds to DNA response elements (REs). Due to the palindromic nature of the consensus binding site, several p53-REs have the potential to form cruciform structures. However, the influence of cruciform formation on the activity of p53-REs has not been evaluated. Therefore, we prepared sets of p53-REs with identical theoretical binding affinity in their linear state, but different probabilities to form extra helical structures, for in vitro and in vivo analyses. Then we evaluated the presence of cruciform structures when inserted into plasmid DNA and employed a yeast-based assay to measure transactivation potential of these p53-REs cloned at a chromosomal locus in isogenic strains. We show that transactivation in vivo correlated more with relative propensity of an RE to form cruciforms than to its predicted in vitro DNA binding affinity for wild type p53. Structural features of p53-REs could therefore be an important determinant of p53 transactivation function.


    Energy Technology Data Exchange (ETDEWEB)

    Hakan Ozaltun; Herman Shen; Pavel Madvedev


    This article presents numerical simulation of dispersion fuel mini plates via fluid–thermal–structural interaction performed by commercial finite element solver COMSOL Multiphysics to identify initial mechanical response under actual operating conditions. Since fuel particles are dispersed in Aluminum matrix, and temperatures during the fabrication process reach to the melting temperature of the Aluminum matrix, stress/strain characteristics of the domain cannot be reproduced by using simplified models and assumptions. Therefore, fabrication induced stresses were considered and simulated via image based modeling techniques with the consideration of the high temperature material data. In order to identify the residuals over the U7Mo particles and the Aluminum matrix, a representative SEM image was employed to construct a microstructure based thermo-elasto-plastic FE model. Once residuals and plastic strains were identified in micro-scale, solution was used as initial condition for subsequent multiphysics simulations at the continuum level. Furthermore, since solid, thermal and fluid properties are temperature dependent and temperature field is a function of the velocity field of the coolant, coupled multiphysics simulations were considered. First, velocity and pressure fields of the coolant were computed via fluidstructural interaction. Computed solution for velocity fields were used to identify the temperature distribution on the coolant and on the fuel plate via fluid-thermal interaction. Finally, temperature fields and residual stresses were used to obtain the stress field of the plates via fluid-thermal-structural interaction.

  11. A distal intergenic region controls pancreatic endocrine differentiation by acting as a transcriptional enhancer and as a polycomb response element (United States)

    Pardanaud-Glavieux, Corinne; García-Hurtado, Javier; Sauty, Claire; Guerci, Aline; Ferrer, Jorge


    Lineage-selective expression of developmental genes is dependent on the interplay between activating and repressive mechanisms. Gene activation is dependent on cell-specific transcription factors that recognize transcriptional enhancer sequences. Gene repression often depends on the recruitment of Polycomb group (PcG) proteins, although the sequences that underlie the recruitment of PcG proteins, also known as Polycomb response elements (PREs), remain poorly understood in vertebrates. While distal PREs have been identified in mammals, a role for positive-acting enhancers in PcG-mediated repression has not been described. Here we have used a highly efficient procedure based on lentiviral-mediated transgenesis to carry out in vivo fine-mapping of, cis-regulatory sequences that control lineage-specific activation of Neurog3, a master regulator of pancreatic endocrine differentiation. Our findings reveal an enhancer region that is sufficient to drive correct spacio-temporal expression of Neurog3 and demonstrate that this same region serves as a PRE in alternative lineages where Neurog3 is inactive. PMID:28225770

  12. Induction of electrophile-responsive element (EpRE)-mediated gene expression by tomato extracts in vitro. (United States)

    Gijsbers, Linda; van Eekelen, Henriëtte D L M; Nguyen, Thuy H; de Haan, Laura H J; van der Burg, Bart; Aarts, Jac M M J G; Rietjens, Ivonne M C M; Bovy, Arnaud G


    The market for food products with additional health benefits is increasing rapidly and tools for identification of bio-functional characteristics of food items are essential. To facilitate the detection of beneficial effects of tomato on gene expression, methods to prepare tomato extracts suitable to test in the EpRE LUX assay and other cell-based reporter gene assays for health-related bioactivity mechanisms, were developed. An isoprenoid-containing chloroform extract of tomato fruit and most individual isoprenoids did not induce electrophile-responsive element (EpRE)-mediated gene expression. A semi-polar extract of tomato fruits, enzymatically hydrolysed to remove the glycosyl residues from the phenolic ingredients was able to induce EpRE-mediated luciferase expression at both mRNA and protein level, which might be partly due to the presence of quercetin, kaempferol, naringenin and naringenin chalcone. It was concluded that induction of EpRE-regulated genes, such as detoxifying phase II and antioxidant enzymes, may contribute to the beneficial health effects of tomato.

  13. Effects of gamma irradiation on the DNA-protein complex between the estrogen response element and the estrogen receptor

    Energy Technology Data Exchange (ETDEWEB)

    Stisova, Viktorie [Department of Radiation Dosimetry, Nuclear Physics Institute AS CR, Na Truhlarce 39/64, 18086 Praha 8 (Czech Republic); Goffinont, Stephane; Spotheim-Maurizot, Melanie [Centre de Biophysique Moleculaire CNRS, rue Charles Sadron, 45071 Orleans Cedex 2 (France); Davidkova, Marie, E-mail: davidkova@ujf.cas.c [Department of Radiation Dosimetry, Nuclear Physics Institute AS CR, Na Truhlarce 39/64, 18086 Praha 8 (Czech Republic)


    Signaling by estrogens, risk factors in breast cancer, is mediated through their binding to the estrogen receptor protein (ER), followed by the formation of a complex between ER and a DNA sequence, called estrogen response element (ERE). Anti-estrogens act as competitive inhibitors by blocking the signal transduction. We have studied in vitro the radiosensitivity of the complex between ERalpha, a subtype of this receptor, and a DNA fragment bearing ERE, as well as the influence of an estrogen (estradiol) or an anti-estrogen (tamoxifen) on this radiosensitivity. We observe that the complex is destabilized upon irradiation with gamma rays in aerated aqueous solution. The analysis of the decrease of binding abilities of the two partners shows that destabilization is mainly due to the damage to the protein. The destabilization is reduced when irradiating in presence of tamoxifen and is increased in presence of estradiol. These effects are due to opposite influences of the ligands on the loss of binding ability of ER. The mechanism that can account for our results is: binding of estradiol or tamoxifen induces distinct structural changes of the ER ligand-binding domain that can trigger (by allostery) distinct structural changes of the ER DNA-binding domains and thus, can differently affect ER-ERE interaction.

  14. The expression of cyclic adenosine monophosphate responsive element modulator in rat sertoli cells following seminal extract administration

    Directory of Open Access Journals (Sweden)

    Muslim Akmal


    Full Text Available Aim: This study aims to determine the effect of seminal vesicle extract on cyclic adenosine monophosphate responsive element modulator (CREM expression in rat Sertoli cells. Materials and Methods: This study examined the expression of CREM on 20 male rats (Rattus norvegicus at 4 months of age, weighing 250-300 g. The rats were divided into four groups: K0, KP1, KP2, and KP3. K0 group was injected with 0.2 ml normal saline; KP1 was injected with 25 mg cloprostenol (Prostavet C, Virbac S. A; KP2 and KP3 were injected with 0.2 and 0.4 ml seminal vesicle extract, respectively. The treatments were conducted 5 times within 12-day interval. At the end of the study, the rats were euthanized by cervical dislocation; then, the testicles were necropsied and processed for histology observation using immunohistochemistry staining. Results: CREM expression in rat Sertoli cells was not altered by the administration of either 0.2 or 0.4 ml seminal vesicle extract. Conclusion: The administration of seminal vesicle extract is unable to increase CREM expression in rat Sertoli cells.

  15. Effect of 2-Hydroxyethyl Methacrylate on Antioxidant Responsive Element-Mediated Transcription: A Possible Indication of Its Cytotoxicity (United States)

    Orimoto, Ai; Suzuki, Takahiro; Ueno, Atsuko; Kawai, Tatsushi; Nakamura, Hiroshi; Kanamori, Takao


    Background The resin monomer 2-hydroxyethyl methacrylate (HEMA) is known to be more cytotoxic than methyl methacrylate (MMA). Using a luciferase reporter assay system, we previously showed that MMA activates the glutathione S-transferase alpha 1 gene (Gsta1) promoter through the anti-oxidant responsive element (ARE). However, it is not known whether HEMA induces ARE-mediated transcription. Methodology/Principal Findings We further developed the reporter system and studied the concentration-dependent effect of HEMA on ARE enhancer activity. The revised system employed HepG2 cells stably transfected with a destabilized luciferase reporter vector carrying 2 copies of the 41-bp ARE region of Gsta1. In this system, MMA increased ARE activity by 244-fold at 30 mM; HEMA augmented ARE activity at 3 mM more intensely than MMA (36-fold versus 11-fold) and was equipotent as MMA at 10 mM (56-fold activation); however, HEMA failed to increase ARE activity at 30 mM. In HepG2 cells, HEMA detectably lowered the cellular glutathione levels at 10 mM and cell viability at 30 mM, but MMA did not. Conclusions These results suggest that the low-concentration effect of HEMA on ARE activity reflects its cytotoxicity. Our reporter system used to examine ARE activity may be useful for evaluating cytotoxicities of resin monomers at concentrations lower than those for which cell viabilities are reduced. PMID:23516576

  16. Effect of beta-ADrenergic Agonist on Cyclic AMP Synthesis in Chicken Skeletal Muscle Cells in Culture (United States)

    Young, R. B.; Bridge, K. Y.; Rose, M. Franklin (Technical Monitor)


    Several beta-adrenergic receptor (bAR) agonists are known to cause hypertrophy of skeletal muscle tissue. Because it seems logical that these agonists exert their action on muscle through stimulation of cAMP synthesis, five bAR agonists encompassing a range in activity from strong to weak were evaluated for their ability to stimulate cAMP accumulation in embryonic chicken skeletal muscle cells in culture. Two strong agonists (epinephrine and isoproterenol), one moderate agonist (albuterol), and two weak agonists known to cause hypertrophy in animals (clenbuterol and cimaterol) were studied. Dose response curves were determined over six orders of magnitude in concentration for each agonist, and values were determined for their maximum stimulation of cAMP synthesis rate (Bmax) and the agonist concentration at which 50% stimulation of cAMP synthesis (EC50) occurred. Bmax values decreased in the following order: isoproterenol, epinephrine, albuterol, cimaterol, clenbuterol. Cimaterol and clenbuterol at their Bmax levels were approximately 15-fold weaker than isoproterenol in stimulating the rate of cAMP synthesis. In addition, the EC50 values for isoproterenol, cimaterol, clenbuterol, epinephrine, and albuterol were 360 nM, 630 nM, 900 nM, 2,470 nM, and 3,650 nM, respectively. Finally, dose response curves show that the concentrations of cimaterol and clenbuterol in culture media at concentrations known to cause significant muscle hypertrophy in animals had no detectable effect on stimulation of CAMP accumulation in chicken skeletal muscle cells.

  17. The cAMP-mediated protein kinase signal transduction pathway is involved in the pyrogenic effect of CRH in rats

    Institute of Scientific and Technical Information of China (English)


    The primary action of corticotropin releasing hormone (CRH) is stimulation of the synthesis and release of adrenocorticotropic hormone and β-endorphin from the pituitary in response to stress. In addition, a number of studies indicate that CRH exerts other physiological actions within the central nervous system which are independent of the pituitary. These include increased body temperature and thermogenesis. However, the intracellular mechanism responsible for pyrogenic action of CRH is still unclear. The purpose of these studies was to determine whether or not cAMP was involved in the pyrogenic action of CRH in the rat. Intracerebroventricular (icv) microinjection of CRH (2.5 μg, 5.0 μg, 10 μg) caused increases in colonic temperature and hypothalamus cAMP level in conscious rats. The pyrogenic effects of CRH were abolished or markedly inhibited by prior injection (icv) of an adenylate cyclase inhibitor, 2,,3,-dideoxyadenosine (DDA, 30 μg) or an inhibitor of cAMP-dependent protein kinase, adenosine-3,,5,-(cyclic) monophosphorothionate (Rp-cAMPs, 15 μg). This is the first report demonstrating the pyrogenic effcet of centrally administration of CRH on the rat via the cAMP-mediated protein kinase signal transduction pathway.

  18. [Phosphodiesterase 3 mediates cross-talk between the protein kinase- and cGMP- dependent pathways and cyclic AMP metabolism]. (United States)

    Makuch, Edyta; Matuszyk, Janusz


    PDE3 is a dual-substrate phosphodiesterase responsible for hydrolyzing both cAMP and cGMP whilst being simultaneously inhibited by cGMP. This feature is related to presence of the 44 amino acid insert in the catalytic domain, which determines the mechanism of introduction of the cyclic nucleotide into the catalytic pocket of the enzyme. Once bound in the catalytic site cGMP results in steric hindrance for cAMP to enter the site. The regulatory domain of PDE3 consists of two hydrophobic regions: NHR1 and NHR2. Their presence defines the enzyme's intracellular localization, thus determining its participation in particular signaling cascades. Due to the properties of PDE3 this enzyme has exceptional importance for the cross-talk between cAMP-dependent signaling and other cascades. There are two different mechanisms of action of PDE3 enzymes in cell signaling pathways. In many signaling cascades assembly of a signalosome is necessary for phosphorylation and activation of the PDE3 proteins. In response to certain hormones and growth factors, PDE3 merges the metabolism of cAMP with protein kinase-dependent signaling pathways. PDE3 also controls the level of cAMP with regard to the alternating concentration of cGMP. This effect occurs in signaling cascades activated by natriuretic peptide.

  19. Hypoxia-response element (HRE)-directed transcriptional regulation of the rat lysyl oxidase gene in response to cobalt and cadmium. (United States)

    Gao, Song; Zhou, Jing; Zhao, Yinzhi; Toselli, Paul; Li, Wande


    Lysyl oxidase (LO) catalyzes crosslink of collagen, elastin, and histone H1, stabilizing the extracellular matrix and cell nucleus. This enzyme displays dual functions for tumorigenesis, i.e., as a tumor suppressor inactivating the ras oncogene and as a tumor promoter enhancing malignant cell metastasis. To elucidate LO transcriptional regulation, we have cloned the 804 base pair region upstream of the translation start site (ATG) of the rat LO gene with the maximal promoter activity. Computer analysis indicated that at least four hypoxia-response element (HRE) consensuses (5'-ACGTG-3') exist in the cloned LO promoter. Treatment of rat lung fibroblasts (RFL6) with CoCl2 (Co, 10-100 μM), a chemical hypoxia reagent, enhanced LO mRNA expression and promoter activities. Overexpression of LO was associated with upregulation of hypoxia-inducible factor (HIF)-1α at mRNA levels in cobalt (Co)-treated cells. Thus, LO is a hypoxia-responsive gene. Dominant negative-HIF-1α inhibited LO promoter activities stimulated by Co. Electrophoretic mobility shift, oligonucleotide competition, and in vitro translated HIF-1α binding assays indicated that only one HRE mapped at -387/-383 relative to ATG was functionally active among four consensuses. Site-directed mutation of this HRE significantly diminished the Co-induced and LO promoter-directed expression of the reporter gene. Cadmium (Cd), an inducer of reactive oxygen species, inhibited HIF-1α mRNA expression and HIF-1α binding to the LO gene in Co-treated cells as revealed by RT-PCR and ChIP assays, respectively. Thus, modulation of the HRE activity by Co and Cd plays a critical role in LO gene transactivation.

  20. Novel cAMP targets in cell proliferation

    NARCIS (Netherlands)

    Kuiperij, Hinke Bertha


    cAMP is a second messenger that plays a role in a wide variety of biological processes, one of which is the regulation of cell proliferation. Adenylate cyclases generate cAMP in the cell upon activation, followed by binding to and activation of its direct targets, PKA and Epac. PKA is a protein kina

  1. Interplay between estrogen response element sequence and ligands controls in vivo binding of estrogen receptor to regulated genes. (United States)

    Krieg, Adam J; Krieg, Sacha A; Ahn, Bonnie S; Shapiro, David J


    To examine the role of the estrogen response element (ERE) sequence in binding of liganded estrogen receptor (ER) to promoters, we analyzed in vivo interaction of liganded ER with the imperfect ERE in the pS2 gene and the composite estrogen-responsive unit (ERU) in the proteinase inhibitor 9 (PI-9) gene. In transient transfections of ER-positive HepG2-ER7 cells, PI-9 was strongly induced by estrogen, moxestrol (MOX), and 4-hydroxytamoxifen (OHT). PI-9 was not induced by raloxifene or ICI 182,780. Quantitative reverse transcriptase-PCR showed that moxestrol strongly induced cellular PI-9 and pS2 mRNAs, whereas OHT moderately induced PI-9 mRNA and weakly induced pS2 mRNA. Chromatin immunoprecipitation experiments demonstrated strong and similar association of 17beta-estradiol-hERalpha and MOX-hERalpha with the PI-9 ERU and with the pS2 ERE. Binding of MOX-hERalpha to the PI-9 ERU and the pS2 ERE was rapid and continuous. Although MOX-hERalpha bound strongly to the PI-9 ERU and less well to the pS2 ERE in chromatin immunoprecipitation, gel shift assays showed that estrogen-hERalpha binds with higher affinity to the deproteinized pS2 ERE than to the PI-9 ERU. Across a broad range of OHT concentrations, OHT-hERalpha associated strongly with the pS2 ERE and weakly with the PI-9 ERU. ICI-hERalpha bound poorly to the PI-9 ERU and effectively to the pS2 ERE. Raloxifene-hERalpha and MOX-hERalpha exhibited similar binding to the PI-9 ERU and the pS2 ERE. These studies demonstrate that ER ligand and ERE sequence work together to regulate in vivo binding of ER to estrogen-responsive promoters.

  2. The RGS protein Crg2 regulates pheromone and cyclic AMP signaling in Cryptococcus neoformans. (United States)

    Shen, Gui; Wang, Yan-Li; Whittington, Amy; Li, Lie; Wang, Ping


    Crg1 and Crg2 are regulators of G-protein signaling homologs found in the human fungal pathogen Cryptococcus neoformans. Crg1 negatively regulates pheromone responses and mating through direct inhibition of Galpha subunits Gpa2 and Gpa3. It has also been proposed that Crg2 has a role in mating, as genetic crosses involving Deltacrg2 mutants resulted in formation of hyperfilaments. We found that mutation of Gpa2 and Gpa3 partially suppressed the hyperfilamentation, mutation of Gpa3 alleviated Deltacrg2-specfic cell swelling, and mutation of the mitogen-activated protein kinase Cpk1 blocked both processes. These findings indicate that Gpa2 and Gpa3 function downstream of Crg2 and that Gpa3 is also epistatic to Crg2 in a Cpk1-dependent morphogenesis process linked to mating. Significantly, we found that Deltacrg2 mutants formed enlarged capsules that mimic cells expressing a constitutively active GPA1(Q284L) allele and that the levels of intracellular cyclic AMP (cAMP) were also elevated, suggesting that Crg2 also negatively regulates the Gpa1-cAMP signaling pathway. We further showed that Crg2 interacted with Gpa3 and Gpa1, but not Gpa2, in a pulldown assay and that Crg2 maintained a higher in vitro GTPase-activating protein activity toward Gpa3 and Gpa1 than to Gpa2. Finally, we found that dysregulation of cAMP due to the Crg2 mutation attenuated virulence in a murine model of cryptococcosis. Taken together, our study reveals Crg2 as an RGS (regulator of G-protein signaling) protein of multiregulatory function, including one that controls mating distinctly from Crg1 and one that serves as a novel inhibitor of Gpa1-cAMP signaling.

  3. cAMP-mediated secretion of brain-derived neurotrophic factor in developing airway smooth muscle. (United States)

    Thompson, Michael A; Britt, Rodney D; Kuipers, Ine; Stewart, Alecia; Thu, James; Pandya, Hitesh C; MacFarlane, Peter; Pabelick, Christina M; Martin, Richard J; Prakash, Y S


    Moderate hyperoxic exposure in preterm infants contributes to subsequent airway dysfunction and to risk of developing recurrent wheeze and asthma. The regulatory mechanisms that can contribute to hyperoxia-induced airway dysfunction are still under investigation. Recent studies in mice show that hyperoxia increases brain-derived neurotrophic factor (BDNF), a growth factor that increases airway smooth muscle (ASM) proliferation and contractility. We assessed the mechanisms underlying effects of moderate hyperoxia (50% O2) on BDNF expression and secretion in developing human ASM. Hyperoxia increased BDNF secretion, but did not alter endogenous BDNF mRNA or intracellular protein levels. Exposure to hyperoxia significantly increased [Ca2+]i responses to histamine, an effect blunted by the BDNF chelator TrkB-Fc. Hyperoxia also increased ASM cAMP levels, associated with reduced PDE4 activity, but did not alter protein kinase A (PKA) activity or adenylyl cyclase mRNA levels. However, 50% O2 increased expression of Epac2, which is activated by cAMP and can regulate protein secretion. Silencing RNA studies indicated that Epac2, but not Epac1, is important for hyperoxia-induced BDNF secretion, while PKA inhibition did not influence BDNF secretion. In turn, BDNF had autocrine effects of enhancing ASM cAMP levels, an effect inhibited by TrkB and BDNF siRNAs. Together, these novel studies suggest that hyperoxia can modulate BDNF secretion, via cAMP-mediated Epac2 activation in ASM, resulting in a positive feedback effect of BDNF-mediated elevation in cAMP levels. The potential functional role of this pathway is to sustain BDNF secretion following hyperoxic stimulus, leading to enhanced ASM contractility and proliferation.

  4. Schistosoma mansoni c-AMP-dependent Protein Kinase (PKA): A Potential New Drug Target (United States)


    after a heavy initial infection and is characterized as a systemic hypersensitivity reaction to migrating schistosomula [27]. Symptoms of Katayama...2001) 8-Cl-adenosine is an active metabolite of 8-Cl-cAMP responsible for its in vitro antiproliferative effects on CHO mutants hypersensitive to...DMEM (with 10% fetal bovine serum and 5% penicillin /streptomycin) and appropriate concentration of inhibitor. Equal amounts of appropriate vehicle

  5. Nitric oxide switches on glycolysis through the AMP protein kinase and 6-phosphofructo-2-kinase pathway


    Almeida Parra, Ángeles; Moncada, Salvador; Bolaños Hernández, Juan Pedro


    El óxido nítrico activa la glucolisis en los astrocitos a través de una cascada de señalización en la que interviene la AMP kinasa, que fosforila (y activa) la fosfofructokinasa-2, enzima responsable de la biosíntesis de fructosa-2, 6-bisfosfato, efector alostérico positivo de la fosfofructokinasa-1. Este mecanismo es citoprotector.

  6. MEK Inhibitors Reverse cAMP-Mediated Anxiety in Zebrafish

    DEFF Research Database (Denmark)

    Lundegaard, Pia R.; Anastasaki, Corina; Grant, Nicola J.;


    Altered phosphodiesterase (PDE)-cyclic AMP (cAMP) activity is frequently associated with anxiety disorders, but current therapies act by reducing neuronal excitability rather than targeting PDE-cAMP-mediated signaling pathways. Here, we report the novel repositioning of anti-cancer MEK inhibitors...... as anxiolytics in a zebrafish model of anxiety-like behaviors. PDE inhibitors or activators of adenylate cyclase cause behaviors consistent with anxiety in larvae and adult zebrafish. Small-molecule screening identifies MEK inhibitors as potent suppressors of cAMP anxiety behaviors in both larvae and adult...... zebrafish, while causing no anxiolytic behavioral effects on their own. The mechanism underlying cAMP-induced anxiety is via crosstalk to activation of the RAS-MAPK signaling pathway. We propose that targeting crosstalk signaling pathways can be an effective strategy for mental health disorders, and advance...

  7. 8-Chloro-cAMP-Related Changes on Mice Uteri

    Directory of Open Access Journals (Sweden)

    Andrea Actis


    Full Text Available Histopathological effects of cAMP analog (8-Chloro-cAMP, tamoxifen, and medroxyprogesterone, alone or combined, upon BALB/c mice uteri are reported. 8-Chloro-cAMP diminished uterine weight, but did not modify its histopathology or estral cycle significantly. Tamoxifen diminished uterine weight showing cystic hyperplasia and an estral cycle arrested at diestrus. Medroxyprogesterone increased uterine weight, caused a swelling of the endometrium and a pseudopregnancy estrus. When combined with 8-Chloro-cAMP, tamoxifen or medroxyprogesterone always had a predominant effect. We concluded that the effects of 8-Chloro-cAMP on mice uteri did not cause significant changes on its histopathology, but diminished its weight.

  8. Control of bacterial exoelectrogenesis by c-AMP-GMP. (United States)

    Nelson, James W; Sudarsan, Narasimhan; Phillips, Grace E; Stav, Shira; Lünse, Christina E; McCown, Phillip J; Breaker, Ronald R


    Major changes in bacterial physiology including biofilm and spore formation involve signaling by the cyclic dinucleotides c-di-GMP and c-di-AMP. Recently, another second messenger dinucleotide, c-AMP-GMP, was found to control chemotaxis and colonization by Vibrio cholerae. We have identified a superregulon of genes controlled by c-AMP-GMP in numerous Deltaproteobacteria, including Geobacter species that use extracellular insoluble metal oxides as terminal electron acceptors. This exoelectrogenic process has been studied for its possible utility in energy production and bioremediation. Many genes involved in adhesion, pilin formation, and others that are important for exoelectrogenesis are controlled by members of a variant riboswitch class that selectively bind c-AMP-GMP. These RNAs constitute, to our knowledge, the first known specific receptors for c-AMP-GMP and reveal that this molecule is used by many bacteria to control specialized physiological processes.

  9. Regulation of Insulin-Response Element Binding Protein-1 in Obesity and Diabetes: Potential Role in Impaired Insulin-Induced Gene Transcription



    One of the major mechanisms by which insulin modulates glucose homeostasis is through regulation of gene expression. Therefore, reduced expression of transcription factors that are required for insulin-regulated gene expression may contribute to insulin resistance. We recently identified insulin response element-binding protein-1 (IRE-BP1) as a transcription factor that binds and transactivates multiple insulin-responsive genes, but the regulation of IRE-BP1 in vivo is largely unknown. In thi...

  10. Influence of Increasing Load on Rat Myocardial ATP, AMP and AMP/ATP%递增负荷对大鼠心肌ATP、AMP及AMP/ATP的影响

    Institute of Scientific and Technical Information of China (English)



    为了探讨逐级递增跑台运动训练方式对大鼠心肌ATP、AMP及AMP/ATP的影响,建立了逐级递增跑台运动训练动物模型,采用高效液相色谱检测AMP、ATP的含量.研究结果表明:随着运动负荷的增加,大鼠心肌AMP含量呈现上升趋势,与对照组相比较,存在显著性差异(P<0.05);ATP含量呈现先下降趋势,存在显著性差异(P<0.05),后期出现的轻微上升趋势没有显著性差异(P>0.05);ATP/AMP呈现上升趋势,存在显著性差异(P<0.05).大鼠心肌对于逐级递增运动呈现适应性强度,运动过程中AMP/ATP比值的改变是ATP与AMP综合变化的效应,ATP变化幅度还是非常的有限,主要是通过增加AMP含量来提升能量供应.%To investigate the influence of the incremental treadmill exercise training on the rat cardiomyocytes ATP,AMP and AMP/ATP 's ,a model of incremental treadmill exercise training for animals was built. High-performance liquid chromatography was used to detect AMP, ATP content. The results:show that with the increase in exercise load, AMP content of rat cardiac increases. Compared with the control group, there was a significant difference (P0. 05) ;with ATP / AMP rising, there was a significant difference (P<0. 05). It can be concluded that the myocardial show adaptability to the intensity of incremental movement. Although the change in the ratio of AMP to ATP results from the change of ATP and AMP, the ATP's variation is limited,mainly by increasing the AMP content to supply energy.

  11. Newly constructed stable reporter cell lines for mechanistic studies on electrophile-responsive element-mediated gene expression reveal a role for flavonoid planarity

    NARCIS (Netherlands)

    Boerboom, A.M.J.F.; Vermeulen, M.; Woude, van der H.; Bremer, B.I.; Lee, Y.Y.; Kampman, E.; Bladeren, van P.J.; Rietjens, I.M.C.M.; Aarts, J.M.M.J.G.


    The electrophile-responsive element (EpRE) is a transcriptional enhancer involved in cancer-chemoprotective gene expression modulation by certain food components. Two stably transfected luciferase reporter cell lines were developed, EpRE(hNQO1)-LUX and EpRE(mGST-Ya)-LUX, based on EpRE sequences from

  12. Newly constructed stable reporter cell lines for mechanistic studies on electrophile-responsive element-mediated gene expression reveal a role for flavonoid planarity.

    NARCIS (Netherlands)

    Boerboom, A.M.A.; Vermeulen, M.; Woude, H. van der; Bremer, B.I.; Lee-Hilz, Y.Y.; Kampman, E.; Bladeren, P.J. van; Rietjens, I.M.C.M.; Aarts, J.


    The electrophile-responsive element (EpRE) is a transcriptional enhancer involved in cancer-chemoprotective gene expression modulation by certain food components. Two stably transfected luciferase reporter cell lines were developed, EpRE(hNQO1)-LUX and EpRE(mGST-Ya)-LUX, based on EpRE sequences from

  13. Potency of isothiocyanates to induce luciferase reporter gene expression via the electrophile-responsive element from murine glutathione S-transferase Ya

    NARCIS (Netherlands)

    Vermeulen, M.; Boerboom, A.M.J.F.; Blankvoort, B.M.G.; Aarts, J.M.M.J.G.; Rietjens, I.; Bladeren, van P.J.; Vaes, W.H.J.


    Isothiocyanates are electrophiles that are able to induce phase II biotransformation enzyme gene expression via an electrophile-responsive element (EpRE) in the gene regulatory region. To study the potency of different isothiocyanates to induce the expression of EpRE-regulated genes, a Hepa-1c1c7 lu

  14. Counteracting roles of AMP deaminase and AMP kinase in the development of fatty liver.

    Directory of Open Access Journals (Sweden)

    Miguel A Lanaspa

    Full Text Available Fatty liver (hepatic steatosis is associated with nucleotide turnover, loss of ATP and generation of adenosine monophosphate (AMP. It is well known that in fatty liver, activity of the AMP-activated kinase (AMPK is reduced and that its stimulation can prevent hepatic steatosis by both enhancing fat oxidation and reducing lipogenesis. Here we show that another AMP dependent enzyme, AMPD2, has opposing effects on fatty acid oxidation when compared to AMPK. In human hepatocytres, AMPD2 activation -either by overexpression or by lowering intracellular phosphate levels with fructose- is associated with a significant reduction in AMPK activity. Likewise, silencing of AMPK spontaneously increases AMPD activity, demonstrating that these enzymes counter-regulate each other. Furthermore, we show that a downstream product of AMP metabolism through AMPD2, uric acid, can inhibit AMPK activity in human hepatocytes. Finally, we show that fructose-induced fat accumulation in hepatocytes is due to a dominant stimulation of AMPD2 despite stimulating AMPK. In this regard, AMPD2-deficient hepatocytes demonstrate a further activation of AMPK after fructose exposure in association with increased fatty acid oxidation, and conversely silencing AMPK enhances AMPD-dependent fat accumulation. In vivo, we show that sucrose fed rats also develop fatty liver that is blocked by metformin in association with both a reduction in AMPD activity and an increase in AMPK activity. In summary, AMPD and AMPK are both important in hepatic fat accumulation and counter-regulate each other. We present the novel finding that uric acid inhibits AMPK kinase activity in fructose-fed hepatocytes thus providing new insights into the pathogenesis of fatty liver.

  15. cAMP-Induced Histones H3 Dephosphorylation Is Independent of PKA and MAP Kinase Activations and Correlates With mTOR Inactivation. (United States)

    Rodriguez, Pedro; Rojas, Juan


    cAMP is a second messenger well documented to be involved in the phosphorylation of PKA, MAP kinase, and histone H3 (H3). Early, we reported that cAMP also induced H3 dephosphorylation in a variety of proliferating cell lines. Herein, it is shown that cAMP elicits a biphasic H3 dephosphorylation independent of PKA activation in cycling cells. H89, a potent inhibitor of PKA catalytic sub-unite, could not abolish this effect. Additionally, H89 induces a rapid and biphasic H3 serine 10 dephosphorylation, while a decline in the basal phosphorylation of CREB/ATF-1 is observed. Rp-cAMPS, an analog of cAMP and specific inhibitor of PKA, is unable to suppress cAMP-mediated H3 dephosphorylation, whereas Rp-cAMPS effectively blocks CREB/ATF-1 hyper-phosphorylation by cAMP and its inducers. Interestingly, cAMP exerts a rapid and profound H3 dephosphorylation at much lower concentration (50-fold lower, 0.125 mM) than the concentration required for maximal CREB/ATF-1 phosphorylation (5 mM). Much higher cAMP concentration is required to fully induce CREB/ATF-1 gain in phosphate (5 mM), which correlates with the inhibition of H3 dephosphorylation. Also, the dephosphorylation of H3 does not overlap at onset of MAP kinase phosphorylation pathways, p38 and ERK. Surprisingly, rapamycin (an mTOR inhibitor), cAMP, and its natural inducer isoproterenol, elicit identical dephosphorylation kinetics on both S6K1 ribosomal kinase (a downstream mTOR target) and H3. Finally, cAMP-induced H3 dephosphorylation is PP1/2-dependent. The results suggest that a pathway, requiring much lower cAMP concentration to that required for CREB/ATF-1 hyper-phosphorylation, is responsible for histone H3 dephosphorylation and may be linked to mTOR down regulation.

  16. Modeling the seismic response of 2D models of asteroid 433 Eros, based on the spectral-element method. (United States)

    Blitz, Celine; Komatitsch, Dimitri; Lognonné, Philippe; Martin, Roland; Le Goff, Nicolas

    The understanding of the interior structure of Near Earth Objects (NEOs) is a fundamental issue to determine their evolution and origin, and also, to design possible mitigation techniques (Walker and Huebner, 2004). Indeed, if an oncoming Potentially Hazardous Object (PHO) were to threaten the Earth, numerous methods are suggested to prevent it from colliding our planet. Such mitigation techniques may involve nuclear explosives on or below the object surface, impact by a projectile, or concentration of solar energy using giant mirrors (Holsapple, 2004). The energy needed in such mitigation techniques highly depends on the porosity of the hazardous threatening object (asteroid or comet), as suggested by Holsapple, 2004. Thus, for a given source, the seismic response of a coherent homogeneous asteroid should be very different from the seismic response of a fractured or rubble-pile asteroid. To assess this hypothesis, we performed numerical simulations of wave propagation in different interior models of the Near Earth Asteroid 433 Eros. The simulations of wave propagation required a shape model of asteroid Eros, kindly provided by A. Cheng and O. Barnouin-Jha (personal communication). A cross-section along the longest axis has been chosen to define our 2D geometrical model, and we study two models of the interior: a homogeneous one, and a complex one characterized by fault networks below the main crosscut craters, and covered by a regolith layer of thickness ranging from 50 m to 150 m. To perform the numerical simulations we use the spectral-element method, which solves the variational weak form of the seismic wave equation (Komatitsch and Tromp, 1999) on the meshes of the 2D models of asteroid Eros. The homogeneous model is composed of an elastic material characterized by a pressure wave velocity Vp = 3000 m.s-1 , a shear wave velocity Vs = 1700 m.s-1 and a density of 2700 kg.m-3 . The fractured model possesses the same characteristics except for the presence of

  17. Activation of Estrogen Response Element-independent ERα signaling protects female mice from diet-induced obesity. (United States)

    Yasrebi, Ali; Rivera, Janelle A; Krumm, Elizabeth A; Yang, Jennifer A; Roepke, Troy A


    17β-estradiol (E2) regulates central and peripheral mechanisms that control energy and glucose homeostasis predominantly through estrogen receptor α (ERα) acting via receptor binding to estrogen response elements (ERE). ERα signaling is also involved in mediating the effects of E2 on diet-induced obesity (DIO), although the roles of ERE-dependent and -independent ERα signaling in ameliorating the effects of DIO remain largely unknown. We hypothesize that ERE-dependent ERα signaling is necessary to ameliorate the effects of DIO. We addressed this question using ERαKO (KO) and ERαKIKO (KIKO) female mice; the latter expressing an ERα that lacks a functional ERE binding domain. Females were ovariectomized, fed low-fat (LFD) or high-fat (HFD) diet, and orally dosed with vehicle or estradiol benzoate (EB, 300 μg/kg). After 9 weeks, body composition, glucose and insulin tolerance, peptide hormone and inflammatory cytokine levels, and hypothalamic arcuate nucleus and liver gene expression were assessed. EB reduced body weight and body fat in WT, regardless of diet, and in HFD-fed KIKO, in part by reducing energy intake and feeding efficiency. EB reduced fasting glucose levels in KIKO mice fed both diets but augmented glucose tolerance only in HFD-fed KIKO. Plasma insulin and IL-6 were elevated in KIKO and KO compared to WT on a LFD. Expression of arcuate neuropeptide and receptor genes and liver fatty acid biosynthesis genes was altered by HFD and by EB through ERE-dependent and -independent mechanisms. Therefore, ERE-independent signaling mechanisms in both the brain and peripheral organs mediate, in part, the effects of E2 during DIO.

  18. Effect of iron stress on Withania somnifera L.: antioxidant enzyme response and nutrient elemental uptake of in vitro grown plants. (United States)

    Rout, Jyoti Ranjan; Behera, Sadhana; Keshari, Nitin; Ram, Shidharth Sankar; Bhar, Subhajit; Chakraborty, Anindita; Sudarshan, Mathummal; Sahoo, Santi Lata


    In the present study the response of antioxidant enzyme activities and the level of expression of their corresponding genes on bioaccumulation of iron (Fe) were investigated. In vitro germinated Withania somnifera L. were grown in Murashige and Skoog's liquid medium with increasing concentrations (0, 25, 50, 100 and 200 µM) of FeSO4 for 7 and 14 days. Root and leaf tissues analyzed for catalase (CAT, EC, superoxide dismutase (SOD, EC and guaiacol peroxidase (GPX, EC, have shown an increase in content with respect to exposure time. Isoforms of CAT, SOD and GPX were separated using non-denaturing polyacrylamide gel electrophoresis and observed that the isoenzymes were greatly affected by higher concentrations of Fe. Reverse transcriptase polymerase chain reaction analysis performed by taking three pairs of genes of CAT (RsCat, Catalase1, Cat1) and SOD (SodCp, TaSOD1.2, MnSOD) to find out the differential expression of antioxidant genes under Fe excess. RsCat from CAT and MnSOD from SOD have exhibited high levels of gene expression under Fe stress, which was consistent with the changes of the activity assayed in solution after 7 days of treatment. Analysis by proton induced X-ray emission exhibited an increasing uptake of Fe in plants by suppressing and expressing of other nutrient elements. The results of the present study suggest that higher concentration of Fe causes disturbance in nutrient balance and induces oxidative stress in plant.

  19. Transrepression of the estrogen receptor promoter by calcitriol in human breast cancer cells via two negative vitamin D response elements. (United States)

    Swami, Srilatha; Krishnan, Aruna V; Peng, Lihong; Lundqvist, Johan; Feldman, David


    Calcitriol (1,25-dihydroxyvitamin D3), the hormonally active metabolite of vitamin D, exerts its anti-proliferative activity in breast cancer (BCa) cells by multiple mechanisms including the downregulation of the expression of estrogen receptor α (ER). We analyzed an ∼3.5 kb ER promoter sequence and demonstrated the presence of two potential negative vitamin D response elements (nVDREs), a newly identified putative nVDRE upstream at -2488 to -2473 bp (distal nVDRE) and a previously published sequence (proximal nVDRE) at -94 to -70 bp proximal to the P1 start site. Transactivation analysis using ER promoter deletion constructs and heterologous promoter-reporter constructs revealed that both nVDREs functioned to mediate calcitriol transrepression. In the electrophoretic mobility shift assay, the vitamin D receptor (VDR) showed strong binding to both nVDREs in the presence of calcitriol, and the chromatin immunoprecipitation assay demonstrated the recruitment of the VDR to the distal nVDRE site. Mutations in the 5' hexameric DNA sequence of the distal nVDRE resulted in the loss of calcitriol-mediated transrepression and the inhibition of protein-DNA complex formation, demonstrating the importance of these nucleotides in VDR DNA binding and transrepression. A putative nuclear factor-Y (NFY) binding site, identified within the distal nVDRE, led to the findings that NFY bound to the distal nVDRE site interfered with the binding of the VDR at the site and reduced calcitriol-mediated transrepression. In conclusion, the ER promoter region contains two negative VDREs that act in concert to bind to the VDR and both nVDREs are required for the maximal inhibition of ER expression by calcitriol. The suppression of ER expression and estrogen-mediated signaling by calcitriol in BCa cells suggests that vitamin D may be useful in the treatment of ER+ BCa.

  20. Foxa2 integrates the transcriptional response of the hepatocyte to fasting. (United States)

    Zhang, Liping; Rubins, Nir E; Ahima, Rexford S; Greenbaum, Linda E; Kaestner, Klaus H


    Survival during prolonged food deprivation depends on the activation of hepatic gluconeogenesis. Inappropriate regulation of this process is a hallmark of diabetes and other metabolic diseases. Activation of the genes encoding gluconeogenic enzymes is mediated by hormone-responsive transcription factors such as the cyclic AMP response element binding protein (CREB) and the glucocorticoid receptor (GR). Here we show using cell-type-specific gene ablation that the winged helix transcription factor Foxa2 is required for activation of the hepatic gluconeogenic program during fasting. Specifically, Foxa2 promotes gene activation both by cyclic AMP, the second messenger for glucagon, and glucocorticoids. Foxa2 mediates these effects by enabling recruitment of CREB and GR to their respective target sites in chromatin. We conclude that Foxa2 is required for execution of the hepatic gluconeogenic program by integrating the transcriptional response of the hepatocyte to hormonal stimulation.

  1. Soil solution chemistry and element fluxes in three European heathlands and their responses to warming and drought

    DEFF Research Database (Denmark)

    Schmidt, I.K.; Tietema, A.; Williams, D.;


    in the first year, but, because of high retention of N in the vegetation or mineral soil, there were no significant effects of warming on seepage water NO3-N and NH4-N. Retention of P was high at all three sites. In several cases, drought increased concentrations of elements momentarily, but element fluxes...

  2. 鲍曼不动杆菌AmpC酶和AmpC耐药基因检测分析%Detection on AmpC b-lactamases and AmpC gene of Acinetobacter baumannii

    Institute of Scientific and Technical Information of China (English)

    张丽梅; 苏丹虹; 徐韫健


    目的 检测临床分离的45株鲍曼不动杆菌的AmpC酶和AmpC耐药基因,并探讨产AmpC酶菌株的耐药情况.方法 采用超声破碎法提取45株细菌的β-内酰胺酶粗提物,进行三维试验,提取45株细菌的总DNA,聚合酶链反应(PCR)扩增AmpC结构基因和ACT-1、CMY-G1、CMY-G2、DHA、FOX耐药基因,最低抑菌浓度药物敏感试验分析菌株的耐药性.结果 45株鲍曼不动杆菌中三维试验和PCR检测AmpC基因同时阳性的有11株,确认产AmpC酶菌株为11株.三维试验方法和PCR基因检测方法比较对AmpC酶的检出率差异无统计学意义(χ2=3.500,P>0.05).ACT-1的检出率为4.4%,未检出FOX 、DHA、CMY-G1、CMY-G2.产AmpC酶菌株的药物敏感度最高是丁胺卡那霉素(90.9%),其次是亚胺培南(54.5%),产AmpC酶菌株耐药率高,对氨苄西林、氨曲南、头孢曲松、头孢替坦、头孢唑啉、呋喃妥因、头孢他啶达到100.0%,产AmpC酶菌株对抗菌药物的耐药性显著高于不产AmpC酶菌株.结论 三维试验方法和PCR基因检测方法对AmpC酶的检出率无明显差别,但仍存在假阳性与假阴性.产AmpC酶菌株耐药情况严重,提示临床应慎重用药.

  3. Lagrangian three-dimensional finite-element formulation for the nonlinear fluid-structural response of reactor components. [LMFBR

    Energy Technology Data Exchange (ETDEWEB)

    Kulak, R. F.; Fiala, C.


    This report presents the formulations used in the NEPTUNE code. Specifically, it describes the finite-element formulation of a three-dimensional hexahedral element for simulating the behavior of either fluid or solid continua. Since the newly developed hexahedral element and the original triangular plate element are finite elements, they are compatible in the sense that they can be combined arbitrarily to simulate complex reactor components in three-dimensional space. Because rate-type constitutive relations are used in conjunction with a velocity-strain tensor, the formulation is applicable to large deformation problems. This development can be used to simulate (1) the fluid adjacent to reactor components and (2) the concrete fill found in large reactor head closures.

  4. Modulation of signaling through GPCR-cAMP-PKA pathways by PDE4 depends on stimulus intensity: Possible implications for the pathogenesis of acrodysostosis without hormone resistance. (United States)

    Motte, Emmanuelle; Le Stunff, Catherine; Briet, Claire; Dumaz, Nicolas; Silve, Caroline


    In acrodysostosis without hormone resistance, a disease caused by phosphodiesterase (PDE)-4D mutations, increased PDE activity leads to bone developmental defects but with normal renal responses to PTH. To identify potential mechanisms for these disparate responses, we compared the effect of PDE activity on hormone signaling through the GPCR-Gsα-cAMP-PKA pathway in cells from two lineages, HEK-293 cells stably overexpressing PTH1R (HEKpthr) and human dermal fibroblasts, including studies evaluating cAMP levels using an Epac-based BRET-sensor for cAMP (CAMYEL). For ligand-induced responses inducing strong cAMP accumulation, the inhibition of PDE4 activity resulted in relatively small further increases. In contrast, when ligand-induced cAMP accumulation was of lesser intensity, the inhibition of PDE4 had a more pronounced effect. Similar results were obtained evaluating downstream events (cellular CREB phosphorylation and CRE-luciferase activity). Thus, the ability of PDE4 to modulate signaling through GPCR-cAMP-PKA pathways can depend on the cell type and stimulus intensity.

  5. The NO/cGMP pathway inhibits transient cAMP signals through the activation of PDE2 in striatal neurons

    Directory of Open Access Journals (Sweden)

    Marina ePolito


    Full Text Available The NO-cGMP signaling plays an important role in the regulation of striatal function although the mechanisms of action of cGMP specifically in medium spiny neurons (MSNs remain unclear. Using genetically encoded fluorescent biosensors, including a novel Epac-based sensor (EPAC-SH150 with increased sensitivity for cAMP, we analyze the cGMP response to NO and whether it affected cAMP/PKA signaling in MSNs. The Cygnet2 sensor for cGMP reported large responses to NO donors in both striatonigral and striatopallidal MSNs, and this cGMP signal was controlled partially by PDE2. At the level of cAMP brief forskolin stimulations produced transient cAMP signals which differed between D1 and D2 medium spiny neurons. NO inhibited these cAMP transients through cGMP-dependent PDE2 activation, an effect that was translated and magnified downstream of cAMP, at the level of PKA. PDE2 thus appears as a critical effector of NO which modulates the post-synaptic response of MSNs to dopaminergic transmission.

  6. c-di-AMP binds the ydaO riboswitch in two pseudo-symmetry-related pockets. (United States)

    Ren, Aiming; Patel, Dinshaw J


    The ydaO riboswitch, involved in sporulation, osmotic stress responses and cell wall metabolism, targets the second messenger cyclic-di-AMP with subnanomolar affinity. We have solved the structure of c-di-AMP bound to the Thermoanaerobacter tengcongensis ydaO riboswitch, thereby identifying a five-helical scaffold containing a zippered-up bubble, a pseudoknot and long-range tertiary base pairs. Highlights include the identification of two c-di-AMP binding pockets on the same face of the riboswitch, related by pseudo-two-fold symmetry, with potential for cross-talk between sites mediated by adjacently positioned base-stacking alignments connecting pockets. The adenine rings of bound c-di-AMP molecules are wedged between bases and stabilized by stacking, base-sugar and sugar-sugar intermolecular hydrogen bonding interactions. The structural studies are complemented by isothermal titration calorimetry-based binding studies of mutants mediating key tertiary intermolecular contacts. The T. tengcongensis ydaO riboswitch, like its Bacillus subtilis counterpart, most likely functions through a transcription termination mechanism, with the c-di-AMP bound state representing an 'off' switch.

  7. Electrical Stimulation Decreases Coupling Efficiency Between Beta-Adrenergic Receptors and Cyclic AMP Production in Cultured Muscle Cells (United States)

    Young, R. B.; Bridge, K. Y.


    Electrical stimulation of skeletal muscle cells in culture is an effective way to simulate the effects of muscle contraction and its effects on gene expression in muscle cells. Expression of the beta-adrenergic receptor and its coupling to cyclic AMP synthesis are important components of the signaling system that controls muscle atrophy and hypertrophy, and the goal of this project was to determine if electrical stimulation altered the beta-adrenergic response in muscle cells. Chicken skeletal muscle cells that had been grown for seven days in culture were subjected to electrical stimulation for an additional two days at a pulse frequency of 0.5 pulses/sec and a pulse duration of 200 msec. At the end of this two-day stimulation period, beta-adrenergic receptor population was measured by the binding of tritium-labeled CGP-12177 to muscle cells, and coupling to cAMP synthesis was measured by Radioimmunoassay (RIA) after treating the cells for 10 min with the potent (beta)AR agonist, isoproterenol. The number of beta adrenergic receptors and the basal levels of intracellular cyclic AMP were not affected by electrical stimulation. However, the ability of these cells to synthesize cyclic AMP was reduced by approximately 50%. Thus, an enhanced level of contraction reduces the coupling efficiency of beta-adrenergic receptors for cyclic AMP production.

  8. Sevoflurane effects on cyclic adenosine monophosphate response element binding protein, phosphorylated cyclic adenosine monophosphate response element binding protein, and Livin expression in the cortex and hippocampus of a vascular cognitive impairment rat model

    Institute of Scientific and Technical Information of China (English)

    Bin Wu; Ling Dan; Xianlin Zhu


    BACKGROUND: Neuronal necrosis and apoptosis play important roles in the pathophysiology of cerebral ischemia and resulting cognitive impairment. However, inhibition of neuronal necrosis and apoptosis has been shown to attenuate cognitive impairment following cerebral ischemia.OBJECTIVE: To investigate the effects of sevoflurane on cyclic adenosine monophosphate response element binding protein (CREB), phosphorylated CREB (pCREB), and Livin expression in the cortex and hippocampus of a rat model of vascular cognitive impairment.DESIGN, TIME AND SETTING: A randomized, controlled experiment was performed in the Chongqing Key Laboratory of Neurology between June 2007 and July 2008.MATERIALS: Sevoflurane was provided by Abbott Laboratory, UK; Morris water maze was provided by Chinese Academy of Medical Sciences, China; goat anti-rat CREB, goat anti-rat pCREB and goat anti-rat Livin antibodies were provided by Biosource International, USA.METHODS: A total of 42 female, Wistar rats were randomly assigned to the following groups: sham operation, vascular cognitive impairment, and sevoflurane treatment. The vascular cognitive impairment rat model was established by permanent bilateral occlusion of both common carotid arteries, and 1.0 MAC sevoflurane was immediately administered by inhalation for 2 hours.MAIN OUTCOME MEASURES: CREB, pCREB, and Livin expression was measured in the cortex and hippocampus by Western blot and reverse transcription-polymerase chain reaction. Behavior was evaluated with Morris water maze.RESULTS: CREB, pCREB, and Livin expression in the sevoflurane treatment group was significantly greater than the vascular cognitive impairment group (P<0.01). However, expression of CREB and pCREB was significantly less in the sevoflurane treatment and vascular cognitive impairment groups, compared with the sham operation group (P<0.01). Livin expression in the sevoflurane treatment and vascular cognitive impairment groups was significantly greater than the sham

  9. The MluI cell cycle box (MCB) motifs, but not damage-responsive elements (DREs), are responsible for the transcriptional induction of the rhp51+ gene in response to DNA replication stress. (United States)

    Sartagul, Wugangerile; Zhou, Xin; Yamada, Yuki; Ma, Ning; Tanaka, Katsunori; Furuyashiki, Tomoyuki; Ma, Yan


    DNA replication stress induces the transcriptional activation of rhp51+, a fission yeast recA homolog required for repair of DNA double strand breaks. However, the mechanism by which DNA replication stress activates rhp51+ transcription is not understood. The promoter region of rhp51+ contains two damage-responsive elements (DREs) and two MluI cell cycle box (MCB) motifs. Using luciferase reporter assays, we examined the role of these elements in rhp51+ transcription. The full-length rhp51+ promoter and a promoter fragment containing MCB motifs only, but not a fragment containing DREs, mediated transcriptional activation upon DNA replication stress. Removal of the MCB motifs from the rhp51+ promoter abolished the induction of rhp51+ transcription by DNA replication stress. Consistent with a role for MCB motifs in rhp51+ transcription activation, deletion of the MBF (MCB-binding factor) co-repressors Nrm1 and Yox1 precluded rhp51+ transcriptional induction in response to DNA replication stress. Using cells deficient in checkpoint signaling molecules, we found that the Rad3-Cds1/Chk1 pathway partially mediated rhp51+ transcription in response to DNA replication stress, suggesting the involvement of unidentified checkpoint signaling pathways. Because MBF is critical for G1/S transcription, we examined how the cell cycle affected rhp51+ transcription. The transcription of rhp51+ and cdc18+, an MBF-dependent G1/S gene, peaked simultaneously in synchronized cdc25-22 cells. Furthermore, DNA replication stress maintained transcription of rhp51+ similarly to cdc18+. Collectively, these results suggest that MBF and its regulators mediate rhp51+ transcription in response to DNA replication stress, and underlie rhp51+ transcription at the G1/S transition.

  10. Identification of cholinergic and non-cholinergic neurons in the pons expressing phosphorylated cyclic adenosine monophosphate response element-binding protein as a function of rapid eye movement sleep. (United States)

    Datta, S; Siwek, D F; Stack, E C


    Recent studies have shown that in the pedunculopontine tegmental nucleus (PPT), increased neuronal activity and kainate receptor-mediated activation of intracellular protein kinase A (PKA) are important physiological and molecular steps for the generation of rapid eye movement (REM) sleep. In the present study performed on rats, phosphorylated cyclic AMP response element-binding protein (pCREB) immunostaining was used as a marker for increased intracellular PKA activation and as a reflection of increased neuronal activity. To identify whether activated cells were either cholinergic or noncholinergic, the PPT and laterodorsal tegmental nucleus (LDT) cells were immunostained for choline acetyltransferase (ChAT) in combination with pCREB or c-Fos. The results demonstrated that during high rapid eye movement sleep (HR, approximately 27%), significantly higher numbers of cells expressed pCREB and c-Fos in the PPT, of which 95% of pCREB-expressing cells were ChAT-positive. With HR, the numbers of pCREB-positive cells were also significantly higher in the medial pontine reticular formation (mPRF), pontine reticular nucleus oral (PnO), and dorsal subcoeruleus nucleus (SubCD) but very few in the locus coeruleus (LC) and dorsal raphe nucleus (DRN). Conversely, with low rapid eye movement sleep (LR, approximately 2%), the numbers of pCREB expressing cells were very few in the PPT, mPRF, PnO, and SubCD but significantly higher in the LC and DRN. The results of regression analyses revealed significant positive relationships between the total percentages of REM sleep and numbers of ChAT+/pCREB+ (Rsqr=0.98) cells in the PPT and pCREB+ cells in the mPRF (Rsqr=0.88), PnO (Rsqr=0.87), and SubCD (Rsqr=0.84); whereas significantly negative relationships were associated with the pCREB+ cells in the LC (Rsqr=0.70) and DRN (Rsqr=0.60). These results provide evidence supporting the hypothesis that during REM sleep, the PPT cholinergic neurons are active, whereas the LC and DRN neurons are

  11. Cyclic AMP Recruits a Discrete Intracellular Ca2+ Store by Unmasking Hypersensitive IP3 Receptors

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    Vera Konieczny


    Full Text Available Inositol 1,4,5-trisphosphate (IP3 stimulates Ca2+ release from the endoplasmic reticulum (ER, and the response is potentiated by 3′,5′-cyclic AMP (cAMP. We investigated this interaction in HEK293 cells using carbachol and parathyroid hormone (PTH to stimulate formation of IP3 and cAMP, respectively. PTH alone had no effect on the cytosolic Ca2+ concentration, but it potentiated the Ca2+ signals evoked by carbachol. Surprisingly, however, the intracellular Ca2+ stores that respond to carbachol alone could be both emptied and refilled without affecting the subsequent response to PTH. We provide evidence that PTH unmasks high-affinity IP3 receptors within a discrete Ca2+ store. We conclude that Ca2+ stores within the ER that dynamically exchange Ca2+ with the cytosol maintain a functional independence that allows one store to be released by carbachol and another to be released by carbachol with PTH. Compartmentalization of ER Ca2+ stores adds versatility to IP3-evoked Ca2+ signals.

  12. The RGS protein Crg2 regulates both pheromone and cAMP signalling in Cryptococcus neoformans. (United States)

    Xue, Chaoyang; Hsueh, Yen-Ping; Chen, Lydia; Heitman, Joseph


    G proteins orchestrate critical cellular functions by transducing extracellular signals into internal signals and controlling cellular responses to environmental cues. G proteins typically function as switches that are activated by G protein-coupled receptors (GPCRs) and negatively controlled by regulator of G protein signalling (RGS) proteins. In the human fungal pathogen Cryptococcus neoformans, three G protein alpha subunits (Gpa1, Gpa2 and Gpa3) have been identified. In a previous study, we identified the RGS protein Crg2 involved in regulating the pheromone response pathway through Gpa2 and Gpa3. In this study, a role for Crg2 was established in the Gpa1-cAMP signalling pathway that governs mating and virulence. We show that Crg2 physically interacts with Gpa1 and crg2 mutations increase cAMP production. crg2 mutations also enhance mating filament hyphae production, but reduce cell-cell fusion and sporulation efficiency during mating. Although crg2 mutations and the Gpa1 dominant active allele GPA1(Q284L) enhanced melanin production under normally repressive conditions, virulence was attenuated in a murine model. We conclude that Crg2 participates in controlling both Gpa1-cAMP-virulence and pheromone-mating signalling cascades and hypothesize it may serve as a molecular interface between these two central signalling conduits.

  13. Cross-talk between signaling pathways can generate robust oscillations in calcium and cAMP.

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    Fernando Siso-Nadal

    Full Text Available BACKGROUND: To control and manipulate cellular signaling, we need to understand cellular strategies for information transfer, integration, and decision-making. A key feature of signal transduction is the generation of only a few intracellular messengers by many extracellular stimuli. METHODOLOGY/PRINCIPAL FINDINGS: Here we model molecular cross-talk between two classic second messengers, cyclic AMP (cAMP and calcium, and show that the dynamical complexity of the response of both messengers increases substantially through their interaction. In our model of a non-excitable cell, both cAMP and calcium concentrations can oscillate. If