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Sample records for amp inducible early

  1. Concerted Regulation of cGMP and cAMP Phosphodiesterases in Early Cardiac Hypertrophy Induced by Angiotensin II

    PubMed Central

    Mokni, Walid; Keravis, Thérèse; Etienne-Selloum, Nelly; Walter, Alison; Kane, Modou O.; Schini-Kerth, Valérie B.; Lugnier, Claire

    2010-01-01

    Left ventricular hypertrophy leads to heart failure and represents a high risk leading to premature death. Cyclic nucleotides (cAMP and cGMP) play a major role in heart contractility and cyclic nucleotide phosphodiesterases (PDEs) are involved in different stages of advanced cardiac diseases. We have investigated their contributions in the very initial stages of left ventricular hypertrophy development. Wistar male rats were treated over two weeks by chronic infusion of angiotensin II using osmotic mini-pumps. Left cardiac ventricles were used as total homogenates for analysis. PDE1 to PDE5 specific activities and protein and mRNA expressions were explored. Rats developed arterial hypertension associated with a slight cardiac hypertrophy (+24%). cAMP-PDE4 activity was specifically increased while cGMP-PDE activities were broadly increased (+130% for PDE1; +76% for PDE2; +113% for PDE5) and associated with increased expressions for PDE1A, PDE1C and PDE5A. The cGMP-PDE1 activation by Ca2+/CaM was reduced. BNP expression was increased by 3.5-fold, while NOX2 expression was reduced by 66% and AMP kinase activation was increased by 64%. In early cardiac hypertrophy induced by angiotensin II, all specific PDE activities in left cardiac ventricles were increased, favoring an increase in cGMP hydrolysis by PDE1, PDE2 and PDE5. Increased cAMP hydrolysis was related to PDE4. We observed the establishment of two cardioprotective mechanisms and we suggest that these mechanisms could lead to increase intracellular cGMP: i) increased expression of BNP could increase “particulate” cGMP pool; ii) increased activation of AMPK, subsequent to increase in PDE4 activity and 5′AMP generation, could elevate “soluble” cGMP pool by enhancing NO bioavailability through NOX2 down-regulation. More studies are needed to support these assumptions. Nevertheless, our results suggest a potential link between PDE4 and AMPK/NOX2 and they point out that cGMP-PDEs, especially PDE1 and PDE2

  2. Cyclic AMP regulation of early gene expression in Dictyostelium discoideum: mediation via the cell surface cyclic AMP receptor.

    PubMed Central

    Mann, S K; Firtel, R A

    1987-01-01

    We examined two sets of genes expressed early in the developmental cycle of Dictyostelium discoideum that appear to be regulated by cyclic AMP (cAMP). The transcripts of both sets of genes were not detectable in vegetative cells. During normal development on filter pads, RNA complementary to these genes could be detected at about 2 h, peaked around 6 to 8 h, and decreased gradually thereafter. Expression of these genes upon starvation in shaking culture was stimulated by pulsing the cells with nanomolar levels of cAMP, a condition that mimics the in vivo pulsing during normal aggregation. Expression was inhibited by caffeine or by continuous levels of cAMP, a condition found later in development when in vivo expression of these genes decreased. The inhibition of caffeine could be overcome by pulsing cells with cAMP. These results suggest that the expression is mediated via the cell surface cAMP receptor, but does not require a rise in intracellular cAMP. mRNA from a gene of the second class was induced upon starvation, peaked by 2.5 h of development, and then declined. In contrast to the other genes, its expression was maintained by continuous levels of cAMP and repressed by cAMP pulses. These and other results on a number of classes of developmentally regulated genes indicates that changing levels of cAMP, acting via the cell surface cAMP receptor, are involved in controlling these groups of genes. We also examined the structure and partial sequence of the cAMP pulse-induced genes. The two tandemly duplicated M3 genes were almost continuously homologous over the sequenced portion of the protein-coding region except for a region near the N-terminal end. The two M3 genes had regions of homology in the 5' flanking sequence and showed slight homology to the same regions in gene D2, another cAMP pulse-induced gene. D2 showed extremely significant homology over its entire sequenced length to an acetylcholinesterase. The results presented here and by others suggest that

  3. cAMP inducibility of transcriptional repressor ICER in developing and mature human T lymphocytes.

    PubMed

    Bodor, J; Spetz, A L; Strominger, J L; Habener, J F

    1996-04-16

    Stimulation of the cAMP-dependent signaling pathway exerts an inhibitory effect on the proliferation and effector functions of T cells. The ability of T cells to form high intracellular levels of cAMP is acquired during development in the human thymus and is retained by the majority of mature peripheral T lymphocytes. Here we show that elevated cAMP levels in T cells correlate with the expression of the potent transcriptional repressor ICER (inducible cAMP early repressor) previously described in the hypothalamic-pituitary-gonadal axis. Further, in transcriptional assays in vivo, ICER inhibits calcineurin-mediated expression of the interleukin 2 promoter as well as Tax-mediated transactivation of the human T-lymphotropic virus type I (HTLV-I) promoter. Thus, the induction of ICER in T cells may play an important role in the cAMP-induced quiescence and the persistent latency of HTLV-I.

  4. Different roles of GNAS and cAMP signaling during early and late stages of osteogenic differentiation.

    PubMed

    Zhang, S; Kaplan, F S; Shore, E M

    2012-09-01

    Progressive osseous heteroplasia (POH) and fibrous dysplasia (FD) are genetic diseases of bone formation at opposite ends of the osteogenic spectrum: imperfect osteogenesis of the skeleton occurs in FD, while heterotopic ossification in skin, subcutaneous fat, and skeletal muscle forms in POH. POH is caused by heterozygous inactivating germline mutations in GNAS, which encodes G-protein subunits regulating the cAMP pathway, while FD is caused by GNAS somatic activating mutations. We used pluripotent mouse ES cells to examine the effects of Gnas dysregulation on osteoblast differentiation. At the earliest stages of osteogenesis, Gnas transcripts Gsα, XLαs and 1A are expressed at low levels and cAMP levels are also low. Inhibition of cAMP signaling (as in POH) by 2',5'-dideoxyadenosine enhanced osteoblast differentiation while conversely, increased cAMP signaling (as in FD), induced by forskolin, inhibited osteoblast differentiation. Notably, increased cAMP was inhibitory for osteogenesis only at early stages after osteogenic induction. Expression of osteogenic and adipogenic markers showed that increased cAMP enhanced adipogenesis and impaired osteoblast differentiation even in the presence of osteogenic factors, supporting cAMP as a critical regulator of osteoblast and adipocyte lineage commitment. Furthermore, increased cAMP signaling decreased BMP pathway signaling, indicating that G protein-cAMP pathway activation (as in FD) inhibits osteoblast differentiation, at least in part by blocking the BMP-Smad pathway, and suggesting that GNAS inactivation as occurs in POH enhances osteoblast differentiation, at least in part by stimulating BMP signaling. These data support that differences in cAMP levels during early stages of cell differentiation regulate cell fate decisions. Supporting information available online at http:/www.thieme-connect.de/ejournals/toc/hmr. PMID:22903279

  5. Hepatic gene expression profiling of 5′-AMP-induced hypometabolism in mice

    PubMed Central

    Miki, Takao; Van Oort-Jansen, Anita; Matsumoto, Tomoko; Loose, David S.; Lee, Cheng Chi

    2011-01-01

    There is currently much interest in clinical applications of therapeutic hypothermia. Hypothermia can be a consequence of hypometabolism. We have recently established a procedure for the induction of a reversible deep hypometabolic state in mice using 5′-adenosine monophosphate (5′-AMP) in conjunction with moderate ambient temperature. The current study aims at investigating the impact of this technology at the gene expression level in a major metabolic organ, the liver. Our findings reveal that expression levels of the majority of genes in liver are not significantly altered by deep hypometabolism. However, among those affected by hypometabolism, more genes are differentially upregulated than downregulated both in a deep hypometabolic state and in the early arousal state. These altered gene expression levels during 5′-AMP induced hypometabolism are largely restored to normal levels within 2 days of the treatment. Our data also suggest that temporal control of circadian genes is largely stalled during deep hypometabolism. PMID:21224422

  6. Glucose-induced hyperaccumulation of cyclic AMP and defective glucose repression in yeast strains with reduced activity of cyclic AMP-dependent protein kinase.

    PubMed

    Mbonyi, K; van Aelst, L; Argüelles, J C; Jans, A W; Thevelein, J M

    1990-09-01

    Addition of glucose or related fermentable sugars to derepressed cells of the yeast Saccharomyces cerevisiae triggers a RAS-mediated cyclic AMP (cAMP) signal that induces a protein phosphorylation cascade. In yeast mutants (tpk1w1, tpk2w1, and tpk3w1) containing reduced activity of cAMP-dependent protein kinase, fermentable sugars, as opposed to nonfermentable carbon sources, induced a permanent hyperaccumulation of cAMP. This finding confirms previous conclusions that fermentable sugars are specific stimulators of cAMP synthesis in yeast cells. Despite the huge cAMP levels present in these mutants, deletion of the gene (BCY1) coding for the regulatory subunit of cAMP-dependent protein kinase severely reduced hyperaccumulation of cAMP. Glucose-induced hyperaccumulation of cAMP was also observed in exponential-phase glucose-grown cells of the tpklw1 and tpk2w1 strains but not the tpk3w1 strain even though addition of glucose to glucose-repressed wild-type cells did not induce a cAMP signal. Investigation of mitochondrial respiration by in vivo 31P nuclear magnetic resonance spectroscopy showed the tpk1w1 and tpk2w1 strains, to be defective in glucose repression. These results are consistent with the idea that the signal transmission pathway from glucose to adenyl cyclase contains a glucose-repressible protein. They also show that a certain level of cAMP-dependent protein phosphorylation is required for glucose repression. Investigation of the glucose-induced cAMP signal and glucose-induced activation of trehalase in derepressed cells of strains containing only one of the wild-type TPK genes indicates that the transient nature of the cAMP signal is due to feedback inhibition by cAMP-dependent protein kinase.

  7. Luteinizing hormone-releasing hormone (LHRH) attenuates morphine-induced inhibition of cyclic AMP (cAMP) in opioid-responsive SK-N-SH cells.

    PubMed

    Ratka, A; Simpkins, J W

    1997-04-01

    SK-N-SH cells were used to assess the effects of luteinizing hormone-releasing hormone (LHRH) on opioid receptor-mediated changes in cyclic AMP (cAMP). Prostaglandin E1 (PGE1, 1 microM) caused a dramatic increase in cAMP levels. Treatment with 10 microM morphine (MOR) significantly inhibited the stimulatory effect of PGE1, LHRH (0.8 microM) caused an increase in the basal level of intracellular cAMP and potentiated the stimulatory effect of PGE1 on cAMP accumulation. In cells pretreated with LHRH the inhibitory effect of MOR on cAMP accumulation was significantly attenuated. An LHRH antagonist had no effect on cAMP. The involvement of pertussis toxin (PTX)-sensitive G proteins in the actions of LHRH was studied. PTX increased the stimulatory effect of PGE1 on cAMP and attenuated the inhibitory effect of MOR. However, PTX pretreatment prevented the effects of LHRH on the intracellular actions of PGE1 but exerted an additive effect with LHRH in blocking the MOR-induced decrease in cAMP levels. We conclude that LHRH attenuates the inhibitory, opioid receptor-mediated effect of MOR on intracellular cAMP accumulation in SK-N-SH cells, and that the G protein-independent mechanism may be involved in LHRH-induced attenuation of the inhibitory effect of MOR on neuronal cAMP.

  8. Ethanol-induced loss of brain cyclic AMP binding proteins: correlation with growth suppression

    SciTech Connect

    Pennington, S.; Kalmus, G.

    1987-05-01

    Brain hypoplasia secondary to maternal ethanol consumption is a common fetal defect observed in all models of fetal alcohol syndrome. The molecular mechanism by which ethanol inhibits growth is unknown but has been hypothesized to involve ethanol-induced changes in the activity of cyclic-AMP stimulated protein kinase. Acute and chronic alcohol exposure elevate cyclic AMP level in many tissues, including brain. This increase in cyclic AMP should increase the phosphorylating activity of kinase by increasing the amount of dissociated (active) kinase catalytic subunit. In 7-day embryonic chick brains, ethanol-induced growth suppression was correlated with increased brain cyclic AMP content but neither basal nor cyclic AMP stimulated kinase catalytic activity was increased. However, the levels of cyclic AMP binding protein (kinase regulatory subunit) were significantly lowered by ethanol exposure. Measured as either /sup 3/H cyclic AMP binding or as 8-azido cyclic AM/sup 32/P labeling, ethanol-exposed brains had significantly less cyclic AMP binding activity (51 +/- 14 versus 29 +/- 10 units/..mu..g protein for 8-azido cyclic AMP binding). These findings suggest that ethanol's effect on kinase activity may involve more than ethanol-induced activation of adenylate cyclase.

  9. Mechanically induced c-fos expression is mediated by cAMP in MC3T3-E1 osteoblasts

    NASA Technical Reports Server (NTRS)

    Fitzgerald, J.; Hughes-Fulford, M.

    1999-01-01

    In serum-deprived MC3T3-E1 osteoblasts, mechanical stimulation caused by mild (287 x g) centrifugation induced a 10-fold increase in mRNA levels of the proto-oncogene, c-fos. Induction of c-fos was abolished by the cAMP-dependent protein kinase inhibitor H-89, suggesting that the transient c-fos mRNA increase is mediated by cAMP. Down-regulation of protein kinase C (PKC) activity by chronic TPA treatment failed to significantly reduce c-fos induction, suggesting that TPA-sensitive isoforms of PKC are not responsible for c-fos up-regulation. In addition, 287 x g centrifugation increased intracellular prostaglandin E2 (PGE2) levels 2.8-fold (P<0. 005). Since we have previously shown that prostaglandin E2 (PGE2) can induce c-fos expression via a cAMP-mediated mechanism, we asked whether the increase in c-fos mRNA was due to centrifugation-induced PGE2 release. Pretreatment with the cyclooxygenase inhibitors indomethacin and flurbiprofen did not hinder the early induction of c-fos by mechanical stimulation. We conclude that c-fos expression induced by mild mechanical loading is dependent primarily on cAMP, not PKC, and initial induction of c-fos is not necessarily dependent on the action of newly synthesized PGE2.

  10. TSH-induced cyclic AMP production in an ovine thyroid cell line: OVNIS 5H.

    PubMed

    Fayet, G; Aouani, A; Hovsépian, S

    1986-01-01

    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. PMID:3000830

  11. cAMP prevents TNF-induced apoptosis through inhibiting DISC complex formation in rat hepatocytes

    SciTech Connect

    Bhattacharjee, Rajesh; Xiang, Wenpei; Wang, Yinna; Zhang, Xiaoying

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer cAMP blocks cell death induced by TNF and actinomycin D in cultured hepatocytes. Black-Right-Pointing-Pointer cAMP blocks NF-{kappa}B activation induced by TNF and actinomycin D. Black-Right-Pointing-Pointer cAMP blocks DISC formation following TNF and actinomycin D exposure. Black-Right-Pointing-Pointer cAMP blocks TNF signaling at a proximal step. -- Abstract: Tumor necrosis factor {alpha} (TNF) is a pleiotropic proinflammatory cytokine that plays a role in immunity and the control of cell proliferation, cell differentiation, and apoptosis. The pleiotropic nature of TNF is due to the formation of different signaling complexes upon the binding of TNF to its receptor, TNF receptor type 1 (TNFR1). TNF induces apoptosis in various mammalian cells when the cells are co-treated with a transcription inhibitor like actinomycin D (ActD). When TNFR1 is activated, it recruits an adaptor protein, TNF receptor-associated protein with death domain (TRADD), through its cytoplasmic death effector domain (DED). TRADD, in turn, recruits other signaling proteins, including TNF receptor-associated protein 2 (TRAF2) and receptor-associated protein kinase (RIPK) 1, to form a complex. Subsequently, this complex combines with FADD and procaspase-8, converts into a death-inducing signaling complex (DISC) to induce apoptosis. Cyclic AMP (cAMP) is a second messenger that regulates various cellular processes such as cell proliferation, gene expression, and apoptosis. cAMP analogues are reported to act as anti-apoptotic agents in various cell types, including hepatocytes. We found that a cAMP analogue, dibutyryl cAMP (db-cAMP), inhibits TNF + ActD-induced apoptosis in rat hepatocytes. The protein kinase A (PKA) inhibitor KT-5720 reverses this inhibitory effect of cAMP on apoptosis. Cytoprotection by cAMP involves down-regulation of various apoptotic signal regulators like TRADD and FADD and inhibition of caspase-8 and caspase-3 cleavage. We also found

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

    SciTech Connect

    Zhang, Wenting; Tingare, Asmita; Ng, David Chi-Heng; Johnson, Hong W.; Schell, Michael J.; Lord, Rebecca L.; Chawla, Sangeeta

    2012-08-24

    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 by

  13. Dibutyryl cAMP effects on thromboxane and leukotriene production in decompression-induced lung injury

    NASA Technical Reports Server (NTRS)

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

    1997-01-01

    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.

  14. cAMP-Induced Histones H3 Dephosphorylation Is Independent of PKA and MAP Kinase Activations and Correlates With mTOR Inactivation.

    PubMed

    Rodriguez, Pedro; Rojas, Juan

    2016-03-01

    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. PMID:26335579

  15. cAMP-Induced Histones H3 Dephosphorylation Is Independent of PKA and MAP Kinase Activations and Correlates With mTOR Inactivation.

    PubMed

    Rodriguez, Pedro; Rojas, Juan

    2016-03-01

    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. Regulation by intracellular Ca sup 2+ and cyclic AMP of the growth factor-induced ruffling membrane formation and stimulation of fluid-phase endocytosis and exocytosis

    SciTech Connect

    Miyata, Yoshihiko Tokyo Metropolitan Inst. of Medical Science ); Nishida, Eisuke; Sakai, Hikoichi ); Koyasu, Shigeo; Yahara, Ichiro )

    1989-04-01

    Insulin, insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF) induce formation of ruffling membranes and stimulate the fluid-phase endocytosis and exocytosis in human epidermoid carcinoma KB cells. An increase in intracellular Ca{sup 2+} concentration by treatment with A23187, a calcium ionophore, or an increase in intracellular cAMP level by treatment with dibutyryl cAMP or forskolin almost completely inhibited the insulin-, IGF-I-, or EGF-induced formation of ruffling membranes. Increases in Ca{sup 2+} or cAMP concentration also inhibited almost completely the stimulation of fluid-phase endocytosis and exocytosis elicited by these growth factors. These results suggest that the growth factor-induced ruffling membrane formation and the stimulation of fluid-phase endocytosis and exocytosis have a common regulatory mechanism involving intracellular concentrations of Ca{sup 2+} and cAMP. {sup 125}I-EGF binding assays and immunoprecipitation experiments with anti-phosphotyrosine antibody revealed that treatment of KB cells with A23187, dibutyryl cAMP, or forskolin did not inhibit the EGF binding to the cells nor subsequent tyrosine autophosphorylation of its receptors. These results indicate that Ca{sup 2+}- and/or cAMP-sensitive intracellular reactions exist downstream from the receptor kinase activation in the process of these early cellular responses.

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

    PubMed

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

    2014-10-01

    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.

  18. Odor-induced cAMP production in Drosophila melanogaster olfactory sensory neurons.

    PubMed

    Miazzi, Fabio; Hansson, Bill S; Wicher, Dieter

    2016-06-15

    Insect odorant receptors are seven transmembrane domain proteins that form cation channels, whose functional properties such as receptor sensitivity are subject to regulation by intracellular signaling cascades. Here, we used the cAMP fluorescent indicator Epac1-camps to investigate the occurrence of odor-induced cAMP production in olfactory sensory neurons (OSNs) of Drosophila melanogaster We show that stimulation of the receptor complex with an odor mixture or with the synthetic agonist VUAA1 induces a cAMP response. Moreover, we show that while the intracellular Ca(2+) concentration influences cAMP production, the OSN-specific receptor OrX is necessary to elicit cAMP responses in Ca(2+)-free conditions. These results provide direct evidence of a relationship between odorant receptor stimulation and cAMP production in olfactory sensory neurons in the fruit fly antenna and show that this method can be used to further investigate the role that this second messenger plays in insect olfaction. PMID:27045092

  19. The role of ventral striatal cAMP signaling in stress-induced behaviors.

    PubMed

    Plattner, Florian; Hayashi, Kanehiro; Hernández, Adan; Benavides, David R; Tassin, Tara C; Tan, Chunfeng; Day, Jonathan; Fina, Maggy W; Yuen, Eunice Y; Yan, Zhen; Goldberg, Matthew S; Nairn, Angus C; Greengard, Paul; Nestler, Eric J; Taussig, Ronald; Nishi, Akinori; Houslay, Miles D; Bibb, James A

    2015-08-01

    The cAMP and cAMP-dependent protein kinase A (PKA) signaling cascade is a ubiquitous pathway acting downstream of multiple neuromodulators. We found that the phosphorylation of phosphodiesterase-4 (PDE4) by cyclin-dependent protein kinase 5 (Cdk5) facilitated cAMP degradation and homeostasis of cAMP/PKA signaling. In mice, loss of Cdk5 throughout the forebrain elevated cAMP levels and increased PKA activity in striatal neurons, and altered behavioral responses to acute or chronic stressors. Ventral striatum- or D1 dopamine receptor-specific conditional knockout of Cdk5, or ventral striatum infusion of a small interfering peptide that selectively targeted the regulation of PDE4 by Cdk5, produced analogous effects on stress-induced behavioral responses. Together, our results demonstrate that altering cAMP signaling in medium spiny neurons of the ventral striatum can effectively modulate stress-induced behavioral states. We propose that targeting the Cdk5 regulation of PDE4 could be a new therapeutic approach for clinical conditions associated with stress, such as depression. PMID:26192746

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

    PubMed Central

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

    2014-01-01

    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. PMID:25154887

  1. The role of ventral striatal cAMP signaling in stress-induced behaviors.

    PubMed

    Plattner, Florian; Hayashi, Kanehiro; Hernández, Adan; Benavides, David R; Tassin, Tara C; Tan, Chunfeng; Day, Jonathan; Fina, Maggy W; Yuen, Eunice Y; Yan, Zhen; Goldberg, Matthew S; Nairn, Angus C; Greengard, Paul; Nestler, Eric J; Taussig, Ronald; Nishi, Akinori; Houslay, Miles D; Bibb, James A

    2015-08-01

    The cAMP and cAMP-dependent protein kinase A (PKA) signaling cascade is a ubiquitous pathway acting downstream of multiple neuromodulators. We found that the phosphorylation of phosphodiesterase-4 (PDE4) by cyclin-dependent protein kinase 5 (Cdk5) facilitated cAMP degradation and homeostasis of cAMP/PKA signaling. In mice, loss of Cdk5 throughout the forebrain elevated cAMP levels and increased PKA activity in striatal neurons, and altered behavioral responses to acute or chronic stressors. Ventral striatum- or D1 dopamine receptor-specific conditional knockout of Cdk5, or ventral striatum infusion of a small interfering peptide that selectively targeted the regulation of PDE4 by Cdk5, produced analogous effects on stress-induced behavioral responses. Together, our results demonstrate that altering cAMP signaling in medium spiny neurons of the ventral striatum can effectively modulate stress-induced behavioral states. We propose that targeting the Cdk5 regulation of PDE4 could be a new therapeutic approach for clinical conditions associated with stress, such as depression.

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

    SciTech Connect

    Hwang, Tsong-Long; Tang, Ming-Chi; Kuo, Liang-Mou; Chang, Wen-De; Chung, Pei-Jen; Chang, Ya-Wen; Fang, Yao-Ching

    2012-04-15

    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.

  3. Prostaglandins and muscarinic agonists induce cyclic AMP attenuation by two distinct mechanisms in the pregnant-rat myometrium. Interaction between cyclic AMP and Ca2+ signals.

    PubMed Central

    Goureau, O; Tanfin, Z; Harbon, S

    1990-01-01

    In pregnant-rat myometrium (day 21 of gestation), isoprenaline-induced cyclic AMP accumulation, resulting from receptor-mediated activation of adenylate cyclase, was negatively regulated by prostaglandins [PGE2, PGF2 alpha; EC50 (concn. giving 50% of maximal response) = 2 nM] and by the muscarinic agonist carbachol (EC50 = 2 microM). PG-induced inhibition was prevented by pertussis-toxin treatment, supporting the idea that it was mediated by the inhibitory G-protein Gi through the inhibitory pathway of the adenylate cyclase. Both isoprenaline-induced stimulation and PG-evoked inhibition of cyclic AMP were insensitive to Ca2+ depletion. By contrast, carbachol-evoked attenuation of cyclic AMP accumulation was dependent on Ca2+ and was insensitive to pertussis toxin. The inhibitory effect of carbachol was mimicked by ionomycin. Indirect evidence was thus provided for the enhancement of cyclic AMP degradation by a Ca2(+)-dependent phosphodiesterase activity in the muscarinic-mediated effect. The attenuation of cyclic AMP elicited by carbachol coincided with carbachol-stimulated inositol phosphate (InsP3, InsP2 and InsP) generation, which displayed an almost identical EC50 (3 microM) and was similarly unaffected by pertussis toxin. Both carbachol effects were reproduced by oxotremorine, whereas pilocarpine (a partial muscarinic agonist) failed to induce any decrease in cyclic AMP accumulation and concurrently was unable to stimulate the generation of inositol phosphates. These data support our proposal for a carbachol-mediated enhancement of a Ca2(+)-dependent phosphodiesterase activity, compatible with the rises in Ca2+ associated with muscarinic-induced increased generation of inositol phosphates. They further illustrate that a cross-talk between the two major transmembrane signalling systems contributed to an ultimate decrease in cyclic AMP in the pregnant-rat myometrium near term. PMID:1700899

  4. Rapid glucocorticoid inhibition of vasoactive intestinal peptide-induced cyclic AMP accumulation and prolactin release in rat pituitary cells in culture.

    PubMed Central

    Rotsztejn, W H; Dussaillant, M; Nobou, F; Rosselin, G

    1981-01-01

    Vasoactive intestinal peptide (VIP) stimulates both adenosine 3',5'-cyclic monophosphate (cAMP) accumulation and prolactin release in normal rat pituitary cells in culture. cAMP accumulation is significant (P less than 0.01) at VIP concentrations as low as 1 nM and reaches a maximum with 0.1 microM. Addition of dexamethasone as early as 15 min before VIP inhibits VIP stimulation of both cAMP production and PRL secretion. The rapid inhibition is dose-dependent: it appears at doses as low as 0.01 pM and is complete at 1 pM dexamethasone. Increasing concentrations of dexamethasone induce a noncompetitive type of inhibition, as shown by the decrease in Vmax with no change in the apparent Km for VIP. Cycloheximide (1 mM) counteracts the inhibitory effect of dexamethasone on VIP-induced cAMP production, which suggests the involvement of a rapid protein synthesis mechanism. Ru-26988, a specific glucocorticoid devoid of any mineralocorticoid activity and which does not bind to intracellular transcortin-like component, also produces an inhibition of VIP-induced cAMP accumulation. Corticosterone also inhibits VIP-induced cAMP production but at concentrations higher than those of dexamethasone. In contrast, aldosterone, progesterone, estradiol, and testosterone have no effect. These results demonstrate that, in normal rat pituitary cells in culture, glucocorticoids at physiological concentrations rapidly inhibit the cAMP production and prolactin release induced by VIP by acting through specific glucocorticoid receptors. PMID:6278481

  5. H2S induces vasoconstriction of rat cerebral arteries via cAMP/adenylyl cyclase pathway.

    PubMed

    Li, Sen; Ping, Na-Na; Cao, Lei; Mi, Yan-Ni; Cao, Yong-Xiao

    2015-12-15

    Hydrogen sulfide (H2S), traditionally known for its toxic effects, is now involved in regulating vascular tone. Here we investigated the vasoconstrictive effect of H2S on cerebral artery and the underlying mechanism. Sodium hydrosulfide (NaHS), a donor of H2S, concentration-dependently induced vasoconstriction on basilar artery, which was enhanced in the presence of isoprenaline, a β-adrenoceptor agonist or forskolin, an adenylyl cyclase activator. Administration of NaHS attenuated the vasorelaxant effects of isoprenaline or forskolin. Meanwhile, the NaHS-induced vasoconstriction was diminished in the presence of 8B-cAMP, an analog of cAMP, but was not affected by Bay K-8644, a selective L-type Ca(2+) channel agonist. These results could be explained by the revised effects of NaHS on isoprenaline-induced cAMP elevation and forskolin-stimulated adenylyl cyclase activity. Additionally, NaHS-induced vasoconstriction was enhanced by removing the endothelium or in the presence of L-NAME, an inhibitor of nitric oxide synthase. L-NAME only partially attenuated the effect of NaHS which was given together with forskolin on the pre-contracted artery. In conclusion, H2S induces vasoconstriction of cerebral artery via, at least in part, cAMP/adenylyl cyclase pathway.

  6. Vv-AMP1, a ripening induced peptide from Vitis vinifera shows strong antifungal activity

    PubMed Central

    de Beer, Abré; Vivier, Melané A

    2008-01-01

    Background Latest research shows that small antimicrobial peptides play a role in the innate defense system of plants. These peptides typically contribute to preformed defense by developing protective barriers around germinating seeds or between different tissue layers within plant organs. The encoding genes could also be upregulated by abiotic and biotic stimuli during active defense processes. The peptides display a broad spectrum of antimicrobial activities. Their potent anti-pathogenic characteristics have ensured that they are promising targets in the medical and agricultural biotechnology sectors. Results A berry specific cDNA sequence designated Vv-AMP1, Vitis vinifera antimicrobial peptide 1, was isolated from Vitis vinifera. Vv-AMP1 encodes for a 77 amino acid peptide that shows sequence homology to the family of plant defensins. Vv-AMP1 is expressed in a tissue specific, developmentally regulated manner, being only expressed in berry tissue at the onset of berry ripening and onwards. Treatment of leaf and berry tissue with biotic or abiotic factors did not lead to increased expression of Vv-AMP1 under the conditions tested. The predicted signal peptide of Vv-AMP1, fused to the green fluorescent protein (GFP), showed that the signal peptide allowed accumulation of its product in the apoplast. Vv-AMP1 peptide, produced in Escherichia coli, had a molecular mass of 5.495 kDa as determined by mass spectrometry. Recombinant Vv-AMP1 was extremely heat-stable and showed strong antifungal activity against a broad spectrum of plant pathogenic fungi, with very high levels of activity against the wilting disease causing pathogens Fusarium oxysporum and Verticillium dahliae. The Vv-AMP1 peptide did not induce morphological changes on the treated fungal hyphae, but instead strongly inhibited hyphal elongation. A propidium iodide uptake assay suggested that the inhibitory activity of Vv-AMP1 might be associated with altering the membrane permeability of the fungal

  7. The role of ventral striatal cAMP signaling in stress-induced behaviors

    PubMed Central

    Plattner, Florian; Hayashi, Kanehiro; Hernandez, Adan; Benavides, David R.; Tassin, Tara C.; Tan, Chunfeng; Day, Jonathan; Fina, Maggy W.; Yuen, Eunice Y.; Yan, Zhen; Goldberg, Matthew S.; Nairn, Angus C.; Greengard, Paul; Nestler, Eric J.; Taussig, Ronald; Nishi, Akinori; Houslay, Miles D.; Bibb, James A.

    2015-01-01

    The cAMP/PKA signaling cascade is a ubiquitous pathway acting downstream of multiple neuromodulators. We found that the phosphorylation of phosphodiesterase-4 (PDE4) by cyclin-dependent protein kinase 5 (Cdk5) facilitates cAMP degradation and homeostasis of cAMP/PKA signaling. In mice, loss of Cdk5 throughout the forebrain elevated cAMP levels and increased PKA activity in striatal neurons, and altered behavioral responses to acute or chronic stressors. Ventral striatum- or D1 dopamine receptor-specific conditional knockout of Cdk5, or ventral striatum infusion of a small interfering peptide that selectively targets the regulation of PDE4 by Cdk5, all produced analogical effects on stress-induced behavioral responses. Together, our results demonstrate that altering cAMP signaling in medium spiny neurons of the ventral striatum can effectively modulate stress-induced behavioral states. We propose that targeting the Cdk5 regulation of PDE4 could be a new therapeutic approach for clinical conditions associated with stress, such as depression. PMID:26192746

  8. Inducible AmpC β-Lactamase of a New Member Enterobacteriaceae

    PubMed Central

    Bonnet, Richard; Chanal, Catherine; Ageron, Elisabeth; Sirot, Danielle; De Champs, Christophe; Grimont, Patrick; Sirot, Jacques

    2002-01-01

    Extensive biochemical testing and 16S rRNA and rpoB sequence analysis revealed that clinical strain CF01Ent1, initially identified as Buttiauxella agrestis by the use of Api 32 biochemical strips, is a new organism in the Enterobacteriaceae family. It produced an inducible AmpC-type β-lactamase whose sequence shares 69 to 72% identity with those of the other AmpC-type β-lactamases of Enterobacteriaceae. This enzyme exhibits an atypical high affinity for all β-lactams tested. PMID:12234870

  9. Trehalase activity and cyclic AMP content during early development of Mucor rouxii spores.

    PubMed Central

    Dewerchin, M A; Van Laere, A J

    1984-01-01

    Incubation of Mucor rouxii sporangiospores in complex medium under aerobic conditions resulted in a transient 20-fold increase in trehalase activity. Maximum activity was reached after 15 min. Simultaneously, the cyclic AMP (cAMP) content increased approximately eightfold, reaching a maximum within 10 min. Increases in trehalase activity and cAMP content were also observed under anaerobic conditions (CO2). The extent of trehalase activation and the changes in cAMP content, during both aerobic and anaerobic incubation, varied with the medium used. Trehalase was activated in vitro by a cAMP- and ATP-dependent process. An even faster activation was obtained when cAMP was replaced by the catalytic subunit of beef heart protein kinase. The coincidence of, and the correlation between, increased cAMP contents and trehalase activities support the involvement of a cAMP-dependent phosphorylation in the in vivo regulation of trehalase activity. PMID:6327611

  10. Autocrine activation of neuronal NMDA receptors by aspartate mediates dopamine- and cAMP-induced CREB-dependent gene transcription.

    PubMed

    Almeida, Luis E F; Murray, Peter D; Zielke, H Ronald; Roby, Clinton D; Kingsbury, Tami J; Krueger, Bruce K

    2009-10-01

    cAMP can stimulate the transcription of many activity-dependent genes via activation of the transcription factor, cAMP response element-binding protein (CREB). However, in mouse cortical neuron cultures, prior to synaptogenesis, neither cAMP nor dopamine, which acts via cAMP, stimulated CREB-dependent gene transcription when NR2B-containing NMDA receptors (NMDARs) were blocked. Stimulation of transcription by cAMP was potentiated by inhibitors of excitatory amino acid uptake, suggesting a role for extracellular glutamate or aspartate in cAMP-induced transcription. Aspartate was identified as the extracellular messenger: enzymatic scavenging of l-aspartate, but not glutamate, blocked stimulation of CREB-dependent gene transcription by cAMP; moreover, cAMP induced aspartate but not glutamate release. Together, these results suggest that cAMP acts via an autocrine or paracrine pathway to release aspartate, which activates NR2B-containing NMDARs, leading to Ca(2+) entry and activation of transcription. This cAMP/aspartate/NMDAR signaling pathway may mediate the effects of transmitters such as dopamine on axon growth and synaptogenesis in developing neurons or on synaptic plasticity in mature neural networks.

  11. Inactivation of the ampD gene causes semiconstitutive overproduction of the inducible Citrobacter freundii beta-lactamase.

    PubMed Central

    Lindberg, F; Lindquist, S; Normark, S

    1987-01-01

    In Citrobacter freundii and Enterobacter cloacae, synthesis of AmpC beta-lactamase is inducible by the addition of beta-lactams to the growth medium. Spontaneous mutants that constitutively overproduce the enzyme occur at a high frequency. When the C. freundii ampC beta-lactamase gene is cloned into Escherichia coli together with the regulatory gene ampR, beta-lactamase expression from the clone is inducible. Spontaneous cefotaxime-resistant mutants were selected from an E. coli strain carrying the cloned C. freundii ampC and ampR genes on a plasmid. Virtually all isolates had chromosomal mutations leading to semiconstitutive overproduction of beta-lactamase. The mutation ampD2 in one such mutant was caused by an IS1 insertion into the hitherto unknown ampD gene, located between nadC and aroP at minute 2.4 on the E. coli chromosome. The wild-type ampD allele cloned on a plasmid could fully trans-complement beta-lactamase-overproducing mutants of both E. coli and C. freundii, restoring the wild-type phenotype of highly inducible enzyme synthesis. This indicates that these E. coli and C. freundii mutants have their lesions in ampD. We hypothesize that induction of beta-lactamase synthesis is caused by blocking of the AmpD function by the beta-lactam inducer and that this leads directly or indirectly to an AmpR-mediated stimulation of ampC expression. PMID:3032901

  12. NO-Evoked macrophage apoptosis is attenuated by cAMP-induced gene expression.

    PubMed Central

    von Knethen, A.; Brockhaus, F.; Kleiter, I.; Brüne, B.

    1999-01-01

    BACKGROUND: Previous work has suggested that an increase in expression of cyclooxygenase-2, concomitant formation of E-type prostanoids, and in turn intracellular cAMP conveys macrophage resistance against apoptosis. MATERIALS AND METHODS: We analyzed the effects of lipophilic cAMP analogs on nitric oxide (NO)-induced apoptosis in RAW 264.7 macrophages and human primary monocyte-derived macrophages. Parameters comprised DNA fragmentation (diphenylamine assay), annexin V staining of phosphatidylserine, caspase activity (quantitated by the cleavage of a fluorogenic caspase-3-like substrate Ac-DEVD-AMC), and mitochondrial membrane depolarization (DeltaPsi), analyzed using DiOC(6)(3). Western blots detected accumulation of the tumor suppressor protein p53, relocation of cytochrome c, and expression of the antiapoptotic protein Bcl-X(L). A cAMP response-element decoy approach confirmed cAMP-dependent gene induction. RESULTS: We verified resistance of murine and human macrophages against NO donors such as S-nitrosoglutathione or spermine-NO by pre-exposing cells to lipophilic cAMP analogs or by pretreatment with lipopolysaccaride, interferon-gamma, and N(G)-nitroarginine-methylester for 15 hr. Cellular prestimulation decreased NO-evoked apoptosis, as apoptotic parameters were basically absent. Macrophage protection was not achieved during a short period of preexposure, i.e., 1 hr. To verify gene induction as the underlying protective principle, we treated RAW cells with oligonucleotides containing a cAMP-responsive element in order to scavenge cAMP response element-binding protein prior to its promoter-activating ability. Decoy oligonucleotides, but not an unrelated control oligonucleotide, weakened cAMP-evoked protection and re-established a p53 response following NO addition. CONCLUSION: Gene induction by cAMP protects macrophages against apoptosis that occurs as a result of excessive NO formation. Decreasing programmed cell death of macrophages may perpetuate

  13. Role of Rac 1 and cAMP in endothelial barrier stabilization and thrombin-induced barrier breakdown.

    PubMed

    Baumer, Y; Spindler, V; Werthmann, R C; Bünemann, M; Waschke, J

    2009-09-01

    Barrier stabilizing effects of cAMP as well as of the small GTPase Rac 1 are well established. Moreover, it is generally believed that permeability-increasing mediators such as thrombin disrupt endothelial barrier functions primarily via activation of Rho A. In this study, we provide evidence that decrease of both cAMP levels and of Rac 1 activity contribute to thrombin-mediated barrier breakdown. Treatment of human dermal microvascular endothelial cells (HDMEC) with Rac 1-inhibitor NSC-23766 decreased transendothelial electrical resistance (TER) and caused intercellular gap formation. These effects were reversed by addition of forskolin/rolipram (F/R) to increase intracellular cAMP but not by the cAMP analogue 8-pCPT-2'-O-Methyl-cAMP (O-Me-cAMP) which primarily stimulates protein kinase A (PKA)-independent signaling via Epac/Rap 1. However, both F/R and O-Me-cAMP did not increase TER above control levels in the presence of NSC-23766 in contrast to experiments without Rac 1 inhibition. Because Rac 1 was required for maintenance of barrier functions as well as for cAMP-mediated barrier stabilization, we tested the role of Rac 1 and cAMP in thrombin-induced barrier breakdown. Thrombin-induced drop of TER and intercellular gap formation were paralleled by a rapid decrease of cAMP as revealed by fluorescence resonance energy transfer (FRET). The efficacy of F/R or O-Me-cAMP to block barrier-destabilizing effects of thrombin was comparable to Y27632-induced inhibition of Rho kinase but was blunted when Rac 1 was inactivated by NSC-23766. Taken together, these data indicate that decrease of cAMP and Rac 1 activity may be an important step in inflammatory barrier disruption.

  14. Exchange factors directly activated by cAMP mediate melanocortin 4 receptor-induced gene expression

    PubMed Central

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

    2016-01-01

    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

  15. Exchange factors directly activated by cAMP mediate melanocortin 4 receptor-induced gene expression.

    PubMed

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

    2016-01-01

    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

  16. Role of the human cytomegalovirus major immediate-early promoter's 19-base-pair-repeat cyclic AMP-response element in acutely infected cells.

    PubMed

    Keller, M J; Wheeler, D G; Cooper, E; Meier, J L

    2003-06-01

    Prior studies have suggested a role of the five copies of the 19-bp-repeat cyclic AMP (cAMP)-response element (CRE) in major immediate-early (MIE) promoter activation, the rate-limiting step in human cytomegalovirus (HCMV) replication. We used two different HCMV genome modification strategies to test this hypothesis in acutely infected cells. We report the following: (i) the CREs do not govern basal levels of MIE promoter activity at a high or low multiplicity of infection (MOI) in human foreskin fibroblast (HFF)- or NTera2-derived neuronal cells; (ii) serum and virion components markedly increase MIE promoter-dependent transcription at a low multiplicity of infection (MOI), but this increase is not mediated by the CREs; (iii) forskolin stimulation of the cAMP signaling pathway induces a two- to threefold increase in MIE RNA levels in a CRE-specific manner at a low MOI in both HFF- and NTera2-derived neuronal cells; and (iv) the CREs do not regulate basal levels of HCMV DNA replication at a high or low MOI in HFF. Their presence does impart a forskolin-induced increase in viral DNA replication at a low MOI but only when basal levels of MIE promoter activity are experimentally diminished. In conclusion, the 19-bp-repeat CREs add to the robust MIE promoter activity that occurs in the acutely infected stimulated cells, although the CREs' greater role may be in other settings.

  17. Integrins and cAMP mediate netrin-induced growth cone collapse

    PubMed Central

    Lemons, M.L.; Abanto, M.L.; Dambrouskas, N.; Clements, C.C.; DeLoughery, Z.; Garozzo, J.; Condic, M.L.

    2013-01-01

    Growth cones integrate a remarkably complex concert of chemical cues to guide axons to their appropriate destinations. Recent work suggests that integrins contribute to axon guidance by interacting with a wide range of extracellular molecules including axon guidance molecules, by mechanisms that are not fully understood. Here, we describe an interaction between integrins and netrin-1 in growth cones that contributes to growth cone collapse. Our data show that netrin-1 causes growth cone collapse in a substratum-specific manner and is integrin-dependent. Netrin-1 causes collapse of cultured chick dorsal root ganglion (DRG) growth cones extending on high levels of laminin-1 (LN) but not growth cones extending on low levels of LN or on fibronectin. Blocking integrin function significantly decreases netrin-induced growth cone collapse on high LN. Netrin-1 and integrins interact on growth cones; netrin-1 causes integrin activation, a conformational shift to a high ligand-affinity state. Netrin-1 directly binds to integrin α3 and α6 peptides, further suggesting a netrin-integrin interaction. Interestingly, our data reveal netrin-1 increases growth cone levels of cAMP in a substratum-specific manner and that netrin-induced growth cone collapse requires increased cAMP in combination with integrin activation. Manipulations that either decrease cAMP levels or integrin activation block netrin-induced collapse. These results imply a common mechanism for growth cone collapse and novel interactions between integrins, netrin-1 and cAMP that contribute to growth cone guidance. PMID:24001590

  18. Glucose Enhances Basal or Melanocortin-Induced cAMP-Response Element Activity in Hypothalamic Cells.

    PubMed

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

    2016-07-01

    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. PMID:27144291

  19. Glucose Enhances Basal or Melanocortin-Induced cAMP-Response Element Activity in Hypothalamic Cells.

    PubMed

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

    2016-07-01

    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.

  20. Binding of the Citrobacter freundii AmpR regulator to a single DNA site provides both autoregulation and activation of the inducible ampC beta-lactamase gene.

    PubMed Central

    Lindquist, S; Lindberg, F; Normark, S

    1989-01-01

    Citrobacter freundii encodes an inducible chromosomal beta-lactamase. Induction requires the product of the ampR gene, which is transcribed in the opposite orientation from the ampC beta-lactamase gene. We show here that the AmpR protein acts as a transcriptional activator by binding to a DNA region immediately upstream of the ampC promoter. The DNase I footprint pattern was not affected by growth in the presence of beta-lactam inducer or by the use of extracts prepared from cells carrying the ampD2 allele leading to semiconstitutive production of beta-lactamase. It is suggested that activation of AmpR facilitates binding or open complex formation for RNA polymerase at the ampC promoter. The AmpR-binding site overlaps the ampR promoter, and beta-galactosidase activity was decreased from an ampR-lacZ transcriptional fusion when AmpR was expressed from a coresident plasmid, suggesting that ampR is autogenously controlled. The AmpR protein belongs to a family of highly homologous transcriptional activators that includes LysR, which regulates the E. coli lysine synthetase gene, and the NodD protein, which regulates expression of a number of genes involved in nodulation in Rhizobium. The lack of sequence homology to any known beta-lactam-binding protein suggests that AmpR does not bind directly to the beta-lactam inducer but interacts with a second messenger of unknown nature. Images PMID:2786868

  1. Dysregulation of hepatic cAMP levels via altered Pde4b expression plays a critical role in alcohol-induced steatosis.

    PubMed

    Avila, Diana V; Barker, David F; Zhang, JingWen; McClain, Craig J; Barve, Shirish; Gobejishvili, Leila

    2016-09-01

    Alcohol-induced hepatic steatosis is a significant risk factor for progressive liver disease. Cyclic adenosine monophosphate (cAMP) signalling has been shown to significantly regulate lipid metabolism; however, the role of altered cAMP homeostasis in alcohol-mediated hepatic steatosis has never been studied. Our previous work demonstrated that increased expression of hepatic phosphodiesterase 4 (Pde4), which specifically hydrolyses and decreases cAMP levels, plays a pathogenic role in the development of liver inflammation/injury. The aim of this study was to examine the role of PDE4 in alcohol-induced hepatic steatosis. C57BL/6 wild-type and Pde4b knockout (Pde4b(-/-) ) mice were pair-fed control or ethanol liquid diets. One group of wild-type mice received rolipram, a PDE4-specific inhibitor, during alcohol feeding. We demonstrate for the first time that an early increase in PDE4 enzyme expression and a resultant decrease in hepatic cAMP levels are associated with the significant reduction in carnitine palmitoyltransferase 1A (Cpt1a) expression. Notably, alcohol-fed (AF) Pde4b(-/-) mice and AF wild-type mice treated with rolipram had significantly lower hepatic free fatty acid content compared with AF wild-type mice. Importantly, PDE4 inhibition in alcohol-fed mice prevented the decrease in hepatic Cpt1a expression via the Pparα/Sirt1/Pgc1α pathway. These results demonstrate that the alcohol- induced increase in hepatic Pde4, specifically Pde4b expression, and compromised cAMP signalling predispose the liver to impaired fatty acid oxidation and the development of steatosis. Moreover, these data also suggest that hepatic PDE4 may be a clinically relevant therapeutic target for the treatment of alcohol-induced hepatic steatosis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. PMID:27287961

  2. Activation of AMP kinase α1 subunit induces aortic vasorelaxation in mice

    PubMed Central

    Goirand, Françoise; Solar, Myriam; Athea, Yoni; Viollet, Benoit; Mateo, Philippe; Fortin, Dominique; Leclerc, Jocelyne; Hoerter, Jacqueline; Ventura-Clapier, Renée; Garnier, Anne

    2007-01-01

    Vasodilatation is a vital mechanism of systemic blood flow regulation that occurs during periods of increased energy demand. The AMP-dependent protein kinase (AMPK) is a serine/threonine kinase that is activated by conditions that increase the AMP-to-ATP ratio, such as exercise and metabolic stress. We hypothesized that AMPK could trigger vasodilatation and participate in blood flow regulation. Rings of thoracic aorta were isolated from C57Bl6 mice and mice deficient in the AMPK catalytic α1 (AMPKα1−/−) or α2 (AMPKα2−/−) subunit and their littermate controls, and mounted in an organ bath. Aortas were preconstricted with phenylephrine (1 μm) and activation of AMPK was induced by addition of increasing concentrations of 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR). AICAR (0.1–3 mm) dose-dependently induced relaxation of precontracted C57BL6, AMPKα1+/+ and α2+/+ aorta (P < 0.001, n = 5–7 per group). This AICAR induced vasorelaxation was not inhibited by the addition of adenosine receptor antagonists. Moreover, when aortic rings were freed of endothelium by gentle rubbing, AICAR still induced aortic ring relaxation, suggesting a direct effect of AICAR on smooth muscle cells. When aortic rings were pretreated with l-NMMA (30 μm) to inhibit nitric oxide synthase activity, AICAR still induced relaxation. Western blot analysis of C57Bl6 mice denuded aorta showed that AMPK was phosphorylated after incubation with AICAR and that AMPKα1 was the main catalytic subunit expressed. Finally, AICAR-induced relaxation of aortic rings was completely abolished in AMPKα1−/− but not AMPKα2−/− mice. Taken together, the results show that activation of AMPKα1 but not AMPKα2 is able to induce aortic relaxation in mice, in an endothelium- and eNOS-independent manner. PMID:17446219

  3. Crystal structure of the AmpR effector binding domain provides insight into the molecular regulation of inducible ampc beta-lactamase.

    PubMed

    Balcewich, Misty D; Reeve, Thomas M; Orlikow, Evan A; Donald, Lynda J; Vocadlo, David J; Mark, Brian L

    2010-07-30

    Hyperproduction of AmpC beta-lactamase (AmpC) is a formidable mechanism of resistance to penicillins and cephalosporins in Gram-negative bacteria such as Pseudomonas aeruginosa and Enterobacteriaceae. AmpC expression is regulated by the LysR-type transcriptional regulator AmpR. ampR and ampC genes form a divergent operon with overlapping promoters to which AmpR binds and regulates the transcription of both genes. AmpR induces ampC by binding to one member of the family of 1,6-anhydro-N-acetylmuramyl peptides, which are cytosolic catabolites of peptidoglycan that accumulate during beta-lactam challenge. To gain structural insights into AmpR regulation, we determined the crystal structure of the effector binding domain (EBD) of AmpR from Citrobacter freundii up to 1.83 A resolution. The AmpR EBD is dimeric and each monomer comprises two subdomains that adopt alpha/beta Rossmann-like folds. Located between the monomer subdomains is a pocket that was found to bind the crystallization buffer molecule 2-(N-morpholino)ethanesulfonic acid. The pocket, together with a groove along the surface of subdomain I, forms a putative effector binding site into which a molecule of 1,6-anhydro-N-acetylmuramyl pentapeptide could be modeled. Amino acid substitutions at the base of the interdomain pocket either were found to render AmpR incapable of inducing ampC (Thr103Val, Ser221Ala and Tyr264Phe) or resulted in constitutive ampC expression (Gly102Glu). While the substitutions that prevented ampC induction did not alter the overall AmpR EBD structure, circular dichroism spectroscopy revealed that the nonconservative Gly102Glu mutation affected EBD secondary structure, confirming previous work suggesting that Gly102Glu induces a conformational change to result in constitutive AmpC production. PMID:20594961

  4. AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B

    PubMed Central

    Johanns, M.; Lai, Y.-C.; Hsu, M.-F.; Jacobs, R.; Vertommen, D.; Van Sande, J.; Dumont, J. E.; Woods, A.; Carling, D.; Hue, L.; Viollet, B.; Foretz, M; Rider, M H

    2016-01-01

    Biguanides such as metformin have previously been shown to antagonize hepatic glucagon-stimulated cyclic AMP (cAMP) signalling independently of AMP-activated protein kinase (AMPK) via direct inhibition of adenylate cyclase by AMP. Here we show that incubation of hepatocytes with the small-molecule AMPK activator 991 decreases glucagon-stimulated cAMP accumulation, cAMP-dependent protein kinase (PKA) activity and downstream PKA target phosphorylation. Moreover, incubation of hepatocytes with 991 increases the Vmax of cyclic nucleotide phosphodiesterase 4B (PDE4B) without affecting intracellular adenine nucleotide concentrations. The effects of 991 to decrease glucagon-stimulated cAMP concentrations and activate PDE4B are lost in hepatocytes deleted for both catalytic subunits of AMPK. PDE4B is phosphorylated by AMPK at three sites, and by site-directed mutagenesis, Ser304 phosphorylation is important for activation. In conclusion, we provide a new mechanism by which AMPK antagonizes hepatic glucagon signalling via phosphorylation-induced PDE4B activation. PMID:26952277

  5. Palmitoleate Reverses High Fat-induced Proinflammatory Macrophage Polarization via AMP-activated Protein Kinase (AMPK)*

    PubMed Central

    Chan, Kenny L.; Pillon, Nicolas J.; Sivaloganathan, Darshan M.; Costford, Sheila R.; Liu, Zhi; Théret, Marine; Chazaud, Benedicte; Klip, Amira

    2015-01-01

    A rise in tissue-embedded macrophages displaying “M1-like” proinflammatory polarization is a hallmark of metabolic inflammation during a high fat diet or obesity. Here we show that bone marrow-derived macrophages (BMDM) from high fat-fed mice retain a memory of their dietary environment in vivo (displaying the elevated proinflammatory genes Cxcl1, Il6, Tnf, Nos2) despite 7-day differentiation and proliferation ex vivo. Notably, 6-h incubation with palmitoleate (PO) reversed the proinflammatory gene expression and cytokine secretion seen in BMDM from high fat-fed mice. BMDM from low fat-fed mice exposed to palmitate (PA) for 18 h ex vivo also showed elevated expression of proinflammatory genes (Cxcl1, Il6, Tnf, Nos2, and Il12b) associated with M1 polarization. Conversely, PO treatment increased anti-inflammatory genes (Mrc1, Tgfb1, Il10, Mgl2) and oxidative metabolism, characteristic of M2 macrophages. Therefore, saturated and unsaturated fatty acids bring about opposite macrophage polarization states. Coincubation of BMDM with both fatty acids counteracted the PA-induced Nos2 expression in a PO dose-dependent fashion. PO also prevented PA-induced IκBα degradation, RelA nuclear translocation, NO production, and cytokine secretion. Mechanistically, PO exerted its anti-inflammatory function through AMP-activated protein kinase as AMP kinase knockout or inhibition by Compound C offset the PO-dependent prevention of PA-induced inflammation. These results demonstrate a nutritional memory of BMDM ex vivo, highlight the plasticity of BMDM polarization in response to saturated and unsaturated fatty acids, and identify the potential to reverse diet- and saturated fat-induced M1-like polarization by administering palmitoleate. These findings could have applicability to reverse obesity-linked inflammation in metabolically relevant tissues. PMID:25987561

  6. Palmitoleate Reverses High Fat-induced Proinflammatory Macrophage Polarization via AMP-activated Protein Kinase (AMPK).

    PubMed

    Chan, Kenny L; Pillon, Nicolas J; Sivaloganathan, Darshan M; Costford, Sheila R; Liu, Zhi; Théret, Marine; Chazaud, Benedicte; Klip, Amira

    2015-07-01

    A rise in tissue-embedded macrophages displaying "M1-like" proinflammatory polarization is a hallmark of metabolic inflammation during a high fat diet or obesity. Here we show that bone marrow-derived macrophages (BMDM) from high fat-fed mice retain a memory of their dietary environment in vivo (displaying the elevated proinflammatory genes Cxcl1, Il6, Tnf, Nos2) despite 7-day differentiation and proliferation ex vivo. Notably, 6-h incubation with palmitoleate (PO) reversed the proinflammatory gene expression and cytokine secretion seen in BMDM from high fat-fed mice. BMDM from low fat-fed mice exposed to palmitate (PA) for 18 h ex vivo also showed elevated expression of proinflammatory genes (Cxcl1, Il6, Tnf, Nos2, and Il12b) associated with M1 polarization. Conversely, PO treatment increased anti-inflammatory genes (Mrc1, Tgfb1, Il10, Mgl2) and oxidative metabolism, characteristic of M2 macrophages. Therefore, saturated and unsaturated fatty acids bring about opposite macrophage polarization states. Coincubation of BMDM with both fatty acids counteracted the PA-induced Nos2 expression in a PO dose-dependent fashion. PO also prevented PA-induced IκBα degradation, RelA nuclear translocation, NO production, and cytokine secretion. Mechanistically, PO exerted its anti-inflammatory function through AMP-activated protein kinase as AMP kinase knockout or inhibition by Compound C offset the PO-dependent prevention of PA-induced inflammation. These results demonstrate a nutritional memory of BMDM ex vivo, highlight the plasticity of BMDM polarization in response to saturated and unsaturated fatty acids, and identify the potential to reverse diet- and saturated fat-induced M1-like polarization by administering palmitoleate. These findings could have applicability to reverse obesity-linked inflammation in metabolically relevant tissues.

  7. MITF mediates cAMP-induced protein kinase C-β expression in human melanocytes

    PubMed Central

    Park, Hee-Young; Wu, Christina; Yonemoto, Laurie; Murphy-Smith, Melissa; Wu, Heng; Stachur, Christina M.; Gilchrest, Barbara A.

    2006-01-01

    The cAMP-dependent pathway up-regulates MITF (microphthalmia-associated transcription factor), important for key melanogenic proteins such as tyrosinase, TRP-1 (tyrosinase-related protein 1) and TRP-2. We asked whether MITF is also a key transcription factor for PKC-β (protein kinase C-β), required to phosphorylate otherwise inactive tyrosinase. When paired cultures of human melanocytes were treated with isobutylmethylxanthine, known to increase intracellular cAMP, both protein and mRNA levels of PKC-β were induced by 24 h. To determine whether MITF modulates PKC-β expression, paired cultures of human melanocytes were transfected with dn-MITF (dominant-negative MITF) or empty control vector. By immunoblotting, PKC-β protein was reduced by 63±3.7% within 48 h. Co-transfection of an expression vector for MITF-M, the MITF isoform specific for pigment cells, or empty control vector with a full-length PKC-β promoter–CAT (chloramphenicol acetyltransferase) reporter construct (PKC-β/CAT) into Cos-7 cells showed >60-fold increase in CAT activity. Melanocytes abundantly also expressed MITF-A, as well as the MITF-B and MITF-H isoforms. However, in contrast with MITF-M, MITF-A failed to transactivate co-expressed PKC-β/CAT or CAT constructs under the control of a full-length tyrosinase promoter. Together, these results demonstrate that MITF, specifically MITF-M, is a key transcription factor for PKC-β, linking the PKC- and cAMP-dependent pathways in regulation of melanogenesis. PMID:16411896

  8. Analysis of a novel cyclic Amp inducible prespore gene in Dictyostelium discoideum: evidence for different patterns of cAMP regulation.

    PubMed

    Agarwal, A; Sloger, M S; Oyama, M; Blumberg, D D

    1994-09-01

    The D7 cDNA clone hybridizes to a 2.8 kb mRNA which first appears at the mound stage of development in the cellular slime mold Dictyostelium discoideum. This gene which is cyclic AMP (cAMP) inducible and is expressed specifically in the prespore cells contains an open reading frame interrupted by only one intron. The predicted amino acid sequence indicates a novel prespore protein which differs from all of the previously described prespore proteins in that it contains no internal repeats and does not share any homology with any of the other prespore genes. The amino acid sequence predicts a protein of 850 amino acids with a molecular weight of 95,343 daltons and an isoelectric point of 4.25. The protein is very rich in glutamine (13.8%), asparagine (10.6%) and glutamic acid (10.4%) with one potential glycosylation site and 28 possible sites for phosphorylation. The amino terminus is hydrophobic with characteristics of a signal sequence while the entire carboxyl half of the protein is notable for its hydrophilicity. Comparison of cAMP regulation of the D7 gene with the regulation of two other cAMP regulated prespore genes, the PL3(SP87) gene and the Psa(D19), reveals some striking differences. Disaggregation in the presence of cAMP results in transient degradation of mRNA for all three genes. The transcription rate for the D7 and PsA(D19) genes remains relatively unaffected by disaggregation but there is a rapid although transient decline in the transcription rate of the PL3(SP87) gene. Although the accumulation of all three mRNAs is first detectable at mound stage, transcription of the D7 and PsA(D19) genes is detected earlier in development, at rippling aggregate stage several hours prior to the earliest time when transcription of the PL3(SP87) gene is detected. Analysis of the promoter region of the D7 gene reveals three CA like boxes flanked by direct repeats as well as four G rich regions that may serve as regulatory elements. PMID:7988791

  9. Curcumin attenuates diet-induced hepatic steatosis by activating AMP-activated protein kinase.

    PubMed

    Um, Min Young; Hwang, Kwang Hyun; Ahn, Jiyun; Ha, Tae Youl

    2013-09-01

    Curcumin is a well-known component of traditional turmeric (Curcuma longa), which has been reported to prevent obesity and diabetes. However, the effect of curcumin on hepatic lipid metabolism remains unclear. The aim of this study was to examine the effects of curcumin on hepatic steatosis in high-fat/cholesterol diet (HFD)-induced obese mice. Male C57BL/6J mice were fed a normal diet (ND), HFD or HFD with 0.15% curcumin (HFD+C) for 11 weeks. We found that curcumin significantly lowered the body-weight and adipose tissue weight of mice in the HFD+C group compared with the findings for the HFD group (p < 0.05). The levels of total cholesterol, fasting glucose and insulin in serum were decreased, and HFD-induced impairment of insulin sensitivity was improved by curcumin supplementation (p < 0.05). Curcumin protected against the development of hepatic steatosis by reducing hepatic fat accumulation. Moreover, curcumin activated AMP-activated protein kinase (AMPK) and elevated the gene expression of peroxisome proliferator-activated receptor alpha. By contrast, curcumin suppressed the HFD-mediated increases in sterol regulatory element-binding protein-1, acetyl-CoA carboxylase 1, fatty acid synthase and cluster of differentiation 36 expression. Taken together, these findings indicate that curcumin attenuates HFD-induced hepatic steatosis by regulating hepatic lipid metabolism via AMPK activation, suggesting its use as a therapeutic for hepatic steatosis.

  10. Activation of AMP-activated protein kinase by tributyltin induces neuronal cell death

    SciTech Connect

    Nakatsu, Yusuke; Kotake, Yaichiro Hino, Atsuko; Ohta, Shigeru

    2008-08-01

    AMP-activated protein kinase (AMPK), a member of the metabolite-sensing protein kinase family, is activated by energy deficiency and is abundantly expressed in neurons. The environmental pollutant, tributyltin chloride (TBT), is a neurotoxin, and has been reported to decrease cellular ATP in some types of cells. Therefore, we investigated whether TBT activates AMPK, and whether its activation contributes to neuronal cell death, using primary cultures of cortical neurons. Cellular ATP levels were decreased 0.5 h after exposure to 500 nM TBT, and the reduction was time-dependent. It was confirmed that most neurons in our culture system express AMPK, and that TBT induced phosphorylation of AMPK. Compound C, an AMPK inhibitor, reduced the neurotoxicity of TBT, suggesting that AMPK is involved in TBT-induced cell death. Next, the downstream target of AMPK activation was investigated. Nitric oxide synthase, p38 phosphorylation and Akt dephosphorylation were not downstream of TBT-induced AMPK activation because these factors were not affected by compound C, but glutamate release was suggested to be controlled by AMPK. Our results suggest that activation of AMPK by TBT causes neuronal death through mediating glutamate release.

  11. Cyclic AMP in prokaryotes.

    PubMed Central

    Botsford, J L; Harman, J G

    1992-01-01

    Cyclic AMP (cAMP) is found in a variety of prokaryotes including both eubacteria and archaebacteria. cAMP plays a role in regulating gene expression, not only for the classic inducible catabolic operons, but also for other categories. In the enteric coliforms, the effects of cAMP on gene expression are mediated through its interaction with and allosteric modification of a cAMP-binding protein (CRP). The CRP-cAMP complex subsequently binds specific DNA sequences and either activates or inhibits transcription depending upon the positioning of the complex relative to the promoter. Enteric coliforms have provided a model to explore the mechanisms involved in controlling adenylate cyclase activity, in regulating adenylate cyclase synthesis, and in performing detailed examinations of CRP-cAMP complex-regulated gene expression. This review summarizes recent work focused on elucidating the molecular mechanisms of CRP-cAMP complex-mediated processes. For other bacteria, less detail is known. cAMP has been implicated in regulating antibiotic production, phototrophic growth, and pathogenesis. A role for cAMP has been suggested in nitrogen fixation. Often the only data that support cAMP involvement in these processes includes cAMP measurement, detection of the enzymes involved in cAMP metabolism, or observed effects of high concentrations of the nucleotide on cell growth. PMID:1315922

  12. Mathematical model of cAMP-dependent signaling pathway in constitutive and UV-induced melanogenesis

    NASA Astrophysics Data System (ADS)

    Stolnitz, Mikhail M.; Peshkova, Anna Y.

    2002-07-01

    Cascade of reactions of cAMP-dependent signaling pathway in melanocytes is investigated by mathematical modeling. Model takes into account (alpha) -melanocyte stimulating hormone binding to melanocortin-1 receptor, adenylate cyclase activation by G-protein, increase of the intracellular cAMP concentration, PKA activation by cAMP, CREB phosphorylation by PKA, microphthalmia gene expression, microphthalmia binding to tyrosinase gene promoter, increase of tyrosinase synthesis. Positive and negative feedback loops of this system are analyzed.

  13. Nicotine induces negative energy balance through hypothalamic AMP-activated protein kinase.

    PubMed

    Martínez de Morentin, Pablo B; Whittle, Andrew J; Fernø, Johan; Nogueiras, Rubén; Diéguez, Carlos; Vidal-Puig, Antonio; López, Miguel

    2012-04-01

    Smokers around the world commonly report increased body weight after smoking cessation as a major factor that interferes with their attempts to quit. Numerous controlled studies in both humans and rodents have reported that nicotine exerts a marked anorectic action. The effects of nicotine on energy homeostasis have been mostly pinpointed in the central nervous system, but the molecular mechanisms controlling its action are still not fully understood. The aim of this study was to investigate the effect of nicotine on hypothalamic AMP-activated protein kinase (AMPK) and its effect on energy balance. Here we demonstrate that nicotine-induced weight loss is associated with inactivation of hypothalamic AMPK, decreased orexigenic signaling in the hypothalamus, increased energy expenditure as a result of increased locomotor activity, increased thermogenesis in brown adipose tissue (BAT), and alterations in fuel substrate utilization. Conversely, nicotine withdrawal or genetic activation of hypothalamic AMPK in the ventromedial nucleus of the hypothalamus reversed nicotine-induced negative energy balance. Overall these data demonstrate that the effects of nicotine on energy balance involve specific modulation of the hypothalamic AMPK-BAT axis. These targets may be relevant for the development of new therapies for human obesity. PMID:22315316

  14. AMP-activated protein kinase induces actin cytoskeleton reorganization in epithelial cells

    SciTech Connect

    Miranda, Lisa; Carpentier, Sarah; Platek, Anna; Hussain, Nusrat; Gueuning, Marie-Agnes; Vertommen, Didier; Ozkan, Yurda; Sid, Brice; Hue, Louis; Courtoy, Pierre J.; Rider, Mark H.; Horman, Sandrine

    2010-06-04

    AMP-activated protein kinase (AMPK), a known regulator of cellular and systemic energy balance, is now recognized to control cell division, cell polarity and cell migration, all of which depend on the actin cytoskeleton. Here we report the effects of A769662, a pharmacological activator of AMPK, on cytoskeletal organization and signalling in epithelial Madin-Darby canine kidney (MDCK) cells. We show that AMPK activation induced shortening or radiation of stress fibers, uncoupling from paxillin and predominance of cortical F-actin. In parallel, Rho-kinase downstream targets, namely myosin regulatory light chain and cofilin, were phosphorylated. These effects resembled the morphological changes in MDCK cells exposed to hyperosmotic shock, which led to Ca{sup 2+}-dependent AMPK activation via calmodulin-dependent protein kinase kinase-{beta}(CaMKK{beta}), a known upstream kinase of AMPK. Indeed, hypertonicity-induced AMPK activation was markedly reduced by the STO-609 CaMKK{beta} inhibitor, as was the increase in MLC and cofilin phosphorylation. We suggest that AMPK links osmotic stress to the reorganization of the actin cytoskeleton.

  15. Nicotine induces negative energy balance through hypothalamic AMP-activated protein kinase.

    PubMed

    Martínez de Morentin, Pablo B; Whittle, Andrew J; Fernø, Johan; Nogueiras, Rubén; Diéguez, Carlos; Vidal-Puig, Antonio; López, Miguel

    2012-04-01

    Smokers around the world commonly report increased body weight after smoking cessation as a major factor that interferes with their attempts to quit. Numerous controlled studies in both humans and rodents have reported that nicotine exerts a marked anorectic action. The effects of nicotine on energy homeostasis have been mostly pinpointed in the central nervous system, but the molecular mechanisms controlling its action are still not fully understood. The aim of this study was to investigate the effect of nicotine on hypothalamic AMP-activated protein kinase (AMPK) and its effect on energy balance. Here we demonstrate that nicotine-induced weight loss is associated with inactivation of hypothalamic AMPK, decreased orexigenic signaling in the hypothalamus, increased energy expenditure as a result of increased locomotor activity, increased thermogenesis in brown adipose tissue (BAT), and alterations in fuel substrate utilization. Conversely, nicotine withdrawal or genetic activation of hypothalamic AMPK in the ventromedial nucleus of the hypothalamus reversed nicotine-induced negative energy balance. Overall these data demonstrate that the effects of nicotine on energy balance involve specific modulation of the hypothalamic AMPK-BAT axis. These targets may be relevant for the development of new therapies for human obesity.

  16. cAMP-Inhibits Cytoplasmic Phospholipase A₂ and Protects Neurons against Amyloid-β-Induced Synapse Damage.

    PubMed

    Bate, Clive; Williams, Alun

    2015-01-01

    A key event in Alzheimer's disease (AD) is the production of amyloid-β (Aβ) peptides and the loss of synapses. In cultured neurons Aβ triggered synapse damage as measured by the loss of synaptic proteins. α-synuclein (αSN), aggregates of which accumulate in Parkinson's disease, also caused synapse damage. Synapse damage was associated with activation of cytoplasmic phospholipase A₂ (cPLA₂), an enzyme that regulates synapse function and structure, and the production of prostaglandin (PG) E₂. In synaptosomes PGE₂ increased concentrations of cyclic adenosine monophosphate (cAMP) which suppressed the activation of cPLA₂ demonstrating an inhibitory feedback system. Thus, Aβ/αSN-induced activated cPLA₂ produces PGE₂ which increases cAMP which in turn suppresses cPLA₂ and, hence, its own production. Neurons pre-treated with pentoxifylline and caffeine (broad spectrum phosphodiesterase (PDE) inhibitors) or the PDE4 specific inhibitor rolipram significantly increased the Aβ/αSN-induced increase in cAMP and consequently protected neurons against synapse damage. The addition of cAMP analogues also inhibited cPLA₂ and protected neurons against synapse damage. These results suggest that drugs that inhibit Aβ-induced activation of cPLA₂ and cross the blood-brain barrier may reduce synapse damage in AD. PMID:26389963

  17. cAMP-Inhibits Cytoplasmic Phospholipase A2 and Protects Neurons against Amyloid-β-Induced Synapse Damage

    PubMed Central

    Bate, Clive; Williams, Alun

    2015-01-01

    A key event in Alzheimer’s disease (AD) is the production of amyloid-β (Aβ) peptides and the loss of synapses. In cultured neurons Aβ triggered synapse damage as measured by the loss of synaptic proteins. α-synuclein (αSN), aggregates of which accumulate in Parkinson’s disease, also caused synapse damage. Synapse damage was associated with activation of cytoplasmic phospholipase A2 (cPLA2), an enzyme that regulates synapse function and structure, and the production of prostaglandin (PG) E2. In synaptosomes PGE2 increased concentrations of cyclic adenosine monophosphate (cAMP) which suppressed the activation of cPLA2 demonstrating an inhibitory feedback system. Thus, Aβ/αSN-induced activated cPLA2 produces PGE2 which increases cAMP which in turn suppresses cPLA2 and, hence, its own production. Neurons pre-treated with pentoxifylline and caffeine (broad spectrum phosphodiesterase (PDE) inhibitors) or the PDE4 specific inhibitor rolipram significantly increased the Aβ/αSN-induced increase in cAMP and consequently protected neurons against synapse damage. The addition of cAMP analogues also inhibited cPLA2 and protected neurons against synapse damage. These results suggest that drugs that inhibit Aβ-induced activation of cPLA2 and cross the blood–brain barrier may reduce synapse damage in AD. PMID:26389963

  18. cAMP signaling inhibits radiation-induced ATM phosphorylation leading to the augmentation of apoptosis in human lung cancer cells

    PubMed Central

    2014-01-01

    Background The ataxia–telangiectasia mutated (ATM) protein kinase plays a central role in coordinating the cellular response to radiation-induced DNA damage. cAMP signaling regulates various cellular responses including metabolism and gene expression. This study aimed to investigate the mechanism through which cAMP signaling regulates ATM activation and cellular responses to ionizing radiation in lung cancer cells. Methods Lung cancer cells were transfected with constitutively active stimulatory G protein (GαsQL), and irradiated with γ-rays. The phosphorylation of ATM and protein phosphatase 2A was analyzed by western blotting, and apoptosis was assessed by western blotting, flow cytometry, and TUNNEL staining. The promoter activity of NF-κB was determined by dual luciferase reporter assay. BALB/c mice were treated with forskolin to assess the effect in the lung tissue. Results Transient expression of GαsQL significantly inhibited radiation-induced ATM phosphorylation in H1299 human lung cancer cells. Treatment with okadaic acid or knock down of PP2A B56δ subunit abolished the inhibitory effect of Gαs on radiation-induced ATM phosphorylation. Expression of GαsQL increased phosphorylation of the B56δ and PP2A activity, and inhibition of PKA blocked Gαs-induced PP2A activation. GαsQL enhanced radiation-induced cleavage of caspase-3 and PARP and increased the number of early apoptotic cells. The radiation-induced apoptosis was increased by inhibition of NF-κB using PDTC or inhibition of ATM using KU55933 or siRNA against ATM. Pretreatment of BALB/c mice with forskolin stimulated phosphorylation of PP2A B56δ, inhibited the activation of ATM and NF-κB, and augmented radiation-induced apoptosis in the lung tissue. GαsQL expression decreased the nuclear levels of the p50 and p65 subunits and NF-κB-dependent activity after γ-ray irradiation in H1299 cells. Pretreatment with prostaglandin E2 or isoproterenol increased B56δ phosphorylation, decreased

  19. Uric acid-dependent inhibition of AMP kinase induces hepatic glucose production in diabetes and starvation: evolutionary implications of the uricase loss in hominids.

    PubMed

    Cicerchi, Christina; Li, Nanxing; Kratzer, James; Garcia, Gabriela; Roncal-Jimenez, Carlos A; Tanabe, Katsuyuki; Hunter, Brandi; Rivard, Christopher J; Sautin, Yuri Y; Gaucher, Eric A; Johnson, Richard J; Lanaspa, Miguel A

    2014-08-01

    Reduced AMP kinase (AMPK) activity has been shown to play a key deleterious role in increased hepatic gluconeogenesis in diabetes, but the mechanism whereby this occurs remains unclear. In this article, we document that another AMP-dependent enzyme, AMP deaminase (AMPD) is activated in the liver of diabetic mice, which parallels with a significant reduction in AMPK activity and a significant increase in intracellular glucose accumulation in human HepG2 cells. AMPD activation is induced by a reduction in intracellular phosphate levels, which is characteristic of insulin resistance and diabetic states. Increased gluconeogenesis is mediated by reduced TORC2 phosphorylation at Ser171 by AMPK in these cells, as well as by the up-regulation of the rate-limiting enzymes PEPCK and G6Pc. The mechanism whereby AMPD controls AMPK activation depends on the production of a specific AMP downstream metabolite through AMPD, uric acid. In this regard, humans have higher uric acid levels than most mammals due to a mutation in uricase, the enzyme involved in uric acid degradation in most mammals, that developed during a period of famine in Europe 1.5 × 10(7) yr ago. Here, working with resurrected ancestral uricases obtained from early hominids, we show that their expression on HepG2 cells is enough to blunt gluconeogenesis in parallel with an up-regulation of AMPK activity. These studies identify a key role AMPD and uric acid in mediating hepatic gluconeogenesis in the diabetic state, via a mechanism involving AMPK down-regulation and overexpression of PEPCK and G6Pc. The uricase mutation in the Miocene likely provided a survival advantage to help maintain glucose levels under conditions of near starvation, but today likely has a role in the pathogenesis of diabetes.

  20. Delineation of the Roles Played by RasG and RasC in cAMP-dependent Signal Transduction during the Early Development of Dictyostelium discoideum

    PubMed Central

    Bolourani, Parvin; Spiegelman, George B.

    2006-01-01

    On starvation, the cellular slime mold Dictyostelium discoideum initiates a program of development leading to formation of multicellular structures. The initial cell aggregation requires chemotaxis to cyclic AMP (cAMP) and relay of the cAMP signal by the activation of adenylyl cyclase (ACA), and it has been shown previously that the Ras protein RasC is involved in both processes. Insertional inactivation of the rasG gene resulted in delayed aggregation and a partial inhibition of early gene expression, suggesting that RasG also has a role in early development. Both chemotaxis and ACA activation were reduced in the rasG− cells, but the effect on chemotaxis was more pronounced. When the responses of rasG− cells to cAMP were compared with the responses of rasC− and rasC−rasG− strains, generated in otherwise isogenic backgrounds, these studies revealed that signal transduction through RasG is more important in chemotaxis and early gene expression, but that signal transduction through RasC is more important in ACA activation. Because the loss of either of the two Ras proteins alone did not result in a total loss of signal output down either of the branches of the cAMP signal-response pathway, there appears to be some overlap of function. PMID:16885420

  1. An AP-2 element acts synergistically with the cyclic AMP- and Phorbol ester-inducible enhancer of the human proenkephalin gene

    SciTech Connect

    Hyman, S.E.; Comb, M.; Pearlberg, J.; Goodman, H.M.

    1989-01-01

    An enhancer with two DNA elements, one containing the sequence CGTCA, is required for cyclic AMP-and phorbol ester-inducible transcription of the human proenkephalin gene. The authors report that an AP-2 element located adjacent to the enhancer acts synergistically with it to confer maximal response to cyclic AMP and phorbol esters.

  2. Exposure to Hydrogen Peroxide Induces Oxidation and Activation of AMP-activated Protein Kinase*

    PubMed Central

    Zmijewski, Jaroslaw W.; Banerjee, Sami; Bae, Hongbeom; Friggeri, Arnaud; Lazarowski, Eduardo R.; Abraham, Edward

    2010-01-01

    Although metabolic conditions associated with an increased AMP/ATP ratio are primary factors in the activation of 5′-adenosine monophosphate-activated protein kinase (AMPK), a number of recent studies have shown that increased intracellular levels of reactive oxygen species can stimulate AMPK activity, even without a decrease in cellular levels of ATP. We found that exposure of recombinant AMPKαβγ complex or HEK 293 cells to H2O2 was associated with increased kinase activity and also resulted in oxidative modification of AMPK, including S-glutathionylation of the AMPKα and AMPKβ subunits. In experiments using C-terminal truncation mutants of AMPKα (amino acids 1–312), we found that mutation of cysteine 299 to alanine diminished the ability of H2O2 to induce kinase activation, and mutation of cysteine 304 to alanine totally abrogated the enhancing effect of H2O2 on kinase activity. Similar to the results obtained with H2O2-treated HEK 293 cells, activation and S-glutathionylation of the AMPKα subunit were present in the lungs of acatalasemic mice or mice treated with the catalase inhibitor aminotriazole, conditions in which intracellular steady state levels of H2O2 are increased. These results demonstrate that physiologically relevant concentrations of H2O2 can activate AMPK through oxidative modification of the AMPKα subunit. The present findings also imply that AMPK activation, in addition to being a response to alterations in intracellular metabolic pathways, is directly influenced by cellular redox status. PMID:20729205

  3. Senescent-induced dysregulation of cAMP/CREB signaling and correlations with cognitive decline

    PubMed Central

    Hansen, Rolf T.; Zhang, Han-Ting

    2013-01-01

    It is well known that alongside senescence there is a gradual decline in cognitive ability, most noticeably certain kinds of memory such as working, episodic, spatial, and long term memory. However, until recently, not much has been known regarding the specific mechanisms responsible for the decline in cognitive ability with age. Over the past decades, researchers have become more interested in cAMP signaling, and its downstream transcription factor cAMP response element binding protein (CREB) in the context of senescence. However, there is still a lack of understanding on what ultimately causes the cognitive deficits observed with senescence. This review will focus on the changes in intracellular signaling in the brain, more specifically, alterations in cAMP/CREB signaling in aging. In addition, the downstream effects of altered cAMP signaling on cognitive ability with age will be further discussed. Overall, understanding the senescent-related changes that occur in cAMP/CREB signaling could be important for the development of novel drug targets for both healthy aging, and pathological aging such as Alzheimer's disease. PMID:23623816

  4. Proteins bound at adjacent DNA elements act synergistically to regulate human proenkephalin cAMP inducible transcription.

    PubMed Central

    Comb, M; Mermod, N; Hyman, S E; Pearlberg, J; Ross, M E; Goodman, H M

    1988-01-01

    Synthesis of the endogenous opioid precursor, proenkephalin, is regulated by neurotransmitters and membrane depolarization. These events act through second messenger dependent signal transduction pathways via a short inducible DNA enhancer to regulate transcription of the proenkephalin gene. Two DNA elements located within this enhancer are essential for the transcriptional response to cAMP and phorbol ester. Inactivation of either element by mutation or by alteration of their stereospecific alignment eliminates inducible enhancer activity. The promoter distal element, ENKCRE-1, in the absence of a functional adjacent ENKCRE-2 element, has no inherent capacity to activate transcription. However, in the presence of a functional ENKCRE-2 element, this element synergistically augments cAMP and phorbol ester inducible transcription. The promoter proximal element, ENKCRE-2, is essential for both basal and regulated enhancer function. Four different protein factors found in HeLa cell nuclear extracts bind in vitro to the enhancer region. ENKTF-1, a novel enhancer binding protein, binds to the DNA region encompassing ENKCRE-1. The transcription factors AP-1 and AP-4 bind to overlapping sites spanning ENKCRE-2, and a fourth transcription factor, AP-2, binds to a site immediately downstream of ENKCRE-2. The binding of ENKTF-1 to mutant ENKCRE-1 sequences in vitro correlates with the in vivo inducibility of the mutant elements suggesting that ENKTF-1 acts in combination with factors that recognize the ENKCRE-2 domain to regulate cAMP inducible transcription. Together, the two DNA elements, ENKCRE-1 and ENKCRE-2 and the protein factors with which they interact, play a critical role in the transduction and reception of signals transmitted from cell surface receptors to the proenkephalin nuclear transcription complex. Images PMID:2850173

  5. Stimulators of the cAMP cascade reverse amnesia induced by intra-amygdala but not intrahippocampal KN-62 administration.

    PubMed

    Barros, D M; Izquierdo, L A; Sant'Anna, M K; Quevedo, J; Medina, J H; McGaugh, J L; Izquierdo, I

    1999-01-01

    Infusion of the calcium-calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-62 (3.5 ng/side) 0 h after training into rat hippocampus CA1 or amygdala has been known for years to cause retrograde amnesia for step-down inhibitory avoidance. On the other hand, drugs that indirectly stimulate protein kinase A (PKA) (8-Br-cAMP, 1.25 microg/side; norepinephrine, 0.3 microg/side; the dopamine D1 receptor agonist, SKF38393, 7.5 microg/side) infused 3 h posttraining into CA1 but not amygdala markedly facilitate retention of this task. Here we find that 8-Br-cAMP, norepinephrine, or SKF38393 given 3 h posttraining into rat CA1 reverses the amnestic effect of KN-62 given into the amygdala 0 h after training, but not that of KN-62 given into CA1 0 h posttraining. The findings bear on the participation of CaMKII and of the cAMP/PKA cascade in memory processes in the hippocampus and the amygdala. Both cascades have been proposed to play a role in memory: CaMKII in the early phase and PKA in the transition between the early phase and long-term memory. Clearly, in CA1, both cascades are involved and are crucial, and the CaMKII cascade must precede the PKA cascade. In contrast, in the amygdala, only the CaMKII cascade is active, and it does not play a central role in memory, inasmuch as its deleterious effect may be fully recovered by stimulation of the PKA cascade in the hippocampus. This further supports the contention that the hippocampus is essential for memory formation of this task, as it is for many others, whereas the amygdala appears to play instead an early modulatory role.

  6. Activation of the cAMP/PKA pathway induces UT-A1 urea transporter monoubiquitination and targets it for lysosomal degradation.

    PubMed

    Su, Hua; Chen, Minguang; Sands, Jeff M; Chen, Guangping

    2013-12-15

    Regulation of urea transporter UT-A1 in the kidney is important for the urinary concentrating mechanism. We previously reported that activation of the cAMP/PKA pathway by forskolin (FSK) leads to UT-A1 ubiquitination, endocytosis, and degradation. In this study, we discovered that FSK-induced UT-A1 ubiquitination is monoubiquitination as judged by immunoblotting with specific ubiquitin antibodies to the different linkages of the ubiquitin chain. UT-A1 monoubiquitination induced by FSK was processed mainly on the cell plasma membrane. Monoubiquitination facilitates UT-A1 endocytosis, and internalized UT-A1 is accumulated in the early endosome. Inhibition of ubiquitination by E1 ubiquitin-activating enzyme inhibitor PYR-41 significantly reduced FSK-induced UT-A1 endocytosis and degradation. Interestingly, FSK-stimulated UT-A1 degradation occurs through a lysosomal protein degradation system. We further found that the PKA phosphorylation sites of UT-A1 at Ser486 and Ser499 are required for FSK-induced UT-A1 monoubiquitination. The physiological significance was confirmed using rat kidney inner medullary collecting duct suspensions, which showed that vasopressin treatment promotes UT-A1 ubiquitination. We conclude that unlike under basal conditions in which UT-A1 is subject to polyubiquitination and proteasome-mediated protein degradation, activation of UT-A1 by FSK induces UT-A1 monoubiquitination and protein lysosomal degradation.

  7. Hydrogen peroxide induces activation of insulin signaling pathway via AMP-dependent kinase in podocytes

    SciTech Connect

    Piwkowska, Agnieszka; Rogacka, Dorota; Angielski, Stefan; Jankowski, Maciej

    2012-11-09

    Highlights: Black-Right-Pointing-Pointer H{sub 2}O{sub 2} activates the insulin signaling pathway and glucose uptake in podocytes. Black-Right-Pointing-Pointer H{sub 2}O{sub 2} induces time-dependent changes in AMPK phosphorylation. Black-Right-Pointing-Pointer H{sub 2}O{sub 2} enhances insulin signaling pathways via AMPK activation. Black-Right-Pointing-Pointer H{sub 2}O{sub 2} stimulation of glucose uptake is AMPK-dependent. -- Abstract: Podocytes are cells that form the glomerular filtration barrier in the kidney. Insulin signaling in podocytes is critical for normal kidney function. Insulin signaling is regulated by oxidative stress and intracellular energy levels. We cultured rat podocytes to investigate the effects of hydrogen peroxide (H{sub 2}O{sub 2}) on the phosphorylation of proximal and distal elements of insulin signaling. We also investigated H{sub 2}O{sub 2}-induced intracellular changes in the distribution of protein kinase B (Akt). Western blots showed that H{sub 2}O{sub 2} (100 {mu}M) induced rapid, transient phosphorylation of the insulin receptor (IR), the IR substrate-1 (IRS1), and Akt with peak activities at 5 min ({Delta} 183%, P < 0.05), 3 min ({Delta} 414%, P < 0.05), and 10 min ({Delta} 35%, P < 0.05), respectively. Immunostaining cells with an Akt-specific antibody showed increased intensity at the plasma membrane after treatment with H{sub 2}O{sub 2}>. Furthermore, H{sub 2}O{sub 2} inhibited phosphorylation of the phosphatase and tensin homologue (PTEN; peak activity at 10 min; {Delta} -32%, P < 0.05) and stimulated phosphorylation of the AMP-dependent kinase alpha subunit (AMPK{alpha}; 78% at 3 min and 244% at 10 min). The stimulation of AMPK was abolished with an AMPK inhibitor, Compound C (100 {mu}M, 2 h). Moreover, Compound C significantly reduced the effect of H{sub 2}O{sub 2} on IR phosphorylation by about 40% (from 2.07 {+-} 0.28 to 1.28 {+-} 0.12, P < 0.05). In addition, H{sub 2}O{sub 2} increased glucose uptake in podocytes

  8. Histamine induced elevation of cyclic AMP phosphodiesterase activity in human monocytes.

    PubMed

    Holden, C A; Chan, S C; Norris, S; Hanifin, J M

    1987-10-01

    We have previously reported histamine desensitization of human blood mononuclear leukocytes resulting in reduced cAMP responses to beta-adrenergic agonists, histamine and prostaglandin E1. This heterologous desensitization occurred at low, micromolar histamine concentrations and was accompanied by elevation of cAMP-phosphodiesterase (PDE) activity in these cells. We have now investigated the activity of PDE in the lymphocyte and monocyte fractions of mononuclear leukocytes to determine the site of histamine effect. PDE activity per cell was higher in monocytes (0.075 +/- 0.070 units) than lymphocytes (0.026 +/- 0.08) units). Monocytes responded to 10(-6) M histamine stimulation with a much greater increase in PDE activity (0.354 +/- 0.1 units) than did lymphocytes (0.047 +/- 0.015 units). Histamine receptor studies, using thiazolylethylamine and chlorpheniramine as H1-agonist and antagonist respectively and dimaprit and cimetidine as H2-agonists and antagonists respectively, indicated that the histamine stimulation of PDE activity is mediated predominantly through H1 histamine receptor in the monocytes and the H1 receptor in the lymphocytes. Previously histamine had been thought to increase cyclic AMP by acting on H2 receptors to activate adenylate cyclase. Our studies show that stimulation of H1 or H2 receptors by low histamine concentration can cause the opposite effect i.e. increased catabolism and a net reduction in cAMP levels. The localization of this effect predominantly to monocytes indicates a potentially important mechanism for histamine action on immune regulation. PMID:2891264

  9. Ecklonia cava Polyphenol Has a Protective Effect against Ethanol-Induced Liver Injury in a Cyclic AMP-Dependent Manner.

    PubMed

    Yamashita, Haruka; Goto, Mayu; Matsui-Yuasa, Isao; Kojima-Yuasa, Akiko

    2015-06-18

    Previously, we showed that Ecklonia cava polyphenol (ECP) treatment suppressed ethanol-induced increases in hepatocyte death by scavenging intracellular reactive oxygen species (ROS) and maintaining intracellular glutathione levels. Here, we examined the effects of ECP on the activities of alcohol-metabolizing enzymes and their regulating mechanisms in ethanol-treated hepatocytes. Isolated hepatocytes were incubated with or without 100 mM ethanol. ECP was dissolved in dimethylsulfoxide. ECP was added to cultured cells that had been incubated with or without ethanol. The cells were incubated for 0-24 h. In cultured hepatocytes, the ECP treatment with ethanol inhibited cytochrome P450 2E1 (CYP2E1) expression and activity, which is related to the production of ROS when large quantities of ethanol are oxidized. On the other hand, ECP treatment with ethanol increased the activity of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase. These changes in activities of CYP2E1 and ADH were suppressed by treatment with H89, an inhibitor of protein kinase A. ECP treatment with ethanol enhanced cyclic AMP concentrations compared with those of control cells. ECP may be a candidate for preventing ethanol-induced liver injury via regulating alcohol metabolic enzymes in a cyclic AMP-dependent manner.

  10. Ecklonia cava Polyphenol Has a Protective Effect against Ethanol-Induced Liver Injury in a Cyclic AMP-Dependent Manner

    PubMed Central

    Yamashita, Haruka; Goto, Mayu; Matsui-Yuasa, Isao; Kojima-Yuasa, Akiko

    2015-01-01

    Previously, we showed that Ecklonia cava polyphenol (ECP) treatment suppressed ethanol-induced increases in hepatocyte death by scavenging intracellular reactive oxygen species (ROS) and maintaining intracellular glutathione levels. Here, we examined the effects of ECP on the activities of alcohol-metabolizing enzymes and their regulating mechanisms in ethanol-treated hepatocytes. Isolated hepatocytes were incubated with or without 100 mM ethanol. ECP was dissolved in dimethylsulfoxide. ECP was added to cultured cells that had been incubated with or without ethanol. The cells were incubated for 0–24 h. In cultured hepatocytes, the ECP treatment with ethanol inhibited cytochrome P450 2E1 (CYP2E1) expression and activity, which is related to the production of ROS when large quantities of ethanol are oxidized. On the other hand, ECP treatment with ethanol increased the activity of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase. These changes in activities of CYP2E1 and ADH were suppressed by treatment with H89, an inhibitor of protein kinase A. ECP treatment with ethanol enhanced cyclic AMP concentrations compared with those of control cells. ECP may be a candidate for preventing ethanol-induced liver injury via regulating alcohol metabolic enzymes in a cyclic AMP-dependent manner. PMID:26096275

  11. p-Hydroxylcinnamaldehyde induces the differentiation of oesophageal carcinoma cells via the cAMP-RhoA-MAPK signalling pathway

    PubMed Central

    Ma, Ming; Zhao, Lian-mei; Yang, Xing-xiao; Shan, Ya-nan; Cui, Wen-xuan; Chen, Liang; Shan, Bao-en

    2016-01-01

    p-Hydroxylcinnamaldehyde (CMSP) has been identified as an inhibitor of the growth of various cancer cells. However, its function in oesophageal squamous cell carcinoma (ESCC) and the underlying mechanism remain unclear. The aim of the present study was to characterize the differentiation effects of CMSP, as well as its mechanism in the differentiation of ESCC Kyse30 and TE-13 cells. The function of CMSP in the viability, colony formation, migration and invasion of Kyse30 and TE-13 cells was determined by MTS, colony-formation, wound healing and transwell assays. Western blotting and pull-down assays were used to investigate the effect of CMSP on the expression level of malignant markers of ESCC, as well as the activity of MAPKs, RhoA and GTP-RhoA in Kyse30 and TE-13 cells. We found that CMSP could inhibit proliferation and migration and induce Kyse30 and TE-13 cell differentiation, characterized by dendrite-like outgrowth, decreased expression of tumour-associated antigens, as well as the decreased expression of malignant markers. Furthermore, increased cAMP, p-P38 and decreased activities of ERK, JNK and GTP-RhoA, were detected after treatment with CMSP. These results indicated that CMSP induced the differentiation of Kyse30 and TE-13 cells through mediating the cAMP-RhoA-MAPK axis, which might provide new potential strategies for ESCC treatment. PMID:27501997

  12. microRNA-208a in an early stage myocardial infarction rat model and the effect on cAMP-PKA signaling pathway

    PubMed Central

    Feng, Gao; Yan, Zhang; Li, Chuanchuan; Hou, Yuemei

    2016-01-01

    The expression level of microRNA-208a (miR-208a) in a rat model with myocardial infarction and the effect of cAMP-PKA signaling pathway in early stage of myocardial infarction in rats were investigated. The early myocardial infarction model was established in 12 male Sprague-Dawley rats by ligation of the anterior descending coronary artery, and 12 rats were selected as the control group (sham operation group). Reverse-transcription quantitative PCR was conducted to detect the expression levels of miR-208a in the myocardium of and the expression levels of miR-208a in the serum of rats in the two groups. Western blot analysis was used to evaluate the expression levels of cAMP-PKA protein in the rat tissues in the two groups. After stimulating high levels of miR-208a expression in human myocardial cells (HCM), western blot analysis was used to detect the cAMP-PKA protein levels. The expression levels of miR-208a in myocardial tissues in rats with myocardial infarction were significantly higher than those in the control group, and the difference was statistically significant (P<0.05). The expression levels of miR-208a in the early stage of myocardial infarction rats were also significantly higher than those in the control group, and the difference was statistically significant (P<0.05). The level of cAMP-PKA protein in myocardial tissue in rats with chronic myocardial infarction was also significantly higher. Transfection of human myocardial cells with miR-208a analogue significantly increased the cAMP-PKA protein levels in human myocardial cells. In conclusion, the over expression of miR-208a in myocardial infarction tissue and the high levels of this miRNA in the serum, may be involved in the process of myocardial infarction by influencing the cAMP-PKA signaling pathway in myocardial cells. PMID:27314868

  13. microRNA-208a in an early stage myocardial infarction rat model and the effect on cAMP-PKA signaling pathway.

    PubMed

    Feng, Gao; Yan, Zhang; Li, Chuanchuan; Hou, Yuemei

    2016-08-01

    The expression level of microRNA-208a (miR-208a) in a rat model with myocardial infarction and the effect of cAMP-PKA signaling pathway in early stage of myocardial infarction in rats were investigated. The early myocardial infarction model was established in 12 male Sprague-Dawley rats by ligation of the anterior descending coronary artery, and 12 rats were selected as the control group (sham operation group). Reverse-transcription quantitative PCR was conducted to detect the expression levels of miR-208a in the myocardium of and the expression levels of miR‑208a in the serum of rats in the two groups. Western blot analysis was used to evaluate the expression levels of cAMP-PKA protein in the rat tissues in the two groups. After stimulating high levels of miR‑208a expression in human myocardial cells (HCM), western blot analysis was used to detect the cAMP-PKA protein levels. The expression levels of miR‑208a in myocardial tissues in rats with myocardial infarction were significantly higher than those in the control group, and the difference was statistically significant (P<0.05). The expression levels of miR‑208a in the early stage of myocardial infarction rats were also significantly higher than those in the control group, and the difference was statistically significant (P<0.05). The level of cAMP-PKA protein in myocardial tissue in rats with chronic myocardial infarction was also significantly higher. Transfection of human myocardial cells with miR‑208a analogue significantly increased the cAMP-PKA protein levels in human myocardial cells. In conclusion, the over-expression of miR-208a in myocardial infarction tissue and the high levels of this miRNA in the serum, may be involved in the process of myocardial infarction by influencing the cAMP-PKA signaling pathway in myocardial cells.

  14. Role of cyclo-oxygenase-2 induction in interleukin-1β induced attenuation of cultured human airway smooth muscle cell cyclic AMP generation in response to isoprenaline

    PubMed Central

    Pang, Linhua; Holland, Elaine; Knox, Alan J

    1998-01-01

    Airway smooth muscle (ASM) in human asthma shows reduced relaxation and cyclic AMP generation in response to β-adrenoceptor agonists. IL-β attenuates cyclic AMP generation but the underlying mechanism is unclear. We have reported that IL-1β induces cyclo-oxygenase-2 (COX-2) in human ASM cells and results in a marked increase in prostanoid generation with PGE2 and PGI2 as the major products.We investigated the role of COX-2 induction and prostanoid release (measured as PGE2) in IL-1β induced attenuation of cyclic AMP generation in response to the β-adrenoceptor agonist isoprenaline (ISO).Pre-treatment of human ASM cells with IL-1β significantly attenuated cyclic AMP generation in response to high concentrations of ISO (1.0–10.0 μM) in a time- and concentration-dependent manner. The effect was accompanied by a high concentration of PGE2 release. The non-selective COX inhibitor indomethacin (Ind), the selective COX-2 inhibitor NS-398, the protein synthesis inhibitors cycloheximide (CHX) and actinomycin D and the steroid dexamethasone (Dex) all abolished the PGE2 release and prevented the attenuated cyclic AMP generation.COX substrate arachidonic acid time- and concentration-dependently mimicked IL-1β induced attenuation and the effect was prevented by the non-selective COX inhibitors Ind and flurbiprofen, but not by NS-398, CHX and Dex.In contrast to IL-1β, TNFα and IFNγ, which are ineffective in inducing COX-2 and releasing PGE2 from human ASM cells, did not affect the cyclic AMP formation.Our study demonstrates that COX-2 induction and the consequent release of prostanoids plays a crucial role in IL-1β induced attenuation of human ASM cell cyclic AMP response to ISO. PMID:9863663

  15. Iloprost- and isoproterenol-induced increases in cAMP are regulated by different phosphodiesterases in erythrocytes of both rabbits and humans.

    PubMed

    Adderley, Shaquria P; Dufaux, Eileen A; Sridharan, Meera; Bowles, Elizabeth A; Hanson, Madelyn S; Stephenson, Alan H; Ellsworth, Mary L; Sprague, Randy S

    2009-05-01

    Activation of the G protein G(s) results in increases in cAMP, a necessary step in the pathway for ATP release from rabbit and human erythrocytes. In all cells, the level of cAMP is the product of its synthesis by adenylyl cyclase and its hydrolysis by phosphodiesterases (PDEs). Both iloprost (Ilo), a PGI(2) analog, and isoproterenol (Iso), a beta-agonist, stimulate receptor-mediated increases in cAMP in rabbit and human erythrocytes. However, the specific PDEs associated with each of these signaling pathways in the erythrocyte have not been fully characterized. Previously, we reported that PDE3B is present in rabbit and human erythrocyte membranes and that PDE3 inhibitors potentiate Ilo-induced increases in cAMP. Here we report that inhibitors of either PDE2 or PDE4, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and rolipram, respectively, potentiate Iso-induced increases in cAMP in rabbit and human erythrocytes. Importantly, these inhibitors had no effect on cAMP increases associated with the incubation of erythrocytes with Ilo. In addition, we establish, for the first time, the presence of PDE2A protein in rabbit and human erythrocyte membranes. Finally, we determined that preincubation of human erythrocytes with EHNA and rolipram together potentiate Iso-induced ATP release, whereas preincubation with cilostazol enhances Ilo-induced release of ATP. These results are consistent with the hypothesis that, in rabbit and human erythrocytes, Ilo-induced increases in cAMP and ATP release are regulated by PDE3, whereas those associated with Iso are regulated by the activities of both PDE2 and PDE4. These studies demonstrate that PDE activity in these cells is localized to specific signaling pathways. PMID:19252089

  16. Phosphorylation of the protein kinase A catalytic subunit is induced by cyclic AMP deficiency and physiological stresses in the fission yeast, Schizosaccharomyces pombe

    SciTech Connect

    McInnis, Brittney; Mitchell, Jessica; Marcus, Stevan

    2010-09-03

    Research highlights: {yields} cAMP deficiency induces phosphorylation of PKA catalytic subunit (Pka1) in S. pombe. {yields} Pka1 phosphorylation is further induced by physiological stresses. {yields} Pka1 phosphorylation is not induced in cells lacking the PKA regulatory subunit. {yields} Results suggest that cAMP-independent Pka1 phosphorylation is stimulatory in nature. -- Abstract: In the fission yeast, Schizosaccharomyces pombe, cyclic AMP (cAMP)-dependent protein kinase (PKA) is not essential for viability under normal culturing conditions, making this organism attractive for investigating mechanisms of PKA regulation. Here we show that S. pombe cells carrying a deletion in the adenylate cyclase gene, cyr1, express markedly higher levels of the PKA catalytic subunit, Pka1, than wild type cells. Significantly, in cyr1{Delta} cells, but not wild type cells, a substantial proportion of Pka1 protein is hyperphosphorylated. Pka1 hyperphosphorylation is strongly induced in cyr1{Delta} cells, and to varying degrees in wild type cells, by both glucose starvation and stationary phase stresses, which are associated with reduced cAMP-dependent PKA activity, and by KCl stress, the cellular adaptation to which is dependent on PKA activity. Interestingly, hyperphosphorylation of Pka1 was not detected in either cyr1{sup +} or cyr1{Delta} S. pombe strains carrying a deletion in the PKA regulatory subunit gene, cgs1, under any of the tested conditions. Our results demonstrate the existence of a cAMP-independent mechanism of PKA catalytic subunit phosphorylation, which we propose could serve as a mechanism for inducing or maintaining specific PKA functions under conditions in which its cAMP-dependent activity is downregulated.

  17. Perturbing microtubule integrity blocks AMP-activated protein kinase-induced meiotic resumption in cultured mouse oocytes

    PubMed Central

    Ya, Ru; Downs, Stephen M.

    2014-01-01

    AMP-activated protein kinase (AMPK) is an important cellular energy sensor that is activated by a low AMP-to-ATP ratio. AMPK is involved in meiotic induction of mouse oocytes and also promotes the completion of maturation, but suppress premature activation. This study was conducted to further examine the activity and localization of AMPK in relation to microtubule (MT) integrity. Immunostaining with tubulin revealed that active AMPK localized with gamma tubulin during metaphase I and II, while it localized at the spindle midzone during anaphase. This discrete localization pattern was dependent on MT integrity. Treatment with nocodazole, which depolymerized spindle MTs, led to disruption of proper spindle pole localization of active AMPK. In contrast, active AMPK was localized at each pole and with small asters when treated with paclitaxel, which induced excessive polymerization of spindle MTs. Disturbing spindle integrity not only influenced active AMPK localization, but also blocked AMPK activity, as both hormone- and AMPK activator- induced oocyte maturation were blocked by MT-disrupting agents. In concert with these data, the treatments inhibited active AMPK germinal vesicle staining as well, which appears before germinal vesicle breakdown (GVB) in meiotically induced oocytes. Targeting actin filament polymerization had only a marginal effect on meiotic induction. Although oocytes stimulated by AMPK activator increased the rate of GVB, spindle formation and PB extrusion, blocking AMPK activity did not influence peripheral movement of the spindle. These data suggest that the meiosis-inducing action and localization of AMPK are regulated by MT spindle integrity during mouse oocyte maturation. PMID:23199370

  18. Matrine-induced autophagy regulated by p53 through AMP-activated protein kinase in human hepatoma cells.

    PubMed

    Xie, Shan-Bu; He, Xing-Xing; Yao, Shu-Kun

    2015-08-01

    Matrine, one of the main extract components of Sophora flavescens, has been shown to exhibit inhibitory effects on some tumors through autophagy. However, the mechanism underlying the effect of matrine remains unclear. The cultured human hepatocellular carcinoma cell line HepG2 and SMMC‑7721 were treated with matrine. Signal transduction and gene expression profile were determined. Matrine stimulated autophagy in SMMC‑7721 cells in a mammalian target of rapamycin (mTOR)-dependent manner, but in an mTOR-independent manner in HepG2 cells. Next, in HepG2 cells, autophagy induced by matrine was regulated by p53 inactivation through AMP-activated protein kinase (AMPK) signaling transduction, then AMPK suppression switched autophagy to apoptosis. Furthermore, the interferon (IFN)-inducible genes, including interferon α-inducible protein 27 (IFI27) and interferon induced transmembrane protein 1 (IFITM1), which are downstream effector of p53, might be modulated by matrine-induced autophagy. In addition, we found that the p53 protein isoforms, p53β, p53γ, ∆133p53, and ∆133p53γ, due to alternative splicing of intron 9, might be regulated by the p53-mediated autophagy. These results show that matrine induces autophagy in human hepatoma cells through a novel mechanism, which is p53/AMPK signaling pathway involvement in matrine-promoted autophagy.

  19. H2O2-induced filamin redistribution in endothelial cells is modulated by the cyclic AMP-dependent protein kinase pathway.

    PubMed

    Hastie, L E; Patton, W F; Hechtman, H B; Shepro, D

    1997-09-01

    Hypoxia/reoxygenation injury in vitro causes endothelial cell cytoskeletal rearrangement that is related to increased monolayer permeability. Nonmuscle filamin (ABP-280) promotes orthogonal branching of F-actin and links microfilaments to membrane glycoproteins. Human umbilical vein endothelial cell monolayers are exposed to H2O2 (100 microM) for 1-60 min, with or without modulators of cAMP-dependent second-messenger pathways, and evaluated for changes in filamin distribution, cAMP levels, and the formation of gaps at interendothelial junctions. Filamin translocates from the membrane-cytoskeletal interface to the cytosol within 1 min of exposure to H2O2. This is associated with a decrease in endothelial cell cAMP levels from 83 pmoles/mg protein to 15 pmoles/mg protein. Intercellular gaps form 15 min after H2O2 treatment and progressively increase in number and diameter through 60 min. Both filamin redistribution and actin redistribution are associated with decreased phosphorylation of filamin and are prevented by activation of the cAMP-dependent protein kinase pathway. A synthetic peptide corresponding to filamin's C-terminal, cAMP-dependent, protein kinase phosphorylation site effectively induces filamin translocation and intercellular gap formation, which suggests that decreased phosphorylation of filamin at this site causes filamin redistribution and destabilization of junctions. These data indicate that H2O2-induced filamin redistribution and interendothelial cell gap formation result from inhibition of the cAMP-dependent protein kinase pathway.

  20. Cyclic AMP-induced G1 phase arrest mediated by an inhibitor (p27Kip1) of cyclin-dependent kinase 4 activation.

    PubMed

    Kato, J Y; Matsuoka, M; Polyak, K; Massagué, J; Sherr, C J

    1994-11-01

    Cyclic AMP (cAMP) blocks the mitogenic effects of colony-stimulating factor 1 (CSF-1) in macrophages, inducing cell cycle arrest in mid-G1 phase. Complexes between cyclin D1 and cyclin-dependent kinase 4 (cdk4) assemble in growth arrested cells, but cdk4 is not phosphorylated in vivo by the cdk-activating kinase (CAK) and remains inactive. Although undetectable in lysates of cAMP-treated cells, active CAK is recovered after antibody precipitation, indicating that it is not the direct target of inhibition. Levels of the cdk inhibitor p27Klp1 increase in cAMP-treated cells, and its immunodepletion from inhibitory lysates restores CAK-mediated cdk4 activation. Kip1 does not bind to CAK, but its association with cyclin D-cdk4 prevents CAK from phosphorylating and activating the holoenzyme. PMID:7954814

  1. T3-induced liver AMP-activated protein kinase signaling: Redox dependency and upregulation of downstream targets

    PubMed Central

    Videla, Luis A; Fernández, Virginia; Cornejo, Pamela; Vargas, Romina; Morales, Paula; Ceballo, Juan; Fischer, Alvaro; Escudero, Nicolás; Escobar, Oscar

    2014-01-01

    AIM: To investigate the redox dependency and promotion of downstream targets in thyroid hormone (T3)-induced AMP-activated protein kinase (AMPK) signaling as cellular energy sensor to limit metabolic stresses in the liver. METHODS: Fed male Sprague-Dawley rats were given a single ip dose of 0.1 mg T3/kg or T3 vehicle (NaOH 0.1 N; controls) and studied at 8 or 24 h after treatment. Separate groups of animals received 500 mg N-acetylcysteine (NAC)/kg or saline ip 30 min prior T3. Measurements included plasma and liver 8-isoprostane and serum β-hydroxybutyrate levels (ELISA), hepatic levels of mRNAs (qPCR), proteins (Western blot), and phosphorylated AMPK (ELISA). RESULTS: T3 upregulates AMPK signaling, including the upstream kinases Ca2+-calmodulin-dependent protein kinase kinase-β and transforming growth factor-β-activated kinase-1, with T3-induced reactive oxygen species having a causal role due to its suppression by pretreatment with the antioxidant NAC. Accordingly, AMPK targets acetyl-CoA carboxylase and cyclic AMP response element binding protein are phosphorylated, with the concomitant carnitine palmitoyltransferase-1α (CPT-1α) activation and higher expression of peroxisome proliferator-activated receptor-γ co-activator-1α and that of the fatty acid oxidation (FAO)-related enzymes CPT-1α, acyl-CoA oxidase 1, and acyl-CoA thioesterase 2. Under these conditions, T3 induced a significant increase in the serum levels of β-hydroxybutyrate, a surrogate marker for hepatic FAO. CONCLUSION: T3 administration activates liver AMPK signaling in a redox-dependent manner, leading to FAO enhancement as evidenced by the consequent ketogenic response, which may constitute a key molecular mechanism regulating energy dynamics to support T3 preconditioning against ischemia-reperfusion injury. PMID:25516653

  2. Viola mandshurica ethanolic extract prevents high-fat-diet-induced obesity in mice by activating AMP-activated protein kinase.

    PubMed

    Sung, Yoon-Young; Kim, Dong-Seon; Kim, Ho Kyoung

    2014-07-01

    Viola mandshurica W. Becker has been used as an expectorant, diuretic, and anti-inflammatory agent. We evaluated the effects of V. mandshurica ethanol extract (VME) on high-fat-diet (HFD)-induced obesity in mice. HPLC analysis showed that the VME contained 11.95 ± 0.37 mg/g esculetin and 0.13 ± 0.01 mg/g scopoletin. Orally administered VME decreased the body weight, adipose tissue mass, adipocyte size, and triglyceride and leptin serum concentrations. In contrast, VME increased serum adiponectin concentrations and adiponectin expression levels in epididymal adipose tissues. VME also significantly reversed the HFD-induced elevation of the mRNA and protein levels of lipogenic genes such as peroxisome proliferator-activated receptor γ, sterol regulatory element-binding protein-1c, fatty-acid synthase, and adipocyte protein 2. Moreover, VME reversed the HFD-induced inhibition of AMP-activated protein kinase (AMPK) and acetyl-coA carboxylase phosphorylation in epididymal adipose tissues. Furthermore, treatment of VME and esculetin in 3T3-L1 cells inhibited adipocyte differentiation and fat accumulation. These results suggest that VME exerts anti-obesity effects in HFD-induced obese mice by activating AMPK and suppressing PPARγ expression in adipose tissues.

  3. Hypoxia induces phosphorylation of the cyclic AMP response element-binding protein by a novel signaling mechanism.

    PubMed

    Beitner-Johnson, D; Millhorn, D E

    1998-07-31

    To investigate signaling mechanisms by which hypoxia regulates gene expression, we examined the effect of hypoxia on the cyclic AMP response element-binding protein (CREB) in PC12 cells. Exposure to physiological levels of hypoxia (5% O2, approximately 50 mm Hg) rapidly induced a persistent phosphorylation of CREB on Ser133, an event that is required for CREB-mediated transcriptional activation. Hypoxia-induced phosphorylation of CREB was more robust than that induced by any other stimulus tested, including forskolin, depolarization, and osmotic stress. Furthermore, this effect was not mediated by any of the previously known signaling pathways that lead to phosphorylation of CREB, including protein kinase A, calcium/calmodulin-dependent protein kinase, protein kinase C, ribosomal S6 kinase-2, and mitogen-activated protein kinase-activated protein kinase-2. Hypoxic activation of a CRE-containing reporter (derived from the 5'-flanking region of the tyrosine hydroxylase gene) was attenuated markedly by mutation of the CRE. Thus, a physiological reduction in O2 levels induces a functional phosphorylation of CREB at Ser133 via a novel signaling pathway. PMID:9677418

  4. Song-induced phosphorylation of cAMP response element-binding protein in the songbird brain.

    PubMed

    Sakaguchi, H; Wada, K; Maekawa, M; Watsuji, T; Hagiwara, M

    1999-05-15

    We have investigated the participation of cAMP response element-binding protein (CREB) in the response of the songbird brain to a natural auditory stimulus, a conspecific song. The cells in the two song control nuclei, the higher vocal center (HVC) and area X of zebra finches (Taeniopygia guttata), were intensely stained with an anti-CREB monoclonal antibody. Double-labeling studies showed that CREB immunoreactivity was detected only in area X-projecting neurons in the HVC. The cloned CREB cDNA from zebra finches (zCREB) is highly homologous to mammalian delta CREB. Phosphorylation of zCREB at Ser119 in area X-projecting HVC neurons was induced by hearing tape-recorded conspecific songs of zebra finches, but not by birdsongs of another species or white noise. These results raise the possibility that zCREB plays a crucial role in the sensory process of song learning.

  5. Song-induced phosphorylation of cAMP response element-binding protein in the songbird brain.

    PubMed

    Sakaguchi, H; Wada, K; Maekawa, M; Watsuji, T; Hagiwara, M

    1999-05-15

    We have investigated the participation of cAMP response element-binding protein (CREB) in the response of the songbird brain to a natural auditory stimulus, a conspecific song. The cells in the two song control nuclei, the higher vocal center (HVC) and area X of zebra finches (Taeniopygia guttata), were intensely stained with an anti-CREB monoclonal antibody. Double-labeling studies showed that CREB immunoreactivity was detected only in area X-projecting neurons in the HVC. The cloned CREB cDNA from zebra finches (zCREB) is highly homologous to mammalian delta CREB. Phosphorylation of zCREB at Ser119 in area X-projecting HVC neurons was induced by hearing tape-recorded conspecific songs of zebra finches, but not by birdsongs of another species or white noise. These results raise the possibility that zCREB plays a crucial role in the sensory process of song learning. PMID:10234027

  6. Nucleotide sequences of fic and fic-1 genes involved in cell filamentation induced by cyclic AMP in Escherichia coli.

    PubMed Central

    Kawamukai, M; Matsuda, H; Fujii, W; Utsumi, R; Komano, T

    1989-01-01

    The nucleotide sequences of fic-1 involved in the cell filamentation induced by cyclic AMP in Escherichia coli and its normal counterpart fic were analyzed. The open reading frame of both fic-1 and fic coded for 200 amino acids. The Gly at position 55 in the Fic protein was changed to Arg in the Fic-1 protein. The promoter activity of fic was confirmed by fusing fic and lacZ. The gene downstream from fic was found to be pabA (p-aminobenzoate). There is an open reading frame (ORF190) coding for 190 amino acids upstream from the fic gene. Computer-assisted analysis showed that Fic has sequence similarity with part of CDC28 of Saccharomyces cerevisiae, CDC2 of Schizosaccharomyces pombe, and FtsA of E. coli. In addition, ORF190 has sequence similarity with the cyclosporin A-binding protein cyclophilin. PMID:2546924

  7. Involvement of AMP-activated protein kinase in mediating pyrrolo-1,5-benzoxazepine-induced apoptosis in neuroblastoma cells.

    PubMed

    Lennon, Jennifer C; Butini, Stefania; Campiani, Giuseppe; O'Meara, Anne; Williams, D Clive; Zisterer, Daniela M

    2016-10-01

    Neuroblastoma, a paediatric malignancy of the sympathetic nervous system, accounts for 15 % of childhood cancer deaths. Despite advances in understanding the biology, it remains one of the most difficult paediatric cancers to treat partly due to the development of multidrug resistance. There is thus a compelling demand for new treatment strategies that can bypass resistance mechanisms. The pyrrolo-1,5-benzoxazepine (PBOX) compounds are a series of novel microtubule-targeting agents that potently induce apoptosis in various tumour models. We have previously reported that PBOX compounds induce apoptosis in drug sensitive and multidrug resistant neuroblastoma cells and synergistically enhance apoptosis induced by chemotherapeutics such as carboplatin. In this study we present further data concerning the molecular basis of PBOX-induced apoptosis in neuroblastoma. We demonstrate that PBOX-6 induced AMP-activated protein kinase (AMPK) activation and downstream acetyl-CoA carboxylase phosphorylation. Increased reactive oxygen species (ROS) appeared to serve as the upstream signal for AMPK activation as pretreatment of cells with the antioxidant N-acetylcysteine inhibited both AMPK activation and PBOX-induced apoptosis. Furthermore, activation of AMPK by PBOX-6 was found to inhibit mTOR complex 1 (mTORC1) signalling. Finally, we demonstrate the efficacy of PBOX-6 in an in vivo xenograft model of neuroblastoma. This study provides new insights into understanding the molecular and cellular mechanisms involved in PBOX-induced cell death in neuroblastoma and further supports their future use as novel anti-cancer agents for the treatment of neuroblastoma.

  8. Involvement of AMP-activated protein kinase in mediating pyrrolo-1,5-benzoxazepine-induced apoptosis in neuroblastoma cells.

    PubMed

    Lennon, Jennifer C; Butini, Stefania; Campiani, Giuseppe; O'Meara, Anne; Williams, D Clive; Zisterer, Daniela M

    2016-10-01

    Neuroblastoma, a paediatric malignancy of the sympathetic nervous system, accounts for 15 % of childhood cancer deaths. Despite advances in understanding the biology, it remains one of the most difficult paediatric cancers to treat partly due to the development of multidrug resistance. There is thus a compelling demand for new treatment strategies that can bypass resistance mechanisms. The pyrrolo-1,5-benzoxazepine (PBOX) compounds are a series of novel microtubule-targeting agents that potently induce apoptosis in various tumour models. We have previously reported that PBOX compounds induce apoptosis in drug sensitive and multidrug resistant neuroblastoma cells and synergistically enhance apoptosis induced by chemotherapeutics such as carboplatin. In this study we present further data concerning the molecular basis of PBOX-induced apoptosis in neuroblastoma. We demonstrate that PBOX-6 induced AMP-activated protein kinase (AMPK) activation and downstream acetyl-CoA carboxylase phosphorylation. Increased reactive oxygen species (ROS) appeared to serve as the upstream signal for AMPK activation as pretreatment of cells with the antioxidant N-acetylcysteine inhibited both AMPK activation and PBOX-induced apoptosis. Furthermore, activation of AMPK by PBOX-6 was found to inhibit mTOR complex 1 (mTORC1) signalling. Finally, we demonstrate the efficacy of PBOX-6 in an in vivo xenograft model of neuroblastoma. This study provides new insights into understanding the molecular and cellular mechanisms involved in PBOX-induced cell death in neuroblastoma and further supports their future use as novel anti-cancer agents for the treatment of neuroblastoma. PMID:27334143

  9. Hormone-induced intercellular signal transfer dissociates cyclic AMP- dependent protein kinase

    PubMed Central

    1984-01-01

    We used co-cultures of porcine ovarian granulosa cells and mouse adrenocortical tumor cells (Y-1) to examine the kinetics of contact- dependent intercellular signal transfer and to assess the molecular mechanisms employed by this process. Exposure to follicle-stimulating hormone (FSH) caused cAMP-dependent protein kinase dissociation in granulosa cells and, with time, in Y-1 cells if, and only if, they contacted a responding granulosa cell. Y-1 cells close to a granulosa cell but not touching it failed to respond similarly. In reciprocal experiments, co-cultures were stimulated with adrenocorticotropic hormone (ACTH). Y-1 cells dissociated protein kinase as did granulosa cells in contact with Y-1 cells; however, granulosa cells that were not in contact with Y-1 cells failed to respond to the hormone. Fluorogenic steroids were secreted by Y-1 cells cultured alone and stimulated with ACTH, but were not secreted by cultures exposed to FSH. Neither hormone caused fluorogenic steroid production by granulosa cells. On the other hand these steroids were secreted in co-cultures stimulated with ACTH and to a lesser degree in co-cultures exposed to FSH. Autoradiography revealed that I125-FSH bound only to granulosa cells, never to Y-1 cells, even if they were in contact with an ovarian cell. The possibility of cell fusion was tested by experiments in which Y-1 cell membranes were labeled with cationized ferritin. These cells were then placed in co-culture with ovarian granulosa cells that had previously been allowed to ingest latex spheres. At regions of gap junctions between Y-1 and granulosa cells ferritin remained attached to the adrenal cell membrane and was never observed to migrate to the granulosa cell membrane. From these data, we conclude that hormone specific stimulation of one cell type leads to protein kinase dissociation in heterotypic partners only if they contact a hormone responsive cell. This signal transfer is bidirectional, exhibits temporal kinetics and

  10. AMP Kinase Activation Alters Oxidant-Induced Stress Granule Assembly by Modulating Cell Signaling and Microtubule Organization.

    PubMed

    Mahboubi, Hicham; Koromilas, Antonis E; Stochaj, Ursula

    2016-10-01

    Eukaryotic cells assemble stress granules (SGs) when translation initiation is inhibited. Different cell signaling pathways regulate SG production. Particularly relevant to this process is 5'-AMP-activated protein kinase (AMPK), which functions as a stress sensor and is transiently activated by adverse physiologic conditions. Here, we dissected the role of AMPK for oxidant-induced SG formation. Our studies identified multiple steps of de novo SG assembly that are controlled by the kinase. Single-cell analyses demonstrated that pharmacological AMPK activation prior to stress exposure changed SG properties, because the granules became more abundant and smaller in size. These altered SG characteristics correlated with specific changes in cell survival, cell signaling, cytoskeletal organization, and the abundance of translation initiation factors. Specifically, AMPK activation increased stress-induced eukaryotic initiation factor (eIF) 2α phosphorylation and reduced the concentration of eIF4F complex subunits eIF4G and eIF4E. At the same time, the abundance of histone deacetylase 6 (HDAC6) was diminished. This loss of HDAC6 was accompanied by increased acetylation of α-tubulin on Lys40. Pharmacological studies further confirmed this novel AMPK-HDAC6 interplay and its importance for SG biology. Taken together, we provide mechanistic insights into the regulation of SG formation. We propose that AMPK activation stimulates oxidant-induced SG formation but limits their fusion into larger granules. PMID:27430620

  11. Redox regulation of cAMP levels by ascorbate in 1,25-dihydroxy- vitamin D3-induced differentiation of HL-60 cells.

    PubMed Central

    López-Lluch, G; Burón, M I; Alcaín, F J; Quesada, J M; Navas, P

    1998-01-01

    1alpha,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] induces differentiation to monocyte-macrophage lineage of several leukaemic cell lines such as HL-60, U937, M1 and Mono Mac 6. Ascorbate also modulates growth and differentiation of different animal cells in culture. We have previously reported the stimulating effect of ascorbate on 1, 25-(OH)2D3-induced HL-60 cell differentiation. We show here that 1, 25-(OH)2D3 induces a transient increase in cAMP levels in these cells, and ascorbate significantly increases these cAMP levels. Ascorbate alone does not have any effect. Other cAMP-increasing agents such as isobutylmethylxanthine, forskolin and prostaglandin E2 maintain high levels of cAMP at 48 h of incubation and also enhance differentiation along the monocytic pathway induced by 1, 25-(OH)2D3, as revealed by specific differentiation markers, demonstrating the importance of cAMP in the differentiation process. It is also shown that the presence of ascorbate and its free radical (AFR) during 1,25-(OH)2D3-induced differentiation significantly decreases cytoplasmic NADH levels compared with those induced by 1,25-(OH)2D3 in HL-60 cells. The results indicate that NADH is an inhibitor of adenylate cyclase in these cells. AFR is an electron acceptor of the trans-plasma-membrane electron-transport system, and NADH is the electron donor. Through this system, ascorbate and AFR keep levels of NADH low, thereby decreasing its inhibitory effect on adenylate cyclase activity and so increasing cAMP synthesis. We also demonstrate that other ascorbate derivatives, such as ascorbate 2-phosphate and dehydroascorbate, both of which are unable to produce AFR, do not alter intracellular NADH levels during 1, 25-(OH)2D3-induced differentiation. Also, ascorbate and AFR increase specific differentiation markers (CD14 and NitroBlue Tetrazolium reduction) but neither ascorbate 2-phosphate nor dehydroascorbate show this enhancing activity. In summary, we propose that the effect of ascorbate on 1

  12. Pancreatic β-Cell Dysfunction in Diet-Induced Obese Mice: Roles of AMP-Kinase, Protein Kinase Cε, Mitochondrial and Cholesterol Metabolism, and Alterations in Gene Expression

    PubMed Central

    Pepin, Émilie; Al-Mass, Anfal; Attané, Camille; Zhang, Kezhuo; Lamontagne, Julien; Lussier, Roxane; Madiraju, S. R. Murthy; Joly, Erik; Ruderman, Neil B.; Sladek, Robert; Prentki, Marc; Peyot, Marie-Line

    2016-01-01

    Diet induced obese (DIO) mice can be stratified according to their weight gain in response to high fat diet as low responders (LDR) and high responders (HDR). This allows the study of β-cell failure and the transitions to prediabetes (LDR) and early diabetes (HDR). C57BL/6N mice were fed for 8 weeks with a normal chow diet (ND) or a high fat diet and stratified as LDR and HDR. Freshly isolated islets from ND, LDR and HDR mice were studied ex-vivo for mitochondrial metabolism, AMPK activity and signalling, the expression and activity of key enzymes of energy metabolism, cholesterol synthesis, and mRNA profiling. Severely compromised glucose-induced insulin secretion in HDR islets, as compared to ND and LDR islets, was associated with suppressed AMP-kinase activity. HDR islets also showed reduced acetyl-CoA carboxylase activity and enhanced activity of 3-hydroxy-3-methylglutaryl-CoA reductase, which led respectively to elevated fatty acid oxidation and increased cholesterol biosynthesis. HDR islets also displayed mitochondrial membrane hyperpolarization and reduced ATP turnover in the presence of elevated glucose. Expression of protein kinase Cε, which reduces both lipolysis and production of signals for insulin secretion, was elevated in DIO islets. Genes whose expression increased or decreased by more than 1.2-fold were minor between LDR and ND islets (17 differentially expressed), but were prominent between HDR and ND islets (1508 differentially expressed). In HDR islets, particularly affected genes were related to cell cycle and proliferation, AMPK signaling, mitochondrial metabolism and cholesterol metabolism. In conclusion, chronically reduced AMPK activity, mitochondrial dysfunction, elevated cholesterol biosynthesis in islets, and substantial alterations in gene expression accompany β-cell failure in HDR islets. The β-cell compensation process in the prediabetic state (LDR) is largely independent of transcriptional adaptive changes, whereas the transition

  13. Pancreatic β-Cell Dysfunction in Diet-Induced Obese Mice: Roles of AMP-Kinase, Protein Kinase Cε, Mitochondrial and Cholesterol Metabolism, and Alterations in Gene Expression.

    PubMed

    Pepin, Émilie; Al-Mass, Anfal; Attané, Camille; Zhang, Kezhuo; Lamontagne, Julien; Lussier, Roxane; Madiraju, S R Murthy; Joly, Erik; Ruderman, Neil B; Sladek, Robert; Prentki, Marc; Peyot, Marie-Line

    2016-01-01

    Diet induced obese (DIO) mice can be stratified according to their weight gain in response to high fat diet as low responders (LDR) and high responders (HDR). This allows the study of β-cell failure and the transitions to prediabetes (LDR) and early diabetes (HDR). C57BL/6N mice were fed for 8 weeks with a normal chow diet (ND) or a high fat diet and stratified as LDR and HDR. Freshly isolated islets from ND, LDR and HDR mice were studied ex-vivo for mitochondrial metabolism, AMPK activity and signalling, the expression and activity of key enzymes of energy metabolism, cholesterol synthesis, and mRNA profiling. Severely compromised glucose-induced insulin secretion in HDR islets, as compared to ND and LDR islets, was associated with suppressed AMP-kinase activity. HDR islets also showed reduced acetyl-CoA carboxylase activity and enhanced activity of 3-hydroxy-3-methylglutaryl-CoA reductase, which led respectively to elevated fatty acid oxidation and increased cholesterol biosynthesis. HDR islets also displayed mitochondrial membrane hyperpolarization and reduced ATP turnover in the presence of elevated glucose. Expression of protein kinase Cε, which reduces both lipolysis and production of signals for insulin secretion, was elevated in DIO islets. Genes whose expression increased or decreased by more than 1.2-fold were minor between LDR and ND islets (17 differentially expressed), but were prominent between HDR and ND islets (1508 differentially expressed). In HDR islets, particularly affected genes were related to cell cycle and proliferation, AMPK signaling, mitochondrial metabolism and cholesterol metabolism. In conclusion, chronically reduced AMPK activity, mitochondrial dysfunction, elevated cholesterol biosynthesis in islets, and substantial alterations in gene expression accompany β-cell failure in HDR islets. The β-cell compensation process in the prediabetic state (LDR) is largely independent of transcriptional adaptive changes, whereas the transition

  14. Piperine, a component of black pepper, decreases eugenol-induced cAMP and calcium levels in non-chemosensory 3T3-L1 cells.

    PubMed

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

    2015-01-01

    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. PMID:25685661

  15. Piperine, a component of black pepper, decreases eugenol-induced cAMP and calcium levels in non-chemosensory 3T3-L1 cells.

    PubMed

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

    2015-01-01

    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.

  16. RNA-dependent protein kinase (PKR) depletes nutrients, inducing phosphorylation of AMP-activated kinase in lung cancer

    PubMed Central

    Guo, Chengcheng; Hao, Chuncheng; Shao, RuPing; Fang, Bingliang; Correa, Arlene M.; Hofstetter, Wayne L.; Roth, Jack A.; Behrens, Carmen; Kalhor, Neda; Wistuba, Ignacio I.; Swisher, Stephen G.; Pataer, Apar

    2015-01-01

    We have demonstrated that RNA-dependent protein kinase (PKR) and its downstream protein p-eIF2α are independent prognostic markers for overall survival in lung cancer. In the current study, we further investigate the interaction between PKR and AMPK in lung tumor tissue and cancer cell lines. We examined PKR protein expression in 55 frozen primary lung tumor tissues by Western blotting and analyzed the association between PKR expression and expresson of 139 proteins on tissue samples examined previously by Reverse Phase Protein Array (RPPA) from the same 55 patients. We observed that biomarkers were either positively (phosphorylated AMP-activated kinaseT172 [p-AMPK]) or negatively (insulin receptor substrate 1, meiotic recombination 11, ATR interacting protein, telomerase, checkpoint kinase 1, and cyclin E1) correlated with PKR. We further confirmed that induction of PKR with expression vectors in lung cancer cells causes activation of the AMPK protein independent of the LKB1, TAK1, and CaMKKβ pathway. We found that PKR causes nutrient depletion, which increases AMP levels and decreases ATP levels, causing AMPK phosphorylation. We further demonstrated that inhibiting AMPK expression with compound C or siRNA enhanced PKR-mediated cell death. We next explored the combination of PKR and p-AMPK expression in NSCLC patients and observed that expression of p-AMPK predicted a poor outcome for adenocarcinoma patients with high PKR expression and a better prognosis for those with low PKR expression. These findings were consistent with our in vitro results. AMPK might rescue cells facing metabolic stresses, such as ATP depletion caused by PKR. Our data indicate that PKR causes nutrient depletion, which induces the phosphorylation of AMPK. AMPK might act as a protective response to metabolic stresses, such as nutrient deprivation. PMID:25798539

  17. Activation of PPARβ/δ prevents hyperglycaemia-induced impairment of Kv7 channels and cAMP-mediated relaxation in rat coronary arteries.

    PubMed

    Morales-Cano, Daniel; Moreno, Laura; Barreira, Bianca; Briones, Ana M; Pandolfi, Rachele; Moral-Sanz, Javier; Callejo, Maria; Mondejar-Parreño, Gema; Cortijo, Julio; Salaices, Mercedes; Duarte, Juan; Perez-Vizcaino, Francisco; Cogolludo, Angel

    2016-10-01

    PPARβ/δ activation protects against endothelial dysfunction in diabetic models. Elevated glucose is known to impair cAMP-induced relaxation and Kv channel function in coronary arteries (CA). Herein, we aimed to analyse the possible protective effects of the PPARβ/δ agonist GW0742 on the hyperglycaemic-induced impairment of cAMP-induced relaxation and Kv channel function in rat CA. As compared with low glucose (LG), incubation under high glucose (HG) conditions attenuated the relaxation induced by the adenylate cyclase activator forskolin in CA and this was prevented by GW0742. The protective effect of GW0742 was supressed by a PPARβ/δ antagonist. In myocytes isolated from CA under LG, forskolin enhanced Kv currents and induced hyperpolarization. In contrast, when CA were incubated with HG, Kv currents were diminished and the electrophysiological effects of forskolin were abolished. These deleterious effects were prevented by GW0742. The protective effects of GW0742 on forskolin-induced relaxation and Kv channel function were confirmed in CA from type-1 diabetic rats. In addition, the differences in the relaxation induced by forskolin in CA incubated under LG, HG or HG + GW0742 were abolished by the Kv7 channel inhibitor XE991. Accordingly, GW0742 prevented the down-regulation of Kv7 channels induced by HG. Finally, the preventive effect of GW0742 on oxidative stress and cAMP-induced relaxation were overcome by the pyruvate dehydrogenase kinase 4 (PDK4) inhibitor dichloroacetate (DCA). Our results reveal that the PPARβ/δ agonist GW0742 prevents the impairment of the cAMP-mediated relaxation in CA under HG. This protective effect was associated with induction of PDK4, attenuation of oxidative stress and preservation of Kv7 channel function. PMID:27413020

  18. Activation of PPARβ/δ prevents hyperglycaemia-induced impairment of Kv7 channels and cAMP-mediated relaxation in rat coronary arteries.

    PubMed

    Morales-Cano, Daniel; Moreno, Laura; Barreira, Bianca; Briones, Ana M; Pandolfi, Rachele; Moral-Sanz, Javier; Callejo, Maria; Mondejar-Parreño, Gema; Cortijo, Julio; Salaices, Mercedes; Duarte, Juan; Perez-Vizcaino, Francisco; Cogolludo, Angel

    2016-10-01

    PPARβ/δ activation protects against endothelial dysfunction in diabetic models. Elevated glucose is known to impair cAMP-induced relaxation and Kv channel function in coronary arteries (CA). Herein, we aimed to analyse the possible protective effects of the PPARβ/δ agonist GW0742 on the hyperglycaemic-induced impairment of cAMP-induced relaxation and Kv channel function in rat CA. As compared with low glucose (LG), incubation under high glucose (HG) conditions attenuated the relaxation induced by the adenylate cyclase activator forskolin in CA and this was prevented by GW0742. The protective effect of GW0742 was supressed by a PPARβ/δ antagonist. In myocytes isolated from CA under LG, forskolin enhanced Kv currents and induced hyperpolarization. In contrast, when CA were incubated with HG, Kv currents were diminished and the electrophysiological effects of forskolin were abolished. These deleterious effects were prevented by GW0742. The protective effects of GW0742 on forskolin-induced relaxation and Kv channel function were confirmed in CA from type-1 diabetic rats. In addition, the differences in the relaxation induced by forskolin in CA incubated under LG, HG or HG + GW0742 were abolished by the Kv7 channel inhibitor XE991. Accordingly, GW0742 prevented the down-regulation of Kv7 channels induced by HG. Finally, the preventive effect of GW0742 on oxidative stress and cAMP-induced relaxation were overcome by the pyruvate dehydrogenase kinase 4 (PDK4) inhibitor dichloroacetate (DCA). Our results reveal that the PPARβ/δ agonist GW0742 prevents the impairment of the cAMP-mediated relaxation in CA under HG. This protective effect was associated with induction of PDK4, attenuation of oxidative stress and preservation of Kv7 channel function.

  19. Activation of Autophagic Flux against Xenoestrogen Bisphenol-A-induced Hippocampal Neurodegeneration via AMP kinase (AMPK)/Mammalian Target of Rapamycin (mTOR) Pathways*

    PubMed Central

    Agarwal, Swati; Tiwari, Shashi Kant; Seth, Brashket; Yadav, Anuradha; Singh, Anshuman; Mudawal, Anubha; Chauhan, Lalit Kumar Singh; Gupta, Shailendra Kumar; Choubey, Vinay; Tripathi, Anurag; Kumar, Amit; Ray, Ratan Singh; Shukla, Shubha; Parmar, Devendra; Chaturvedi, Rajnish Kumar

    2015-01-01

    The human health hazards related to persisting use of bisphenol-A (BPA) are well documented. BPA-induced neurotoxicity occurs with the generation of oxidative stress, neurodegeneration, and cognitive dysfunctions. However, the cellular and molecular mechanism(s) of the effects of BPA on autophagy and association with oxidative stress and apoptosis are still elusive. We observed that BPA exposure during the early postnatal period enhanced the expression and the levels of autophagy genes/proteins. BPA treatment in the presence of bafilomycin A1 increased the levels of LC3-II and SQSTM1 and also potentiated GFP-LC3 puncta index in GFP-LC3-transfected hippocampal neural stem cell-derived neurons. BPA-induced generation of reactive oxygen species and apoptosis were mitigated by a pharmacological activator of autophagy (rapamycin). Pharmacological (wortmannin and bafilomycin A1) and genetic (beclin siRNA) inhibition of autophagy aggravated BPA neurotoxicity. Activation of autophagy against BPA resulted in intracellular energy sensor AMP kinase (AMPK) activation, increased phosphorylation of raptor and acetyl-CoA carboxylase, and decreased phosphorylation of ULK1 (Ser-757), and silencing of AMPK exacerbated BPA neurotoxicity. Conversely, BPA exposure down-regulated the mammalian target of rapamycin (mTOR) pathway by phosphorylation of raptor as a transient cell's compensatory mechanism to preserve cellular energy pool. Moreover, silencing of mTOR enhanced autophagy, which further alleviated BPA-induced reactive oxygen species generation and apoptosis. BPA-mediated neurotoxicity also resulted in mitochondrial loss, bioenergetic deficits, and increased PARKIN mitochondrial translocation, suggesting enhanced mitophagy. These results suggest implication of autophagy against BPA-mediated neurodegeneration through involvement of AMPK and mTOR pathways. Hence, autophagy, which arbitrates cell survival and demise during stress conditions, requires further assessment to be

  20. Prolonged AICAR-induced AMP-kinase activation promotes energy dissipation in white adipocytes: novel mechanisms integrating HSL and ATGL.

    PubMed

    Gaidhu, Mandeep P; Fediuc, Sergiu; Anthony, Nicole M; So, Mandy; Mirpourian, Mani; Perry, Robert L S; Ceddia, Rolando B

    2009-04-01

    This study was designed to investigate the effects of prolonged activation of AMP-activated protein kinase (AMPK) on lipid partitioning and the potential molecular mechanisms involved in these processes in white adipose tissue (WAT). Rat epididymal adipocytes were incubated with 5'-aminoimidasole-4-carboxamide-1-beta-d-ribofuranoside (AICAR;0.5 mM) for 15 h. Also, epididymal adipocytes were isolated 15 h after AICAR was injected (i.p. 0.7 g/kg body weight) in rats. Adipocytes were utilized for various metabolic assays and for determination of gene expression and protein content. Time-dependent in vivo plasma NEFA concentrations were determined. AICAR treatment significantly increased AMPK activation, inhibited lipogenesis, and increased FA oxidation. This was accompanied by upregulation of peroxisome proliferator-activated receptor (PPAR)alpha, PPARdelta, and PPARgamma-coactivator-1alpha (PGC-1alpha) mRNA levels. Lipolysis was first suppressed, but then increased, both in vitro and in vivo, with prolonged AICAR treatment. Exposure to AICAR increased adipose triglyceride lipase (ATGL) content and FA release, despite inhibition of basal and epinephrine-stimulated hormone-sensitive lipase (HSL) activity. Here, we provide evidence that prolonged AICAR-induced AMPK activation can remodel adipocyte metabolism by upregulating pathways that favor energy dissipation versus lipid storage in WAT. Additionally, we show novel time-dependent effects of AICAR-induced AMPK activation on lipolysis, which involves antagonistic modulation of HSL and ATGL.

  1. The light-induced increase of carbohydrate metabolism in glial cells of the honeybee retina is not mediated by K+ movement nor by cAMP.

    PubMed

    Evêquoz-Mercier, V; Tsacopoulos, M

    1991-09-01

    The retina of the honeybee drone is a nervous tissue in which glial cells and photoreceptor neurons constitute two distinct metabolic compartments. The phosphorylation of glucose and its subsequent incorporation into glycogen occur essentially in glia, whereas O2 consumption occurs in the photoreceptors. After [3H] glucose loading of superfused retinal slices, light stimulation induced a significant rise in [3H] glycogen turnover in the glia. This occurs without a concomitant covalent modification of glycogen enzymes. Probably only an increase or a decrease of the availability of [3H] glycosyls that are incorporated into glycogen is necessary. As only photoreceptors are directly excitable by light, we searched for a signal that stimulates glycogen metabolism in the glia. Although K+ in extracellular space and glia increases after repetitive light stimulation, increasing bath K+ in the dark did not mimic the metabolic effects of light, despite an equivalent increase of K+ in the extracellular space and glia. We subsequently explored the role of cAMP, a universal intracellular second messenger. Exposure of retinal slices to the adenylate-cyclase activator forskolin induced an expected increase in the rate of formation of cAMP, but only partially mimicked the metabolic effects of light. Furthermore, light stimulation failed to induce a rise in the rate of formation of cAMP. We conclude that in this nervous system, without synapses, neither K+ nor cAMP mediates the effect of light stimulation on intraglial glucose metabolism.

  2. AMP-activated protein kinase (AMPK) regulates the insulin-induced activation of the nitric oxide synthase in human platelets.

    PubMed

    Fleming, Ingrid; Schulz, Christian; Fichtlscherer, Birgit; Kemp, Bruce E; Fisslthaler, Beate; Busse, Rudi

    2003-11-01

    Little is known about the signaling cascades that eventually regulate the activity of the endothelial nitric oxide synthase (eNOS) in platelets. Here, we investigated the effects of insulin on the phosphorylation and activation of eNOS in washed human platelets and in endothelial cells. Insulin activated the protein kinase Akt in cultured endothelial cells and increased the phosphorylation of eNOS on Ser(1177) but failed to increase endothelial cyclic GMP levels or to elicit the relaxation of endothelium-intact porcine coronary arteries. In platelets, insulin also elicited the activation of Akt as well as the phosphorylation of eNOS and initiated NO production which was associated with increased cyclic GMP levels and the inhibition of thrombin-induced aggregation. The insulin-induced inhibition of aggregation was accompanied by a decreased Ca(2+) response to thrombin and was also prevented by N(omega) nitro-L-arginine. In platelets, but not in endothelial cells, insulin induced the activation of the AMP-activated protein kinase (AMPK), a metabolic stress-sensing kinase which was sensitive to the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin and the AMPK inhibitor iodotubercidin. Moreover, the insulin-mediated inhibition of thrombin-induced aggregation was prevented by iodotubercidin. Insulin-independent activation of the AMPK using 5-aminoimidazole-4-carboxamide ribonucleoside, increased platelet eNOS phosphorylation, increased cyclic GMP levels and attenuated platelet aggregation. These results highlight the differences in the signal transduction cascade activated by insulin in endothelial cells and platelets, and demonstrate that insulin stimulates the formation of NO in human platelets, in the absence of an increase in Ca(2+), by acti-vating PI3-K and AMPK which phosphorylates eNOS on Ser(1177).

  3. Suppression of LPS-Induced TNF-α Production in Macrophages by cAMP Is Mediated by PKA-AKAP95-p105

    PubMed Central

    Wall, Estelle A.; Zavzavadjian, Joelle R.; Chang, Mi Sook; Randhawa, Baljinder; Zhu, Xiaocui; Hsueh, Robert C.; Liu, Jamie; Driver, Adrienne; Bao, Xiaoyan Robert; Sternweis, Paul C.; Simon, Melvin I.; Fraser, Iain D. C.

    2009-01-01

    The activation of macrophages through Toll-like receptor (TLR) pathways leads to the production of a broad array of cytokines and mediators that coordinate the immune response. The inflammatory potential of this response can be reduced by compounds, such as prostaglandin E2, that induce the production of cyclic adenosine monophosphate (cAMP). Through experiments with cAMP analogs and multigene RNA interference (RNAi), we showed that key anti-inflammatory effects of cAMP were mediated specifically by cAMP-dependent protein kinase (PKA). Selective inhibitors of PKA anchoring, time-lapse microscopy, and RNAi screening suggested that differential mechanisms of PKA action existed. We showed a specific role for A kinase-anchoring protein 95 in suppressing the expression of the gene encoding tumor necrosis factor-α, which involved phosphorylation of p105 (also known as Nfkb1) by PKA at a site adjacent to the region targeted by inhibitor of nuclear factor κB kinases. These data suggest that crosstalk between the TLR4 and cAMP pathways in macrophages can be coordinated through PKA-dependent scaffolds that localize specific pools of the kinase to distinct substrates. PMID:19531803

  4. Reversible cAMP-induced translocation of cytoskeleton-associated 300- to 350-kDa proteins from nucleus to cytoplasm

    SciTech Connect

    Nakayama, Tokiko; Nishizawa, Kimiko; Sato, Chicako )

    1988-08-01

    The authors previously reported that treatment of SV-3Y1 cells in an exponential growth state with db-cAMP plus theophylline induced reversible disappearance of nuclear dots stained by monoclonal anti-microtubule-associated protein (MAP)-1 antibody. In the present study, the authors examined the relation between the intracellular localization and phosphorylation of 300- to 350-kDa proteins that are intracellular antigens for our anti-Map-1 and -2 antibodies. Treatment with db-cAMP plus theophylline was found to result in a reversible decrease in immunofluorescent staining of the nucleus with polyclonal MAP-1 or -2 antibody, and a reversible increase in that of the cytoplasm. Simultaneous treatment with colchicine, colcemid, putrescine, or {alpha}-naphthyl phosphate in the presence of db-cAMP plus theophylline almost prevented this effect of db-cAMP plus theophylline. They examined the cytoplasmic and nuclear fractions by immunoperoxidase staining, immunoprecipitation, and {sup 125}I-protein A with anti-MAP-1 and -2 antibodies. The present research indicated that treatment with db-cAMP plus theophylline resulted in the reversible translocation of 300- to 350-kDa proteins from the nucleus to the cytoplasm accompanied by the dephosphorylation of these proteins.

  5. 14-Deoxyandrographolide alleviates ethanol-induced hepatosteatosis through stimulation of AMP-activated protein kinase activity in rats.

    PubMed

    Mandal, Samir; Mukhopadhyay, Sibabrata; Bandhopadhyay, Sukdeb; Sen, Gargi; Biswas, Tuli

    2014-03-01

    Andrographis paniculata (AP) is a traditional medicinal plant of Ayurveda. It grows widely in Asia and is prescribed in the treatment of liver diseases. Here we have investigated the beneficial role of 14-deoxyandrographolide (14-DAG), a bioactive diterpenoid from AP, against alcoholic steatosis in rats. 14-DAG was extracted from aerial parts (leaves and stems) of AP. Rats were fed with ethanol for 8 weeks. Animals were treated with 14-DAG during the last 4 weeks of ethanol treatment. In vitro studies were undertaken in a human hepatocellular liver carcinoma cell line culture. Hepatosteatosis was assessed from histopathological studies of liver sections. Acetyl-CoA, malonyl-CoA, and triglyceride contents were determined using commercially available kits. Fatty acid synthesis was evaluated from incorporation of 1-(14)C acetate. Regulation of fatty acid oxidation and lipogenesis were monitored with immunoblotting and immunoprecipitation studies. Ethanol exposure led to hepatotoxicity, as evident from the marked enhancement in the levels of AST and ALT. The values decreased almost to control levels in response to 14-DAG treatment. Results showed that ethanol feeding induced deactivation of AMP-activated protein kinase (AMPK) that led to enhanced lipid synthesis and decreased fatty acid oxidation, culminating in hepatic fat accumulation. Treatment with 14-DAG activated AMPK through induction of cyclic AMP-protein kinase A pathway. Activation of AMPK was followed by down-regulation of sterol regulatory element binding protein-1c, acetyl-CoA carboxylase, and fatty acid synthase, leading to suppression of lipogenesis. This was associated with up-regulation of sirtuin 1 and depletion of malonyl-CoA, in favor of increased fatty acid oxidation. 14-DAG controlled ethanol-induced hepatosteatosis by interfering with dysregulation of lipid metabolism. In conclusion, our results indicated that 14-DAG was capable of preventing the development of fatty liver through AMPK

  6. Involvement of the CDC25 gene product in the signal transmission pathway of the glucose-induced RAS-mediated cAMP signal in the yeast Saccharomyces cerevisiae.

    PubMed

    van Aelst, L; Jans, A W; Thevelein, J M

    1991-02-01

    Addition of glucose or related fermentable sugars to derepressed cells of the yeast Saccharomyces cerevisiae triggers a RAS-protein-mediated cAMP signal, which induces a protein phosphorylation cascade. Yeast strains without a functional CDC25 gene were deficient in basal cAMP synthesis and in the glucose-induced cAMP signal. Addition of dinitrophenol, which in wild-type strains strongly stimulates in vivo cAMP synthesis by lowering intracellular pH, did not enhance the cAMP level. cdc25 disruption mutants, in which the basal cAMP level was restored by the RAS2val19 oncogene or by disruption of the gene (PDE2) coding for the high-affinity phosphodiesterase, were still deficient in the glucose- and acidification-induced cAMP responses. These results indicate that the CDC25 gene product is required not only for basal cAMP synthesis in yeast but also for specific activation of cAMP synthesis by the signal transmission pathway leading from glucose to adenyl cyclase. They also show that intracellular acidification stimulates the pathway at or upstream of the CDC25 protein. When shifted to the restrictive temperature, cells with the temperature sensitive cdc25-5 mutation lost their cAMP content within a few minutes. After prolonged incubation at the restrictive temperature, cells with this mutation, and also those with the temperature sensitive cdc25-1 mutation, arrested at the 'start' point (in G1) of the cell cycle, and subsequently accumulated in the resting state G0. In contrast with cdc25-5 cells, however, the cAMP level did not decrease and normal glucose- and acidification-induced cAMP responses were observed when cdc25-1 cells were shifted to the restrictive temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. The role of AMP-activated protein kinase in the androgenic potentiation of cannabinoid-induced changes in energy homeostasis

    PubMed Central

    Borgquist, Amanda; Meza, Cecilia

    2014-01-01

    Orexigenic mediators can impact the hypothalamic feeding circuitry via the activation of AMP-dependent protein kinase (AMPK). Given that testosterone is an orexigenic hormone, we hypothesized that androgenic changes in energy balance are due to enhanced cannabinoid-induced inhibition of anorexigenic proopiomelanocortin (POMC) neurons via activation of AMPK. To this end, whole animal experiments were carried out in gonadectomized male guinea pigs treated subcutaneously with either testosterone propionate (TP; 400 μg) or its sesame oil vehicle (0.1 ml). TP-treated animals displayed increases in energy intake associated with increases in meal size. TP also increased several indices of energy expenditure as well as the p-AMPK/AMPK ratio in the arcuate nucleus (ARC) measured 2 and 24 h posttreatment. Subcutaneous administration of the CB1 receptor antagonist AM251 (3 mg/kg) rapidly blocked the hyperphagic effect of TP. This was mimicked largely upon third ventricular administration of AM251 (10 μg). Electrophysiological studies revealed that TP potentiated the ability of the cannabinoid receptor agonist WIN 55,212-2 to decrease the frequency of miniature excitatory postsynaptic currents in ARC neurons. TP also increased the basal frequency of miniature inhibitory postsynaptic currents. In addition, depolarization-induced suppression (DSE) is potentiated in cells from TP-treated animals and blocked by AM251. The AMPK inhibitor compound C attenuated DSE from TP-treated animals, whereas the AMPK activator metformin enhanced DSE from vehicle-treated animals. These effects occurred in a sizable number of identified POMC neurons. Collectively, these results indicate that the androgen-induced increases in energy intake are mediated via an AMPK-dependent augmentation in endocannabinoid tone onto POMC neurons. PMID:25550281

  8. Coenzyme Q10 Attenuates High Glucose-Induced Endothelial Progenitor Cell Dysfunction through AMP-Activated Protein Kinase Pathways.

    PubMed

    Tsai, Hsiao-Ya; Lin, Chih-Pei; Huang, Po-Hsun; Li, Szu-Yuan; Chen, Jia-Shiong; Lin, Feng-Yen; Chen, Jaw-Wen; Lin, Shing-Jong

    2016-01-01

    Coenzyme Q10 (CoQ10), an antiapoptosis enzyme, is stored in the mitochondria of cells. We investigated whether CoQ10 can attenuate high glucose-induced endothelial progenitor cell (EPC) apoptosis and clarified its mechanism. EPCs were incubated with normal glucose (5 mM) or high glucose (25 mM) environment for 3 days, followed by treatment with CoQ10 (10 μM) for 24 hr. Cell proliferation, nitric oxide (NO) production, and JC-1 assay were examined. The specific signal pathways of AMP-activated protein kinase (AMPK), eNOS/Akt, and heme oxygenase-1 (HO-1) were also assessed. High glucose reduced EPC functional activities, including proliferation and migration. Additionally, Akt/eNOS activity and NO production were downregulated in high glucose-stimulated EPCs. Administration of CoQ10 ameliorated high glucose-induced EPC apoptosis, including downregulation of caspase 3, upregulation of Bcl-2, and increase in mitochondrial membrane potential. Furthermore, treatment with CoQ10 reduced reactive oxygen species, enhanced eNOS/Akt activity, and increased HO-1 expression in high glucose-treated EPCs. These effects were negated by administration of AMPK inhibitor. Transplantation of CoQ10-treated EPCs under high glucose conditions into ischemic hindlimbs improved blood flow recovery. CoQ10 reduced high glucose-induced EPC apoptosis and dysfunction through upregulation of eNOS, HO-1 through the AMPK pathway. Our findings provide a potential treatment strategy targeting dysfunctional EPC in diabetic patients.

  9. Nicotinamide ameliorates palmitate-induced ER stress in hepatocytes via cAMP/PKA/CREB pathway-dependent Sirt1 upregulation.

    PubMed

    Li, Jiaxin; Dou, Xiaobing; Li, Songtao; Zhang, Ximei; Zeng, Yong; Song, Zhenyuan

    2015-11-01

    Nicotinamide (NAM) is the amide of nicotinic acid and a predominant precursor for NAD(+) biosynthesis via the salvage pathway. Sirt1 is a NAD(+)-dependent deacetylase, playing an important role in regulating cellular functions. Although hepatoprotective effect of NAM has been reported, the underlying mechanism remains elusive. ER stress, induced by saturated fatty acids, in specific palmitate, plays a pathological role in the development of nonalcoholic fatty liver disease. This study aims to determine the effect of NAM on palmitate-induced ER stress in hepatocytes and to elucidate molecular mechanisms behind. Both HepG2 cells and primary mouse hepatocytes were exposed to palmitate (conjugated to BSA at a 2:1 M ratio), NAM, or their combination for different durations. Cellular NAD(+) level, Sirt1 expression/activity, ER stress, as well as cAMP/PKA/CREB pathway activation were determined. NAM increased Sirt1 expression and enzymatic activity, which contributes to the ameliorative effect of NAM on palmitate-triggered ER stress. NAM increased intracellular NAD(+) level in hepatocytes, however, blocking the salvage pathway, a pathway for NAD(+) synthesis from NAM, only partially prevented NAM-induced Sirt1 upregulation while completely prevented NAD+ increase in response to NAM. Further mechanistic investigations revealed that NAM elevated intracellular cAMP level via suppressing PDE activity, leading to downstream PKA and CREB activation. Importantly, cAMP/PKA/CREB pathway blockade abolished not only NAM-induced Sirt1 upregulation, but also its protective effect against ER stress. Our results demonstrate that NAM protects hepatocytes against palmitate-induced ER stress in hepatocytes via upregulating Sirt1. Activation of the cAMP/PKA/CREB pathway plays a key role in NAM-induced Sirt1 upregulation. PMID:26352206

  10. Prostaglandin E2 induces vascular endothelial growth factor secretion in prostate cancer cells through EP2 receptor-mediated cAMP pathway.

    PubMed

    Wang, Xingya; Klein, Russell D

    2007-11-01

    Prostaglandin E2 (PGE2) has been shown to induce expression of vascular endothelial growth factor (VEGF) and other signaling molecules in several cancers. PGE2 elicits its functions though four G-protein coupled membrane receptors (EP1-4). In this study, we investigated the role of EP receptors in PGE2-induced molecular events in prostate cancer cells. qRT-PCR analysis revealed that PC-3 cells express a substantially higher level of EP2 and moderately higher EP4 than DU145 and LNCaP cells. LNCaP cells had virtually no detectable EP2 mRNA. EP1 and EP3 mRNAs were not detected in these cells. Treatment of prostate cancer cells with PGE2 (1 nM-10 microM) increased both VEGF secretion and cyclic adenosine monophosphate (cAMP) production. Levels of induction in PC-3 cells were greater than in DU145 and LNCaP cells. The selective EP2 agonist CAY10399 also significantly increased VEGF secretion and cAMP production in PC-3 cells, but not in DU145 and LNCaP cells. Moreover, PGE2 and CAY10399 increased mitogen activated protein kinase/extracellular signal regulated kinase (MAPK/Erk) and Akt phosphorylation in PC-3 and DU145 cells, but not in LNCaP cells. However, neither the MAPK/Erk inhibitor U0126 nor the PI3K/Akt inhibitor LY294002 abolished PGE2-induced VEGF secretion in PC-3 cells. We further demonstrated that the adenylate cyclase activator forskolin and the cAMP anologue 8-bromo-cAMP mimicked the effects of PGE2 on VEGF secretion in PC-3 cells. Meanwhile, the adenylate cyclase inhibitor 2'5'-dideoxyadenosine, at concentrations that inhibited PGE2-induced cAMP, significantly blocked PGE2-induced VEGF secretion in PC-3 cells. We conclude that PGE2-induced VEGF secretion in prostate cancer cells is mediated through EP2-, and possibly EP4-, dependent cAMP signaling pathways. PMID:17427962

  11. A novel link between Fic (filamentation induced by cAMP)-mediated adenylylation/AMPylation and the unfolded protein response.

    PubMed

    Sanyal, Anwesha; Chen, Andy J; Nakayasu, Ernesto S; Lazar, Cheri S; Zbornik, Erica A; Worby, Carolyn A; Koller, Antonius; Mattoo, Seema

    2015-03-27

    The maintenance of endoplasmic reticulum (ER) homeostasis is a critical aspect of determining cell fate and requires a properly functioning unfolded protein response (UPR). We have discovered a previously unknown role of a post-translational modification termed adenylylation/AMPylation in regulating signal transduction events during UPR induction. A family of enzymes, defined by the presence of a Fic (filamentation induced by cAMP) domain, catalyzes this adenylylation reaction. The human genome encodes a single Fic protein, called HYPE (Huntingtin yeast interacting protein E), with adenylyltransferase activity but unknown physiological target(s). Here, we demonstrate that HYPE localizes to the lumen of the endoplasmic reticulum via its hydrophobic N terminus and adenylylates the ER molecular chaperone, BiP, at Ser-365 and Thr-366. BiP functions as a sentinel for protein misfolding and maintains ER homeostasis. We found that adenylylation enhances BiP's ATPase activity, which is required for refolding misfolded proteins while coping with ER stress. Accordingly, HYPE expression levels increase upon stress. Furthermore, siRNA-mediated knockdown of HYPE prevents the induction of an unfolded protein response. Thus, we identify HYPE as a new UPR regulator and provide the first functional data for Fic-mediated adenylylation in mammalian signaling. PMID:25601083

  12. Rheum palmatum L. Attenuates High Fat Diet-Induced Hepatosteatosis by Activating AMP-Activated Protein Kinase.

    PubMed

    Yang, Mingxing; Li, Xiumin; Zeng, Xin; Ou, Zhimin; Xue, Mei; Gao, Dehong; Liu, Suhuan; Li, Xuejun; Yang, Shuyu

    2016-01-01

    Nonalcoholic fatty liver disease (NAFLD) is a common metabolic disorder characterized by the accumulation of excess fat in the liver. Rheum palmatumL. (RP) decoctions have been reported to ameliorate NAFLD. The aim of the present study was to investigate the effects and underlying mechanisms of RP in fatty liver disease induced by a high-fat diet (HFD) in rats. Low and high doses of aqueous RP extraction were orally administered to HFD-fed rats for six weeks. Body weight, tissue weight, glucose tolerance, insulin tolerance, hepatic morphology, and liver triglyceride (TG) content were assessed. The effects of RP on the expressions of lipogenic and lipolysis genes were measured by quantitative real-time PCR. The phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) was determined by Western blotting. Treatment with low-dose RP significantly reduced liver weight, liver TG content, and improved glucose tolerance in HFD-fed rats. Consistently, RP attenuated excess fat accumulation and downregulated the expression of lipogenic genes in the liver. Further, an increased phosphorylation of AMPK and ACC was observed. These findings suggest that low-dose RP alleviates hepatosteatosis, at least in part, by stimulating AMPK activity.

  13. Histamine-induced inhibition of leukotriene biosynthesis in human neutrophils: involvement of the H2 receptor and cAMP

    PubMed Central

    Flamand, Nicolas; Plante, Hendrick; Picard, Serge; Laviolette, Michel; Borgeat, Pierre

    2004-01-01

    Histamine is generally regarded as a pro-inflammatory mediator in diseases such as allergy and asthma. A growing number of studies, however, suggest that this autacoid is also involved in the downregulation of human polymorphonuclear leukocyte (PMN) functions and inflammatory responses through activation of the Gs-coupled histamine H2 receptor. We report here that histamine inhibits thapsigargin- and ligand (PAF and fMLP)-induced leukotriene (LT) biosynthesis in human PMN in a dose-dependent manner. The suppressive effect of histamine on LT biosynthesis was abrogated by the histamine H2 receptor antagonists cimetidine, ranitidine, and tiotidine. In contrast, the histamine H1, H3, and H4 receptor antagonists used in this study were ineffective in counteracting the inhibitory effect of histamine on the biosynthesis of LT in activated human PMN. The inhibition of LT biosynthesis by histamine was characterized by decreased arachidonic acid release and 5-lipoxygenase translocation to the nuclear membrane. Incubation of PMN with the cAMP-dependent protein kinase (PKA) inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide prevented the inhibitory effect of histamine on LT biosynthesis, suggesting an important role for PKA in this effect of histamine on LT biosynthesis in PMN. These data provide the first evidences that, similarly to adenosine and prostaglandin E2, histamine is a potent suppressor of LT biosynthesis, and support the concept that histamine may play a dual role in the regulation of inflammation. PMID:14744809

  14. Involvement of cAMP in the Human Serum-Induced Migration of Adipose-Derived Stem Cells

    PubMed Central

    Lee, Minji; Koh, Wonyoung; Kim, Bomee; Chung, Hyeju; Cho, Gahyang; Kim, Haekwon

    2016-01-01

    Previously we observed that human adipose-derived stem cells (hADSCs) could form aggregation during culture in the presence of human serum (HS). In the present study, we have examined if the aggregation might result from the cell migration and analyzed the difference of cell adhesivity after culture in various conditions. When cells were cultured in fetal bovine serum (FBS) alone, there was no morphological change. Similarly, cells pretreated with FBS for 1 day or cultured in a mixture of FBS and HS showed little change. In contrast, cells cultured in HS alone exhibited formation of cell-free area (spacing) and/or cell aggregation. When cells cultured in FBS or pretreated with FBS were treated with 0.06% trypsin, almost cells remained attached to the dish surfaces. In contrast, when cells cultured in HS alone were examined, most cells detached from the dish by the same treatment. Treatment of cells with forskolin, isobutylmethyl xanthine (IBMX) or LY294002 inhibited the formation of spacing whereas H89 or Y27632 showed little effect. When these cells were treated with 0.06% trypsin after culture, most cells detached from the dishes as cells cultured in HS alone did. However, cells treated with IBMX exhibited weaker adhesivity than HS alone. Based on these observations, it is suggested that HS treatment might decrease the adhesivity and induce three-dimensional migration of hADSCs, in the latter of which cAMP signaling could be involved.

  15. Involvement of cAMP in the Human Serum-Induced Migration of Adipose-Derived Stem Cells

    PubMed Central

    Lee, Minji; Koh, Wonyoung; Kim, Bomee; Chung, Hyeju; Cho, Gahyang; Kim, Haekwon

    2016-01-01

    Previously we observed that human adipose-derived stem cells (hADSCs) could form aggregation during culture in the presence of human serum (HS). In the present study, we have examined if the aggregation might result from the cell migration and analyzed the difference of cell adhesivity after culture in various conditions. When cells were cultured in fetal bovine serum (FBS) alone, there was no morphological change. Similarly, cells pretreated with FBS for 1 day or cultured in a mixture of FBS and HS showed little change. In contrast, cells cultured in HS alone exhibited formation of cell-free area (spacing) and/or cell aggregation. When cells cultured in FBS or pretreated with FBS were treated with 0.06% trypsin, almost cells remained attached to the dish surfaces. In contrast, when cells cultured in HS alone were examined, most cells detached from the dish by the same treatment. Treatment of cells with forskolin, isobutylmethyl xanthine (IBMX) or LY294002 inhibited the formation of spacing whereas H89 or Y27632 showed little effect. When these cells were treated with 0.06% trypsin after culture, most cells detached from the dishes as cells cultured in HS alone did. However, cells treated with IBMX exhibited weaker adhesivity than HS alone. Based on these observations, it is suggested that HS treatment might decrease the adhesivity and induce three-dimensional migration of hADSCs, in the latter of which cAMP signaling could be involved. PMID:27660827

  16. Involvement of cAMP in the Human Serum-Induced Migration of Adipose-Derived Stem Cells.

    PubMed

    Lee, Minji; Koh, Wonyoung; Kim, Bomee; Chung, Hyeju; Cho, Gahyang; Kim, Haekwon

    2016-06-01

    Previously we observed that human adipose-derived stem cells (hADSCs) could form aggregation during culture in the presence of human serum (HS). In the present study, we have examined if the aggregation might result from the cell migration and analyzed the difference of cell adhesivity after culture in various conditions. When cells were cultured in fetal bovine serum (FBS) alone, there was no morphological change. Similarly, cells pretreated with FBS for 1 day or cultured in a mixture of FBS and HS showed little change. In contrast, cells cultured in HS alone exhibited formation of cell-free area (spacing) and/or cell aggregation. When cells cultured in FBS or pretreated with FBS were treated with 0.06% trypsin, almost cells remained attached to the dish surfaces. In contrast, when cells cultured in HS alone were examined, most cells detached from the dish by the same treatment. Treatment of cells with forskolin, isobutylmethyl xanthine (IBMX) or LY294002 inhibited the formation of spacing whereas H89 or Y27632 showed little effect. When these cells were treated with 0.06% trypsin after culture, most cells detached from the dishes as cells cultured in HS alone did. However, cells treated with IBMX exhibited weaker adhesivity than HS alone. Based on these observations, it is suggested that HS treatment might decrease the adhesivity and induce three-dimensional migration of hADSCs, in the latter of which cAMP signaling could be involved. PMID:27660827

  17. Adiponectin protects the rats liver against chronic intermittent hypoxia induced injury through AMP-activated protein kinase pathway

    PubMed Central

    Ding, Wenxiao; Zhang, Qiang; Dong, Yanbin; Ding, Ning; Huang, Hanpeng; Zhu, Xianji; Hutchinson, Sean; Gao, Xingya; Zhang, Xilong

    2016-01-01

    This study was performed to assess the effect of chronic intermittent hypoxia (CIH) on the liver, the associated mechanisms and the potential therapeutic roles of adiponectin (Ad). Sixty rats were randomly assigned to four groups: the normal control (NC), NC and Ad supplement (NC + Ad), CIH, and CIH and Ad supplement (CIH + Ad) groups. The rats in the CIH and CIH + Ad groups were exposed to a hypoxic environment for 4 months. Rats in the NC + Ad and CIH + Ad groups were also treated with an intravenous injection of Ad (10 ug), twice a week. The plasma levels of hepatic enzymes, serum triglyceride, liver triglyceride, fasting blood glucose and hepatic cell apoptosis in hepatic tissue, were higher in the CIH group than in the NC and NC + Ad groups. However, the Ad supplementation in the CIH + Ad group rescued the hepatic tissue insult by activating the AMP-activated protein kinase (AMPK) pathway. In conclusion, Ad could protect against CIH-induced hepatic injury partly through the AMPK pathway. PMID:27678302

  18. Activation of the cAMP Pathway Induces RACK1-Dependent Binding of β-Actin to BDNF Promoter

    PubMed Central

    Neasta, Jeremie; Fiorenza, Anna; He, Dao-Yao; Phamluong, Khanhky; Kiely, Patrick A.; Ron, Dorit

    2016-01-01

    RACK1 is a scaffolding protein that contributes to the specificity and propagation of several signaling cascades including the cAMP pathway. As such, RACK1 participates in numerous cellular functions ranging from cell migration and morphology to gene transcription. To obtain further insights on the mechanisms whereby RACK1 regulates cAMP-dependent processes, we set out to identify new binding partners of RACK1 during activation of the cAMP signaling using a proteomics strategy. We identified β-actin as a direct RACK1 binding partner and found that the association between β-actin and RACK1 is increased in response to the activation of the cAMP pathway. Furthermore, we show that cAMP-dependent increase in BDNF expression requires filamentous actin. We further report that β-actin associates with the BDNF promoter IV upon the activation of the cAMP pathway and present data to suggest that the association of β-actin with BDNF promoter IV is RACK1-dependent. Taken together, our data suggest that β-actin is a new RACK1 binding partner and that the RACK1 and β-actin association participate in the cAMP-dependent regulation of BDNF transcription. PMID:27505161

  19. Bacillus bombysepticus α-Toxin Binding to G Protein-Coupled Receptor Kinase 2 Regulates cAMP/PKA Signaling Pathway to Induce Host Death

    PubMed Central

    Lin, Ping; Cheng, Tingcai; Ma, Sanyuan; Gao, Junping; Jin, Shengkai; Jiang, Liang; Xia, Qingyou

    2016-01-01

    Bacterial pathogens and their toxins target host receptors, leading to aberrant behavior or host death by changing signaling events through subversion of host intracellular cAMP level. This is an efficient and widespread mechanism of microbial pathogenesis. Previous studies describe toxins that increase cAMP in host cells, resulting in death through G protein-coupled receptor (GPCR) signaling pathways by influencing adenylyl cyclase or G protein activity. G protein-coupled receptor kinase 2 (GRK2) has a central role in regulation of GPCR desensitization. However, little information is available about the pathogenic mechanisms of toxins associated with GRK2. Here, we reported a new bacterial toxin-Bacillus bombysepticus (Bb) α-toxin that was lethal to host. We showed that Bb α-toxin interacted with BmGRK2. The data demonstrated that Bb α-toxin directly bound to BmGRK2 to promote death by affecting GPCR signaling pathways. This mechanism involved stimulation of Gαs, increase level of cAMP and activation of protein kinase A (PKA). Activated cAMP/PKA signal transduction altered downstream effectors that affected homeostasis and fundamental biological processes, disturbing the structural and functional integrity of cells, resulting in death. Preventing cAMP/PKA signaling transduction by inhibitions (NF449 or H-89) substantially reduced the pathogenicity of Bb α-toxin. The discovery of a toxin-induced host death specifically linked to GRK2 mediated signaling pathway suggested a new model for bacterial toxin action. Characterization of host genes whose expression and function are regulated by Bb α-toxin and GRK2 will offer a deeper understanding of the pathogenesis of infectious diseases caused by pathogens that elevate cAMP. PMID:27022742

  20. Follicle-stimulating hormone and cyclic AMP induce transcription from the human urokinase promoter in primary cultures of mouse Sertoli cells.

    PubMed

    Rossi, P; Grimaldi, P; Blasi, F; Geremia, R; Verde, P

    1990-06-01

    The hormonal regulation of the human urokinase type plasminogen activator (uPA) gene has been studied by introducing into mouse and rat Sertoli cell primary cultures a recombinant plasmid, in which the transcription regulatory elements of the cloned human uPA gene drive the expression of the bacterial chloramphenicol-acetyl-transferase gene. It was found to be expressed and regulated by FSH and (Bu)2cAMP in the mouse cells only, in agreement with data on the expression of the endogenous gene in rat and mouse gonads. The stimulation of transcription by FSH was evident in cultures from 13-day-old but not from 18-day-old mice, even though (Bu)2cAMP induction could be observed at both ages. Phorbol-myristate acetate was found to activate the human uPA promoter in Sertoli cell cultures from mice of both ages, even though the effect was less evident in cultures of 18-day-old animals. Deletion analysis of the human uPA 5'-flanking region showed that the distal enhancer element is not needed for (Bu)2cAMP induction, and that at least two promoter regions are involved in (Bu)2cAMP induced transcription. One of these cAMP responsive regions lies between nucleotides -72 and -29 from the CAP site. The sequence of this region would suggest the binding of transcription factor AP-2, a cell-specific mediator of both cAMP and phorbol esters action on gene expression. However, these sequences do not mediate phorbol ester activation of human uPA promoter in mouse Sertoli cells.

  1. Elevation of cAMP Levels Inhibits Doxorubicin-Induced Apoptosis in Pre- B ALL NALM- 6 Cells Through Induction of BAD Phosphorylation and Inhibition of P53 Accumulation.

    PubMed

    Fatemi, Ahmad; Kazemi, Ahmad; Kashiri, Meysam; Safa, Majid

    2015-01-01

    Recognition of the molecular mechanisms of cAMP action against DNA damage-induced apoptosis can be useful to improve the efficacy of DNA damaging therapeutic agents. Considering the critical role of bcl-2-associated death promoter (BAD) and p53 proteins in DNA damage -induced apoptosis, the aim of this study was to assess the effect of cAMP-elevating agents on these proteins in doxorubicin-treated pre-B acute lymphoblastic leukemia (pre-B ALL) NALM-6 cells.The pre-B ALL cell line NALM-6 was cultured and treated with doxorubicin in combination with or without cAMP-elevating agents forskolin and 3-isobutyl-1-methylxanthine (IBMX). Cell viability was measured by trypan blue staining and MTT assay. For evaluation of apoptosis, annexin-V staining by flow cytometry and caspase-3 activity assay were used. Protein expression of p53, BAD and phoshorylated BAD was detected by western blotting analysis.cAMP-increasing agents diminished the doxorubicin-mediated cytotoxicity in NALM-6 cells as indicated by the viability assays. Annexin-V apoptosis assay showed that the cAMP-elevating agents decreased doxorubicin-induced apoptosis. Moreover, doxorubicin-induced caspase-3 activity was attenuated in the presence of cAMP-increasing agents. Western blot results revealed the reduced expression of p53 protein in cells treated with combination of cAMP-elevating agents and doxorubicin in contrast to cells treated with doxorubicin alone. Expression of total BAD protein was not affected by doxorubicin and cAMP-elevating agents. However, phosphorylation of BAD protein was induced in the presence of cAMP-elevating agents. Our study suggests that elevated cAMP levels inhibit doxorubicin-induced apoptosis in pre-B ALL cells through induction of BAD phosphorylation and abrogation of p53 accumulation.

  2. 17beta-estradiol potently suppresses cAMP-induced insulin-like growth factor-I gene activation in primary rat osteoblast cultures

    NASA Technical Reports Server (NTRS)

    McCarthy, T. L.; Ji, C.; Shu, H.; Casinghino, S.; Crothers, K.; Rotwein, P.; Centrella, M.

    1997-01-01

    Insulin-like growth factor-I (IGF-I) is a key factor in bone remodeling. In osteoblasts, IGF-I synthesis is enhanced by parathyroid hormone and prostaglandin E2 (PGE2) through cAMP-activated protein kinase. In rats, estrogen loss after ovariectomy leads to a rise in serum IGF-I and an increase in bone remodeling, both of which are reversed by estrogen treatment. To examine estrogen-dependent regulation of IGF-I expression at the molecular level, primary fetal rat osteoblasts were co-transfected with the estrogen receptor (hER, to ensure active ER expression), and luciferase reporter plasmids controlled by promoter 1 of the rat IGF-I gene (IGF-I P1), used exclusively in these cells. As reported, 1 microM PGE2 increased IGF-I P1 activity by 5-fold. 17beta-Estradiol alone had no effect, but dose-dependently suppressed the stimulatory effect of PGE2 by up to 90% (ED50 approximately 0.1 nM). This occurred within 3 h, persisted for at least 16 h, required ER, and appeared specific, since 17alpha-estradiol was 100-300-fold less effective. By contrast, 17beta-estradiol stimulated estrogen response element (ERE)-dependent reporter expression by up to 10-fold. 17beta-Estradiol also suppressed an IGF-I P1 construct retaining only minimal promoter sequence required for cAMP-dependent gene activation, but did not affect the 60-fold increase in cAMP induced by PGE2. There is no consensus ERE in rat IGF-I P1, suggesting novel downstream interactions in the cAMP pathway that normally enhances IGF-I expression in skeletal cells. To explore this, nuclear extract from osteoblasts expressing hER were examined by electrophoretic mobility shift assay using the atypical cAMP response element in IGF-I P1. Estrogen alone did not cause DNA-protein binding, while PGE2 induced a characteristic gel shift complex. Co-treatment with both hormones caused a gel shift greatly diminished in intensity, consistent with their combined effects on IGF-I promoter activity. Nonetheless, hER did not bind

  3. Spinal Activation of the cAMP-PKA Pathway Induces Respiratory Motor Recovery Following High Cervical Spinal Cord Injury

    PubMed Central

    Kajana, S.; Goshgarian, H.G.

    2008-01-01

    The present study investigated the involvement of the adenosine 3’5’-cyclic monophosphate-dependent protein kinase A (cAMP-PKA) pathway in the activation of the crossed phrenic pathways after left C2 spinal cord hemisection. Experiments were conducted on left C2 spinal cord hemisected, anesthetized, vagotomized, pancuronium paralyzed, and artificially ventilated male Sprague-Dawley rats. One week post-injury, the ipsilateral phrenic nerve exhibited no respiratory-related activity indicating a functionally complete hemisection. Intrathecal spinal cord administration of the cAMP analog, 8-Br-cAMP at the level of the phrenic nucleus resulted in an enhancement of contralateral phrenic nerve output and a restoration of respiratory-related activity in the phrenic nerve ipsilateral to the hemisection. Furthermore, pretreatment with Rp-8-Br-cAMP, a PKA inhibitor, abolished the effects of 8-Br-cAMP. These results suggest that PKA activation is necessary for the cAMP-mediated respiratory recovery following high cervical spinal cord injury and that activation of intracellular signaling cascades may represent an important strategy for improving respiratory function after spinal cord injury. PMID:18656458

  4. Hypertonicity-induced transmitter release at Drosophila neuromuscular junctions is partly mediated by integrins and cAMP/protein kinase A

    NASA Technical Reports Server (NTRS)

    Suzuki, Kazuhiro; Grinnell, Alan D.; Kidokoro, Yoshiaki

    2002-01-01

    The frequency of quantal transmitter release increases upon application of hypertonic solutions. This effect bypasses the Ca(2+) triggering step, but requires the presence of key molecules involved in vesicle fusion, and hence could be a useful tool for dissecting the molecular process of vesicle fusion. We have examined the hypertonicity response at neuromuscular junctions of Drosophila embryos in Ca(2+)-free saline. Relative to wild-type, the response induced by puff application of hypertonic solution was enhanced in a mutant, dunce, in which the cAMP level is elevated, or in wild-type embryos treated with forskolin, an activator of adenylyl cyclase, while protein kinase A (PKA) inhibitors decreased it. The response was also smaller in a mutant, DC0, which lacks the major subunit of PKA. Thus the cAMP/PKA cascade is involved in the hypertonicity response. Peptides containing the sequence Arg-Gly-Asp (RGD), which inhibit binding of integrins to natural ligands, reduced the response, whereas a peptide containing the non-binding sequence Arg-Gly-Glu (RGE) did not. A reduced response persisted in a mutant, myospheroid, which expresses no integrins, and the response in DC0 was unaffected by RGD peptides. These data indicate that there are at lease two components in the hypertonicity response: one that is integrin mediated and involves the cAMP/PKA cascade, and another that is not integrin mediated and does not involve the cAMP/PKA cascade.

  5. AMP-activated protein kinase controls exercise training- and AICAR-induced increases in SIRT3 and MnSOD.

    PubMed

    Brandauer, Josef; Andersen, Marianne A; Kellezi, Holti; Risis, Steve; Frøsig, Christian; Vienberg, Sara G; Treebak, Jonas T

    2015-01-01

    The mitochondrial protein deacetylase sirtuin (SIRT) 3 may mediate exercise training-induced increases in mitochondrial biogenesis and improvements in reactive oxygen species (ROS) handling. We determined the requirement of AMP-activated protein kinase (AMPK) for exercise training-induced increases in skeletal muscle abundance of SIRT3 and other mitochondrial proteins. Exercise training for 6.5 weeks increased SIRT3 (p < 0.01) and superoxide dismutase 2 (MnSOD; p < 0.05) protein abundance in quadriceps muscle of wild-type (WT; n = 13-15), but not AMPK α2 kinase dead (KD; n = 12-13) mice. We also observed a strong trend for increased MnSOD abundance in exercise-trained skeletal muscle of healthy humans (p = 0.051; n = 6). To further elucidate a role for AMPK in mediating these effects, we treated WT (n = 7-8) and AMPK α2 KD (n = 7-9) mice with 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR). Four weeks of daily AICAR injections (500 mg/kg) resulted in AMPK-dependent increases in SIRT3 (p < 0.05) and MnSOD (p < 0.01) in WT, but not AMPK α2 KD mice. We also tested the effect of repeated AICAR treatment on mitochondrial protein levels in mice lacking the transcriptional coactivator peroxisome proliferator-activated receptor γ-coactivator 1α (PGC-1α KO; n = 9-10). Skeletal muscle SIRT3 and MnSOD protein abundance was reduced in sedentary PGC-1α KO mice (p < 0.01) and AICAR-induced increases in SIRT3 and MnSOD protein abundance was only observed in WT mice (p < 0.05). Finally, the acetylation status of SIRT3 target lysine residues on MnSOD (K122) or oligomycin-sensitivity conferring protein (OSCP; K139) was not altered in either mouse or human skeletal muscle in response to acute exercise. We propose an important role for AMPK in regulating mitochondrial function and ROS handling in skeletal muscle in response to exercise training. PMID:25852572

  6. AMP-activated protein kinase controls exercise training- and AICAR-induced increases in SIRT3 and MnSOD

    PubMed Central

    Brandauer, Josef; Andersen, Marianne A.; Kellezi, Holti; Risis, Steve; Frøsig, Christian; Vienberg, Sara G.; Treebak, Jonas T.

    2015-01-01

    The mitochondrial protein deacetylase sirtuin (SIRT) 3 may mediate exercise training-induced increases in mitochondrial biogenesis and improvements in reactive oxygen species (ROS) handling. We determined the requirement of AMP-activated protein kinase (AMPK) for exercise training-induced increases in skeletal muscle abundance of SIRT3 and other mitochondrial proteins. Exercise training for 6.5 weeks increased SIRT3 (p < 0.01) and superoxide dismutase 2 (MnSOD; p < 0.05) protein abundance in quadriceps muscle of wild-type (WT; n = 13–15), but not AMPK α2 kinase dead (KD; n = 12–13) mice. We also observed a strong trend for increased MnSOD abundance in exercise-trained skeletal muscle of healthy humans (p = 0.051; n = 6). To further elucidate a role for AMPK in mediating these effects, we treated WT (n = 7–8) and AMPK α2 KD (n = 7–9) mice with 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR). Four weeks of daily AICAR injections (500 mg/kg) resulted in AMPK-dependent increases in SIRT3 (p < 0.05) and MnSOD (p < 0.01) in WT, but not AMPK α2 KD mice. We also tested the effect of repeated AICAR treatment on mitochondrial protein levels in mice lacking the transcriptional coactivator peroxisome proliferator-activated receptor γ-coactivator 1α (PGC-1α KO; n = 9–10). Skeletal muscle SIRT3 and MnSOD protein abundance was reduced in sedentary PGC-1α KO mice (p < 0.01) and AICAR-induced increases in SIRT3 and MnSOD protein abundance was only observed in WT mice (p < 0.05). Finally, the acetylation status of SIRT3 target lysine residues on MnSOD (K122) or oligomycin-sensitivity conferring protein (OSCP; K139) was not altered in either mouse or human skeletal muscle in response to acute exercise. We propose an important role for AMPK in regulating mitochondrial function and ROS handling in skeletal muscle in response to exercise training. PMID:25852572

  7. A possible signal-coupling role for cyclic AMP during endocytosis in Amoeba proteus.

    PubMed

    Prusch, R D; Roscoe, J C

    1993-01-01

    Cytoplasmic levels of cAMP in Amoeba proteus were measured utilizing radioimmunoassays under control conditions and when stimulated by inducers of either pinocytosis or phagocytosis. In control cells, cytoplasmic cAMP levels were approximately 0.39 pM/mg cells. When exposed to either chemotactic peptide or mannose which stimulate phagocytosis in the amoeba, there is a rapid doubling of the cAMP level within 45 sec of stimulation which then returns to the control level within 3-5 min. Theophylline prolongs the elevation of cytoplasmic cAMP in stimulated cells and is also capable of eliciting food vacuole formation in the amoeba. In addition isoproterenol also causes food vacuole formation in the amoeba as well as a large and prolonged increase in cytoplasmic cAMP levels. Inducers of pinocytosis (BSA and Na Cl) also elicit changes in cytoplasmic cAMP in the amoeba, but the response appears to differ from that elicited by inducers of phagocytosis in that the peak cAMP levels are broader and biphasic. It is concluded that cAMP plays a signal-coupling role during the early phases of both forms of endocytosis in Amoeba proteus.

  8. Role of LRP1 and ERK and cAMP Signaling Pathways in Lactoferrin-Induced Lipolysis in Mature Rat Adipocytes

    PubMed Central

    Ikoma-Seki, Keiko; Nakamura, Kanae; Morishita, Satoru; Ono, Tomoji; Sugiyama, Keikichi; Nishino, Hoyoku; Hirano, Hisashi; Murakoshi, Michiaki

    2015-01-01

    Lactoferrin (LF) is a multifunctional glycoprotein present in milk. A clinical study showed that enteric-coated bovine LF tablets decrease visceral fat accumulation. Furthermore, animal studies revealed that ingested LF is partially delivered to mesenteric fat, and in vitro studies showed that LF promotes lipolysis in mature adipocytes. The aim of the present study was to determine the mechanism underlying the induction of lipolysis in mature adipocytes that is induced by LF. To address this question, we used proteomics techniques to analyze protein expression profiles. Mature adipocytes from primary cultures of rat mesenteric fat were collected at various times after exposure to LF. Proteomic analysis revealed that the expression levels of hormone-sensitive lipase (HSL), which catalyzes the rate-limiting step of lipolysis, were upregulated and that HSL was activated by protein kinase A within 15 min after the cells were treated with LF. We previously reported that LF increases the intracellular concentration of cyclic adenosine monophosphate (cAMP), suggesting that LF activates the cAMP signaling pathway. In this study, we show that the expression level and the activity of the components of the extracellular signal-regulated kinase (ERK) signaling pathway were upregulated. Moreover, LF increased the activity of the transcription factor cAMP response element binding protein (CREB), which acts downstream in the cAMP and ERK signaling pathways and regulates the expression levels of adenylyl cyclase and HSL. Moreover, silencing of the putative LF receptor low-density lipoprotein receptor-related protein 1 (LRP1) attenuated lipolysis in LF-treated adipocytes. These results suggest that LF promoted lipolysis in mature adipocytes by regulating the expression levels of proteins involved in lipolysis through controlling the activity of cAMP/ERK signaling pathways via LRP1. PMID:26506094

  9. Involvement of platelet cyclic GMP but not cyclic AMP suppression in leukocyte-dependent platelet adhesion to endothelial cells induced by platelet-activating factor in vitro.

    PubMed Central

    Hirafuji, M.; Nezu, A.; Shinoda, H.; Minami, M.

    1996-01-01

    1. Incubation of endothelial cells with platelets in the absence or the presence of PAF (10 nM) markedly increased platelet cyclic AMP levels, which were significantly decreased by indomethacin (3 microM). Co-incubation of endothelial cells and platelets with polymorphonuclear leukocytes (PMNs) did not change the platelet cyclic AMP levels. 2. Incubation of endothelial cells with platelets in the absence of PAF increased platelet cyclic GMP levels, which were increased 3.5 fold by PAF. These cyclic GMP levels were significantly decreased by NG-nitro-L-arginine (100 microM), and completely by methylene blue (10 microM). When endothelial cells and platelets were co-incubated with PMNs, the cyclic GMP level in the cell mixture was 42.5 and 65.3% lower than that in endothelial cells and platelets without and with PAF stimulation, respectively. 3. PAF induced platelet adhesion to endothelial cells only when PMNs were present. Methylene blue dose-dependently potentiated the PMN-dependent platelet adhesion induced by PAF, although it had no effect in the absence of PMNs. 4. Sodium nitroprusside and 8-bromo cyclic GMP but not dibutyryl cyclic AMP significantly, although partially, inhibited the platelet adhesion. Inhibition of cyclic GMP-specific phosphodiesterase by zaprinast slightly inhibited the PMN-induced platelet adhesion and potentiated the inhibitory effect of 8-bromo cyclic GMP, while these drugs markedly inhibited the adhesion of platelet aggregates induced by PMN sonicates. 5. These results suggest that the impairment by activated PMNs of EDRF-induced platelet cyclic GMP formation is involved in part in the mechanism of PMN-dependent platelet adhesion to endothelial cells induced by PAF in vitro. The precise mechanism still remains to be clarified. PMID:8789382

  10. The sequential mechanism of guanidine hydrochloride-induced denaturation of cAMP receptor protein from Escherichia coli. A fluorescent study using 8-anilino-1-naphthalenesulfonic acid.

    PubMed

    Malecki, J; Wasylewski, Z

    1998-11-01

    cAMP receptor protein (CRP) regulates expression of a number of genes in Escherichia coli. The protein is a homodimer and each monomer is folded into two structural domains. The biological activation of CRP upon cAMP binding may involve the subunit realignment as well as reorientation between the domains within each subunit. In order to study the interactions between the subunits or domains, we performed stopped-flow measurements of the guanidine hydrochloride (GuHCI)-induced denaturation of CRP. The changes in CRP structure induced by GuHCl were monitored using both intrinsic Trp fluorescence as well as the fluorescence of an extrinsic probe, 8-anilino-1-Naphthalenesulfonic acid (ANS). Results of CRP denaturation using Trp fluorescence detection are consistent with a two-step model [Malecki, and Wasylewski, (1997), Eur. J. Biochem. 243, 660], where the dissociation of dimer into subunits is followed by the monomer unfolding. The denaturation of CRP monitored by ANS fluorescence reveals the existence of two additional processes. One occurs before the dissociation of CRP into subunits, whereas the second takes place after the dissociation, but prior to proper subunit unfolding. These additional processes suggest that CRP denaturation is described by a more complicated mechanism than a simple three-state equilibrium and may involve additional changes in both inter- and intrasubunit interactions. We also report the effect of cAMP on the kinetics of CRP subunit unfolding and refolding. PMID:9988521

  11. AMP-activated protein kinase regulates CO2-induced alveolar epithelial dysfunction in rats and human cells by promoting Na,K-ATPase endocytosis

    PubMed Central

    Vadász, István; Dada, Laura A.; Briva, Arturo; Trejo, Humberto E.; Welch, Lynn C.; Chen, Jiwang; Tóth, Péter T.; Lecuona, Emilia; Witters, Lee A.; Schumacker, Paul T.; Chandel, Navdeep S.; Seeger, Werner; Sznajder, Jacob I.

    2008-01-01

    Hypercapnia (elevated CO2 levels) occurs as a consequence of poor alveolar ventilation and impairs alveolar fluid reabsorption (AFR) by promoting Na,K-ATPase endocytosis. We studied the mechanisms regulating CO2-induced Na,K-ATPase endocytosis in alveolar epithelial cells (AECs) and alveolar epithelial dysfunction in rats. Elevated CO2 levels caused a rapid activation of AMP-activated protein kinase (AMPK) in AECs, a key regulator of metabolic homeostasis. Activation of AMPK was mediated by a CO2-triggered increase in intracellular Ca2+ concentration and Ca2+/calmodulin-dependent kinase kinase-β (CaMKK-β). Chelating intracellular Ca2+ or abrogating CaMKK-β function by gene silencing or chemical inhibition prevented the CO2-induced AMPK activation in AECs. Activation of AMPK or overexpression of constitutively active AMPK was sufficient to activate PKC-ζ and promote Na,K-ATPase endocytosis. Inhibition or downregulation of AMPK via adenoviral delivery of dominant-negative AMPK-α1 prevented CO2-induced Na,K-ATPase endocytosis. The hypercapnia effects were independent of intracellular ROS. Exposure of rats to hypercapnia for up to 7 days caused a sustained decrease in AFR. Pretreatment with a β-adrenergic agonist, isoproterenol, or a cAMP analog ameliorated the hypercapnia-induced impairment of AFR. Accordingly, we provide evidence that elevated CO2 levels are sensed by AECs and that AMPK mediates CO2-induced Na,K-ATPase endocytosis and alveolar epithelial dysfunction, which can be prevented with β-adrenergic agonists and cAMP. PMID:18188452

  12. Expression of orphan G-protein coupled receptor GPR174 in CHO cells induced morphological changes and proliferation delay via increasing intracellular cAMP

    SciTech Connect

    Sugita, Kazuya; Yamamura, Chiaki; Tabata, Ken-ichi; Fujita, Norihisa

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Expression of GPR174 in CHO cells induces morphological changes and proliferation delay. Black-Right-Pointing-Pointer These are due to increase in intracellular cAMP concentration. Black-Right-Pointing-Pointer Lysophosphatidylserine was identified to stimulate GPR174 leading to activate ACase. Black-Right-Pointing-Pointer The potencies of fatty acid moiety on LysoPS were oleoyl Greater-Than-Or-Slanted-Equal-To stearoyl > palmitoyl. Black-Right-Pointing-Pointer We propose that GPR174 is a lysophosphatidylserine receptor. -- Abstract: We established cell lines that stably express orphan GPCR GPR174 using CHO cells, and studied physiological and pharmacological features of the receptor. GPR174-expressing cells showed cell-cell adhesion with localization of actin filaments to cell membrane, and revealed significant delay of cell proliferation. Since the morphological changes of GPR174-cells were very similar to mock CHO cells treated with cholera toxin, we measured the concentration of intracellular cAMP. The results showed the concentration was significantly elevated in GPR174-cells. By measuring intracellular cAMP concentration in GPR174-cells, we screened lipids and nucleotides to identify ligands for GPR174. We found that lysophosphatidylserine (LysoPS) stimulated increase in intracellular cAMP in a dose-dependent manner. Moreover, phosphorylation of Erk was elevated by LysoPS in GPR174 cells. These LysoPS responses were inhibited by NF449, an inhibitor of G{alpha}{sub s} protein. These results suggested that GPR174 was a putative LysoPS receptor conjugating with G{alpha}{sub s}, and its expression induced morphological changes in CHO cells by constitutively activating adenylyl cycles accompanied with cell conjunctions and delay of proliferation.

  13. The cAMP signaling system inhibits the repair of {gamma}-ray-induced DNA damage by promoting Epac1-mediated proteasomal degradation of XRCC1 protein in human lung cancer cells

    SciTech Connect

    Cho, Eun-Ah; Juhnn, Yong-Sung

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer cAMP signaling system inhibits repair of {gamma}-ray-induced DNA damage. Black-Right-Pointing-Pointer cAMP signaling system inhibits DNA damage repair by decreasing XRCC1 expression. Black-Right-Pointing-Pointer cAMP signaling system decreases XRCC1 expression by promoting its proteasomal degradation. Black-Right-Pointing-Pointer The promotion of XRCC1 degradation by cAMP signaling system is mediated by Epac1. -- Abstract: Cyclic AMP is involved in the regulation of metabolism, gene expression, cellular growth and proliferation. Recently, the cAMP signaling system was found to modulate DNA-damaging agent-induced apoptosis by regulating the expression of Bcl-2 family proteins and inhibitors of apoptosis. Thus, we hypothesized that the cAMP signaling may modulate DNA repair activity, and we investigated the effects of the cAMP signaling system on {gamma}-ray-induced DNA damage repair in lung cancer cells. Transient expression of a constitutively active mutant of stimulatory G protein (G{alpha}sQL) or treatment with forskolin, an adenylyl cyclase activator, augmented radiation-induced DNA damage and inhibited repair of the damage in H1299 lung cancer cells. Expression of G{alpha}sQL or treatment with forskolin or isoproterenol inhibited the radiation-induced expression of the XRCC1 protein, and exogenous expression of XRCC1 abolished the DNA repair-inhibiting effect of forskolin. Forskolin treatment promoted the ubiquitin and proteasome-dependent degradation of the XRCC1 protein, resulting in a significant decrease in the half-life of the protein after {gamma}-ray irradiation. The effect of forskolin on XRCC1 expression was not inhibited by PKA inhibitor, but 8-pCPT-2 Prime -O-Me-cAMP, an Epac-selective cAMP analog, increased ubiquitination of XRCC1 protein and decreased XRCC1 expression. Knockdown of Epac1 abolished the effect of 8-pCPT-2 Prime -O-Me-cAMP and restored XRCC1 protein level following {gamma}-ray irradiation. From

  14. Exchange protein activated by cAMP (Epac) induces vascular relaxation by activating Ca2+-sensitive K+ channels in rat mesenteric artery

    PubMed Central

    Roberts, Owain Llŷr; Kamishima, Tomoko; Barrett-Jolley, Richard; Quayle, John M; Dart, Caroline

    2013-01-01

    Vasodilator-induced elevation of intracellular cyclic AMP (cAMP) is a central mechanism governing arterial relaxation but is incompletely understood due to the diversity of cAMP effectors. Here we investigate the role of the novel cAMP effector exchange protein directly activated by cAMP (Epac) in mediating vasorelaxation in rat mesenteric arteries. In myography experiments, the Epac-selective cAMP analogue 8-pCPT-2′-O-Me-cAMP-AM (5 μm, subsequently referred to as 8-pCPT-AM) elicited a 77.6 ± 7.1% relaxation of phenylephrine-contracted arteries over a 5 min period (mean ± SEM; n= 6). 8-pCPT-AM induced only a 16.7 ± 2.4% relaxation in arteries pre-contracted with high extracellular K+ over the same time period (n= 10), suggesting that some of Epac's relaxant effect relies upon vascular cell hyperpolarization. This involves Ca2+-sensitive, large-conductance K+ (BKCa) channel opening as iberiotoxin (100 nm) significantly reduced the ability of 8-pCPT-AM to reverse phenylephrine-induced contraction (arteries relaxed by only 35.0 ± 8.5% over a 5 min exposure to 8-pCPT-AM, n= 5; P < 0.05). 8-pCPT-AM increased Ca2+ spark frequency in Fluo-4-AM-loaded mesenteric myocytes from 0.045 ± 0.008 to 0.103 ± 0.022 sparks s-1μm-1 (P < 0.05) and reversibly increased both the frequency (0.94 ± 0.25 to 2.30 ± 0.72 s−1) and amplitude (23.9 ± 3.3 to 35.8 ± 7.7 pA) of spontaneous transient outward currents (STOCs) recorded in isolated mesenteric myocytes (n= 7; P < 0.05). 8-pCPT-AM-activated STOCs were sensitive to iberiotoxin (100 nm) and to ryanodine (30 μm). Current clamp recordings of isolated myocytes showed a 7.9 ± 1.0 mV (n= 10) hyperpolarization in response to 8-pCPT-AM that was sensitive to iberiotoxin (n= 5). Endothelial disruption suppressed 8-pCPT-AM-mediated relaxation in phenylephrine-contracted arteries (24.8 ± 4.9% relaxation after 5 min of exposure, n= 5; P < 0.05), as did apamin and TRAM-34, blockers of Ca2+-sensitive, small- and intermediate

  15. Exchange protein activated by cAMP (Epac) induces vascular relaxation by activating Ca2+-sensitive K+ channels in rat mesenteric artery.

    PubMed

    Roberts, Owain Llŷr; Kamishima, Tomoko; Barrett-Jolley, Richard; Quayle, John M; Dart, Caroline

    2013-10-15

    Vasodilator-induced elevation of intracellular cyclic AMP (cAMP) is a central mechanism governing arterial relaxation but is incompletely understood due to the diversity of cAMP effectors. Here we investigate the role of the novel cAMP effector exchange protein directly activated by cAMP (Epac) in mediating vasorelaxation in rat mesenteric arteries. In myography experiments, the Epac-selective cAMP analogue 8-pCPT-2-O-Me-cAMP-AM (5 μM, subsequently referred to as 8-pCPT-AM) elicited a 77.6 ± 7.1% relaxation of phenylephrine-contracted arteries over a 5 min period (mean ± SEM; n = 6). 8-pCPT-AM induced only a 16.7 ± 2.4% relaxation in arteries pre-contracted with high extracellular K(+) over the same time period (n = 10), suggesting that some of Epac's relaxant effect relies upon vascular cell hyperpolarization. This involves Ca(2+)-sensitive, large-conductance K(+) (BK(Ca)) channel opening as iberiotoxin (100 nM) significantly reduced the ability of 8-pCPT-AM to reverse phenylephrine-induced contraction (arteries relaxed by only 35.0 ± 8.5% over a 5 min exposure to 8-pCPT-AM, n = 5; P < 0.05). 8-pCPT-AM increased Ca(2+) spark frequency in Fluo-4-AM-loaded mesenteric myocytes from 0.045 ± 0.008 to 0.103 ± 0.022 sparks s(-1) μm(-1) (P < 0.05) and reversibly increased both the frequency (0.94 ± 0.25 to 2.30 ± 0.72 s(-1)) and amplitude (23.9 ± 3.3 to 35.8 ± 7.7 pA) of spontaneous transient outward currents (STOCs) recorded in isolated mesenteric myocytes (n = 7; P < 0.05). 8-pCPT-AM-activated STOCs were sensitive to iberiotoxin (100 nM) and to ryanodine (30 μM). Current clamp recordings of isolated myocytes showed a 7.9 ± 1.0 mV (n = 10) hyperpolarization in response to 8-pCPT-AM that was sensitive to iberiotoxin (n = 5). Endothelial disruption suppressed 8-pCPT-AM-mediated relaxation in phenylephrine-contracted arteries (24.8 ± 4.9% relaxation after 5 min of exposure, n = 5; P < 0.05), as did apamin and TRAM-34, blockers of Ca(2+)-sensitive, small- and

  16. Exendin-4 alleviates angiotensin II-induced senescence in vascular smooth muscle cells by inhibiting Rac1 activation via a cAMP/PKA-dependent pathway.

    PubMed

    Zhao, Liang; Li, Ai Q; Zhou, Teng F; Zhang, Meng Q; Qin, Xiao M

    2014-12-15

    Vascular aging has been implicated in the progression of diabetes and age-related cardiovascular disorders. Glucagon-like peptide-1 (GLP-1) is an incretin hormone capable of cytoprotective actions in addition to its glucose-lowering effect. The present study was undertaken to examine whether Exendin-4, a specific ligand for the GLP-1 receptor, could prevent angiotensin (ANG) II-induced premature senescence in vascular smooth muscle cells (VSMCs) and to determine the underlying mechanism involved. Senescence-associated β-galactosidase (SA β-gal) assay showed that ANG II induced premature senescence of VSMCs. Pretreatment with Exendin-4 significantly attenuated ANG II-induced generation of H2O2 and the subsequent VSMC senescence. These effects were, however, reversed in the presence of exendin fragment 9-39, a GLP-1 receptor antagonist, or PKI14-22. Moreover, a marked increase in the levels of p53 and p21 induced by ANG II was blunted by the treatment with Exendin-4. Nevertheless, Exendin-4 failed to decrease ANG II-induced expression of NAD(P)H oxidase 1 (Nox1), NAD(P)H oxidase 4 (Nox4), p22(phox), or p47(phox) in VSMCs. Mechanistically, Exendin-4 blocked ANG II-induced Rac1 activation through the cAMP/PKA signaling cascade. Specifically, NSC23766, a Rac1 inhibitor, abrogated the suppressive effects of Exendin-4 on ANG II-induced premature senescence and H2O2 generation, respectively. Thus Exendin-4 confers resistance to ANG II-induced superoxide anion generation from NAD(P)H oxidase and the resultant VSMC senescence by inhibiting Rac1 activation via a cAMP/PKA-dependent pathway. These findings demonstrate that GLP-1 as well as its analogs (GLP-1-related reagents) may hold therapeutic potential in the treatment of diabetes with cardiovascular disease.

  17. Cyclic AMP-elevating agents down-regulate the oxidative burst induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) in adherent neutrophils.

    PubMed Central

    Ottonello, L; Morone, M P; Dapino, P; Dallegri, F

    1995-01-01

    Human neutrophils, plated on fibronectin-precoated wells, were found to release large quantities of superoxide anion (O2-) in response to GM-CSF. O2- production was reduced by prostaglandin E2 (PGE2) and the phosphodiesterase type IV (PDE IV) inhibitor RO 20-1724. Both agents are known to increase intracellular cyclic AMP (cAMP) levels by inducing its production (PGE2) or blocking its catabolism (RO 20-1724). When added in combination, PGE2 and RO 20-1724 had a marked synergistic inhibitory effect, which was reproduced by replacing PGE2 with a direct activator of adenylate cyclase, i.e. forskolin (FK). Moreover, the neutrophil response to GM-CSF was inhibited by a membrane-permeable analogue of cAMP in a dose-dependent manner. As GM-CSF and PGE2 are known to be generated at tissue sites of inflammation, the results suggest the existence of a PGE2-dependent regulatory pathway potentially capable of controlling the neutrophil response to GM-CSF, in turn limiting the risk of local oxidative tissue injury. Moreover, owing to its susceptibility to amplification by RO 20-1724, the PGE2-dependent pathway and in particular PDE-IV may represent a pharmacological target to reduce the generation of histotoxic oxidants by GM-CSF-responding neutrophils. PMID:7664497

  18. Modulation of dog atrial swelling-induced chloride current by cAMP: protein kinase A-dependent and -independent pathways.

    PubMed Central

    Du, X Y; Sorota, S

    1997-01-01

    1. The modulation of dog atrial swelling-induced chloride current (I(Cl,swelling)) by cAMP-elevating agents was studied. Forskolin (10 microM) or isoprenaline (1 microM) exerted multiple effects. Although the pattern between cells was variable, there was, in general, a stimulatory action and a more slowly developing inhibitory effect. 2. In any given cell, the response to forskolin or isoprenaline was qualitatively similar suggesting that all of the responses were dependent on stimulation of adenylyl cyclase. The effects of forskolin or isoprenaline on I(Cl,swelling) were inhibited by intracellular dialysis with a P-site inhibitor of adenylyl cyclase, 2'-deoxyadenosine 3'-monophosphate (300 microM). 3. Intracellular dialysis with a peptide inhibitor of protein kinase A (PKI(6-22); 100 microM) blocked the inhibitory response to forskolin or isoprenaline and all cells responded with a monophasic stimulation of I(Cl,swelling). 4. After intracellular dialysis of cells with PKI(6-22) (100 microM) and cAMP (100 microM), current amplitude was not further stimulated by forskolin. 5. After intracellular dialysis with PKI(6-22) and adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), forskolin stimulated I(Cl,swelling) and the effect of forskolin subsided after it was washed out. 6. In conclusion, there are dual pathways by which cAMP can modulate dog atrial cell I(Cl,swelling). Inhibition results from protein kinase A (PKA)-dependent phosphorylation. In addition, a stimulatory pathway exists that is independent of phosphorylation by PKA or other cellular kinases. Although alternative explanations are possible, the stimulatory effect of cAMP may represent a direct modulation of I(Cl,swelling). Images Figure 1 PMID:9097937

  19. cAMP and in vivo hypoxia induce tob, ifr1, and fos expression in erythroid cells of the chick embryo.

    PubMed

    Dragon, Stefanie; Offenhäuser, Nina; Baumann, Rosemarie

    2002-04-01

    During avian embryonic development, terminal erythroid differentiation occurs in the circulation. Some of the key events, such as the induction of erythroid 2,3-bisphosphoglycerate (2,3-BPG), carbonic anhydrase (CAII), and pyrimidine 5'-nucleotidase (P5N) synthesis are oxygen dependent (Baumann R, Haller EA, Schöning U, and Weber M, Dev Biol 116: 548-551, 1986; Dragon S and Baumann R, Am J Physiol Regulatory Integrative Comp Physiol 280: R870-R878, 2001; Dragon S, Carey C, Martin K, and Baumann R, J Exp Biol 202: 2787-2795, 1999; Dragon S, Glombitza S, Götz R, and Baumann R, Am J Physiol Regulatory Integrative Comp Physiol 271: R982-R989, 1996; Dragon S, Hille R, Götz R, and Baumann R, Blood 91: 3052-3058, 1998; Million D, Zillner P, and Baumann R, Am J Physiol Regulatory Integrative Comp Physiol 261: R1188-R1196, 1991) in an indirect way: hypoxia stimulates the release of norepinephrine (NE)/adenosine into the circulation (Dragon et al., J Exp Biol 202: 2787-2795, 1999; Dragon et al., Am J Physiol Regulatory Integrative Comp Physiol 271: R982-R989, 1996). This leads via erythroid beta-adrenergic/adenosine A(2) receptor activation to a cAMP signal inducing several proteins in a transcription-dependent manner (Dragon et al., Am J Physiol Regulatory Integrative Comp Physiol 271: R982-R989, 1996; Dragon et al., Blood 91: 3052-3058, 1998; Glombitza S, Dragon S, Berghammer M, Pannermayr M, and Baumann R, Am J Physiol Regulatory Integrative Comp Physiol 271: R973-R981, 1996). To understand how the cAMP-dependent processes are initiated, we screened an erythroid cDNA library for cAMP-regulated genes. We detected three genes that were strongly upregulated (>5-fold) by cAMP in definitive and primitive red blood cells. They are homologous to the mammalian Tob, Ifr1, and Fos proteins. In addition, the genes are induced in the intact embryo during short-term hypoxia. Because the genes are regulators of proliferation and differentiation in other cell types, we suggest that cAMP

  20. cAMP mediators of pulsatile insulin secretion from glucose-stimulated single beta-cells.

    PubMed

    Idevall-Hagren, Olof; Barg, Sebastian; Gylfe, Erik; Tengholm, Anders

    2010-07-23

    Pulsatile insulin release from glucose-stimulated beta-cells is driven by oscillations of the Ca(2+) and cAMP concentrations in the subplasma membrane space ([Ca(2+)](pm) and [cAMP](pm)). To clarify mechanisms by which cAMP regulates insulin secretion, we performed parallel evanescent wave fluorescence imaging of [cAMP](pm), [Ca(2+)](pm), and phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) in the plasma membrane. This lipid is formed by autocrine insulin receptor activation and was used to monitor insulin release kinetics from single MIN6 beta-cells. Elevation of the glucose concentration from 3 to 11 mm induced, after a 2.7-min delay, coordinated oscillations of [Ca(2+)](pm), [cAMP](pm), and PIP(3). Inhibitors of protein kinase A (PKA) markedly diminished the PIP(3) response when applied before glucose stimulation, but did not affect already manifested PIP(3) oscillations. The reduced PIP(3) response could be attributed to accelerated depolarization causing early rise of [Ca(2+)](pm) that preceded the elevation of [cAMP](pm). However, the amplitude of the PIP(3) response after PKA inhibition was restored by a specific agonist to the cAMP-dependent guanine nucleotide exchange factor Epac. Suppression of cAMP formation with adenylyl cyclase inhibitors reduced already established PIP(3) oscillations in glucose-stimulated cells, and this effect was almost completely counteracted by the Epac agonist. In cells treated with small interfering RNA targeting Epac2, the amplitudes of the glucose-induced PIP(3) oscillations were reduced, and the Epac agonist was without effect. The data indicate that temporal coordination of the triggering [Ca(2+)](pm) and amplifying [cAMP](pm) signals is important for glucose-induced pulsatile insulin release. Although both PKA and Epac2 partake in initiating insulin secretion, the cAMP dependence of established pulsatility is mediated by Epac2.

  1. Phosphorylation of CREB affects its binding to high and low affinity sites: implications for cAMP induced gene transcription.

    PubMed Central

    Nichols, M; Weih, F; Schmid, W; DeVack, C; Kowenz-Leutz, E; Luckow, B; Boshart, M; Schütz, G

    1992-01-01

    Cyclic AMP treatment of hepatoma cells leads to increased protein binding at the cyclic AMP response element (CRE) of the tyrosine aminotransferase (TAT) gene in vivo, as revealed by genomic footprinting, whereas no increase is observed at the CRE of the phosphoenolpyruvate carboxykinase (PEPCK) gene. Several criteria establish that the 43 kDa CREB protein is interacting with both of these sites. Two classes of CRE with different affinity for CREB are described. One class, including the TATCRE, is characterized by asymmetric and weak binding sites (CGTCA), whereas the second class containing symmetrical TGACGTCA sites shows a much higher binding affinity for CREB. Both classes show an increase in binding after phosphorylation of CREB by protein kinase A (PKA). An in vivo phosphorylation-dependent change in binding of CREB increases the occupancy of weak binding sites used for transactivation, such as the TATCRE, while high affinity sites may have constitutive binding of transcriptionally active and inactive CREB dimers, as demonstrated by in vivo footprinting at the PEPCK CRE. Thus, lower basal level and higher relative stimulation of transcription by cyclic AMP through low affinity CREs should result, allowing finely tuned control of gene activation. Images PMID:1354612

  2. 5-aminoimidazole-4-carboxamide Riboside Induces Apoptosis Through AMP-activated Protein Kinase-independent and NADPH Oxidase-dependent Pathways.

    PubMed

    Wi, Sae Mi; Lee, Ki-Young

    2014-10-01

    It is debatable whether AMP-activated protein kinase (AMPK) activation is involved in anti-apoptotic or pro-apoptotic signaling. AICAR treatment increases AMPK-α1 phosphorylation, decreases intracellular reactive oxygen species (ROS) levels, and significantly increases Annexin V-positive cells, DNA laddering, and caspase activity in human myeloid cell. AMPK activation is therefore implicated in apoptosis. However, AMPK-α1-knockdown THP-1 cells are more sensitive to apoptosis than control THP-1 cells are, suggesting that the apoptosis is AMPK-independent. Low doses of AICAR induce cell proliferation, whereas high doses of AICAR suppress cell proliferation. Moreover, these effects are significantly correlated with the downregulation of intracellular ROS, strongly suggesting that AICAR-induced apoptosis is critically associated with the inhibition of NADPH oxidase by AICAR. Collectively, our results demonstrate that in AICAR-induced apoptosis, intracellular ROS levels are far more relevant than AMPK activation.

  3. Early ALS-type gait abnormalities in AMP-dependent protein kinase-deficient mice suggest a role for this metabolic sensor in early stages of the disease.

    PubMed

    Vergouts, Maxime; Marinangeli, Claudia; Ingelbrecht, Caroline; Genard, Geraldine; Schakman, Olivier; Sternotte, Anthony; Calas, André-Guilhem; Hermans, Emmanuel

    2015-12-01

    Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective loss of motoneurons. While the principal cause of the disease remains so far unknown, the onset and progression of the pathology are increasingly associated with alterations in the control of cell metabolism. On the basis of the well-known key roles of 5'-adenosine monophosphate-activated protein kinase (AMPK) in sensing and regulating the intracellular energy status, we hypothesized that mice with a genetic deletion of AMPK would develop locomotor abnormalities that bear similarity with those detected in the very early disease stage of mice carrying the ALS-associated mutated gene hSOD1(G93A). Using an automated gait analysis system (CatWalk), we here show that hSOD1(G93A) mice and age-matched mice lacking the neuronal and skeletal muscle predominant α2 catalytic subunit of AMPK showed an altered gait, clearly different from wild type control mice. Double mutant mice lacking AMPK α2 and carrying hSOD1(G93A) showed the same early gait abnormalities as hSOD1(G93A) mice over an age span of 8 to 16 weeks. Taken together, these data support the concept that altered AMPK function and associated bioenergetic abnormalities could constitute an important component in the early pathogenesis of ALS. Therapeutic interventions acting on metabolic pathways could prove beneficial on early locomotor deficits, which are sensitively detectable in rodent models using the CatWalk system. PMID:26152932

  4. A noble function of BAY 11-7082: Inhibition of platelet aggregation mediated by an elevated cAMP-induced VASP, and decreased ERK2/JNK1 phosphorylations.

    PubMed

    Lee, Hyun-Sub; Kim, Sung Dae; Lee, Whi Min; Endale, Mehari; Kamruzzaman, S M; Oh, Won Jun; Cho, Jae Youl; Kim, Sang Keun; Cho, Hyun-Jeong; Park, Hwa-Jin; Rhee, Man Hee

    2010-02-10

    Platelets, though anucleated, possess several transcription factors, including NF-kappaB, that exert non-genomic functions regulating platelet activation. Since platelets have not only been recognized as central players of homeostasis, but also participated in pathological conditions such as thrombosis, atherosclerosis, and inflammation, we examined rat platelet NF-kappaB expression and evaluated the effects of anti-inflammatory drug BAY 11-7082, an inhibitor of NF-kappaB activation, in platelet physiology. Western blotting revealed that rat platelets express NF-kappaB. BAY 11-7082, dose dependently, inhibited collagen- or thrombin-induced-platelet aggregation. ATP release, TXB(2) formation, P-selectin expression, and intercellular Ca(2+) concentration activated by collagen were reduced in BAY 11-7082-treated platelets. BAY 11-7082 elevated intracellular levels of cAMP, but not cGMP, and its co-incubation with cAMP-activating agent (forskolin) or its hydrolyzing enzyme inhibitor (3-isobutyl-1-methylxanthine, IBMX), synergistically inhibited collagen-induced-platelet aggregation. In addition, vasodilator-stimulated-phosphoprotein (VASP) phosphorylation was enhanced in BAY 11-7082-treated platelets, which was partially inhibited by a protein kinase A (PKA) inhibitor, H-89. Moreover, while p38 mitogen-activated protein kinase (MAPK) was not affected, BAY 11-7082 attenuated c-Jun N-terminal kinase 1 (JNK1) and extracellular-signal-regulated protein kinase 2 (ERK2) phosphorylations. In conclusion, BAY 11-7082 inhibits platelet activation, granule secretion, and aggregation, and that this effect is mediated by inhibition of JNK1 and ERK2 phosphorylations, and partially by stimulation of cAMP-dependent PKA VASP phosphorylation. The ability of BAY 11-7082 to inhibit platelet function might be relevant in cases involving aberrant platelet activation where the drug is considered as anti-atherothrombosis, and anti-inflammatory therapy.

  5. Phosphodiesterase-3B-cAMP pathway of leptin signalling in the hypothalamus is impaired during the development of diet-induced obesity in FVB/N mice.

    PubMed

    Sahu, M; Anamthathmakula, P; Sahu, A

    2015-04-01

    The phosphodiesterase-3B (PDE3B)-cAMP pathway plays an important role in transducing the action of leptin in the hypothalamus. Obesity is usually associated with hyperleptinaemia and resistance to anorectic and body weight-reducing effects of leptin. To determine whether the hypothalamic PDE3B-cAMP pathway of leptin signalling is impaired during the development of diet-induced obesity (DIO), we fed male FVB/N mice a high-fat diet (HFD: 58% kcal as fat) or low-fat diet (LFD: 6% kcal as fat) for 4 weeks. HFD fed mice developed DIO in association with hyperphagia, hyperleptinaemia and hyperinsulinaemia. Leptin (i.p.) significantly increased hypothalamic PDE3B activity and phosphorylated (p)-Akt levels in LFD-fed but not in HFD-fed mice. However, basal p-Akt levels in hypothalamus were increased in DIO mice. Additionally, amongst six-microdissected brain nuclei examined, leptin selectively decreased cAMP levels in the arcuate nucleus (ARC) of LFD-fed mice but failed to do so in HFD-fed mice. We next tested whether both the PBE3B and Akt pathways of leptin signalling remained impaired in DIO mice on the HFD for 12 weeks (long-term). DIO mice were hyperinsulinaemic and hyperleptinaemic in association with impaired glucose and insulin tolerance. Although, in LFD-fed mice, leptin significantly increased PDE3B activity and p-Akt levels in the hypothalamus, it failed to do so in HFD-fed mice. Also, basal p-Akt levels in the hypothalamus were increased in DIO mice and leptin had no further effect. Similarly, immunocytochemistry showed that leptin increased the number of p-Akt-positive cells in the ARC of LFD-fed but not in HFD-fed mice, and there was an increased basal number of p-Akt positive cells in the ARC of DIO mice. These results suggest that the PDE3B-cAMP- and Akt-pathways of leptin signalling in the hypothalamus are impaired during the development of DIO. Thus, a defect in the regulation by leptin of the hypothalamic PDE3B-cAMP pathway and Akt signalling may be one

  6. Effects of taurine on myocardial cGMP/cAMP ratio, antioxidant ability, and ultrastructure in cardiac hypertrophy rats induced by isoproterenol.

    PubMed

    Yang, Qunhui; Yang, Jiancheng; Wu, Gaofeng; Feng, Ying; Lv, Qiufeng; Lin, Shumei; Hu, Jianmin

    2013-01-01

    Taurine is the most abundant free amino acid in the human body and accounts for more than 50% of the total amino acid pool in the mammalian heart. To investigate the preventive effects of taurine on cardiac hypertrophy in rats, myocardial injury was established by hypodermic injection of isoprenaline (ISO) (10 mg/kg d) for 7 days. The preventive effects of taurine (100 mg/kg d, 200 mg/kg d, and 300 mg/kg d, i.p) on heart coefficient; ultrastructure of cardiac muscle; the levels of creatine kinase heart isoenzyme (CK-MB), cAMP, and cGMP; and antioxidant ability were investigated. The results showed that taurine could significantly prevent the increase of heart coefficient induced by ISO. Compared with the model group, 100 mg/kg and 200 mg/kg taurine significantly decrease the levels of cAMP and cGMP, while 300 mg/kg taurine could significantly decrease the levels of cAMP in myocardium, and all the three concentrations of taurine could significantly increase the ratio of cGMP/cAMP. The level of serum CK-MB was significantly increased by ISO; 200 mg/kg taurine could significantly decrease it, but 100 mg/kg and 300 mg/kg taurine had no significant effect. As for the antioxidant ability, ISO administration could significantly increase the myocardial level of MDA but had no significant effects on the myocardial levels of SOD, GSH, GSH-Px, and T-AOC. However, taurine administration could significantly decrease the myocardial level of MDA and increase the levels of GSH and T-AOC compared with the model group. The serum levels of SOD, GSH-Px, GSH, and T-AOC were significantly reduced by ISO administration, but the level of MDA showed no significant changes compared with the control group. Taurine administration could significantly increase the serum levels of SOD, GSH-Px, GSH, and T-AOC and decrease the level of MDA compared with the model group. All the results indicated that 200 mg/kg taurine had better effects. The ultrastructure of cardiomyocytes showed that taurine

  7. Enhancement by lithium of cAMP-induced CRE/CREB-directed gene transcription conferred by TORC on the CREB basic leucine zipper domain

    PubMed Central

    Böer, Ulrike; Eglins, Julia; Krause, Doris; Schnell, Susanne; Schöfl, Christof; Knepel, Willhart

    2007-01-01

    The molecular mechanism of the action of lithium salts in the treatment of bipolar disorder is not well understood. As their therapeutic action requires chronic treatment, adaptive neuronal processes are suggested to be involved. The molecular basis of this are changes in gene expression regulated by transcription factors such as CREB (cAMP-response-element-binding protein). CREB contains a transactivation domain, in which Ser119 is phosphorylated upon activation, and a bZip (basic leucine zipper domain). The bZip is involved in CREB dimerization and DNA-binding, but also contributes to CREB transactivation by recruiting the coactivator TORC (transducer of regulated CREB). In the present study, the effect of lithium on CRE (cAMP response element)/CREB-directed gene transcription was investigated. Electrically excitable cells were transfected with CRE/CREB-driven luciferase reporter genes. LiCl (6 mM or higher) induced an up to 4.7-fold increase in 8-bromo-cAMP-stimulated CRE/CREB-directed transcription. This increase was not due to enhanced Ser119 phosphorylation or DNA-binding of CREB. Also, the known targets inositol monophosphatase and GSK3β (glycogen-synthase-kinase 3β) were not involved as specific GSK3β inhibitors and inositol replenishment did not mimic and abolish respectively the effect of lithium. However, lithium no longer enhanced CREB activity when the CREB-bZip was deleted or the TORC-binding site inside the CREB-bZip was specifically mutated (CREB-R300A). Otherwise, TORC overexpression conferred lithium responsiveness on CREB-bZip or the CRE-containing truncated rat somatostatin promoter. This indicates that lithium enhances cAMP-induced CRE/CREB-directed transcription, conferred by TORC on the CREB-bZip. We thus support the hypothesis that lithium salts modulate CRE/CREB-dependent gene transcription and suggest the CREB coactivator TORC as a new molecular target of lithium. PMID:17696880

  8. Compartmentalized accumulation of cAMP near complexes of multidrug resistance protein 4 (MRP4) and cystic fibrosis transmembrane conductance regulator (CFTR) contributes to drug-induced diarrhea.

    PubMed

    Moon, Changsuk; Zhang, Weiqiang; Ren, Aixia; Arora, Kavisha; Sinha, Chandrima; Yarlagadda, Sunitha; Woodrooffe, Koryse; Schuetz, John D; Valasani, Koteswara Rao; de Jonge, Hugo R; Shanmukhappa, Shiva Kumar; Shata, Mohamed Tarek M; Buddington, Randal K; Parthasarathi, Kaushik; Naren, Anjaparavanda P

    2015-05-01

    Diarrhea is one of the most common adverse side effects observed in ∼7% of individuals consuming Food and Drug Administration (FDA)-approved drugs. The mechanism of how these drugs alter fluid secretion in the gut and induce diarrhea is not clearly understood. Several drugs are either substrates or inhibitors of multidrug resistance protein 4 (MRP4), such as the anti-colon cancer drug irinotecan and an anti-retroviral used to treat HIV infection, 3'-azido-3'-deoxythymidine (AZT). These drugs activate cystic fibrosis transmembrane conductance regulator (CFTR)-mediated fluid secretion by inhibiting MRP4-mediated cAMP efflux. Binding of drugs to MRP4 augments the formation of MRP4-CFTR-containing macromolecular complexes that is mediated via scaffolding protein PDZK1. Importantly, HIV patients on AZT treatment demonstrate augmented MRP4-CFTR complex formation in the colon, which defines a novel paradigm of drug-induced diarrhea.

  9. Levodopa-induced dyskinesias are associated with transient down-regulation of cAMP and cGMP in the caudate-putamen of hemiparkinsonian rats: reduced synthesis or increased catabolism?

    PubMed

    Sancesario, Giuseppe; Morrone, Luigi Antonio; D'Angelo, Vincenza; Castelli, Valentina; Ferrazzoli, Davide; Sica, Francesco; Martorana, Alessandro; Sorge, Roberto; Cavaliere, Federica; Bernardi, Giorgio; Giorgi, Mauro

    2014-12-01

    Second messenger cAMP and cGMP represent a key step in the action of dopamine that modulates directly or indirectly their synthesis. We aimed to verify whether levodopa-induced dyskinesias are associated with changes of the time course of levodopa/dopamine stimulated cAMP and cGMP levels, and/or with changes of their catabolism by phosphodiesterase activity in rats with experimental hemiparkinsonism. Microdialysis and tissue homogenates of the striatal tissues demonstrated that extracellular and intracellular cAMP/cGMP levels were lower in dyskinetic animals during the increasing phase of dyskinesias compared to eukinetic animals, but cAMP/cGMP levels increased in dyskinetic animals during the phase of decreasing and extinction of dyskinesias. Dyskinesias and the abnormal lowering of striatal cGMP and cAMP after levodopa were prevented by pretreatment with the multipotent drug amantadine, outlining the inverse relationship of cAMP/cGMP to dyskinesias. Moreover, dyskinetic animals showed higher striatal hydrolyzing cGMP-phosphodiesterase but not hydrolyzing cAMP-phosphodiesterase activity, suggesting that low cGMP but not cAMP levels could be due to increased catabolism. However, expressions of isozyme phosphodiesterase-1B and -10A highly and specifically located in the basal ganglia were not changed after levodopa in dyskinetic and eukinetic animals: accordingly, selective inhibitors of phosphodiesterase-1B and -10A were ineffective on levodopa dyskinesias. Therefore, the isozyme(s) expressing higher cGMP-phosphodiesterase activity in the striatum of dyskinetic animal should be determined. These observations suggest that dopamine-mediated processes of synthesis and/or degradation of cAMP/cGMP could be acutely impaired in levodopa dyskinesias, opening new ways to understanding physiopathology and treatment.

  10. Defect-Related Luminescent Hydroxyapatite-Enhanced Osteogenic Differentiation of Bone Mesenchymal Stem Cells Via an ATP-Induced cAMP/PKA Pathway.

    PubMed

    Wang, Chao; Liu, Dandan; Zhang, Cuimiao; Sun, Jiadong; Feng, Weipei; Liang, Xing-Jie; Wang, Shuxiang; Zhang, Jinchao

    2016-05-11

    Novel defect-related hydroxyapatite (DHAP), which combines the advantages of HAP and defect-related luminescence, has the potential application in tissue engineering and biomedical area, because of its excellent capability of monitoring the osteogenic differentiation and material biodegradation. Although the extracellular mechanism of DHAP minerals and PO4(3-) functioning in osteogenic differentiation has been widely studied, the intracellular molecular mechanism through which PO4(3-) mediates osteogenesis of bone mesenchymal stem cells (BMSCs) is not clear. We examined a previously unknown molecular mechanism through which PO4(3-) promoted osteogenesis of BMSCs with an emphasis on adenosine-triphosphate (ATP)-induced cAMP/PKA pathway. Our studies showed that DHAP could be uptaken into lysosome, in which PO4(3-) was released from DHAP, because of the acid environment of lysosome. The released PO4(3-) interacted with ADP to form ATP, and then degraded into adenosine, an ATP metabolite, which interacted with A2b adenosine receptor to activate the cAMP/PKA pathway, resulting in the high expression of osteogenesis-related genes, such as Runx2, BMP-2, and OCN. These findings first revealed the function of ATP-metabolism in bone physiological homeostasis, which may be developed to cure bone metabolic diseases. PMID:27088570

  11. A Novel Fic (Filamentation Induced by cAMP) Protein from Clostridium difficile Reveals an Inhibitory Motif-independent Adenylylation/AMPylation Mechanism.

    PubMed

    Dedic, Emil; Alsarraf, Husam; Welner, Ditte Hededam; Østergaard, Ole; Klychnikov, Oleg I; Hensbergen, Paul J; Corver, Jeroen; van Leeuwen, Hans C; Jørgensen, René

    2016-06-17

    Filamentation induced by cAMP (Fic) domain proteins have been shown to catalyze the transfer of the AMP moiety from ATP onto a protein target. This type of post-translational modification was recently shown to play a crucial role in pathogenicity mediated by two bacterial virulence factors. Herein we characterize a novel Fic domain protein that we identified from the human pathogen Clostridium difficile The crystal structure shows that the protein adopts a classical all-helical Fic fold, which belongs to class II of Fic domain proteins characterized by an intrinsic N-terminal autoinhibitory α-helix. A conserved glutamate residue in the inhibitory helix motif was previously shown in other Fic domain proteins to prevent proper binding of the ATP γ-phosphate. However, here we demonstrate that both ATP binding and autoadenylylation activity of the C. difficile Fic domain protein are independent of the inhibitory motif. In support of this, the crystal structure of a mutant of this Fic protein in complex with ATP reveals that the γ-phosphate adopts a conformation unique among Fic domains that seems to override the effect of the inhibitory helix. These results provide important structural insight into the adenylylation reaction mechanism catalyzed by Fic domains. Our findings reveal the presence of a class II Fic domain protein in the human pathogen C. difficile that is not regulated by autoinhibition and challenge the current dogma that all class I-III Fic domain proteins are inhibited by the inhibitory α-helix. PMID:27076635

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

    PubMed

    Choi, Sekyu; Kim, Wonho; Chung, Jongkyeong

    2011-01-28

    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.

  13. Role of cyclic AMP sensor Epac1 in masseter muscle hypertrophy and myosin heavy chain transition induced by β2-adrenoceptor stimulation.

    PubMed

    Ohnuki, Yoshiki; Umeki, Daisuke; Mototani, Yasumasa; Jin, Huiling; Cai, Wenqian; Shiozawa, Kouichi; Suita, Kenji; Saeki, Yasutake; Fujita, Takayuki; Ishikawa, Yoshihiro; Okumura, Satoshi

    2014-12-15

    The predominant isoform of β-adrenoceptor (β-AR) in skeletal muscle is β2-AR and that in the cardiac muscle is β1-AR. We have reported that Epac1 (exchange protein directly activated by cAMP 1), a new protein kinase A-independent cAMP sensor, does not affect cardiac hypertrophy in response to pressure overload or chronic isoproterenol (isoprenaline) infusion. However, the role of Epac1 in skeletal muscle hypertrophy remains poorly understood. We thus examined the effect of disruption of Epac1, the major Epac isoform in skeletal muscle, on masseter muscle hypertrophy induced by chronic β2-AR stimulation with clenbuterol (CB) in Epac1-null mice (Epac1KO). The masseter muscle weight/tibial length ratio was similar in wild-type (WT) and Epac1KO at baseline and was significantly increased in WT after CB infusion, but this increase was suppressed in Epac1KO. CB treatment significantly increased the proportion of myosin heavy chain (MHC) IIb at the expense of that of MHC IId/x in both WT and Epac1KO, indicating that Epac1 did not mediate the CB-induced MHC isoform transition towards the faster isoform. The mechanism of suppression of CB-mediated hypertrophy in Epac1KO is considered to involve decreased activation of Akt signalling. In addition, CB-induced histone deacetylase 4 (HDAC4) phosphorylation on serine 246 mediated by calmodulin kinase II (CaMKII), which plays a role in skeletal muscle hypertrophy, was suppressed in Epac1KO. Our findings suggest that Epac1 plays a role in β2-AR-mediated masseter muscle hypertrophy, probably through activation of both Akt signalling and CaMKII/HDAC4 signalling. PMID:25344550

  14. Tonic modulatory role of mouse cerebellar α- and β-adrenergic receptors in the expression of ethanol-induced ataxia: role of AC-cAMP.

    PubMed

    Dar, M Saeed; Al-Rejaie, Salim

    2013-03-15

    To further study neurochemical basis of ethanol-induced ataxia (EIA), we investigated role of cerebellar α and β-adrenergic receptors. Male CD-1 mice received intracerebellar microinfusion of adrenergic drugs to evaluate their effect on EIA (2g/kg; ip) by Rotorod. Isoproterenol, phenylephrine (4, 8, 16 ng each), methoxamine (8 ng), and atenolol (2, 4, 8 ng), propranolol (4, 8, 16 ng), markedly attenuated and accentuated, respectively, EIA indicating the tonic nature of modulation. The attenuation of EIA by isoproterenol is β(1)-receptor mediated because it is blocked by atenolol. Tonic β(1) modulation is functionally correlated with EIA potentiation by atenolol and propranolol. The prazosin-induced attenuation of EIA, initially thought of α(1)-receptor mediated, appeared instead β(1)-receptor modulated because of: (i) blockade by atenolol; and (ii) phosphodiesterase inhibition by prazosin. The phenylephrine/methoxamine-induced attenuation of EIA seems paradoxical as the response is similar to antagonist prazosin. However, functionally the attenuation seems β(1) receptor-mediated since atenolol blocked it but prazosin did not. Also norepinephrine (NE) attenuated EIA that was inhibited by atenolol suggesting role of β(1) receptors. Similarly yohimbine and rauwolscine attenuated EIA that indicates α(2)-receptor modulation associated with stimulation of AC-cAMP pathway. The results of study support the hypothesis that attenuation and potentiation of EIA is mediated by activation and inhibition of AC-cAMP pathway, respectively, in agreement with our previous reports, via direct and/or indirect activation of β-receptor.

  15. Role of cyclic AMP sensor Epac1 in masseter muscle hypertrophy and myosin heavy chain transition induced by β2-adrenoceptor stimulation

    PubMed Central

    Ohnuki, Yoshiki; Umeki, Daisuke; Mototani, Yasumasa; Jin, Huiling; Cai, Wenqian; Shiozawa, Kouichi; Suita, Kenji; Saeki, Yasutake; Fujita, Takayuki; Ishikawa, Yoshihiro; Okumura, Satoshi

    2014-01-01

    The predominant isoform of β-adrenoceptor (β-AR) in skeletal muscle is β2-AR and that in the cardiac muscle is β1-AR. We have reported that Epac1 (exchange protein directly activated by cAMP 1), a new protein kinase A-independent cAMP sensor, does not affect cardiac hypertrophy in response to pressure overload or chronic isoproterenol (isoprenaline) infusion. However, the role of Epac1 in skeletal muscle hypertrophy remains poorly understood. We thus examined the effect of disruption of Epac1, the major Epac isoform in skeletal muscle, on masseter muscle hypertrophy induced by chronic β2-AR stimulation with clenbuterol (CB) in Epac1-null mice (Epac1KO). The masseter muscle weight/tibial length ratio was similar in wild-type (WT) and Epac1KO at baseline and was significantly increased in WT after CB infusion, but this increase was suppressed in Epac1KO. CB treatment significantly increased the proportion of myosin heavy chain (MHC) IIb at the expense of that of MHC IId/x in both WT and Epac1KO, indicating that Epac1 did not mediate the CB-induced MHC isoform transition towards the faster isoform. The mechanism of suppression of CB-mediated hypertrophy in Epac1KO is considered to involve decreased activation of Akt signalling. In addition, CB-induced histone deacetylase 4 (HDAC4) phosphorylation on serine 246 mediated by calmodulin kinase II (CaMKII), which plays a role in skeletal muscle hypertrophy, was suppressed in Epac1KO. Our findings suggest that Epac1 plays a role in β2-AR-mediated masseter muscle hypertrophy, probably through activation of both Akt signalling and CaMKII/HDAC4 signalling. PMID:25344550

  16. Functional characterization of the recombinant antimicrobial peptide Trx-Ace-AMP1 and its application on the control of tomato early blight disease.

    PubMed

    Wu, Yin; He, Yue; Ge, Xiaochun

    2011-05-01

    Ace-AMP1 is a potent antifungal peptide found in onion (Allium cepa) seeds with sequence similarity to plant lipid transfer proteins. Transgenic plants over-expressing Ace-AMP1 gene have enhanced disease resistance to some fungal pathogens. However, mass production in heterologous systems and in vitro application of this peptide have not been reported. In this study, Ace-AMP1 was highly expressed in a prokaryotic Escherichia coli system as a fusion protein. The purified protein inhibited the growth of many plant fungal pathogens, especially Alternaria solani, Fusarium oxysporum f. sp. vasinfectum, and Verticillium dahliae. The inhibitory effect was accompanied by hyphal hyperbranching for V. dahliae but not for F. oxysporum f. sp. vasinfectum and A. solani, suggesting that the mode of action of Ace-AMP1 on different fungi might be different. Application of Ace-AMP1 on tomato leaves showed that the recombinant protein conferred strong resistance to the tomato pathogen A. solani and could be used as an effective fungicide. PMID:21380518

  17. Glucocorticoids antagonize cAMP-induced Star transcription in Leydig cells through the orphan nuclear receptor NR4A1.

    PubMed

    Martin, Luc J; Tremblay, Jacques J

    2008-09-01

    It is well established that stress, either physical or psychosocial, causes a decrease in testosterone production by Leydig cells. Glucocorticoids (Gc) are the main mediators of stress response and they convey their repressive effect on Leydig cells through the glucocorticoid receptor (GR). So far, various mechanisms have been proposed to explain the mechanism of action of Gc on Leydig cell steroidogenesis including repression of genes involved in testosterone biosynthesis. Several steroidogenic genes, including steroidogenic acute regulatory (STAR) protein, have been shown to be repressed by Gc in a GR-dependent manner but the underlying mechanisms remain to be fully elucidated. Here, we found that dexamethasone (Dex), a potent synthetic Gc, partly antagonizes the cAMP-dependent stimulation of the mouse Star promoter in MA-10 Leydig cells as revealed by transient transfection assays. This repression requires an element located at -95 bp previously implicated in the activation of the Star promoter by the nuclear receptors, NR4A1 and NR5A1. Dex was found to inhibit NR4A1-dependent transactivation of the Star promoter in Leydig cells by decreasing NR4A1, but not NR5A1, recruitment to the proximal Star promoter as determined by chromatin immunoprecipitation assay. Western blots revealed that Dex did not affect NR4A1 or NR5A1 expression in response to cAMP. These data suggest that NR4A1 would be associated with the GR in a transcriptionally inactive complex as previously demonstrated in pituitary corticotrope cells. Thus, our data provide new molecular insights into the stress-mediated suppression of testosterone production in testicular Leydig cells.

  18. Cinnamon extract enhances glucose uptake in 3T3-L1 adipocytes and C2C12 myocytes by inducing LKB1-AMP-activated protein kinase signaling.

    PubMed

    Shen, Yan; Honma, Natsumi; Kobayashi, Katsuya; Jia, Liu Nan; Hosono, Takashi; Shindo, Kazutoshi; Ariga, Toyohiko; Seki, Taiichiro

    2014-01-01

    We previously demonstrated that cinnamon extract (CE) ameliorates type 1 diabetes induced by streptozotocin in rats through the up-regulation of glucose transporter 4 (GLUT4) translocation in both muscle and adipose tissues. This present study was aimed at clarifying the detailed mechanism(s) with which CE increases the glucose uptake in vivo and in cell culture systems using 3T3-L1 adipocytes and C2C12 myotubes in vitro. Specific inhibitors of key enzymes in insulin signaling and AMP-activated protein kinase (AMPK) signaling pathways, as well as small interference RNA, were used to examine the role of these kinases in the CE-induced glucose uptake. The results showed that CE stimulated the phosphorylation of AMPK and acetyl-CoA carboxylase. An AMPK inhibitor and LKB1 siRNA blocked the CE-induced glucose uptake. We also found for the first time that insulin suppressed AMPK activation in the adipocyte. To investigate the effect of CE on type 2 diabetes in vivo, we further performed oral glucose tolerance tests and insulin tolerance tests in type 2 diabetes model rats administered with CE. The CE improved glucose tolerance in oral glucose tolerance tests, but not insulin sensitivity in insulin tolerance test. In summary, these results indicate that CE ameliorates type 2 diabetes by inducing GLUT4 translocation via the AMPK signaling pathway. We also found insulin antagonistically regulates the activation of AMPK. PMID:24551069

  19. Prostaglandin E2 induces chloride secretion through crosstalk between cAMP and calcium signaling in mouse inner medullary collecting duct cells.

    PubMed

    Rajagopal, Madhumitha; Thomas, Sheela V; Kathpalia, Paru P; Chen, Yu; Pao, Alan C

    2014-02-01

    Under conditions of high dietary salt intake, prostaglandin E2 (PGE2) production is increased in the collecting duct and promotes urinary sodium chloride (NaCl) excretion; however, the molecular mechanisms by which PGE2 increases NaCl excretion in this context have not been clearly defined. We used the mouse inner medullary collecting duct (mIMCD)-K2 cell line to characterize mechanisms underlying PGE2-regulated NaCl transport. When epithelial Na(+) channels were inhibited, PGE2 exclusively stimulated basolateral EP4 receptors to increase short-circuit current (Isc(PGE2)). We found that Isc(PGE2) was sensitive to inhibition by H-89 and CFTR-172, indicating that EP4 receptors signal through protein kinase A to induce Cl(-) secretion via cystic fibrosis transmembrane conductance regulator (CFTR). Unexpectedly, we also found that Isc(PGE2) was sensitive to inhibition by BAPTA-AM (Ca(2+) chelator), 2-aminoethoxydiphenyl borate (2-APB) (inositol triphosphate receptor blocker), and flufenamic acid (FFA) [Ca(2+)-activated Cl(-) channel (CACC) inhibitor], suggesting that EP4 receptors also signal through Ca(2+) to induce Cl(-) secretion via CACC. Additionally, we observed that PGE2 stimulated an increase in Isc through crosstalk between cAMP and Ca(2+) signaling; BAPTA-AM or 2-APB inhibited a component of Isc(PGE2) that was sensitive to CFTR-172 inhibition; H-89 inhibited a component of Isc(PGE2) that was sensitive to FFA inhibition. Together, our findings indicate that PGE2 activates basolateral EP4 receptors and signals through both cAMP and Ca(2+) to stimulate Cl(-) secretion in IMCD-K2 cells. We propose that these signaling pathways, and the crosstalk between them, may provide a concerted mechanism for enhancing urinary NaCl excretion under conditions of high dietary NaCl intake.

  20. Cadmium-induced decrement of the LH receptor expression and cAMP levels in the testis of rats.

    PubMed

    Gunnarsson, David; Nordberg, Gunnar; Lundgren, Per; Selstam, Gunnar

    2003-02-01

    Cadmium (Cd) is a widespread environmental pollutant, characterized by its ability to affect various organs. Adverse effect of Cd on the testis including decreased testosterone production are well-known phenomena, but the cellular events explaining these effects have not yet been established. In the present study the initial steps of gonadotropin mediated testosterone biosynthesis were examined in vivo in rats, in relation to Cd dose and time after injection. In the dose-response experiment Male Sprague-Dawley rats received a single subcutaneous (s.c.) injection of CdCl(2) (1, 5 or 10 micromol/kg body weight) and were sacrificed 48 h after injection. A statistically significant decrease in luteinizing hormone (LH) receptor mRNA level in the testicular tissue was demonstrated at the highest dose (10 micromol/kg). In the temporal-response experiment rats were given 10 micromol/kg of CdCl(2) s.c. and sacrificed 0.48, 4.8, 48 or 144 h after injection. LH receptor mRNA levels as well as cyclic adenosine monophosphate (cAMP) levels were found to be significantly lowered at 48 and 144 h. These observations of the mechanisms whereby Cd exerts its effect on the initial steps of testosterone biosynthesis are the first from in vivo experiments. PMID:12504342

  1. Ca/sup + +/- and cyclic AMP-induced changes in intact cell phosphorylation of ileal microvillus membrane proteins

    SciTech Connect

    Sharp, G.W.G.; Hannah, C.M.; Cohen, M.; Donowitz, M.

    1986-03-05

    Pieces of rabbit distal ileal mucosa, with the muscularis propria and serosa removed, were incubated for 90 minutes in Krebs-Ringer bicarborate buffer (KRB) with /sup 32/PO/sub 4/ to label the intracellular nucleotide pools. After rinsing, the mucosal pieces were transferred to KRB in the absence and presence of 10 ..mu..M A23187 or 10 mM theophylline. After a further 10 minutes the cells were scraped off and microvillus membranes prepared. The membranes were solubilized, subjected to two dimensional gel electrophoresis and autoradiography, and analyzed by densitometry. A23187 increased the phosphorylation of four microvillus membrane proteins with M/sub r/ of 32, 52, 110 and 116K. Increased phosphorylation of the 52 and 116K proteins has also been detected in microvillus membranes subjected to Ca/sup + +/ and calmodulin in the presence of ..gamma..-/sup 32/P-ATP. Theophylline increased the phosphorylation of the same 32 and 52K proteins and, additionally, of a second 32K peptide. While any of these proteins could be involved in the control of electrolyte transport, it is noteworthy that increased Ca/sup + +/, and increased cyclic AMP levels exert similar effects upon intestinal electrolyte transport. That A23187 and theophylline both increase the phosphorylation of the 32 and 52K proteins increases the possibility that these are involved in ion transport.

  2. Adiponectin Inhibits LPS-Induced HMGB1 Release through an AMP Kinase and Heme Oxygenase-1-Dependent Pathway in RAW 264 Macrophage Cells

    PubMed Central

    Kaede, Ryuji; Okamatsu-Ogura, Yuko

    2016-01-01

    High mobility group protein B1 (HMGB1) is a late inflammatory mediator that exaggerates septic symptoms. Adiponectin, an adipokine, has potent anti-inflammatory properties. However, possible effects of adiponectin on lipopolysaccharide- (LPS-) induced HMGB1 release are unknown. The aim of this study was to investigate effects of full length adiponectin on HMGB1 release in LPS-stimulated RAW 264 macrophage cells. Treatment of the cells with LPS alone significantly induced HMGB1 release associated with HMGB1 translocation from the nucleus to the cytosol. However, prior treatment with adiponectin suppressed LPS-induced HMGB1 release and translocation. The anti-inflammatory cytokine interleukin- (IL-) 10 similarly suppressed LPS-induced HMGB1 release. Adiponectin treatment decreased toll-like receptor 4 (TLR4) mRNA expression and increased heme oxygenase- (HO-) 1 mRNA expression without inducing IL-10 mRNA, while IL-10 treatment decreased TLR2 and HMGB1 mRNA expression and increased the expression of IL-10 and HO-1 mRNA. Treatment with the HO-1 inhibitor ZnPP completely prevented the suppression of HMGB1 release by adiponectin but only partially inhibited that induced by IL-10. Treatment with compound C, an AMP kinase (AMPK) inhibitor, abolished the increase in HO-1 expression and the suppression of HMGB1 release mediated by adiponectin. In conclusion, our results indicate that adiponectin suppresses HMGB1 release by LPS through an AMPK-mediated and HO-1-dependent IL-10-independent pathway. PMID:27313399

  3. 24-hydroxyursolic acid from the leaves of the Diospyros kaki (Persimmon) induces apoptosis by activation of AMP-activated protein kinase.

    PubMed

    Khanal, Prem; Oh, Won-Keun; Thuong, Phuong Thien; Cho, Sung Dae; Choi, Hong Seok

    2010-05-01

    There are multiple lines of evidence that persimmon extract and its constituents have potent antitumor activity against human cancer cells. However, the molecular mechanisms of 24-hydroxyursolic acid, a triterpenoid found in persimmon, on antitumor activities are not yet understood. Here, we demonstrate that 24-hydroxyursolic acid inhibited cell proliferation, strongly activated AMP-activated protein kinase (AMPK) and mediated critical anticancer effects by inhibition of cyclooxygenase (COX-2) expression in HT-29 cells. In addition, 24-hydroxyursolic acid induced cellular apoptosis by activation of poly(ADP-ribose) polymerase (PARP), caspase-3, and phosphorylation of p53 at Ser15. It also strongly induced DNA fragmentation in HT-29 cells and thereby significantly inhibited colony formation of HT-29 cells in soft agar. In addition, 24-hydroxyursolic acid blocked the EGF-induced ERKs phosphorylation and led to the inhibition of AP-1 activity and cell transformation in JB6 CL41 cells. Collectively, these findings are the first to reveal a molecular basis for the anticarcinogenic action of 24-hydroxyursolic acid and might account for the reported chemopreventive and chemotherapic effects of persimmon extracts.

  4. Role of Deleted in Breast Cancer 1 (DBC1) Protein in SIRT1 Deacetylase Activation Induced by Protein Kinase A and AMP-activated Protein Kinase*

    PubMed Central

    Nin, Veronica; Escande, Carlos; Chini, Claudia C.; Giri, Shailendra; Camacho-Pereira, Juliana; Matalonga, Jonathan; Lou, Zhenkun; Chini, Eduardo N.

    2012-01-01

    The NAD+-dependent deacetylase SIRT1 is a key regulator of several aspects of metabolism and aging. SIRT1 activation is beneficial for several human diseases, including metabolic syndrome, diabetes, obesity, liver steatosis, and Alzheimer disease. We have recently shown that the protein deleted in breast cancer 1 (DBC1) is a key regulator of SIRT1 activity in vivo. Furthermore, SIRT1 and DBC1 form a dynamic complex that is regulated by the energetic state of the organism. Understanding how the interaction between SIRT1 and DBC1 is regulated is therefore essential to design strategies aimed to activate SIRT1. Here, we investigated which pathways can lead to the dissociation of SIRT1 and DBC1 and consequently to SIRT1 activation. We observed that PKA activation leads to a fast and transient activation of SIRT1 that is DBC1-dependent. In fact, an increase in cAMP/PKA activity resulted in the dissociation of SIRT1 and DBC1 in an AMP-activated protein kinase (AMPK)-dependent manner. Pharmacological AMPK activation led to SIRT1 activation by a DBC1-dependent mechanism. Indeed, we found that AMPK activators promote SIRT1-DBC1 dissociation in cells, resulting in an increase in SIRT1 activity. In addition, we observed that the SIRT1 activation promoted by PKA and AMPK occurs without changes in the intracellular levels of NAD+. We propose that PKA and AMPK can acutely activate SIRT1 by inducing dissociation of SIRT1 from its endogenous inhibitor DBC1. Our experiments provide new insight on the in vivo mechanism of SIRT1 regulation and a new avenue for the development of pharmacological SIRT1 activators targeted at the dissociation of the SIRT1-DBC1 complex. PMID:22553202

  5. Effects of carvedilol treatment on cardiac cAMP response element binding protein expression and phosphorylation in acute coxsackievirus B3-induced myocarditis

    PubMed Central

    2013-01-01

    Background The role of β-adrenergic stimulation on viral myocarditis has been investigated in animal models of viral myocarditis. Excess stimulation of β-adrenergic receptors by catecholamines causes phosphorylation/activation of cAMP response element binding protein (CREB) by the cAMP signaling pathway. CREB as an important regulator of gene expression mediates the cardiovascular remodeling process and promotes anti-inflammatory immune responses. However, the CREB expression and phosphorylation have not been studied, and the effects of carvedilol (a nonselective β-adrenoceptor antagonist) on the CREB has not been investigated in the setting of acute viral myocarditis. Methods This study was therefore designed to examine the effects of carvedilol on the transcriptional factor CREB in a murine model of acute viral myocarditis. In a coxsackievirus B3 murine myocarditis model (Balb/c), effects of carvedilol on plasma noradrenaline, heart rate and blood pressure, myocardial histopathological changes and fibrosis, cardiomyocyte apoptosis, cardiac CREB and phosphorylated CREB, cytokine levels, and viral RNA were studied. Results The expression and phosphorylation of CREB were decreased with concomitant increase of IL-6 and TNF-α in murine coxsackievirus-induced acute viral myocarditis. The levels of IL-6 and TNF-α were correlated with the expression of CREB or phosphorylated CREB. Carvedilol increased the cardiac CREB expression and phosphorylation and decreased the plasma catecholamine levels and the production of IL-6 and TNF-α with amelioration of acute viral myocarditis. Conclusion These results show that CREB may be involved in the pathophysiology of viral myocarditis and carvedilol exerts some of its beneficial effects by increasing the CREB expression and phosphorylation. PMID:24225056

  6. Raloxifene induces autophagy-dependent cell death in breast cancer cells via the activation of AMP-activated protein kinase.

    PubMed

    Kim, Dong Eun; Kim, Yunha; Cho, Dong-Hyung; Jeong, Seong-Yun; Kim, Sung-Bae; Suh, Nayoung; Lee, Jung Shin; Choi, Eun Kyung; Koh, Jae-Young; Hwang, Jung Jin; Kim, Choung-Soo

    2015-01-01

    Raloxifene is a selective estrogen receptor modulator (SERM) that binds to the estrogen receptor (ER), and exhibits potent anti-tumor and autophagy-inducing effects in breast cancer cells. However, the mechanism of raloxifene-induced cell death and autophagy is not well-established. So, we analyzed mechanism underlying death and autophagy induced by raloxifene in MCF-7 breast cancer cells. Treatment with raloxifene significantly induced death in MCF-7 cells. Raloxifene accumulated GFP-LC3 puncta and increased the level of autophagic marker proteins, such as LC3-II, BECN1, and ATG12-ATG5 conjugates, indicating activated autophagy. Raloxifene also increased autophagic flux indicators, the cleavage of GFP from GFP-LC3 and only red fluorescence-positive puncta in mRFP-GFP-LC3-expressing cells. An autophagy inhibitor, 3-methyladenine (3-MA), suppressed the level of LC3-II and blocked the formation of GFP-LC3 puncta. Moreover, siRNA targeting BECN1 markedly reversed cell death and the level of LC3-II increased by raloxifene. Besides, raloxifene-induced cell death was not related to cleavage of caspases-7, -9, and PARP. These results indicate that raloxifene activates autophagy-dependent cell death but not apoptosis. Interestingly, raloxifene decreased the level of intracellular adenosine triphosphate (ATP) and activated the AMPK/ULK1 pathway. However it was not suppressed the AKT/mTOR pathway. Addition of ATP decreased the phosphorylation of AMPK as well as the accumulation of LC3-II, finally attenuating raloxifene-induced cell death. Our current study demonstrates that raloxifene induces autophagy via the activation of AMPK by sensing decreases in ATP, and that the overactivation of autophagy promotes cell death and thereby mediates the anti-cancer effects of raloxifene in breast cancer cells.

  7. Modulation of a human lymphoblastoid B cell line by cyclic AMP. Ig secretion and phosphatidylcholine metabolism

    SciTech Connect

    Shearer, W.T.; Patke, C.L.; Gilliam, E.B.; Rosenblatt, H.M.; Barron, K.S.; Orson, F.M.

    1988-09-01

    A transformed human B cell line, LA350, was found to be sensitive to cAMP-elevating agents by responding with rapid (0 to 2 h) severalfold elevations of intracellular cAMP to treatment with cholera toxin, isobutylmethylxanthine (IBMX), forskolin, and dibutyryl cAMP (all p less than 0.001). These cAMP-elevating agents also produced significant inhibitions of subsequent (48 to 72 h) Ig secretion by the same B cells as measured by a reverse hemolytic plaque assay and an enzyme-linked immunoadsorbent assay for IgM (both p less than 0.001). PMA- and IBMX-treated cells were particularly responsive to the effects of cholera toxin, showing a doubling of cAMP content and profound decrease in Ig production (p less than 0.001). Because our previous studies had correlated activation of the metabolic turnover of the phosphatidylcholine (PC) fraction of membrane phospholipids with enhanced Ig secretion, we examined the sensitivity of PC metabolism to cAMP in control and PMA-stimulated cells. Formation of PC was found to be inhibited by forskolin and IBMX (both p less than 0.002) but breakdown of PC was stimulated (p less than 0.001). These findings imply that as the enzymatic products of PC, choline phosphate and diacylglycerol, are depleted due to the combined effects of cAMP upon synthesis and turnover of PC, there is a decrease in Ig secretion. Since diacylglycerol activates protein kinase C, it appears reasonable that Ig secretion is at least partially regulated by cAMP-responsive alterations in PC metabolism produced by protein kinase C-induced phosphorylation. We conclude that the early cAMP-sensitive changes in PC metabolism in this activated B cell line may signal for subsequent alterations in Ig secretion.

  8. cAMP controls rod photoreceptor sensitivity via multiple targets in the phototransduction cascade

    PubMed Central

    Astakhova, Luba A.; Samoiliuk, Evgeniia V.; Govardovskii, Victor I.

    2012-01-01

    In early studies, both cyclic AMP (cAMP) and cGMP were considered as potential secondary messengers regulating the conductivity of the vertebrate photoreceptor plasma membrane. Later discovery of the cGMP specificity of cyclic nucleotide–gated channels has shifted attention to cGMP as the only secondary messenger in the phototransduction cascade, and cAMP is not considered in modern schemes of phototransduction. Here, we report evidence that cAMP may also be involved in regulation of the phototransduction cascade. Using a suction pipette technique, we recorded light responses of isolated solitary rods from the frog retina in normal solution and in the medium containing 2 µM of adenylate cyclase activator forskolin. Under forskolin action, flash sensitivity rose more than twofold because of a retarded photoresponse turn-off. The same concentration of forskolin lead to a 2.5-fold increase in the rod outer segment cAMP, which is close to earlier reported natural day/night cAMP variations. Detailed analysis of cAMP action on the phototransduction cascade suggests that several targets are affected by cAMP increase: (a) basal dark phosphodiesterase (PDE) activity decreases; (b) at the same intensity of light background, steady background-induced PDE activity increases; (c) at light backgrounds, guanylate cyclase activity at a given fraction of open channels is reduced; and (d) the magnitude of the Ca2+ exchanger current rises 1.6-fold, which would correspond to a 1.6-fold elevation of [Ca2+]in. Analysis by a complete model of rod phototransduction suggests that an increase of [Ca2+]in might also explain effects (b) and (c). The mechanism(s) by which cAMP could regulate [Ca2+]in and PDE basal activity is unclear. We suggest that these regulations may have adaptive significance and improve the performance of the visual system when it switches between day and night light conditions. PMID:23008435

  9. cAMP controls rod photoreceptor sensitivity via multiple targets in the phototransduction cascade.

    PubMed

    Astakhova, Luba A; Samoiliuk, Evgeniia V; Govardovskii, Victor I; Firsov, Michael L

    2012-10-01

    In early studies, both cyclic AMP (cAMP) and cGMP were considered as potential secondary messengers regulating the conductivity of the vertebrate photoreceptor plasma membrane. Later discovery of the cGMP specificity of cyclic nucleotide-gated channels has shifted attention to cGMP as the only secondary messenger in the phototransduction cascade, and cAMP is not considered in modern schemes of phototransduction. Here, we report evidence that cAMP may also be involved in regulation of the phototransduction cascade. Using a suction pipette technique, we recorded light responses of isolated solitary rods from the frog retina in normal solution and in the medium containing 2 µM of adenylate cyclase activator forskolin. Under forskolin action, flash sensitivity rose more than twofold because of a retarded photoresponse turn-off. The same concentration of forskolin lead to a 2.5-fold increase in the rod outer segment cAMP, which is close to earlier reported natural day/night cAMP variations. Detailed analysis of cAMP action on the phototransduction cascade suggests that several targets are affected by cAMP increase: (a) basal dark phosphodiesterase (PDE) activity decreases; (b) at the same intensity of light background, steady background-induced PDE activity increases; (c) at light backgrounds, guanylate cyclase activity at a given fraction of open channels is reduced; and (d) the magnitude of the Ca(2+) exchanger current rises 1.6-fold, which would correspond to a 1.6-fold elevation of [Ca(2+)](in). Analysis by a complete model of rod phototransduction suggests that an increase of [Ca(2+)](in) might also explain effects (b) and (c). The mechanism(s) by which cAMP could regulate [Ca(2+)](in) and PDE basal activity is unclear. We suggest that these regulations may have adaptive significance and improve the performance of the visual system when it switches between day and night light conditions. PMID:23008435

  10. Detection of early caries by laser-induced breakdown spectroscopy

    NASA Astrophysics Data System (ADS)

    Sasazawa, Shuhei; Kakino, Satoko; Matsuura, Yuji

    2015-07-01

    To improve sensitivity of dental caries detection by laser-induced breakdown spectroscopy (LIBS) analysis, it is proposed to utilize emission peaks in the ultraviolet. We newly focused on zinc whose emission peaks exist in ultraviolet because zinc exists at high concentration in the outer layer of enamel. It was shown that by using ratios between heights of an emission peak of Zn and that of Ca, the detection sensitivity and stability are largely improved. It was also shown that early caries are differentiated from healthy part by properly setting a threshold in the detected ratios. The proposed caries detection system can be applied to dental laser systems such as ones based on Er:YAG-lasers. When ablating early caries part by laser light, the system notices the dentist that the ablation of caries part is finished. We also show the intensity of emission peaks of zinc decreased with ablation with Er:YAG laser light.

  11. AMP-Activated Protein Kinase Attenuates High Salt-Induced Activation of Epithelial Sodium Channels (ENaC) in Human Umbilical Vein Endothelial Cells

    PubMed Central

    Li, Xin-Yuan; Hu, Qing-Qing; Ma, He-Ping

    2016-01-01

    Recent studies suggest that the epithelial sodium channel (ENaC) is expressed in the endothelial cells. To test whether high salt affects the NO production via regulation of endothelial ENaC, human umbilical vein endothelial cells (HUVECs) were incubated in solutions containing either normal or high sodium (additional 20 mM NaCl). Our data showed that high sodium treatment significantly increased α-, β-, and γ-ENaC expression levels in HUVECs. Using the cell-attached patch-clamp technique, we demonstrated that high sodium treatment significantly increased ENaC open probability (PO). Moreover, nitric oxide synthase (eNOS) phosphorylation (Ser 1177) levels and NO production were significantly decreased by high sodium in HUVECs; the effects of high sodium on eNOS phosphorylation and NO production were inhibited by a specific ENaC blocker, amiloride. Our results showed that high sodium decreased AMP-activated kinase (AMPK) phosphorylation in endothelial cells. On the other hand, metformin, an AMPK activator, prevented high sodium-induced upregulation of ENaC expression and PO. Moreover, metformin prevented high salt-induced decrease in NO production and eNOS phosphorylation. These results suggest that high sodium stimulates ENaC activation by negatively modulating AMPK activity, thereby leading to reduction in eNOS activity and NO production in endothelial cells.

  12. AMP-Activated Protein Kinase Attenuates High Salt-Induced Activation of Epithelial Sodium Channels (ENaC) in Human Umbilical Vein Endothelial Cells.

    PubMed

    Zheng, Wei-Wan; Li, Xin-Yuan; Liu, Hui-Bin; Wang, Zi-Rui; Hu, Qing-Qing; Li, Yu-Xia; Song, Bin-Lin; Lou, Jie; Wang, Qiu-Shi; Ma, He-Ping; Zhang, Zhi-Ren

    2016-01-01

    Recent studies suggest that the epithelial sodium channel (ENaC) is expressed in the endothelial cells. To test whether high salt affects the NO production via regulation of endothelial ENaC, human umbilical vein endothelial cells (HUVECs) were incubated in solutions containing either normal or high sodium (additional 20 mM NaCl). Our data showed that high sodium treatment significantly increased α-, β-, and γ-ENaC expression levels in HUVECs. Using the cell-attached patch-clamp technique, we demonstrated that high sodium treatment significantly increased ENaC open probability (P O ). Moreover, nitric oxide synthase (eNOS) phosphorylation (Ser 1177) levels and NO production were significantly decreased by high sodium in HUVECs; the effects of high sodium on eNOS phosphorylation and NO production were inhibited by a specific ENaC blocker, amiloride. Our results showed that high sodium decreased AMP-activated kinase (AMPK) phosphorylation in endothelial cells. On the other hand, metformin, an AMPK activator, prevented high sodium-induced upregulation of ENaC expression and P O . Moreover, metformin prevented high salt-induced decrease in NO production and eNOS phosphorylation. These results suggest that high sodium stimulates ENaC activation by negatively modulating AMPK activity, thereby leading to reduction in eNOS activity and NO production in endothelial cells. PMID:27635187

  13. Inactivation of MARK4, an AMP-activated protein kinase (AMPK)-related kinase, leads to insulin hypersensitivity and resistance to diet-induced obesity.

    PubMed

    Sun, Chao; Tian, Liang; Nie, Jia; Zhang, Hai; Han, Xiao; Shi, Yuguang

    2012-11-01

    MARK4, also known as Par-1d/MarkL1, is a member of the AMP-activated protein kinase (AMPK)-related family of kinases, which are implicated in the regulation of dynamic biological functions, including glucose and energy homeostasis. However, the physiological function of MARK4 in mammals remains elusive. Here, we investigated a role for MARK4 in regulating energy homeostasis by generating mice with targeted inactivation of the mark4 gene. We show that MARK4 deficiency in mice caused hyperphagia, hyperactivity, and hypermetabolism, leading to protection from diet-induced obesity and its related metabolic complications through up-regulation of brown fat activity. Consequently, MARK4 deficiency mitigated insulin resistance associated with diet-induced obesity by dramatically enhancing insulin-stimulated AKT phosphorylation in major metabolic tissues. Ablation of MARK4 also significantly improved glucose homeostasis by up-regulating the activity and expression of AMPK kinase in key metabolic tissues. Taken together, these data identify a key role of MARK4 in energy metabolism, implicating the kinase as a novel drug target for the treatment of obesity and type 2 diabetes. PMID:22992738

  14. AMP-Activated Protein Kinase Attenuates High Salt-Induced Activation of Epithelial Sodium Channels (ENaC) in Human Umbilical Vein Endothelial Cells

    PubMed Central

    Li, Xin-Yuan; Hu, Qing-Qing; Ma, He-Ping

    2016-01-01

    Recent studies suggest that the epithelial sodium channel (ENaC) is expressed in the endothelial cells. To test whether high salt affects the NO production via regulation of endothelial ENaC, human umbilical vein endothelial cells (HUVECs) were incubated in solutions containing either normal or high sodium (additional 20 mM NaCl). Our data showed that high sodium treatment significantly increased α-, β-, and γ-ENaC expression levels in HUVECs. Using the cell-attached patch-clamp technique, we demonstrated that high sodium treatment significantly increased ENaC open probability (PO). Moreover, nitric oxide synthase (eNOS) phosphorylation (Ser 1177) levels and NO production were significantly decreased by high sodium in HUVECs; the effects of high sodium on eNOS phosphorylation and NO production were inhibited by a specific ENaC blocker, amiloride. Our results showed that high sodium decreased AMP-activated kinase (AMPK) phosphorylation in endothelial cells. On the other hand, metformin, an AMPK activator, prevented high sodium-induced upregulation of ENaC expression and PO. Moreover, metformin prevented high salt-induced decrease in NO production and eNOS phosphorylation. These results suggest that high sodium stimulates ENaC activation by negatively modulating AMPK activity, thereby leading to reduction in eNOS activity and NO production in endothelial cells. PMID:27635187

  15. Nitric oxide protects neuroblastoma cells from apoptosis induced by serum deprivation through cAMP-response element-binding protein (CREB) activation.

    PubMed

    Ciani, Elisabetta; Guidi, Sandra; Della Valle, Giuliano; Perini, Giovanni; Bartesaghi, Renata; Contestabile, Antonio

    2002-12-20

    The transcription factor cAMP-response element-binding protein (CREB) mediates survival in many cells, including neurons. Recently, death of cerebellar granule neurons due to nitric oxide (NO) deprivation was shown to be accompanied by down-regulation of CREB activity (). We now provide evidence that overproduction of endogenous NO or supplementation with exogenous NO renders SK-N-BE human neuroblastoma cells more resistant to apoptosis induced by serum deprivation. Parental cells underwent apoptosis after 24 h of serum deprivation, an outcome largely absent in clones overexpressing human neuronal nitric oxide synthase (nNOS). This protective effect was reversed by the inhibition of NOS itself or soluble guanylyl cyclase, pointing at cGMP as an intermediate effector of NO-mediated rescue. A slow-releasing NO donor protected parental cells to a significant extent, thus confirming the survival effect of NO. The impaired viability of serum-deprived parental cells was accompanied by a strong decrease of CREB phosphorylation and transcriptional activity, effects significantly attenuated in nNOS-overexpressing clones. To confirm the role of CREB in survival, the ectopic expression of CREB and/or protein kinase A largely counteracted serum deprivation-induced cell death of SK-N-BE cells, whereas transfection with a CREB negative mutant was ineffective. These experiments indicate that CREB activity is an important step for NO-mediated survival in neuronal cells.

  16. AMP-activated protein kinase α2 protects against liver injury from metastasized tumors via reduced glucose deprivation-induced oxidative stress.

    PubMed

    Qiu, Shu-Lan; Xiao, Zhi-Cheng; Piao, Chun-Mei; Xian, Ying-Lin; Jia, Li-Xin; Qi, Yong-Fen; Han, Jia-Huai; Zhang, You-Yi; Du, Jie

    2014-03-28

    It is well known that tumors damage affected tissues; however, the specific mechanism underlying such damage remains elusive. AMP-activated protein kinase (AMPK) senses energetic changes and regulates glucose metabolism. In this study, we examined the mechanisms by which AMPK promotes metabolic adaptation in the tumor-bearing liver using a murine model of colon cancer liver metastasis. Knock-out of AMPK α2 significantly enhanced tumor-induced glucose deprivation in the liver and increased the extent of liver injury and hepatocyte death. Mechanistically, we observed that AMPK α2 deficiency resulted in elevated reactive oxygen species, reduced mitophagy, and increased cell death in response to tumors or glucose deprivation in vitro. These results imply that AMPK α2 is essential for attenuation of liver injury during tumor metastasis via hepatic glucose deprivation and mitophagy-mediated inhibition of reactive oxygen species production. Therefore, AMPK α2 might represent an important therapeutic target for colon cancer metastasis-induced liver injury. PMID:24515110

  17. Nitric oxide protects neuroblastoma cells from apoptosis induced by serum deprivation through cAMP-response element-binding protein (CREB) activation.

    PubMed

    Ciani, Elisabetta; Guidi, Sandra; Della Valle, Giuliano; Perini, Giovanni; Bartesaghi, Renata; Contestabile, Antonio

    2002-12-20

    The transcription factor cAMP-response element-binding protein (CREB) mediates survival in many cells, including neurons. Recently, death of cerebellar granule neurons due to nitric oxide (NO) deprivation was shown to be accompanied by down-regulation of CREB activity (). We now provide evidence that overproduction of endogenous NO or supplementation with exogenous NO renders SK-N-BE human neuroblastoma cells more resistant to apoptosis induced by serum deprivation. Parental cells underwent apoptosis after 24 h of serum deprivation, an outcome largely absent in clones overexpressing human neuronal nitric oxide synthase (nNOS). This protective effect was reversed by the inhibition of NOS itself or soluble guanylyl cyclase, pointing at cGMP as an intermediate effector of NO-mediated rescue. A slow-releasing NO donor protected parental cells to a significant extent, thus confirming the survival effect of NO. The impaired viability of serum-deprived parental cells was accompanied by a strong decrease of CREB phosphorylation and transcriptional activity, effects significantly attenuated in nNOS-overexpressing clones. To confirm the role of CREB in survival, the ectopic expression of CREB and/or protein kinase A largely counteracted serum deprivation-induced cell death of SK-N-BE cells, whereas transfection with a CREB negative mutant was ineffective. These experiments indicate that CREB activity is an important step for NO-mediated survival in neuronal cells. PMID:12368293

  18. The stellate vascular smooth muscle cell phenotype is induced by IL-1β via the secretion of PGE2 and subsequent cAMP-dependent protein kinase A activation.

    PubMed

    Blirando, Karl; Blaise, Régis; Gorodnaya, Natalia; Rouxel, Clotilde; Meilhac, Olivier; Vincent, Pierre; Limon, Isabelle

    2015-12-01

    Atherosclerosis development is associated with morphological changes to intimal cells, leading to a stellate cell phenotype. In this study, we aimed to determine whether and how key pro-atherogenic cytokines present in atherosclerotic plaques (IL-1β, TNFα and IFNγ) could induce this phenotype, as these molecules are known to trigger the transdifferentiation of vascular smooth muscle cells (VSMCs). We found that, IL-1β was the only major inflammatory mediator tested capable of inducing a stellate morphology in VSMCs. This finding was confirmed by staining for F-actin and vinculin at focal adhesions, as these two markers were disrupted only by IL-1β. We then investigated the possible association of this IL-1β-dependent change in morphology with an increase in intracellular cAMP concentration ([cAMP]), using the FRET-based biosensor for cAMP (T)Epac(VV). Experiments in the presence of IL-1β or medium conditioned by IL-1β-treated VSMCs and pharmacological tools demonstrated that the long-term increase in intracellular cAMP concentration was induced by the secretion of an autocrine/paracrine mediator, prostaglandin E₂(PGE₂), acting through the EP4 receptor. Finally, by knocking down the expression of the regulatory subunit PKAR1α, thereby reproducing the effects of IL-1β and PGE₂ on VSMCs, we demonstrated the contribution of PKA activity to the observed behavior of VSMCs. PMID:26403276

  19. Aspirin-induced AMP-activated protein kinase activation regulates the proliferation of vascular smooth muscle cells from spontaneously hypertensive rats

    SciTech Connect

    Sung, Jin Young; Choi, Hyoung Chul

    2011-05-06

    Highlights: {yields} Aspirin-induced AMPK phosphorylation was greater in VSMC from SHR than WKY. {yields} Aspirin-induced AMPK phosphorylation inhibited proliferation of VSMC from SHR. {yields} Low basal AMPK phosphorylation in SHR elicits increased VSMC proliferation. {yields} Inhibition of AMPK restored decreased VSMC proliferation by aspirin in SHR. {yields} Aspirin exerts anti-proliferative effect through AMPK activation in VSMC from SHR. -- Abstract: Acetylsalicylic acid (aspirin), used to reduce risk of cardiovascular disease, plays an important role in the regulation of cellular proliferation. However, mechanisms responsible for aspirin-induced growth inhibition are not fully understood. Here, we investigated whether aspirin may exert therapeutic effects via AMP-activated protein kinase (AMPK) activation in vascular smooth muscle cells (VSMC) from wistar kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Aspirin increased AMPK and acetyl-CoA carboxylase phosphorylation in a time- and dose-dependent manner in VSMCs from WKY and SHR, but with greater efficacy in SHR. In SHR, a low basal phosphorylation status of AMPK resulted in increased VSMC proliferation and aspirin-induced AMPK phosphorylation inhibited proliferation of VSMCs. Compound C, an AMPK inhibitor, and AMPK siRNA reduced the aspirin-mediated inhibition of VSMC proliferation, this effect was more pronounced in SHR than in WKY. In VSMCs from SHR, aspirin increased p53 and p21 expression and inhibited the expression of cell cycle associated proteins, such as p-Rb, cyclin D, and cyclin E. These results indicate that in SHR VSMCs aspirin exerts anti-proliferative effects through the induction of AMPK phosphorylation.

  20. Reciprocal roles between caffeine and estrogen on bone via differently regulating cAMP/PKA pathway: the possible mechanism for caffeine-induced osteoporosis in women and estrogen's antagonistic effects.

    PubMed

    Zhou, Yi; Zhu, Zhuo-Li; Guan, Xiao-Xu; Hou, Wei-Wei; Yu, Hai-Yang

    2009-07-01

    Caffeine is consumed by most people in Europe and North America. As a risk factor for osteoporosis, caffeine has been reported to decrease bone mineral density, negatively influence calcium absorption and increase the risk of bone fracture in women. Except for the epidemiological observations and several studies which proved caffeine's unfavorable effects on osteoblast proliferation and impaired ability to form bone, little mechanism is known for the caffeine-induced osteoporosis. Since our unpublished studies showed that the precursor cells of osteoblasts, bone marrow-derived mesenchymal stem cells (BMSCs), were more sensitive than osteoblasts when exposed to the same dose of caffeine. We herein hypothesize that MSCs may be the primary target cells for caffeine-induced osteoporosis. It is well established that increasing cyclic 3',5'-adenosine monophosphate (cAMP) can regulate the expression of key genes involved in bone metabolism, including Cbfa1, PPARgamma, RANKL and OPG. We thereby propose the hypothesis that caffeine, a known inhibitor of cAMP phosphodiesterase, may affect bone metabolism by activating cAMP-dependent protein kinase A (PKA) pathway. In addition, considering the fact observed in epidemiology that caffeine's negative effects on bone only occurred in postmenopausal women and the inverse roles of caffeine and estrogen on bone metabolism, we postulate that caffeine may exert its undesirable influences on bone only in absence or low level of estrogen in vivo and estrogen may antagonize the adverse effect of caffeine on bone. Since several studies have demonstrated that estrogen may have ability to temper the biological effects of cAMP stimulators' roles on bone through cAMP to regulate some important genes' expression in bone metabolism. We assume that estrogen may block cAMP-dependent PKA pathway which is shared by caffeine, to exhibit its antagonistic roles. PMID:19278793

  1. AMP-Activated Kinase (AMPK) Activation by AICAR in Human White Adipocytes Derived from Pericardial White Adipose Tissue Stem Cells Induces a Partial Beige-Like Phenotype.

    PubMed

    Abdul-Rahman, Omar; Kristóf, Endre; Doan-Xuan, Quang-Minh; Vida, András; Nagy, Lilla; Horváth, Ambrus; Simon, József; Maros, Tamás; Szentkirályi, István; Palotás, Lehel; Debreceni, Tamás; Csizmadia, Péter; Szerafin, Tamás; Fodor, Tamás; Szántó, Magdolna; Tóth, Attila; Kiss, Borbála; Bacsó, Zsolt; Bai, Péter

    2016-01-01

    Beige adipocytes are special cells situated in the white adipose tissue. Beige adipocytes, lacking thermogenic cues, morphologically look quite similar to regular white adipocytes, but with a markedly different response to adrenalin. White adipocytes respond to adrenergic stimuli by enhancing lipolysis, while in beige adipocytes adrenalin induces mitochondrial biogenesis too. A key step in the differentiation and function of beige adipocytes is the deacetylation of peroxisome proliferator-activated receptor (PPARγ) by SIRT1 and the consequent mitochondrial biogenesis. AMP-activated protein kinase (AMPK) is an upstream activator of SIRT1, therefore we set out to investigate the role of AMPK in beige adipocyte differentiation using human adipose-derived mesenchymal stem cells (hADMSCs) from pericardial adipose tissue. hADMSCs were differentiated to white and beige adipocytes and the differentiation medium of the white adipocytes was supplemented with 100 μM [(2R,3S,4R,5R)-5-(4-Carbamoyl-5-aminoimidazol-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl dihydrogen phosphate (AICAR), a known activator of AMPK. The activation of AMPK with AICAR led to the appearance of beige-like morphological properties in differentiated white adipocytes. Namely, smaller lipid droplets appeared in AICAR-treated white adipocytes in a similar fashion as in beige cells. Moreover, in AICAR-treated white adipocytes the mitochondrial network was more fused than in white adipocytes; a fused mitochondrial system was characteristic to beige adipocytes. Despite the morphological similarities between AICAR-treated white adipocytes and beige cells, functionally AICAR-treated white adipocytes were similar to white adipocytes. We were unable to detect increases in basal or cAMP-induced oxygen consumption rate (a marker of mitochondrial biogenesis) when comparing control and AICAR-treated white adipocytes. Similarly, markers of beige adipocytes such as TBX1, UCP1, CIDEA, PRDM16 and TMEM26 remained the same when

  2. AMP-Activated Kinase (AMPK) Activation by AICAR in Human White Adipocytes Derived from Pericardial White Adipose Tissue Stem Cells Induces a Partial Beige-Like Phenotype

    PubMed Central

    Abdul-Rahman, Omar; Kristóf, Endre; Doan-Xuan, Quang-Minh; Vida, András; Nagy, Lilla; Horváth, Ambrus; Simon, József; Maros, Tamás; Szentkirályi, István; Palotás, Lehel; Debreceni, Tamás; Csizmadia, Péter; Szerafin, Tamás; Fodor, Tamás; Szántó, Magdolna; Tóth, Attila; Kiss, Borbála; Bacsó, Zsolt; Bai, Péter

    2016-01-01

    Beige adipocytes are special cells situated in the white adipose tissue. Beige adipocytes, lacking thermogenic cues, morphologically look quite similar to regular white adipocytes, but with a markedly different response to adrenalin. White adipocytes respond to adrenergic stimuli by enhancing lipolysis, while in beige adipocytes adrenalin induces mitochondrial biogenesis too. A key step in the differentiation and function of beige adipocytes is the deacetylation of peroxisome proliferator-activated receptor (PPARγ) by SIRT1 and the consequent mitochondrial biogenesis. AMP-activated protein kinase (AMPK) is an upstream activator of SIRT1, therefore we set out to investigate the role of AMPK in beige adipocyte differentiation using human adipose-derived mesenchymal stem cells (hADMSCs) from pericardial adipose tissue. hADMSCs were differentiated to white and beige adipocytes and the differentiation medium of the white adipocytes was supplemented with 100 μM [(2R,3S,4R,5R)-5-(4-Carbamoyl-5-aminoimidazol-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl dihydrogen phosphate (AICAR), a known activator of AMPK. The activation of AMPK with AICAR led to the appearance of beige-like morphological properties in differentiated white adipocytes. Namely, smaller lipid droplets appeared in AICAR-treated white adipocytes in a similar fashion as in beige cells. Moreover, in AICAR-treated white adipocytes the mitochondrial network was more fused than in white adipocytes; a fused mitochondrial system was characteristic to beige adipocytes. Despite the morphological similarities between AICAR-treated white adipocytes and beige cells, functionally AICAR-treated white adipocytes were similar to white adipocytes. We were unable to detect increases in basal or cAMP-induced oxygen consumption rate (a marker of mitochondrial biogenesis) when comparing control and AICAR-treated white adipocytes. Similarly, markers of beige adipocytes such as TBX1, UCP1, CIDEA, PRDM16 and TMEM26 remained the same when

  3. Acute activation of AMP-activated protein kinase prevents H2O2-induced premature senescence in primary human keratinocytes.

    PubMed

    Ido, Yasuo; Duranton, Albert; Lan, Fan; Cacicedo, Jose M; Chen, Tai C; Breton, Lionel; Ruderman, Neil B

    2012-01-01

    We investigated the effects of AMPK on H(2)O(2)-induced premature senescence in primary human keratinocytes. Incubation with 50 µM H(2)O(2) for 2 h resulted in premature senescence with characteristic increases in senescence-associated ß-galactosidase (SA-gal) staining 3 days later and no changes in AMPK or p38 MAPK activity. The increase in SA-gal staining was preceded by increases in both p53 phosphorylation (S15) (1 h) and transactivation (6 h) and the abundance of the cyclin inhibitor p21(CIP1) (16 h). Incubation with AICAR or resveratrol, both of which activated AMPK, prevented the H(2)O(2)-induced increases in both SA-Gal staining and p21 abundance. In addition, AICAR diminished the increase in p53 transactivation. The decreases in SA-Gal expression induced by resveratrol and AICAR were prevented by the pharmacological AMPK inhibitor Compound C, expression of a DN-AMPK or AMPK knock-down with shRNA. Likewise, both knockdown of AMPK and expression of DN-AMPK were sufficient to induce senescence, even in the absence of exogenous H(2)O(2). As reported by others, we found that AMPK activation by itself increased p53 phosphorylation at S15 in embryonic fibroblasts (MEF), whereas under the same conditions it decreased p53 phosphorylation in the keratinocytes, human aortic endothelial cells, and human HT1080 fibrosarcoma cells. In conclusion, the results indicate that H(2)O(2) at low concentrations causes premature senescence in human keratinocytes by activating p53-p21(CIP1) signaling and that these effects can be prevented by acute AMPK activation and enhanced by AMPK downregulation. They also suggest that this action of AMPK may be cell or context-specific. PMID:22514710

  4. Nordihydroguaiaretic acid protects against high-fat diet-induced fatty liver by activating AMP-activated protein kinase in obese mice

    SciTech Connect

    Lee, Myoung-Su; Kim, Daeyoung; Jo, Keunae; Hwang, Jae-Kwan

    2010-10-08

    Research highlights: {yields} NDGA decreases high-fat diet-induced body weight gain and adiposity. {yields} NDGA reduces high-fat diet-induced triglyceride accumulation in liver. {yields} NDGA improves lipid storage in vitro through altering lipid regulatory proteins. {yields} Inhibition of lipid storage in vivo and in vitro is mediated by AMPK activation. -- Abstract: Nonalcoholic fatty liver disease, one of the most common causes of chronic liver disease, is strongly associated with metabolic syndrome. Nordihydroguaiaretic acid (NDGA) has been reported to inhibit lipoprotein lipase; however, the effect of NDGA on hepatic lipid metabolism remains unclear. We evaluated body weight, adiposity, liver histology, and hepatic triglyceride content in high-fat diet (HFD)-fed C57BL/6J mice treated with NDGA. In addition, we characterized the underlying mechanism of NDGA's effects in HepG2 hepatocytes by Western blot and RT-PCR analysis. NDGA (100 or 200 mg/kg/day) reduced weight gain, fat pad mass, and hepatic triglyceride accumulation, and improved serum lipid parameters in mice fed a HFD for 8 weeks. NDGA significantly increased AMP-activated protein kinase (AMPK) phosphorylation in the liver and in HepG2 hepatocytes. NDGA downregulated the level of mature SREBP-1 and its target genes (acetyl-CoA carboxylase and fatty acid synthase), but, it upregulated expression of genes involved in fatty acid oxidation, such as peroxisome proliferator-activated receptor (PPAR){alpha}, PPAR{gamma} coactivator-1, carnitine palmitoyl transferase-1, and uncoupling protein-2. The specific AMPK inhibitor compound C attenuated the effects of NDGA on expression of lipid metabolism-related proteins in HepG2 hepatocytes. The beneficial effects of NDGA on HFD-induced hepatic triglyceride accumulation are mediated through AMPK signaling pathways, suggesting a potential target for preventing NAFLD.

  5. Antiaging Gene Klotho Deficiency Promoted High-Fat Diet-Induced Arterial Stiffening via Inactivation of AMP-Activated Protein Kinase.

    PubMed

    Lin, Yi; Chen, Jianglei; Sun, Zhongjie

    2016-03-01

    Klotho was originally discovered as an aging-suppressor gene. The objective of this study is to investigate whether klotho gene deficiency affects high-fat diet (HFD)-induced arterial stiffening. Heterozygous Klotho-deficient (KL(+/-)) mice and WT littermates were fed on HFD or normal diet. HFD increased pulse wave velocity within 5 weeks in KL(+/-) mice but not in wild-type mice, indicating that klotho deficiency accelerates and exacerbates HFD-induced arterial stiffening. A greater increase in blood pressure was found in KL(+/-) mice fed on HFD. Protein expressions of phosphorylated AMP-activated protein kinase-α (AMPKα), phosphorylated endothelial nitric oxide synthase (eNOS), and manganese-dependent superoxide dismutase (Mn-SOD) were decreased, whereas protein expressions of collagen I, transforming growth factor-β1, and Runx2 were increased in aortas of KL(+/-) mice fed on HFD. Interestingly, daily injections of an AMPKα activator, 5-aminoimidazole-4-carboxamide-3-ribonucleoside, abolished the increases in pulse wave velocity, blood pressure, and blood glucose in KL(+/-) mice fed on HFD. Treatment with 5-aminoimidazole-4-carboxamide-3-ribonucleoside for 2 weeks not only abolished the downregulation of phosphorylated AMPKα, phosphorylated eNOS, and Mn-SOD levels but also attenuated the increased levels of collagen I, transforming growth factor-β1, Runx2, superoxide, elastic lamellae breaks, and calcification in aortas of KL(+/-) mice fed on HFD. In cultured mouse aortic smooth muscle cells, cholesterol plus KL-deficient serum decreased phosphorylation levels of AMPKα and LKB1 (an important upstream regulator of AMPKα activity) but increased collagen I synthesis, which can be eliminated by activation of AMPKα by 5-aminoimidazole-4-carboxamide-3-ribonucleoside. In conclusions, Klotho deficiency promoted HFD-induced arterial stiffening and hypertension via downregulation of AMPKα activity. PMID:26781278

  6. Metformin-induced mitochondrial function and ABCD2 up-regulation in X-linked adrenoleukodystrophy involves AMP-activated protein kinase.

    PubMed

    Singh, Jaspreet; Olle, Brittany; Suhail, Hamid; Felicella, Michelle M; Giri, Shailendra

    2016-07-01

    X-linked adrenoleukodystrophy (X-ALD) is a progressive neurometabolic disease caused by mutations/deletions in the Abcd1 gene. Similar mutations/deletions in the Abcd1 gene often result in diagonally opposing phenotypes of mild adrenomyeloneuropathy and severe neuroinflammatory cerebral adrenoleukodystrophy (ALD), which suggests involvement of downstream modifier genes. We recently documented the first evidence of loss of AMP-activated protein kinase α1 (AMPKα1) in ALD patient-derived cells. Here, we report the novel loss of AMPKα1 in postmortem brain white matter of patients with ALD phenotype. Pharmacological activation of AMPK can rescue the mitochondrial dysfunction and inhibit the pro-inflammatory response. The FDA approved anti-diabetic drug Metformin, a well-known AMPK activator, induces mitochondrial biogenesis and is documented for its anti-inflammatory role. We observed a dose-dependent activation of AMPKα1 in metformin-treated X-ALD patient-derived fibroblasts. Metformin also induced mitochondrial oxidative phosphorylation and ATP levels in X-ALD patient-derived fibroblasts. Metformin treatment decreased very long chain fatty acid levels and pro-inflammatory cytokine gene expressions in X-ALD patient-derived cells. Abcd2 [adrenoleukodystrophy protein-related protein] levels were increased in metformin-treated X-ALD patient-derived fibroblasts and Abcd1-KO mice primary mixed glial cells. Abcd2 induction was AMPKα1-dependent since metformin failed to induce Abcd2 levels in AMPKα1-KO mice-derived primary mixed glial cells. In vivo metformin (100 mg/Kg) in drinking water for 60 days induced Abcd2 levels and mitochondrial oxidative phosphorylation protein levels in the brain and spinal cord of Abcd1-KO mice. Taken together, these results provide proof-of-principle for therapeutic potential of metformin as a useful strategy for correcting the metabolic and inflammatory derangements in X-ALD by targeting AMPK. There is no effective therapy for inherited

  7. Metformin-induced mitochondrial function and ABCD2 up-regulation in X-linked adrenoleukodystrophy involves AMP-activated protein kinase.

    PubMed

    Singh, Jaspreet; Olle, Brittany; Suhail, Hamid; Felicella, Michelle M; Giri, Shailendra

    2016-07-01

    X-linked adrenoleukodystrophy (X-ALD) is a progressive neurometabolic disease caused by mutations/deletions in the Abcd1 gene. Similar mutations/deletions in the Abcd1 gene often result in diagonally opposing phenotypes of mild adrenomyeloneuropathy and severe neuroinflammatory cerebral adrenoleukodystrophy (ALD), which suggests involvement of downstream modifier genes. We recently documented the first evidence of loss of AMP-activated protein kinase α1 (AMPKα1) in ALD patient-derived cells. Here, we report the novel loss of AMPKα1 in postmortem brain white matter of patients with ALD phenotype. Pharmacological activation of AMPK can rescue the mitochondrial dysfunction and inhibit the pro-inflammatory response. The FDA approved anti-diabetic drug Metformin, a well-known AMPK activator, induces mitochondrial biogenesis and is documented for its anti-inflammatory role. We observed a dose-dependent activation of AMPKα1 in metformin-treated X-ALD patient-derived fibroblasts. Metformin also induced mitochondrial oxidative phosphorylation and ATP levels in X-ALD patient-derived fibroblasts. Metformin treatment decreased very long chain fatty acid levels and pro-inflammatory cytokine gene expressions in X-ALD patient-derived cells. Abcd2 [adrenoleukodystrophy protein-related protein] levels were increased in metformin-treated X-ALD patient-derived fibroblasts and Abcd1-KO mice primary mixed glial cells. Abcd2 induction was AMPKα1-dependent since metformin failed to induce Abcd2 levels in AMPKα1-KO mice-derived primary mixed glial cells. In vivo metformin (100 mg/Kg) in drinking water for 60 days induced Abcd2 levels and mitochondrial oxidative phosphorylation protein levels in the brain and spinal cord of Abcd1-KO mice. Taken together, these results provide proof-of-principle for therapeutic potential of metformin as a useful strategy for correcting the metabolic and inflammatory derangements in X-ALD by targeting AMPK. There is no effective therapy for inherited

  8. Inflammatory Role of ROS-Sensitive AMP-Activated Protein Kinase in the Hypersensitivity of Lung Vagal C Fibers Induced by Intermittent Hypoxia in Rats

    PubMed Central

    Yang, Chang-Huan; Shen, Yan-Jhih; Lai, Ching Jung; Kou, Yu Ru

    2016-01-01

    Obstructive sleep apnea (OSA), manifested by airway exposure to intermittent hypoxia (IH), is associated with excess reactive oxygen species (ROS) production in airways, airway inflammation, and hyperreactive airway diseases. The cause-effect relationship for these events remains unclear. We investigated the inflammatory role of ROS-sensitive AMP-activated protein kinase (AMPK) in IH-induced airway hypersensitivity mediated by lung vagal C fibers (LVCFs) in rats. Conscious rats were exposed to room air (RA) or IH with or without treatment with N-acetyl-L-cysteine (NAC, an antioxidant), Compound C (an AMPK inhibitor), ibuprofen (a cyclooxygenase inhibitor), or their vehicles. Immediately after exposure (24 h), we found that intravenous capsaicin, phenylbiguanide, or α,β-methylene-ATP evoked augmented LVCF-mediated apneic responses and LVCF afferent responses in rats subjected to IH exposure in comparison with those in RA rats. The potentiating effect of IH on LVCF responses decreased at 6 h after and vanished at 12 h after the termination of IH exposure. The potentiating effect of IH on LVCF-mediated apneic and LVCF afferent responses was significantly attenuated by treatment with NAC, compound C, or ibuprofen, but not by their vehicles. Further biochemical analysis revealed that rats exposed to IH displayed increased lung levels of lipid peroxidation (an index of oxidative stress), AMPK phosphorylation (an index of AMPK activation), and prostaglandin E2 (a cyclooxygenase metabolite), compared with those exposed to RA. IH-induced increase in lipid peroxidation was considerably suppressed by treatment with NAC but not by compound C or ibuprofen. IH-induced increase in AMPK phosphorylation was totally abolished by NAC or compound C but not by ibuprofen. IH-induced increase in prostaglandin E2 was considerably prevented by any of these three inhibitor treatments. The vehicles of these inhibitors exerted no significant effect on the three IH-induced responses. These

  9. Chronic Glutathione Depletion Confers Protection against Alcohol-induced Steatosis: Implication for Redox Activation of AMP-activated Protein Kinase Pathway.

    PubMed

    Chen, Ying; Singh, Surendra; Matsumoto, Akiko; Manna, Soumen K; Abdelmegeed, Mohamed A; Golla, Srujana; Murphy, Robert C; Dong, Hongbin; Song, Byoung-Joon; Gonzalez, Frank J; Thompson, David C; Vasiliou, Vasilis

    2016-01-01

    The pathogenesis of alcoholic liver disease (ALD) is not well established. However, oxidative stress and associated decreases in levels of glutathione (GSH) are known to play a central role in ALD. The present study examines the effect of GSH deficiency on alcohol-induced liver steatosis in Gclm knockout (KO) mice that constitutively have ≈15% normal hepatic levels of GSH. Following chronic (6 week) feeding with an ethanol-containing liquid diet, the Gclm KO mice were unexpectedly found to be protected against steatosis despite showing increased oxidative stress (as reflected in elevated levels of CYP2E1 and protein carbonyls). Gclm KO mice also exhibit constitutive activation of liver AMP-activated protein kinase (AMPK) pathway and nuclear factor-erythroid 2-related factor 2 target genes, and show enhanced ethanol clearance, altered hepatic lipid profiles in favor of increased levels of polyunsaturated fatty acids and concordant changes in expression of genes associated with lipogenesis and fatty acid oxidation. In summary, our data implicate a novel mechanism protecting against liver steatosis via an oxidative stress adaptive response that activates the AMPK pathway. We propose redox activation of the AMPK may represent a new therapeutic strategy for preventing ALD. PMID:27403993

  10. Chronic Glutathione Depletion Confers Protection against Alcohol-induced Steatosis: Implication for Redox Activation of AMP-activated Protein Kinase Pathway

    PubMed Central

    Chen, Ying; Singh, Surendra; Matsumoto, Akiko; Manna, Soumen K.; Abdelmegeed, Mohamed A.; Golla, Srujana; Murphy, Robert C.; Dong, Hongbin; Song, Byoung-Joon; Gonzalez, Frank J.; Thompson, David C.; Vasiliou, Vasilis

    2016-01-01

    The pathogenesis of alcoholic liver disease (ALD) is not well established. However, oxidative stress and associated decreases in levels of glutathione (GSH) are known to play a central role in ALD. The present study examines the effect of GSH deficiency on alcohol-induced liver steatosis in Gclm knockout (KO) mice that constitutively have ≈15% normal hepatic levels of GSH. Following chronic (6 week) feeding with an ethanol-containing liquid diet, the Gclm KO mice were unexpectedly found to be protected against steatosis despite showing increased oxidative stress (as reflected in elevated levels of CYP2E1 and protein carbonyls). Gclm KO mice also exhibit constitutive activation of liver AMP-activated protein kinase (AMPK) pathway and nuclear factor-erythroid 2–related factor 2 target genes, and show enhanced ethanol clearance, altered hepatic lipid profiles in favor of increased levels of polyunsaturated fatty acids and concordant changes in expression of genes associated with lipogenesis and fatty acid oxidation. In summary, our data implicate a novel mechanism protecting against liver steatosis via an oxidative stress adaptive response that activates the AMPK pathway. We propose redox activation of the AMPK may represent a new therapeutic strategy for preventing ALD. PMID:27403993

  11. Piperine potentiates the effects of trans-resveratrol on stress-induced depressive-like behavior: involvement of monoaminergic system and cAMP-dependent pathway.

    PubMed

    Xu, Ying; Zhang, Chong; Wu, Feiyan; Xu, Xiaoxiao; Wang, Gang; Lin, Mengmeng; Yu, Yingcong; An, Yiran; Pan, Jianchun

    2016-08-01

    Stress can act as a precipitation factor in the onset of emotional disorders, particularly depression. Trans-resveratrol is a polyphenolic compound enriched in polygonum cuspidatum and has been found to exert antidepressant-like effects in our previous studies. In present study, we assessed the effects of trans-resveratrol used in combination with piperine, commonly known as a bioavailability enhancer, on chronic unpredictable mild stress-induced depressive-like behaviors and relevant molecular targets. Trans-resveratrol used alone reduced the immobility time of rats in the forced swimming test, with the maximal effects of trans-resveratrol around 60 % inhibition at the highest dose tested, 40 mg/kg. However, when a subthreshold dose of piperine, 2.5 mg/kg was used in combination with trans-resveratrol, the minimum effective dose of trans-resveratrol in reducing the immobility time was reduced to 20 mg/kg. Further evidence from neurochemical (monoamines in the frontal cortex and the hippocampus), biochemical (monoamine oxidase, MAO activities) and molecular biological (cAMP, PKA, CREB and BDNF) assays supported the findings in the behavioral studies. These results suggest that the co-treatment strategy with trans-resveratrol and piperine might be an alternative therapy that provides efficacious protection against chronic stress.

  12. Piperine potentiates the effects of trans-resveratrol on stress-induced depressive-like behavior: involvement of monoaminergic system and cAMP-dependent pathway.

    PubMed

    Xu, Ying; Zhang, Chong; Wu, Feiyan; Xu, Xiaoxiao; Wang, Gang; Lin, Mengmeng; Yu, Yingcong; An, Yiran; Pan, Jianchun

    2016-08-01

    Stress can act as a precipitation factor in the onset of emotional disorders, particularly depression. Trans-resveratrol is a polyphenolic compound enriched in polygonum cuspidatum and has been found to exert antidepressant-like effects in our previous studies. In present study, we assessed the effects of trans-resveratrol used in combination with piperine, commonly known as a bioavailability enhancer, on chronic unpredictable mild stress-induced depressive-like behaviors and relevant molecular targets. Trans-resveratrol used alone reduced the immobility time of rats in the forced swimming test, with the maximal effects of trans-resveratrol around 60 % inhibition at the highest dose tested, 40 mg/kg. However, when a subthreshold dose of piperine, 2.5 mg/kg was used in combination with trans-resveratrol, the minimum effective dose of trans-resveratrol in reducing the immobility time was reduced to 20 mg/kg. Further evidence from neurochemical (monoamines in the frontal cortex and the hippocampus), biochemical (monoamine oxidase, MAO activities) and molecular biological (cAMP, PKA, CREB and BDNF) assays supported the findings in the behavioral studies. These results suggest that the co-treatment strategy with trans-resveratrol and piperine might be an alternative therapy that provides efficacious protection against chronic stress. PMID:26946512

  13. Pin1 Associates with and Induces Translocation of CRTC2 to the Cytosol, Thereby Suppressing cAMP-responsive Element Transcriptional Activity*

    PubMed Central

    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

    2010-01-01

    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, Ser136 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. PMID:20675384

  14. Pin1 associates with and induces translocation of CRTC2 to the cytosol, thereby suppressing cAMP-responsive element transcriptional activity.

    PubMed

    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

    2010-10-22

    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.

  15. Stimulation of StAR expression by cAMP is controlled by inhibition of highly inducible SIK1 via CRTC2, a co-activator of CREB.

    PubMed

    Lee, Jinwoo; Tong, Tiegang; Takemori, Hiroshi; Jefcoate, Colin

    2015-06-15

    In mouse steroidogenic cells the activation of cholesterol metabolism is mediated by steroidogenic acute regulatory protein (StAR). Here, we visualized a coordinated regulation of StAR transcription, splicing and post-transcriptional processing, which are synchronized by salt inducible kinase (SIK1) and CREB-regulated transcription coactivator (CRTC2). To detect primary RNA (pRNA), spliced primary RNA (Sp-RNA) and mRNA in single cells, we generated probe sets by using fluorescence in situ hybridization (FISH). These methods allowed us to address the nature of StAR gene expression and to visualize protein-nucleic acid interactions through direct detection. We show that SIK1 represses StAR expression in Y1 adrenal and MA10 testis cells through inhibition of processing mediated by CRTC2. Digital image analysis matches qPCR analyses of the total cell culture. Evidence is presented for spatially separate accumulation of StAR pRNA and Sp-RNA at the gene loci in the nucleus. These findings establish that cAMP, SIK and CRTC mediate StAR expression through activation of individual StAR gene loci.

  16. Cell cycle regulation of cyclin A gene expression by the cyclic AMP-responsive transcription factors CREB and CREM.

    PubMed Central

    Desdouets, C; Matesic, G; Molina, C A; Foulkes, N S; Sassone-Corsi, P; Brechot, C; Sobczak-Thepot, J

    1995-01-01

    Cyclin A is a pivotal regulatory protein which, in mammalian cells, is involved in the S phase of the cell cycle. Transcription of the human cyclin A gene is cell cycle regulated. We have investigated the role of the cyclic AMP (cAMP)-dependent signalling pathway in this cell cycle-dependent control. In human diploid fibroblasts (Hs 27), induction of cyclin A gene expression at G1/S is stimulated by 8-bromo-cAMP and suppressed by the protein kinase A inhibitor H89, which was found to delay S phase entry. Transfection experiments showed that the cyclin A promoter is inducible by activation of the adenylyl cyclase signalling pathway. Stimulation is mediated predominantly via a cAMP response element (CRE) located at positions -80 to -73 with respect to the transcription initiation site and is able to bind CRE-binding proteins and CRE modulators. Moreover, activation by phosphorylation of the activators CRE-binding proteins and CRE modulator tau and levels of the inducible cAMP early repressor are cell cycle regulated, which is consistent with the pattern of cyclin A inducibility by cAMP during the cell cycle. These results suggest that the CRE is, at least partly, implicated in stimulation of cyclin A transcription at G1/S. PMID:7760825

  17. Piperidine alkaloids from Piperretrofractum Vahl. protect against high-fat diet-induced obesity by regulating lipid metabolism and activating AMP-activated protein kinase

    SciTech Connect

    Kim, Kyung Jin; Lee, Myoung-Su; Jo, Keunae; Hwang, Jae-Kwan

    2011-07-22

    Highlights: {yields} Piperidine alkaloids from Piperretrofractum Vahl. (PRPAs), including piperine, pipernonaline, and dehydropipernonaline, are isolated as the anti-obesity constituents. {yields} PRPA administration significantly reduces body weight gain without altering food intake and fat pad mass. {yields} PRPA reduces high-fat diet-induced triglyceride accumulation in liver. {yields} PRPAs attenuate HFD-induced obesity by activating AMPK and PPAR{delta}, and regulate lipid metabolism, suggesting their potential anti-obesity effects. -- Abstract: The fruits of Piperretrofractum Vahl. have been used for their anti-flatulent, expectorant, antitussive, antifungal, and appetizing properties in traditional medicine, and they are reported to possess gastroprotective and cholesterol-lowering properties. However, their anti-obesity activity remains unexplored. The present study was conducted to isolate the anti-obesity constituents from P. retrofractum Vahl. and evaluate their effects in high-fat diet (HFD)-induced obese mice. Piperidine alkaloids from P. retrofractum Vahl. (PRPAs), including piperine, pipernonaline, and dehydropipernonaline, were isolated as the anti-obesity constituents through a peroxisome proliferator-activated receptor {delta} (PPAR{delta}) transactivation assay. The molecular mechanism was investigated in 3T3-L1 adipocytes and L6 myocytes. PRPA treatment activated AMP-activated protein kinase (AMPK) signaling and PPAR{delta} protein and also regulated the expression of lipid metabolism-related proteins. In the animal model, oral PRPA administration (50, 100, or 300 mg/kg/day for 8 weeks) significantly reduced HFD-induced body weight gain without altering the amount of food intake. Fat pad mass was reduced in the PRPA treatment groups, as evidenced by reduced adipocyte size. In addition, elevated serum levels of total cholesterol, low-density lipoprotein cholesterol, total lipid, leptin, and lipase were suppressed by PRPA treatment. PRPA also

  18. Intracellular mechanisms involved in copper-gonadotropin-releasing hormone (Cu-GnRH) complex-induced cAMP/PKA signaling in female rat anterior pituitary cells in vitro.

    PubMed

    Gajewska, Alina; Zielinska-Gorska, Marlena; Wolinska-Witort, Ewa; Siawrys, Gabriela; Baran, Marta; Kotarba, Grzegorz; Biernacka, Katarzyna

    2016-01-01

    The copper-gonadotropin-releasing hormone molecule (Cu-GnRH) is a GnRH analog, which preserves its amino acid sequence, but which contains a Cu(2+) ion stably bound to the nitrogen atoms including that of the imidazole ring of Histidine(2). A previous report indicated that Cu-GnRH was able to activate cAMP/PKA signaling in anterior pituitary cells in vitro, but raised the question of which intracellular mechanism(s) mediated the Cu-GnRH-induced cAMP synthesis in gonadotropes. To investigate this mechanism, in the present study, female rat anterior pituitary cells in vitro were pretreated with 0.1 μM antide, a GnRH antagonist; 0.1 μM cetrorelix, a GnRH receptor antagonist; 0.1 μM PACAP6-38, a PAC-1 receptor antagonist; 2 μM GF109203X, a protein kinase C inhibitor; 50 mM PMA, a protein kinase C activator; the protein kinase A inhibitors H89 (30 μM) and KT5720 (60 nM); factors affecting intracellular calcium activity: 2.5 mM EGTA; 2 μM thapsigargin; 5 μM A23187, a Ca(2+) ionophore; or 10 μg/ml cycloheximide, a protein synthesis inhibitor. After one of the above pretreatments, cells were incubated in the presence of 0.1 μM Cu-GnRH for 0.5, 1, and 3 h. Radioimmunoassay analysis of cAMP confirmed the functional link between Cu-GnRH stimulation and cAMP/PKA signal transduction in rat anterior pituitary cells, demonstrating increased intracellular cAMP, which was reduced in the presence of specific PKA inhibitors. The stimulatory effect of Cu-GnRH on cAMP production was partly dependent on GnRH receptor activation. In addition, an indirect and Ca(2+)-dependent mechanism might be involved in intracellular adenylate cyclase stimulation. Neither activation of protein kinase C nor new protein synthesis was involved in the Cu-GnRH-induced increase of cAMP in the rat anterior pituitary primary cultures. Presented data indicate that conformational changes of GnRH molecule resulting from cooper ion coordination affect specific pharmacological properties of Cu

  19. Activation of AMP-Activated Protein Kinase Prevents TGF-β1–Induced Epithelial-Mesenchymal Transition and Myofibroblast Activation

    PubMed Central

    Thakur, Sachin; Viswanadhapalli, Suryavathi; Kopp, Jeffrey B.; Shi, Qian; Barnes, Jeffrey L.; Block, Karen; Gorin, Yves; Abboud, Hanna E.

    2016-01-01

    Transforming growth factor (TGF)-β contributes to tubulointerstitial fibrosis. We investigated the mechanism by which TGF-β exerts its profibrotic effects and specifically the role of AMP-activated protein kinase (AMPK) in kidney tubular epithelial cells and interstitial fibroblasts. In proximal tubular epithelial cells, TGF-β1 treatment causes a decrease in AMPK phosphorylation and activation together with increased fibronectin and α-smooth muscle actin expression and decreased in E-cadherin. TGF-β1 causes similar changes in interstitial fibroblasts. Activation of AMPK with 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside, metformin, or overexpression of constitutively active AMPK markedly attenuated TGF-β1 functions. Conversely, inhibition of AMPK with adenine 9-β-d-arabinofuranoside or siRNA-mediated knockdown of AMPK (official name PRKAA1) mimicked the effect of TGF-β1 and enhanced basal and TGF-β1–induced phenotypic changes. Importantly, we found that tuberin contributed to the protective effects of AMPK and that TGF-β1 promoted cell injury by blocking AMPK-mediated tuberin phosphorylation and activation. In the kidney cortex of TGF-β transgenic mice, the significant decrease in AMPK phosphorylation and tuberin phosphorylation on its AMPK-dependent activating site was associated with an increase in mesenchymal markers and a decrease in E-cadherin. Collectively, the data indicate that TGF-β exerts its profibrotic action in vitro and in vivo via inactivation of AMPK. AMPK and tuberin activation prevent tubulointerstitial injury induced by TGF-β. Activators of AMPK provide potential therapeutic strategy to prevent kidney fibrosis and progressive kidney disease. PMID:26071397

  20. Berberine Reduces cAMP-Induced Chloride Secretion in T84 Human Colonic Carcinoma Cells through Inhibition of Basolateral KCNQ1 Channels

    PubMed Central

    Alzamora, Rodrigo; O’Mahony, Fiona; Ko, Wing-Hung; Yip, Tiffany Wai-Nga; Carter, Derek; Irnaten, Mustapha; Harvey, Brian Joseph

    2011-01-01

    Berberine is a plant alkaloid with multiple pharmacological actions, including antidiarrhoeal activity and has been shown to inhibit Cl− secretion in distal colon. The aims of this study were to determine the molecular signaling mechanisms of action of berberine on Cl− secretion and the ion transporter targets. Monolayers of T84 human colonic carcinoma cells grown in permeable supports were placed in Ussing chambers and short-circuit current measured in response to secretagogues and berberine. Whole-cell current recordings were performed in T84 cells using the patch-clamp technique. Berberine decreased forskolin-induced short-circuit current in a concentration-dependent manner (IC50 80 ± 8 μM). In apically permeabilized monolayers and whole-cell current recordings, berberine inhibited a cAMP-dependent and chromanol 293B-sensitive basolateral membrane K+ current by 88%, suggesting inhibition of KCNQ1 K+ channels. Berberine did not affect either apical Cl− conductance or basolateral Na+–K+-ATPase activity. Berberine stimulated p38 MAPK, PKCα and PKA, but had no effect on p42/p44 MAPK and PKCδ. However, berberine pre-treatment prevented stimulation of p42/p44 MAPK by epidermal growth factor. The inhibitory effect of berberine on Cl− secretion was partially blocked by HBDDE (∼65%), an inhibitor of PKCα and to a smaller extent by inhibition of p38 MAPK with SB202190 (∼15%). Berberine treatment induced an increase in association between PKCα and PKA with KCNQ1 and produced phosphorylation of the channel. We conclude that berberine exerts its inhibitory effect on colonic Cl− secretion through inhibition of basolateral KCNQ1 channels responsible for K+ recycling via a PKCα-dependent pathway. PMID:21747769

  1. Functional analysis of chloroplast early light inducible proteins (ELIPs)

    SciTech Connect

    Wetzel, Carolyn M

    2005-02-22

    The objectives of this project were to characterize gene expression patterns of early light inducible protein (ELIP) genes in Arabidopsis thaliana and in Lycopersicon esculentum, to identify knock mutants of the 2 ELIP genes in Arabidopsis, and to characterize the effects of the knockouts. Expression in Arabidopsis was studied in response to thylakoid electron transport chain (PETC) capacity, where it was found that there is a signal for expression associated with reduction of the PETC. Expression in response to salt was also studied, with different responses of the two gene copies. Knockout lines for ELIP1 and ELIP2 have been identified and are being characterized. In tomato, it was found that the single-copy ELIP gene is highly expressed in ripening fruit during the chloroplast-to-chromoplast transition. Studies of expression in tomato ripening mutants are ongoing.

  2. Early Renal Histological Changes in Alloxan-Induced Diabetic Rats

    PubMed Central

    Pourghasem, Mohsen; Nasiri, Ebrahim; Shafi, Hamid

    2014-01-01

    Diabetes mellitus is a progressive disease. Most investigators have focused on glomerular changes in diabetic kidney and non-glomerular alterations have been less attended. The present study has been conducted to find early non-glomerular histological changes in diabetic renal tissue. Twenty male Wistar rats weighting 200-250 g were used for the diabetic group. Diabetes mellitus was induced by single injection of Alloxan. After 8 weeks, paraffin embedded blocks of kidneys were prepared for evaluating the histological changes due to diabetes. Histological study showed the deposit of eosinophilic materials in the intermediate substantial of medulla and thickening of renal arterial wall in the kidney of 70% of diabetic rats. The average weight of kidneys increased when compared to non diabetic animals. Furthermore, the amount of blood flow in arteries of all diabetic kidneys has been enhanced. The present study demonstrates some early renal histological changes in diabetes mellitus which were earlier compared to those reported previously. Diabetic nephropathy is a progressive disease and renal care design can help better prognosis achievement. PMID:24551816

  3. Metformin inhibits early stage diethylnitrosamine‑induced hepatocarcinogenesis in rats.

    PubMed

    Jo, Woori; Yu, Eun-Sil; Chang, Minsun; Park, Hyun-Kyu; Choi, Hyun-Ji; Ryu, Jae-Eun; Jang, Sungwoong; Lee, Hyo-Ju; Jang, Ja-June; Son, Woo-Chan

    2016-01-01

    Antitumor effects of metformin have recently emerged despite its original use for type II diabetes. In the present study, the effects of metformin on the development and recurrence of hepatocellular carcinoma (HCC) were investigated using the diethylnitrosamine (DEN)‑induced rat model of HCC. Tumor foci were characterized by gross examination and by histopathological characteristics, including proliferation, hepatic progenitor cell content and the expression of hepatocarcinoma‑specific molecular markers. Potential target molecules of metformin were investigated to determine the molecular mechanism underlying the inhibitory effects of metformin on chemically induced liver tumorigenesis. The antitumor effects of metformin were increased by the reduction of surface nodules and decreased the incidence of altered hepatocellular foci, hepatocellular adenoma and carcinoma. Also, decreased expression levels of glutathione S‑transferase placental form, proliferating cell nuclear antigen and cytokeratin 8 described the inhibitory effects of metformin on HCC. In the present study, Wistar rats receiving treatment with DEN were administered metformin for 16 weeks. In addition, metformin suppressed liver tumorigenesis via an AMPK‑dependent pathway. These results suggested that metformin has promising effects on the early stage of HCC in rats. Therefore, metformin may be used for the prevention of HCC recurrence following primary chemotherapy for HCC and/or for high‑risk patients, including chronic hepatitis and cirrhosis.

  4. Metformin inhibits early stage diethylnitrosamine-induced hepatocarcinogenesis in rats

    PubMed Central

    JO, WOORI; YU, EUN-SIL; CHANG, MINSUN; PARK, HYUN-KYU; CHOI, HYUN-JI; RYU, JAE-EUN; JANG, SUNGWOONG; LEE, HYO-JU; JANG, JA-JUNE; SON, WOO-CHAN

    2016-01-01

    Antitumor effects of metformin have recently emerged despite its original use for type II diabetes. In the present study, the effects of metformin on the development and recurrence of hepatocellular carcinoma (HCC) were investigated using the diethylnitrosamine (DEN)-induced rat model of HCC. Tumor foci were characterized by gross examination and by histopathological characteristics, including proliferation, hepatic progenitor cell content and the expression of hepatocarcinoma-specific molecular markers. Potential target molecules of metformin were investigated to determine the molecular mechanism underlying the inhibitory effects of metformin on chemically induced liver tumorigenesis. The antitumor effects of metformin were increased by the reduction of surface nodules and decreased the incidence of altered hepatocellular foci, hepatocellular adenoma and carcinoma. Also, decreased expression levels of glutathione S-transferase placental form, proliferating cell nuclear antigen and cytokeratin 8 described the inhibitory effects of metformin on HCC. In the present study, Wistar rats receiving treatment with DEN were administered metformin for 16 weeks. In addition, metformin suppressed liver tumorigenesis via an AMPK-dependent pathway. These results suggested that metformin has promising effects on the early stage of HCC in rats. Therefore, metformin may be used for the prevention of HCC recurrence following primary chemotherapy for HCC and/or for high-risk patients, including chronic hepatitis and cirrhosis. PMID:26548419

  5. Early Administration of Carvedilol Protected against Doxorubicin-Induced Cardiomyopathy.

    PubMed

    Chen, Yung-Lung; Chung, Sheng-Ying; Chai, Han-Tan; Chen, Chih-Hung; Liu, Chu-Feng; Chen, Yi-Ling; Huang, Tien-Hung; Zhen, Yen-Yi; Sung, Pei-Hsun; Sun, Cheuk-Kwan; Chua, Sarah; Lu, Hung-I; Lee, Fan-Yen; Sheu, Jiunn-Jye; Yip, Hon-Kan

    2015-12-01

    This study tested for the benefits of early administration of carvedilol as protection against doxorubicin (DOX)-induced cardiomyopathy. Thirty male, adult B6 mice were categorized into group 1 (untreated control), group 2 [DOX treatment (15 mg/every other day for 2 weeks, i.p.], and group 3 [carvedilol (15 mg/kg/d, from day 7 after DOX treatment for 28 days)], and euthanized by day 35 after DOX treatment. By day 35, the left ventricular ejection fraction (LVEF) was significantly lower in group 2 than in groups 1 and 3, and significantly lower in group 3 than in group 1, whereas the left ventricular (LV) end-diastolic and LV end-systolic dimensions showed an opposite pattern to the LVEF among the three groups. The protein expressions of fibrotic (Smad3, TGF-β), apoptotic (BAX, cleaved caspase 3, PARP), DNA damage (γ-H2AX), oxidative stress (oxidized protein), mitochondrial damage (cytosolic cytochrome-C), heart failure (brain natriuretic peptide), and hypertrophic (β-MHC) biomarkers of the LV myocardium showed an opposite pattern to the LVEF among the three groups. The protein expressions of antifibrotic (BMP-2, Smad1/5), α-MHC, and phosphorylated-Akt showed an identical pattern to the LVEF among the three groups. The microscopic findings of fibrotic and collagen-deposition areas and the numbers of γ-H2AX(+) and 53BP1(+) cells in the LV myocardium exhibited an opposite pattern, whereas the numbers of endothelial cell (CD31(+), vWF(+)) markers showed an identical pattern to the LVEF among the three groups. Cardiac stem cell markers (C-kit(+) and Sca-1(+) cells) were significantly and progressively increased from group 1 to group 3. Additionally, the in vitro study showed carvedilol treatment significantly inhibited DOX-induced cardiomyoblast DNA (CD90/XRCC1(+), CD90/53BP1(+), and r-H2AX(+) cells) damage. Early carvedilol therapy protected against DOX-induced DNA damage and cardiomyopathy.

  6. Early Administration of Carvedilol Protected against Doxorubicin-Induced Cardiomyopathy.

    PubMed

    Chen, Yung-Lung; Chung, Sheng-Ying; Chai, Han-Tan; Chen, Chih-Hung; Liu, Chu-Feng; Chen, Yi-Ling; Huang, Tien-Hung; Zhen, Yen-Yi; Sung, Pei-Hsun; Sun, Cheuk-Kwan; Chua, Sarah; Lu, Hung-I; Lee, Fan-Yen; Sheu, Jiunn-Jye; Yip, Hon-Kan

    2015-12-01

    This study tested for the benefits of early administration of carvedilol as protection against doxorubicin (DOX)-induced cardiomyopathy. Thirty male, adult B6 mice were categorized into group 1 (untreated control), group 2 [DOX treatment (15 mg/every other day for 2 weeks, i.p.], and group 3 [carvedilol (15 mg/kg/d, from day 7 after DOX treatment for 28 days)], and euthanized by day 35 after DOX treatment. By day 35, the left ventricular ejection fraction (LVEF) was significantly lower in group 2 than in groups 1 and 3, and significantly lower in group 3 than in group 1, whereas the left ventricular (LV) end-diastolic and LV end-systolic dimensions showed an opposite pattern to the LVEF among the three groups. The protein expressions of fibrotic (Smad3, TGF-β), apoptotic (BAX, cleaved caspase 3, PARP), DNA damage (γ-H2AX), oxidative stress (oxidized protein), mitochondrial damage (cytosolic cytochrome-C), heart failure (brain natriuretic peptide), and hypertrophic (β-MHC) biomarkers of the LV myocardium showed an opposite pattern to the LVEF among the three groups. The protein expressions of antifibrotic (BMP-2, Smad1/5), α-MHC, and phosphorylated-Akt showed an identical pattern to the LVEF among the three groups. The microscopic findings of fibrotic and collagen-deposition areas and the numbers of γ-H2AX(+) and 53BP1(+) cells in the LV myocardium exhibited an opposite pattern, whereas the numbers of endothelial cell (CD31(+), vWF(+)) markers showed an identical pattern to the LVEF among the three groups. Cardiac stem cell markers (C-kit(+) and Sca-1(+) cells) were significantly and progressively increased from group 1 to group 3. Additionally, the in vitro study showed carvedilol treatment significantly inhibited DOX-induced cardiomyoblast DNA (CD90/XRCC1(+), CD90/53BP1(+), and r-H2AX(+) cells) damage. Early carvedilol therapy protected against DOX-induced DNA damage and cardiomyopathy. PMID:26511374

  7. Nectandrin B, a lignan isolated from nutmeg, inhibits liver X receptor-α-induced hepatic lipogenesis through AMP-activated protein kinase activation.

    PubMed

    Choi, Du Gon; Kim, Eun Kyung; Yang, Jin Won; Song, Jae Sook; Kim, Young-Mi

    2015-11-01

    Nonalcoholic fatty liver disease is recognized as the most commonly occurring chronic liver disease. Liver X receptor α (LXRα) and sterol regulatory element-binding protein (SREBP)-1c play a central role in de novo fatty acid synthesis. This study investigated pharmacological effects of nectandrin B, a lignan isolated from nutmeg extract, on hepatic lipogenesis stimulated by LXRα-SREBP-1c-mediated pathway and the possible molecular basis. The reporter gene assay revealed that nectandrin B completely represses LXRα activity enhanced by a synthetic LXRα ligand (T0901317) in HepG2 cells. The inhibitory effect was further supported by the suppression of mRNA expression of LXRα target genes, SREBP-1c and LXRα itself. Nectandrin B also inhibited the increase in SREBP-1c expression promoted by insulin plus high glucose, major contributors to hepatic lipid accumulation. LXRα-SREBP-1c-mediated induction of acetyl-CoA carboxylase 1 and fatty acid synthase, major genes for de novo lipogenesis, was suppressed by nectandrin B. Moreover, Oil Red O staining showed that nectandrin B notably attenuates LXRα-induced lipid accumulation. AMP-activated protein kinase (AMPK) inhibits the activities of LXRα and SREBP-1c. Nectandrin B strongly activated AMPK signaling in HepG2 cells. Taken together, the suppressive effects of nectandrin B on lipogenic gene expression and lipid accumulation in hepatocytes may be due to its inhibitory effect on the LXRα-SREBP-1c pathway presumably via AMPK activation. These results suggest the potential of nectandrin B as a therapeutic candidate for fatty liver disease. PMID:26790190

  8. Head-down posture induces PERG alterations in early glaucoma

    PubMed Central

    Ventura, Lori M.; Golubev, Iuri; Lee, William; Nose, Izuru; Parel, Jean-Marie; Feuer, William J.; Porciatti, Vittorio

    2011-01-01

    Purpose To probe susceptibility of retinal ganglion cells (RGC) to physiological stressors associated with moderate head-down body tilt in patients with suspicion of glaucoma or early manifest glaucoma. Methods One hundred nine subjects with best corrected visual acuity ≥20/20 and no disease other than glaucoma (glaucoma suspects, GS= 79, early manifest glaucoma, EMG=14, normal controls, NC= 16) and comparable age range were tested. Non-contact IOP, pattern electroretinogram (PERG), and brachial blood pressure/heart rate measurements were performed in three consecutive (~8 minutes apart) conditions: seated (baseline), −10 deg whole body head-down (HDT), and seated again (recovery). PERG amplitude and latency, IOP, and systolic/diastolic blood pressures, heart rate, calculated mean central retinal artery pressure, ocular perfusion pressure, and systolic/diastolic perfusion pressures were evaluated. Results During HDT, IOP significantly (P<0.001) increased in all groups approximately to the same extent (~20%). PERG amplitude did not change in NC but decreased significantly (P<0.001) in patients (GS, −25%, EMG −23%). PERG phase become delayed in NC (− 1.6%, P=0.04) but more so in patients (GS, −2.7%, P<0.001; EMG, −6.0%, P<0.001). The proportion of patients with PERG alterations significantly (P<0.05) exceeding those occurring in age- and baseline-adjusted NC were, GS: amplitude 20%, phase 15%; EMG: amplitude 14%, phase 50%. All measures recovered baseline values after HDT. Conclusions Moderate HDT induces temporary worsening of RGC function in a subpopulation of GS and EMG patients. This non-invasive protocol may help disclose abnormal susceptibility of RGCs in a subset of the patients at risk of glaucoma. PMID:22138883

  9. AMPED Program Overview

    ScienceCinema

    Gur, Ilan

    2016-07-12

    An overview presentation about ARPA-E's AMPED program. AMPED projects seek to develop advanced sensing, control, and power management technologies that redefine the way we think about battery management. Energy storage can significantly improve U.S. energy independence, efficiency, and security by enabling a new generation of electric vehicles. While rapid progress is being made in new battery materials and storage technologies, few innovations have emerged in the management of advanced battery systems. AMPED aims to unlock enormous untapped potential in the performance, safety, and lifetime of today's commercial battery systems exclusively through system-level innovations, and is thus distinct from existing efforts to enhance underlying battery materials and architectures.

  10. AMPED Program Overview

    SciTech Connect

    Gur, Ilan

    2014-03-04

    An overview presentation about ARPA-E's AMPED program. AMPED projects seek to develop advanced sensing, control, and power management technologies that redefine the way we think about battery management. Energy storage can significantly improve U.S. energy independence, efficiency, and security by enabling a new generation of electric vehicles. While rapid progress is being made in new battery materials and storage technologies, few innovations have emerged in the management of advanced battery systems. AMPED aims to unlock enormous untapped potential in the performance, safety, and lifetime of today's commercial battery systems exclusively through system-level innovations, and is thus distinct from existing efforts to enhance underlying battery materials and architectures.

  11. Pituitary adenylate cyclase-activating peptide induces long-lasting neuroprotection through the induction of activity-dependent signaling via the cyclic AMP response element-binding protein-regulated transcription co-activator 1

    PubMed Central

    Baxter, Paul S; Martel, Marc-Andre; McMahon, Aoife; Kind, Peter C; Hardingham, Giles E

    2011-01-01

    Pituitary adenylate cyclase-activating peptide (PACAP) is a neuroprotective peptide which exerts its effects mainly through the cAMP-protein kinase A (PKA) pathway. Here, we show that in cortical neurons, PACAP-induced PKA signaling exerts a major part of its neuroprotective effects indirectly, by triggering action potential (AP) firing. Treatment of cortical neurons with PACAP induces a rapid and sustained PKA-dependent increase in AP firing and associated intracellular Ca2+ transients, which are essential for the anti-apoptotic actions of PACAP. Transient exposure to PACAP induces long-lasting neuroprotection in the face of apoptotic insults which is reliant on AP firing and the activation of cAMP response element (CRE) binding protein (CREB)-mediated gene expression. Although direct, activity-independent PKA signaling is sufficient to trigger phosphorylation on CREB’s activating serine-133 site, this is insufficient for activation of CREB-mediated gene expression. Full activation is dependent on CREB-regulated transcription co-activator 1 (CRTC1), whose PACAP-induced nuclear import is dependent on firing activity-dependent calcineurin signaling. Over-expression of CRTC1 is sufficient to rescue PACAP-induced CRE-mediated gene expression in the face of activity-blockade, while dominant negative CRTC1 interferes with PACAP-induced, CREB-mediated neuroprotection. Thus, the enhancement of AP firing may play a significant role in the neuroprotective actions of PACAP and other adenylate cyclase-coupled ligands. PMID:21623792

  12. Limonin, a Component of Dictamni Radicis Cortex, Inhibits Eugenol-Induced Calcium and cAMP Levels and PKA/CREB Signaling Pathway in Non-Neuronal 3T3-L1 Cells.

    PubMed

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

    2015-12-10

    Limonin, one of the major components in dictamni radicis cortex (DRC), has been shown to play various biological roles in cancer, inflammation, and obesity in many different cell types and tissues. Recently, the odorant-induced signal transduction pathway (OST) has gained attention not only because of its function in the perception of smell but also because of its numerous physiological functions in non-neuronal cells. However, little is known about the effects of limonin and DRC on the OST pathway in non-neuronal cells. We investigated odorant-stimulated increases in Ca(2+) and cAMP, major second messengers in the OST pathway, in non-neuronal 3T3-L1 cells pretreated with limonin and ethanol extracts of DRC. Limonin and the extracts significantly decreased eugenol-induced Ca(2+) and cAMP levels and upregulated phosphorylation of CREB and PKA. Our results demonstrated that limonin and DRC extract inhibit the OST pathway in non-neuronal cells by modulating Ca(2+) and cAMP levels and phosphorylation of CREB.

  13. Early corticosteroid administration in experimental radiation-induced heart disease

    SciTech Connect

    Reeves, W.C.; Stryker, J.A.; Abt, A.A.; Chung, C.K.; Whitesell, L.; Zelis, R.

    1980-02-01

    The ability of dexamethasone (DEX) to reduce the severity of the late stage of radiation-induced heart disease (RIHD) was assessed in 25 New Zealand white rabbits. Ten rabbits served as unirradiated controls (CONT). In Group A, seven rabbits received intravenous DEX prior to irradiation and every 24 hours for three consecutive days. DEX was not administered to the eight rabbits in Group B. At 100 days postirradiation, the severity of the late state was determined by microscopic examination (MICRO) for myocardial fibrosis and determination of myocardial hydroxyproline content (MHP). Myocardial fibrosis was evident in groups A (40%) and B (80%) while none was present in CONT by MICRO. One rabbit in Group B with no fibrosis by MICRO had abnormally increased MHP. MHP was significantly increased in Groups A and B, as compared to CONT (p < 0.01). In addition to less fibrosis by MICRO, Group A demonstrated a significant reduction of MHP when compared to Group B (p < 0.05). Determination of MHP may be superior to MICRO in the detection of the late stage of RIHD. Also, early DEX administration appears to reduce myocardial collagen content (fibrosis) in this experimental model.

  14. Early light-induced proteins protect Arabidopsis from photooxidative stress.

    PubMed

    Hutin, Claire; Nussaume, Laurent; Moise, Nicolae; Moya, Ismaël; Kloppstech, Klaus; Havaux, Michel

    2003-04-15

    The early light-induced proteins (ELIPs) belong to the multigenic family of light-harvesting complexes, which bind chlorophyll and absorb solar energy in green plants. ELIPs accumulate transiently in plants exposed to high light intensities. By using an Arabidopsis thaliana mutant (chaos) affected in the posttranslational targeting of light-harvesting complex-type proteins to the thylakoids, we succeeded in suppressing the rapid accumulation of ELIPs during high-light stress, resulting in leaf bleaching and extensive photooxidative damage. Constitutive expression of ELIP genes in chaos before light stress resulted in ELIP accumulation and restored the phototolerance of the plants to the wild-type level. Free chlorophyll, a generator of singlet oxygen in the light, was detected by chlorophyll fluorescence lifetime measurements in chaos leaves before the symptoms of oxidative stress appeared. Our findings indicate that ELIPs fulfill a photoprotective function that could involve either the binding of chlorophylls released during turnover of pigment-binding proteins or the stabilization of the proper assembly of those proteins during high-light stress. PMID:12676998

  15. Cyclic AMP efflux inhibitors as potential therapeutic agents for leukemia

    PubMed Central

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

    2016-01-01

    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

  16. Directed evolution of the Escherichia coli cAMP receptor protein at the cAMP pocket.

    PubMed

    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

    2015-10-30

    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.

  17. cAMP-induced phosphorylation of 26S proteasomes on Rpn6/PSMD11 enhances their activity and the degradation of misfolded proteins.

    PubMed

    Lokireddy, Sudarsanareddy; Kukushkin, Nikolay Vadimovich; Goldberg, Alfred Lewis

    2015-12-29

    Although rates of protein degradation by the ubiquitin-proteasome pathway (UPS) are determined by their rates of ubiquitination, we show here that the proteasome's capacity to degrade ubiquitinated proteins is also tightly regulated. We studied the effects of cAMP-dependent protein kinase (PKA) on proteolysis by the UPS in several mammalian cell lines. Various agents that raise intracellular cAMP and activate PKA (activators of adenylate cyclase or inhibitors of phosphodiesterase 4) promoted degradation of short-lived (but not long-lived) cell proteins generally, model UPS substrates having different degrons, and aggregation-prone proteins associated with major neurodegenerative diseases, including mutant FUS (Fused in sarcoma), SOD1 (superoxide dismutase 1), TDP43 (TAR DNA-binding protein 43), and tau. 26S proteasomes purified from these treated cells or from control cells and treated with PKA degraded ubiquitinated proteins, small peptides, and ATP more rapidly than controls, but not when treated with protein phosphatase. Raising cAMP levels also increased amounts of doubly capped 26S proteasomes. Activated PKA phosphorylates the 19S subunit, Rpn6/PSMD11 (regulatory particle non-ATPase 6/proteasome subunit D11) at Ser14. Overexpression of a phosphomimetic Rpn6 mutant activated proteasomes similarly, whereas a nonphosphorylatable mutant decreased activity. Thus, proteasome function and protein degradation are regulated by cAMP through PKA and Rpn6, and activation of proteasomes by this mechanism may be useful in treating proteotoxic diseases.

  18. Different effect of prostaglandin E2 on B-cell activation by two distinct B-cell differentiation factors, B151-TRF1/IL-5 and B151-TRF2: selective inhibition of B151-TRF2-induced antibody response through increases in intracellular cyclic AMP levels

    PubMed Central

    Ishihara, K.; Ono, S.; Takahama, Y.; Hirayama, F.; Hirano, H.; Itoh, K.; Dobashi, K.; Murakami, S.; Katoh, Y.; Yamaguchi, M.; Hamaoka, T.

    1989-01-01

    Effects of prostaglandin E2 (PGE2) on murine B-cell activation induced by two distinct B-cell differentiation factors, B151-TRF1/IL-5 and B151-TRF2, were examined. A final differentiation of unprimed B cells into IgM-producing cells induced by B151-TRF2 was markedly inhibited by PGE2 at physiological concentrations (around 10-8 M), whereas B151-TRF1/IL-5-induced antibody responses of unprimed as well as activated B cells were not affected by PGE2, even at 10-6 M. B-cell responses induced by B151-TRF2-like factors from autoimmune-prone MRL/1pr mice were also inhibited by PGE2. Biphasic increases in intracellular cyclic AMP (cAMP) levels were induced by culturing B cells with 10-6 or 10-8 M PGE2: rapid increases within 8 min and delayed increases around 16 hr. The direct addition of dibutyryl cAMP to cultures of B cells resulted in marked inhibition of antibody responses when stimulated with B151-TRF2 but not with B151-TRF1/IL-5. The B151-TRF2-induced antibody responses were also inhibited by cAMP-elevating reagents such as forskolin, cholera toxin and theophyline. Furthermore, 2′, 5′-dideoxyadenosine, which is an inhibitor of adenylate cyclase, prevented the PGE2-mediated cAMP accumulation in unprimed B cells as well as the PGE2-mediated inhibition of B151-TRF2-induced B-cell responses when added at the initiation of culture. These results suggest that PGE2 inhibits B151-TRF2-induced antibody responses through the activation of adenylate cyclase and subsequent accumulation of intracellular cAMP, whereas B151-TRF1/IL-5-responsive B cells are resistant to the inhibitory effect of PGE2 and cAMP. PMID:2553585

  19. Early life exposure to air pollution induces adult cardiac dysfunction.

    PubMed

    Gorr, Matthew W; Velten, Markus; Nelin, Timothy D; Youtz, Dane J; Sun, Qinghua; Wold, Loren E

    2014-11-01

    Exposure to ambient air pollution contributes to the progression of cardiovascular disease, particularly in susceptible populations. The objective of the present study was to determine whether early life exposure to air pollution causes persistent cardiovascular consequences measured at adulthood. Pregnant FVB mice were exposed to filtered (FA) or concentrated ambient particulate matter (PM2.5) during gestation and nursing. Mice were exposed to PM2.5 at an average concentration of 51.69 μg/m(3) from the Columbus, OH region for 6 h/day, 7 days/wk in utero until weaning at 3 wk of age. Birth weight was reduced in PM2.5 pups compared with FA (1.36 ± 0.12 g FA, n = 42 mice; 1.30 ± 0.15 g PM2.5, n = 67 P = 0.012). At adulthood, mice exposed to perinatal PM2.5 had reduced left ventricular fractional shortening compared with FA-exposed mice (43.6 ± 2.1% FA, 33.2 ± 1.6% PM2.5, P = 0.001) with greater left ventricular end systolic diameter. Pressure-volume loops showed reduced ejection fraction (79.1 ± 3.5% FA, 35.5 ± 9.5% PM2.5, P = 0.005), increased end-systolic volume (10.4 ± 2.5 μl FA, 39.5 ± 3.8 μl PM2.5, P = 0.001), and reduced dP/dt maximum (11,605 ± 200 μl/s FA, 9,569 ± 800 μl/s PM2.5, P = 0.05) and minimum (-9,203 ± 235 μl/s FA, -7,045 ± 189 μl/s PM2.5, P = 0.0005) in PM2.5-exposed mice. Isolated cardiomyocytes from the hearts of PM2.5-exposed mice had reduced peak shortening (%PS, 8.53 ± 2.82% FA, 6.82 ± 2.04% PM2.5, P = 0.003), slower calcium reuptake (τ, 0.22 ± 0.09 s FA, 0.26 ± 0.07 s PM2.5, P = 0.048), and reduced response to β-adrenergic stimulation compared with cardiomyocytes isolated from mice that were exposed to FA. Histological analyses revealed greater picro-sirius red-positive-stained areas in the PM2.5 vs. FA group, indicative of increased collagen deposition. We concluded that these data demonstrate the detrimental role of early life exposure to ambient particulate air pollution in programming of adult cardiovascular

  20. Coupling gene expression to cAMP signalling: role of CREB and CREM.

    PubMed

    Sassone-Corsi, P

    1998-01-01

    Several endocrine and neuronal functions are governed by the cAMP-dependent pathway. Transcriptional regulation upon stimulation of this pathway is mediated by a family of cAMP-responsive nuclear factors. This family consists of a large number of members, which may act as activators or repressors. These factors contain the basic domain/leucine zipper motifs and bind as dimers to cAMP-response elements (CRE). CRE-binding protein (CREBs) function is modulated by phosphorylation by several kinases. Direct activation of gene expression by CREB requires phosphorylation by the cAMP-dependent PKA to serine 133. Among the repressors, ICER (Inducible cAMP Early Repressor) deserves special mention. ICER is generated from an alternative CREM promoter and is the only inducible CRE-binding protein. ICER negatively autoregulates the alternative promoter, generating a feedback loop. ICER expression is tissue specific and developmentally regulated. The kinetics of ICER expression are characteristic of an early response gene. CREM plays a key physiological and developmental role within the hypothalamic-pituitary-gonadal axis. The transcriptional activator CREM is highly expressed in postmeiotic cells. The role of CREM in spermiogenesis was addressed using CREM knock-out mice. Spermatogenesis stops at the first step of spermiogenesis in the mutants and there is a significant increase in apoptotic germ cells. This phenotype is reminiscent of cases of human infertility. ICER is regulated in a circadian manner in the pineal gland, the site of the hormone melatonin production. This night-day oscillation is driven by the endogenous clock (located in the suprachiasmatic nucleus). The synthesis of melatonin is regulated by a rate-limiting enzyme, serotonin N-acetyltransferase (NAT). Analysis of the CREM-null mice and of the promoter of the NAT gene revealed that ICER controls the amplitude and rhythmicity of NAT, and thus the oscillation in the hormonal synthesis of melatonin.

  1. Thymosin alpha 1 antagonizes dexamethasone and CD3-induced apoptosis of CD4+ CD8+ thymocytes through the activation of cAMP and protein kinase C dependent second messenger pathways.

    PubMed

    Baumann, C A; Badamchian, M; Goldstein, A L

    1997-03-01

    It is well established that glucocorticoid hormones and anti-CD3 monoclonal antibodies induce apoptosis in immature developing thymocytes. This process can be modulated by soluble factors, anti-oxidants and adhesion receptors. Previously we have demonstrated that thymosin alpha 1 (T alpha 1), a 28-amino acid thymic peptide hormone, is a dose and time dependent antagonist of dexamethasone (DEX) and CD# induced DNA fragmentation of murine thymocytes in vitro. To further investigate the mechanism of T alpha 1 action we determined a T alpha 1 sensitive thymocyte population and examined some of the molecular events associated with T alpha 1 anti-apoptotic activity. Phenotypic analysis of the sub-populations of thymocytes, based on CD4 and CD8 expression, revealed that T alpha 1 exerts its effect on CD4+ CD8+ immature thymocytes. T alpha 1 treatment of thymocytes delays the production of free radicals and the subsequent consumption of glutathione, that is observed during both DEX and CD3 induced apoptosis. We further demonstrate that T alpha 1 stimulates the production of cAMP and activates PKC in thymocytes. These data suggest that T alpha 1 exerts an influence on the development of a population of immature T-cells in the thymus by effecting the sensitivity of thymocytes to apoptosis during the pre-selection stages of thymic development. Our studies also suggest that the mechanism of T alpha 1 action involves the induction of both cAMP and PKC dependent second messenger pathways.

  2. The local corticotropin-releasing hormone receptor 2 signalling pathway partly mediates hypoxia-induced increases in lipolysis via the cAMP-protein kinase A signalling pathway in white adipose tissue.

    PubMed

    Xiong, Yanlei; Qu, Zhuan; Chen, Nan; Gong, Hui; Song, Mintao; Chen, Xuequn; Du, Jizeng; Xu, Chengli

    2014-07-01

    Our objective was to investigate the mechanisms by which the endogenous CRHR2 in white adipose tissue (WAT) regulates metabolic activities associated with lipogenesis and lipolysis under continuous exposure to hypoxia. We found that hypobaric hypoxia at a simulated altitude of 5000 m significantly reduced the body weight, food intake, and WAT mass of rats. Hypoxia also accelerated lipolysis and suppressed lipogenesis in WAT. Pretreatment with astressin 2B, a selective CRHR2 antagonist, partly but significantly attenuated the hypoxia-induced reductions in body weight and WAT mass by blocking the cAMP-protein kinase A (PKA)-hormone-sensitive lipase (HSL)/perilipin signalling pathway. Astressin 2B treatment failed to attenuate hypoxia induced lipogenic inhibition. In conclusion, activation of endogenous WAT Ucn2/3 autocrine/paracrine pathway was involved in hypoxia induced lipolysis via CRHR2 - cAMP-PKA signalling pathway. This study provides the novel understanding of local CRHR2 signaling pathway playing important role in WAT loss and lipid metabolism under hypoxia.

  3. Chrysin abrogates early hepatocarcinogenesis and induces apoptosis in N-nitrosodiethylamine-induced preneoplastic nodules in rats

    SciTech Connect

    Khan, Mahaboob S.; Devaraj, Halagowder; Devaraj, Niranjali

    2011-02-15

    Flavonoids possess strong anti-oxidant and cancer chemopreventive activities. Chrysin (5,7-dihydroxyflavone) occurs naturally in many plants, honey, and propolis. In vitro, chrysin acts as a general anti-oxidant, causes cell cycle arrest and promotes cell death. However, the mechanism by which chrysin inhibits cancer cell growth and the subcellular pathways activated remains poorly understood. Effect of dietary supplementation with chrysin on proliferation and apoptosis during diethylnitrosamine (DEN)-induced early hepatocarcinogenesis was investigated in male Wistar rats. To induce hepatocarcinogenesis, rats were given DEN injections (i.p., 200 mg/kg) three times at a 15 day interval. An oral dose of chrysin (250 mg/kg bodyweight) was given three times weekly for 3 weeks, commencing 1 week after the last dose of DEN. Changes in the mRNA expression of COX-2, NFkB p65, p53, Bcl-xL and {beta}-arrestin-2 were assessed by quantitative real-time PCR. Changes in the protein levels were measured by western blotting. Chrysin administration significantly (P < 0.001) reduced the number and size of nodules formed. Also, a significant (P < 0.01) reduction in serum activities of AST, ALT, ALP, LDH and {gamma}GT was noticed. Expression of COX-2 and NFkB p65 was significantly reduced whereas that of p53, Bax and caspase 3 increased at the mRNA and protein levels. Likewise, a decrease in levels of {beta}-arrestin and the anti-apoptotic marker Bcl-xL was also noted. These findings suggest that chrysin exerts global hepato-protective effect and its chemopreventive activity is associated with p53-mediated apoptosis during early hepatocarcinogenesis.

  4. Inflammation-induced microvascular insulin resistance is an early event in diet-induced obesity.

    PubMed

    Zhao, Lina; Fu, Zhuo; Wu, Jing; Aylor, Kevin W; Barrett, Eugene J; Cao, Wenhong; Liu, Zhenqi

    2015-12-01

    Endothelial dysfunction and vascular insulin resistance usually coexist and chronic inflammation engenders both. In the present study, we investigate the temporal relationship between vascular insulin resistance and metabolic insulin resistance. We assessed insulin responses in all arterial segments, including aorta, distal saphenous artery and the microvasculature, as well as the metabolic insulin responses in muscle in rats fed on a high-fat diet (HFD) for various durations ranging from 3 days to 4 weeks with or without sodium salicylate treatment. Compared with controls, HFD feeding significantly blunted insulin-mediated Akt (protein kinase B) and eNOS [endothelial nitric oxide (NO) synthase] phosphorylation in aorta in 1 week, blunted vasodilatory response in small resistance vessel in 4 weeks and microvascular recruitment in as early as 3 days. Insulin-stimulated whole body glucose disposal did not begin to progressively decrease until after 1 week. Salicylate treatment fully inhibited vascular inflammation, prevented microvascular insulin resistance and significantly improved muscle metabolic responses to insulin. We conclude that microvascular insulin resistance is an early event in diet-induced obesity and insulin resistance and inflammation plays an essential role in this process. Our data suggest microvascular insulin resistance contributes to the development of metabolic insulin resistance in muscle and muscle microvasculature is a potential therapeutic target in the prevention and treatment of diabetes and its related complications.

  5. Applying Mathematical Processes (AMP)

    ERIC Educational Resources Information Center

    Kathotia, Vinay

    2011-01-01

    This article provides insights into the "Applying Mathematical Processes" resources, developed by the Nuffield Foundation. It features Nuffield AMP activities--and related ones from Bowland Maths--that were designed to support the teaching and assessment of key processes in mathematics--representing a situation mathematically, analysing,…

  6. Inducible expression of the chromosomal cdiA from Citrobacter diversus NF85, encoding an ambler class A beta-lactamase, is under similar genetic control to the chromosomal ampC, encoding an ambler class C enzyme, from Citrobacter freundii OS60.

    PubMed

    Jones, M E; Bennett, P M

    1995-01-01

    This study aimed to characterize the molecular basis of beta-lactamase induction in Citrobacter diversus. The chromosomal beta-lactamase encoding region from C. diversus, strain NF85, was cloned and expressed in Escherichia coli. The cloned region was sequenced and open-reading frames encoding a class A beta-lactamase, designated cdiA, and a putative LysR-type transcriptional regulator protein, divergently transcribed from the beta-lactamase gene and designated cdiR, were identified. The nucleotide sequence of the NF85 cdiA was identical to that of the published C. diversus ULA27 ampC sequence. A putative helix-turn-helix DNA-binding motif was located at the N-terminus of CdiR, and homology with enterobacterial AmpR proteins was noted. CdiR was demonstrated to bind to the C. diversus cdiAR intergenic region but not to the C. freundii ampCR intergenic region. A putative CdiR binding motif was identified in the cdiAR intergenic region. The cloned cdiAR region was inducible in E. coli strains SNO3 and HfrH. The inducible phenotype was dependent on the E. coli ampD and ampG gene products. We conclude that the molecular basis of inducible cdiA expression in C. diversus is similar to that of C. freundii ampC.

  7. Calmodulin-dependent protein kinase II/cAMP response element-binding protein/Wnt/β-catenin signaling cascade regulates angiotensin II-induced podocyte injury and albuminuria.

    PubMed

    Jiang, Lei; Xu, Lingling; Song, Yuxian; Li, Jianzhong; Mao, Junhua; Zhao, Allan Zijian; He, Weichun; Yang, Junwei; Dai, Chunsun

    2013-08-01

    Angiotensin II (Ang II) plays a pivotal role in promoting podocyte dysfunction and albuminuria, however, the underlying mechanisms have not been fully delineated. In this study, we found that Ang II induced Wnt1 expression and β-catenin nuclear translocation in cultured mouse podocytes. Blocking Wnt signaling with Dickkopf-1 (Dkk1) or β-catenin siRNA attenuated Ang II-induced podocyte injury. Ang II could also induce the phosphorylation of calmodulin-dependent protein kinase (CaMK) II and cAMP response element-binding protein (CREB) in cultured podocytes. Blockade of this pathway with CK59 or CREB siRNA could significantly inhibit Ang II-induced Wnt/β-catenin signaling and podocyte injury. In in vivo studies, administration of Ang II promoted Wnt/β-catenin signaling, aggregated podocyte damage, and albuminuria in mice. CK59 could remarkably ameliorate Ang II-induced podocyte injury and albuminuria. Furthermore, ectopic expression of exogenous Dkk1 also attenuated Ang II-induced podocytopathy in mice. Taken together, this study demonstrates that the CaMK II/CREB/Wnt/β-catenin signaling cascade plays an important role in regulating Ang II-induced podocytopathy. Targeting this signaling pathway may offer renal protection against the development of proteinuric kidney diseases. PMID:23803607

  8. A Fluorescent Transport Assay Enables Studying AmpG Permeases Involved in Peptidoglycan Recycling and Antibiotic Resistance.

    PubMed

    Perley-Robertson, G Evan; Yadav, Anuj K; Winogrodzki, Judith L; Stubbs, Keith A; Mark, Brian L; Vocadlo, David J

    2016-09-16

    Inducible AmpC β-lactamases deactivate a broad-spectrum of β-lactam antibiotics and afford antibiotic resistance in many Gram-negative bacteria. The disturbance of peptidoglycan recycling caused by β-lactam antibiotics leads to accumulation of GlcNAc-1,6-anhydroMurNAc-peptides, which are transported by AmpG to the cytoplasm where they are processed into AmpC inducers. AmpG transporters are poorly understood; however, their loss restores susceptibility toward β-lactam antibiotics, highlighting AmpG as a potential target for resistance-attenuating therapeutics. We prepare a GlcNAc-1,6-anhydroMurNAc-fluorophore conjugate and, using live E. coli spheroplasts, quantitatively analyze its transport by AmpG and inhibition of this process by a competing substrate. Further, we use this transport assay to evaluate the function of two AmpG homologues from Pseudomonas aeruginosa and show that P. aeruginosa AmpG (Pa-AmpG) but not AmpP (Pa-AmpP) transports this probe substrate. We corroborate these results by AmpC induction assays with Pa-AmpG and Pa-AmpP. This fluorescent AmpG probe and spheroplast-based transport assay will enable improved understanding of PG recycling and of permeases from the major facilitator superfamily of transport proteins and may aid in identification of AmpG antagonists that combat AmpC-mediated resistance toward β-lactam antibiotics.

  9. Angiotensin-converting enzyme inhibition prevents myocardial infarction-induced increase in renal cortical cGMP and cAMP phosphodiesterase activities.

    PubMed

    Clauss, François; Charloux, Anne; Piquard, François; Doutreleau, Stéphane; Talha, Samy; Zoll, Joffrey; Lugnier, Claire; Geny, Bernard

    2015-08-01

    We investigated whether myocardial infarction (MI) enhances renal phosphodiesterases (PDE) activities, investigating particularly the relative contribution of PDE1-5 isozymes in total PDE activity involved in both cGMP and cAMP pathways, and whether angiotensin-converting enzyme inhibition (ACEi) decreases such renal PDE hyperactivities. We also investigated whether ACEi might thereby improve atrial natriuretic peptide (ANP) efficiency. We studied renal cortical PDE1-5 isozyme activities in sham (SH)-operated, MI rats and in MI rats treated with perindopril (ACEi) 1 month after coronary artery ligation. Circulating atrial natriuretic peptide (ANP), its second intracellular messenger cyclic guanosine monophosphate (cGMP) and cGMP/ANP ratio were also determined. Cortical cGMP-PDE2 (80.3 vs. 65.1 pmol/min/mg) and cGMP-PDE1 (50.7 vs. 30.1 pmol/min/mg), and cAMP-PDE2 (161 vs. 104.1 pmol/min/mg) and cAMP-PDE4 (307.5 vs. 197.2 pmol/min/mg) activities were higher in MI than in SH rats. Despite increased ANP plasma level, ANP efficiency tended to be decreased in MI compared to SH rats. Perindopril restored PDE activities and tended to improve ANP efficiency in MI rats. One month after coronary ligation, perindopril treatment of MI rats prevents the increase in renal cortical PDE activities. This may contribute to increase renal ANP efficiency in MI rats.

  10. Angiotensin-converting enzyme inhibition prevents myocardial infarction-induced increase in renal cortical cGMP and cAMP phosphodiesterase activities.

    PubMed

    Clauss, François; Charloux, Anne; Piquard, François; Doutreleau, Stéphane; Talha, Samy; Zoll, Joffrey; Lugnier, Claire; Geny, Bernard

    2015-08-01

    We investigated whether myocardial infarction (MI) enhances renal phosphodiesterases (PDE) activities, investigating particularly the relative contribution of PDE1-5 isozymes in total PDE activity involved in both cGMP and cAMP pathways, and whether angiotensin-converting enzyme inhibition (ACEi) decreases such renal PDE hyperactivities. We also investigated whether ACEi might thereby improve atrial natriuretic peptide (ANP) efficiency. We studied renal cortical PDE1-5 isozyme activities in sham (SH)-operated, MI rats and in MI rats treated with perindopril (ACEi) 1 month after coronary artery ligation. Circulating atrial natriuretic peptide (ANP), its second intracellular messenger cyclic guanosine monophosphate (cGMP) and cGMP/ANP ratio were also determined. Cortical cGMP-PDE2 (80.3 vs. 65.1 pmol/min/mg) and cGMP-PDE1 (50.7 vs. 30.1 pmol/min/mg), and cAMP-PDE2 (161 vs. 104.1 pmol/min/mg) and cAMP-PDE4 (307.5 vs. 197.2 pmol/min/mg) activities were higher in MI than in SH rats. Despite increased ANP plasma level, ANP efficiency tended to be decreased in MI compared to SH rats. Perindopril restored PDE activities and tended to improve ANP efficiency in MI rats. One month after coronary ligation, perindopril treatment of MI rats prevents the increase in renal cortical PDE activities. This may contribute to increase renal ANP efficiency in MI rats. PMID:25939307

  11. Impact induced surface heating by planetesimals on early Mars

    NASA Astrophysics Data System (ADS)

    Maindl, T. I.; Dvorak, R.; Lammer, H.; Güdel, M.; Schäfer, C.; Speith, R.; Odert, P.; Erkaev, N. V.; Kislyakova, K. G.; Pilat-Lohinger, E.

    2015-02-01

    Aims: We investigate the influence of impacts of large planetesimals and small planetary embryos on the early Martian surface on the hydrodynamic escape of an early steam atmosphere that is exposed to the high soft X-ray and extreme-ultraviolet (EUV) flux of the young Sun. Methods: Impact statistics in terms of number, masses, velocities, and angles of asteroid impacts onto early Mars are determined via n-body integrations. Based on these statistics, smoothed particle hydrodynamics (SPH) simulations result in estimates of energy transfer into the planetary surface material and the resulting surface heating. For the estimation of the atmospheric escape rates we applied a soft X-ray and EUV absorption model and a 1D upper atmosphere hydrodynamic model to a magma ocean-related catastrophically outgassed steam atmosphere with surface pressure values of 52 bar H2O and 11 bar CO2. Results: The estimated impact rates and energy deposition onto an early Martian surface can account for substantial heating. The energy influx and conversion rate into internal energy is probably sufficient to keep a shallow magma ocean liquid for an extended period of time. Higher surface temperatures keep the outgassed steam atmosphere longer in vapor form and therefore enhance its escape to space within ~0.6 Myr after its formation.

  12. Intranasal Delivery of Influenza rNP Adjuvanted with c-di-AMP Induces Strong Humoral and Cellular Immune Responses and Provides Protection against Virus Challenge

    PubMed Central

    Sanchez, Maria Victoria; Ebensen, Thomas; Schulze, Kai; Cargnelutti, Diego; Blazejewska, Paulina; Scodeller, Eduardo A.; Guzmán, Carlos A.

    2014-01-01

    There is a critical need for new influenza vaccines able to protect against constantly emerging divergent virus strains. This will be sustained by the induction of vigorous cellular responses and humoral immunity capable of acting at the portal of entry of this pathogen. In this study we evaluate the protective efficacy of intranasal vaccination with recombinant influenza nucleoprotein (rNP) co-administrated with bis-(3′,5′)-cyclic dimeric adenosine monophosphate (c-di-AMP) as adjuvant. Immunization of BALB/c mice with two doses of the formulation stimulates high titers of NP-specific IgG in serum and secretory IgA at mucosal sites. This formulation also promotes a strong Th1 response characterized by high secretion of INF-γ and IL-2. The immune response elicited promotes efficient protection against virus challenge. These results suggest that c-di-AMP is a potent mucosal adjuvant which may significantly contribute towards the development of innovative mucosal vaccines against influenza. PMID:25140692

  13. Caffeine protects against alcohol-induced liver fibrosis by dampening the cAMP/PKA/CREB pathway in rat hepatic stellate cells.

    PubMed

    Wang, Qi; Dai, Xuefei; Yang, Wanzhi; Wang, He; Zhao, Han; Yang, Feng; Yang, Yan; Li, Jun; Lv, Xiongwen

    2015-04-01

    Alcoholic liver fibrosis (ALF) is characterized by hyperplasia of extracellular matrix under long-term alcohol stimulation. Hepatic stellate cell (HSC) activation plays an important role in promoting hepatic fibrogenesis. Caffeine, as the main active component of coffee and tea, was widely consumed in daily life. It was always a thought that caffeine can reduce the probability of suffering from liver diseases. In this study, we attempt to validate the hypothesis that caffeine inhibits activation of HSCs which were isolated from rat ALF model. The rats were gavaged by ethanol to establish ALF model and then treated with different concentrations of caffeine or colchicine. Serum was collected to measure the contents of serum alanine aminotransferase (ALT), aspartate transaminase (AST), hyaluronic acid (HA), laminin (LN), N-terminal peptide of type III procollagen (PIIINP) and type IV collagen (CIV). Then liver tissues were obtained for hematoxylin-eosin staining and Sirius-red staining. Others were treated through liver perfusion to isolate primary rat HSCs. Interestingly, we found that caffeine significantly decreased ALT, AST, HA, LN, PIIINP and CIV levels and reversed liver fibrosis in rat ALF models. Results of immunohistochemistry, real-time PCR and western blot indicated that caffeine could reduce fibrosis and inhibit cAMP/PKA/CREB signal pathway in HSC. Caffeine has a preventive effect on ALF. The mechanism may be interpreted that caffeine inhibits the cAMP/PKA/CREB signal pathway through adenosine A2A receptors in HSC.

  14. Resveratrol induces AMPK-dependent MDR1 inhibition in colorectal cancer HCT116/L-OHP cells by preventing activation of NF-κB signaling and suppressing cAMP-responsive element transcriptional activity.

    PubMed

    Wang, Ziyuan; Zhang, Long; Ni, Zhenhua; Sun, Jian; Gao, Hong; Cheng, Zhuoan; Xu, Jianhua; Yin, Peihao

    2015-12-01

    Resveratrol, a natural polyphenolic compound found in foods and beverages, has attracted increasing attention in recent years because of its potent chemopreventive and anti-tumor effects. In this study, the effects of resveratrol on the expression of P-glycoprotein/multi-drug resistance protein 1 (P-gp/MDR1), and the underlying molecular mechanisms, were investigated in oxaliplatin (L-OHP)-resistant colorectal cancer cells (HCT116/L-OHP). Resveratrol downregulated MDR1 protein and mRNA expression levels and reduced MDR1 promoter activity. It also enhanced the intracellular accumulation of rhodamine 123, suggesting that resveratrol can reverse multi-drug resistance by downregulating MDR1 expression and reducing drug efflux. Resveratrol treatment also reduced nuclear factor-κB (NF-κB) activity, reduced phosphorylation levels of IκBα, and reduced nuclear translocation of the NF-κB subunit p65. Moreover, downregulation of MDR1 expression and promoter activity was mediated by resveratrol-induced AMP-activated protein kinase (AMPK) phosphorylation. The inhibitory effects of resveratrol on MDR1 expression and cAMP-responsive element-binding protein (CREB) phosphorylation were reversed by AMPKα siRNA transfection. We found that the transcriptional activity of cAMP-responsive element (CRE) was inhibited by resveratrol. These results demonstrated that the inhibitory effects of resveratrol on MDR1 expression in HCT116/L-OHP cells were closely associated with the inhibition of NF-κB signaling and CREB activation in an AMPK-dependent manner.

  15. Naphazoline-induced neuroendocrine changes: increases in ANP and cGMP levels, but suppression of NE, 3H-NE, and cAMP levels in rabbit eyes.

    PubMed

    Ogidigben, Miller J; Chu, Teh-Ching; Potter, David E

    2002-07-01

    The objective of this study was to determine whether naphazoline, an alpha 2 (alpha2)/imidazoline (I1) agonist, can alter endogenous levels of atrial natriuretic peptide (ANP) and norepinephrine (NE) in aqueous humor and cyclic nucleotide (cAMP, cGMP) accumulation and NE overflow in the iris-ciliary body (ICB) of the rabbit eye. Topical naphazoline (25, 75, and 250 microg) caused a dose-dependent elevation of the ANP levels (36, 54, and 137 pg/ml, respectively) in aqueous humor. This effect was antagonized by pretreatment with efaroxan, an antagonist of I1/alpha2 receptors. Another alpha2/I1 agonist, moxonidine (75 microg topically), caused significant increases in ANP levels in aqueous humor, whereas other relatively selective alpha2-adrenergic receptor agonists, brimonidine (50 microg topically) and oxymetazoline (75 microg topically), did not. In naphazoline (75 microg) pretreated eyes, the NE levels in aqueous humor were attenuated by 36% (from 6.0 to 3.8 pg/ml). Furthermore, naphazoline (1, 10, and 100 micromol/l) caused a dose-related inhibition of NE release from ICBs: 25, 45, and 80%, respectively. The isoproterenol (1 micromol/l) stimulated cAMP accumulation was inhibited 53% by naphazoline (100 micromol/l). In contrast, naphazoline significantly increased the cGMP levels in ICBs. These data demonstrate that naphazoline acts on I1 receptors to increase ANP and to reduce NE levels in aqueous humor. The former effect could also contribute to elevation of cGMP levels and inhibition of cAMP accumulation in the ICB. Further studies will be required to determine if elevation of ANP levels is a critical component of naphazoline-induced alteration of aqueous humor dynamics.

  16. Localized cyclic AMP-dependent protein kinase activity is required for myogenic cell fusion

    SciTech Connect

    Mukai, Atsushi; Hashimoto, Naohiro

    2008-01-15

    Multinucleated myotubes are formed by fusion of mononucleated myogenic progenitor cells (myoblasts) during terminal skeletal muscle differentiation. In addition, myoblasts fuse with myotubes, but terminally differentiated myotubes have not been shown to fuse with each other. We show here that an adenylate cyclase activator, forskolin, and other reagents that elevate intracellular cyclic AMP (cAMP) levels induced cell fusion between small bipolar myotubes in vitro. Then an extra-large myotube, designated a 'myosheet,' was produced by both primary and established mouse myogenic cells. Myotube-to-myotube fusion always occurred between the leading edge of lamellipodia at the polar end of one myotube and the lateral plasma membrane of the other. Forskolin enhanced the formation of lamellipodia where cAMP-dependent protein kinase (PKA) was accumulated. Blocking enzymatic activity or anchoring of PKA suppressed forskolin-enhanced lamellipodium formation and prevented fusion of multinucleated myotubes. Localized PKA activity was also required for fusion of mononucleated myoblasts. The present results suggest that localized PKA plays a pivotal role in the early steps of myogenic cell fusion, such as cell-to-cell contact/recognition through lamellipodium formation. Furthermore, the localized cAMP-PKA pathway might be involved in the specification of the fusion-competent areas of the plasma membrane in lamellipodia of myogenic cells.

  17. Transcranial magnetic stimulation-induced 'visual echoes' are generated in early visual cortex.

    PubMed

    Jolij, Jacob; Lamme, Victor A F

    2010-11-01

    Transcranial magnetic stimulation (TMS) of the early visual areas can trigger perception of a flash of light, a so-called phosphene. Here we show that a very brief presentation of a stimulus can modulate features of a subsequent TMS-induced phosphene, to a level that participants mistake phosphenes for real stimuli, inducing 'visual echoes' of a previously seen stimulus. These 'echoes' are modulated by visual context at the moment of magnetic stimulation, showing that they are generated in early visual areas, and that the brain processes these 'echoes' as if they are factually presented stimuli. This shows that TMS can re-activate weak visual representations in early visual areas. Based on the pattern of contextual modulation of visual echoes, we theorize that perception of these echoes is not a passive reactivation of residual activity in early visual cortex, but an active interpretation of the combined activity of TMS-induced neural noise and cortical state. PMID:20732388

  18. CREM: a master-switch in the transcriptional response to cAMP.

    PubMed

    Lamas, M; Monaco, L; Zazopoulos, E; Lalli, E; Tamai, K; Penna, L; Mazzucchelli, C; Nantel, F; Foulkes, N S; Sassone-Corsi, P

    1996-04-29

    The CREM gene encodes both repressors and activators of cAMP-dependent transcription in a tissue and developmentally regulated manner. In addition, multiple and cooperative phosphorylation events regulate the function of the CREM proteins. CREM plays a key physiological and developmental role within the hypothalamic-pituitary axis. There is a functional switch in CREM expression during the development of male germ cells which is directed by the pituitary hormone FSH. The CREM protein in germ cells is a powerful activator which appears to function as a master-switch in the regulation of postmeiotic genes. CREM is inducible by activation of the cAMP signalling pathway with the kinetics of an early response gene. The induction is transient, cell-specific, does not involve increased transcript stability and does not require protein synthesis. The subsequent decline in CREM expression requires de novo protein synthesis. The induced transcript encodes ICER and is generated from an alternative, intronic promoter. ICER functions as a powerful repressor of cAMP-induced transcription, and represses the activity of its own promoter, thus constituting a negative autoregulatory loop.

  19. Early plasmapheresis in patients with severe hypertriglyceridemia induced acute pancreatitis.

    PubMed

    Nasa, Prashant; Alexander, George; Kulkarni, Amitabh; Juneja, Deven; Sehra, Sudhish; Agarwal, Rajesh; Koul, Kandy

    2015-08-01

    Hypertriglyceridemia can cause severe diseases such as acute pancreatitis (AP) and coronary artery disease. The routine management of hypertriglyceridemia is dietary restriction of fat and lipid-lowering medications to manage the secondary or precipitating causes of hypertriglyceridemia. However, in cases of AP with severe hypertriglyceridemia (SHTG) (triglycerides [TG] >1000 mg/dl) rapid reduction of TG levels to well below 1000 mg/dl can improve outcome and prevent further episodes of pancreatitis. Plasmapheresis is a therapeutic option in such medical emergencies. We discussed 2 cases of severe AP with SHTG where we used early plsmapheresis along with other supportive management. PMID:26321811

  20. The histamine H3 receptor antagonist clobenpropit enhances GABA release to protect against NMDA-induced excitotoxicity through the cAMP/protein kinase A pathway in cultured cortical neurons.

    PubMed

    Dai, Haibin; Fu, Qiuli; Shen, Yao; Hu, Weiwei; Zhang, Zhongmiao; Timmerman, Henk; Leurs, Rob; Chen, Zhong

    2007-06-01

    Using the histamine H3 receptor antagonist clobenpropit, the roles of histamine H3 receptors in NMDA-induced necrosis were investigated in rat cultured cortical neurons. Clobenpropit reversed the neurotoxicity in a concentration-dependent manner, and showed peak protection at a concentration of 10(-7) M. This protection was antagonized by the histamine H3 receptor agonist (R)-alpha-methylhistamine, but not by the histamine H1 receptor antagonist pyrilamine or the histamine H2 receptor antagonist cimetidine. In addition, the protection by clobenpropit was inhibited by the GABAA receptor antagonists picrotoxin and bicuculline. Further study demonstrated that the protection by clobenpropit was due to increased GABA release. The inducible GABA release was also inhibited by (R)-alpha-methylhistamine, but not by pyrilamine or cimetidine. Furthermore, both the adenylyl cyclase inhibitor SQ-22536 and the protein kinase A (PKA) inhibitor H-89 reversed the protection and the GABA release by clobenpropit. In addition, clobenpropit reversed the NMDA-induced increase in intracellular calcium level, which was antagonized by (R)-alpha-methylhistamine. These results indicate that clobenpropit enhanced GABA release to protect against NMDA-induced excitotoxicity, which was induced through the cAMP/PKA pathway, and reduction of intracellular calcium level may also be involved.

  1. Early Dexamethasone Treatment Induces Placental Apoptosis in Sheep

    PubMed Central

    Meng, Wenbin; Shang, Hongkai; Li, Shaofu; Sloboda, Deborah M.; Ehrlich, Loreen; Lange, Karolin; Xu, Huaisheng; Henrich, Wolfgang; Dudenhausen, Joachim W.; Plagemann, Andreas; Newnham, John P.; Challis, John R. G.

    2015-01-01

    Glucocorticoid treatment given in late pregnancy in sheep resulted in altered placental development and function. An imbalance of placental survival and apoptotic factors resulting in an increased rate of apoptosis may be involved. We have now investigated the effects of dexamethasone (DEX) in early pregnancy on binucleate cells (BNCs), placental apoptosis, and fetal sex as a determinant of these responses. Pregnant ewes carrying singleton fetuses (n = 105) were randomized to control (n = 56, 2 mL saline/ewe) or DEX treatment (n = 49, intramuscular injections of 0.14 mg/kg ewe weight per 12 hours over 48 hours) at 40 to 41 days of gestation (dG). Placentomes were collected at 50, 100, 125, and 140 dG. At 100 dG, DEX in females reduced BNC numbers, placental antiapoptotic (proliferating cell nuclear antigen), and increased proapoptotic factors (Bax, p53), associated with a temporarily decrease in fetal growth. At 125 dG, BNC numbers and apoptotic markers were restored to normal. In males, ovine placental lactogen-protein levels after DEX were increased at 50 dG, but at 100 and 140 dG significantly decreased compared to controls. In contrast to females, these changes were independent of altered BNC numbers or apoptotic markers. Early DEX was associated with sex-specific, transient alterations in BNC numbers, which may contribute to changes in placental and fetal development. Furthermore, in females, altered placental apoptosis markers may be involved. PMID:25063551

  2. Possible methane-induced polar warming in the early Eocene.

    PubMed

    Sloan, L C; Walker, J C; Moore, T C; Rea, D K; Zachos, J C

    1992-05-28

    Reconstructions of early Eocene climate depict a world in which the polar environments support mammals and reptiles, deciduous forests, warm oceans and rare frost conditions. At the same time, tropical sea surface temperatures are interpreted to have been the same as or slightly cooler than present values. The question of how to warm polar regions of Earth without noticeably warming the tropics remains unresolved; increased amounts of greenhouse gases would be expected to warm all latitudes equally. Oceanic heat transport has been postulated as a mechanism for heating high latitudes, but it is difficult to explain the dynamics that would achieve this. Here we consider estimates of Eocene wetland areas and suggest that the flux of methane, an important greenhouse gas, may have been substantially greater during the Eocene than at present. Elevated methane concentrations would have enhanced early Eocene global warming, and also might specifically have prevented severe winter cooling of polar regions because of the potential of atmospheric methane to promote the formation of optically thick, polar stratospheric ice clouds.

  3. Fluoride-induced early teeth wearing in Argentinian cattle.

    PubMed

    Lopez, T A; Busetti, M R; Fort, M C; Bedotti, D O

    1994-09-01

    The relationship between observed early teeth wearing in cattle and the existence of large areas where excess fluoride in underground water prevails in Argentina, but has never been studied. Average values of bone fluoride exceeding 5000 ppm and well water containing up to 10.5 ppm of this element were found in a farm (ETWF) where early teeth wearing is observed, while in a control farm (NETWF), those values were 1480 ppm and 3.0 ppm respectively. Urine samples from animals from the ETWF contained almost 4 times more fluoride than those from the latter. Forage in both farms contained about the same levels of fluoride (15 to 25 ppm). None of the farms was exposed to industrial, volcanic, or geothermal activity. Lactating and adult animals were transferred from one farm to the other. After 30 months, adult animals from the NETWF showed no damage in teeth, while adults from the ETWF continued a rapid teeth wearing. Young animals from the NETWF developed severely damaged permanent teeth, while young animals from the ETWF developed normal teeth. We concluded that the problem exists in the ETWF and being a standard farm, the condition could be extrapolated to other areas.

  4. Regulatory components in Citrobacter freundii ampC beta-lactamase induction.

    PubMed

    Lindberg, F; Westman, L; Normark, S

    1985-07-01

    Citrobacter freundii encodes an inducible chromosomal beta-lactamase similar to the constitutively expressed ampC beta-lactamase of Escherichia coli. In the latter species the ampC gene is located next to the fumarate reductase (frd) operon, whereas in C. freundii the ampC gene is known to be separated from frd by 1100 base pairs. This intervening DNA segment carries a gene, ampR, coding for a 31-kilodalton polypeptide. The cloned C. freundii OS60 ampC gene is inducible by beta-lactam antibiotics in E. coli, but only in the presence of an intact ampR gene. In the absence of inducer the AmpR protein represses C. freundii ampC synthesis 2.5-fold. Addition of beta-lactams induced expression from the cloned ampC beta-lactamase gene 11-fold. Thus, the AmpR protein has a positive effect on ampC expression in the presence of inducing beta-lactams. Two spontaneous mutants of C. freundii were isolated that constitutively overproduce the ampC beta-lactamase. The mutations in both these strains occurred outside the frd-amp region, suggesting that there is at least one additional component in the regulatory system. With the cloned C. freundii ampC gene in E. coli, mutants with the same phenotype could be obtained. These mutations were located on the E. coli chromosome. The constitutive beta-lactamase overproduction in these mutants requires the presence of an intact ampR gene.

  5. Observable induced gravitational waves from an early matter phase

    SciTech Connect

    Alabidi, Laila; Sasaki, Misao; Kohri, Kazunori; Sendouda, Yuuiti E-mail: kohri@post.kek.jp E-mail: sendouda@cc.hirosaki-u.ac.jp

    2013-05-01

    Assuming that inflation is succeeded by a phase of matter domination, which corresponds to a low temperature of reheating T{sub r} < 10{sup 9}GeV, we evaluate the spectra of gravitational waves induced in the post-inflationary universe. We work with models of hilltop-inflation with an enhanced primordial scalar spectrum on small scales, which can potentially lead to the formation of primordial black holes. We find that a lower reheat temperature leads to the production of gravitational waves with energy densities within the ranges of both space and earth based gravitational wave detectors.

  6. Calcitriol inhibits bleomycin-induced early pulmonary inflammatory response and epithelial-mesenchymal transition in mice.

    PubMed

    Tan, Zhu-Xia; Chen, Yuan-Hua; Xu, Shen; Qin, Hou-Ying; Zhang, Cheng; Zhao, Hui; Xu, De-Xiang

    2016-01-01

    Early pulmonary inflammation and epithelial-mesenchymal transition (EMT) play important roles during lung fibrosis. Increasing evidence demonstrates that calcitriol, the active form of vitamin D3, has anti-inflammatory activities. The aim of this study was to investigate the effects of calcitriol on bleomycin (BLM)-induced early pulmonary inflammation and subsequent EMT. Mice were intratracheally injected with BLM (3.0mg/kg). In three calcitriol+BLM groups, mice were intraperitoneal (i.p.) injected with different doses of calcitriol (0.2, 1.0 or 5.0 μg/kg) daily, beginning at 48 h before BLM injection. Twenty-four hours, seven and fourteen days after BLM injection, pulmonary inflammation and EMT were evaluated. As expected, BLM-induced infiltration of inflammatory cells in the lungs was attenuated by calcitriol. BLM-induced pulmonary inflammatory cytokines were repressed by calcitriol. Moreover, BLM-induced nuclear translocation of nuclear factor kappa B (NF-κB) p65 was blocked by calcitriol. In addition, BLM-induced phosphorylation of pulmonary p38 MAPK and protein kinase B (Akt) was inhibited by calcitriol. Further analysis showed that BLM-induced α-smooth muscle actin (α-SMA), a marker for EMT in the lungs, was significantly attenuated by calcitriol. BLM-induced transforming growth factor-beta 1 (TGF-β1) up-regulation and Smad phosphorylation were attenuated by calcitriol. In conclusion, calcitriol inhibits BLM-induced early pulmonary inflammation and subsequent EMT. PMID:26520185

  7. Textile dyes induce toxicity on zebrafish early life stages.

    PubMed

    de Oliveira, Gisele Augusto Rodrigues; de Lapuente, Joaquín; Teixidó, Elisabet; Porredón, Constança; Borràs, Miquel; de Oliveira, Danielle Palma

    2016-02-01

    Textile manufacturing is one of the most polluting industrial sectors because of the release of potentially toxic compounds, such as synthetic dyes, into the environment. Depending on the class of the dyes, their loss in wastewaters can range from 2% to 50% of the original dye concentration. Consequently, uncontrolled use of such dyes can negatively affect human health and the ecological balance. The present study assessed the toxicity of the textile dyes Direct Black 38 (DB38), Reactive Blue 15 (RB15), Reactive Orange 16 (RO16), and Vat Green 3 (VG3) using zebrafish (Danio rerio) embryos for 144 h postfertilization (hpf). At the tested conditions, none of the dyes caused significant mortality. The highest RO16 dose significantly delayed or inhibited the ability of zebrafish embryos to hatch from the chorion after 96 hpf. From 120 hpf to 144 hpf, all the dyes impaired the gas bladder inflation of zebrafish larvae, DB38 also induced curved tail, and VG3 led to yolk sac edema in zebrafish larvae. Based on these data, DB38, RB15, RO16, and VG3 can induce malformations during embryonic and larval development of zebrafish. Therefore, it is essential to remove these compounds from wastewater or reduce their concentrations to safe levels before discharging textile industry effluents into the aquatic environment.

  8. Fraxetin Induces Heme Oxygenase-1 Expression by Activation of Akt/Nrf2 or AMP-activated Protein Kinase α/Nrf2 Pathway in HaCaT Cells

    PubMed Central

    Kundu, Juthika; Chae, In Gyeong; Chun, Kyung-Soo

    2016-01-01

    Background Fraxetin (7,8-dihydroxy-6-methoxy coumarin), a coumarin derivative, has been reported to possess antioxidative, anti-inflammatory and neuroprotective effects. A number of recent observations suggest that the induction of heme oxygenase-1 (HO-1) inhibits inflammation and tumorigenesis. In the present study, we determined the effect of fraxetin on HO-1 expression in HaCaT human keratinocytes and investigated its underlying molecular mechanisms. Methods Reverse transcriptase-PCR and Western blot analysis were performed to detect HO-1 mRNA and protein expression, respectively. Cell viability was measured by the MTS test. The induction of intracellular reactive oxygen species (ROS) by fraxetin was evaluated by 2′,7′-dichlorofluorescin diacetate staining. Results Fraxetin upregulated mRNA and protein expression of HO-1. Incubation with fraxetin induced the localization of nuclear factor-erythroid-2-related factor-2 (Nrf2) in the nucleus and increased the antioxidant response element-reporter gene activity. Fraxetin also induced the phosphorylation of Akt and AMP-activated protein kinase (AMPK)α and diminished the expression of phosphatase and tensin homolog, a negative regulator of Akt. Pharmacological inhibition of Akt and AMPKα abrogated fraxetin-induced expression of HO-1 and nuclear localization of Nrf2. Furthermore, fraxetin generated ROS in a concentration-dependent manner. Conclusions Fraxetin induces HO-1 expression through activation of Akt/Nrf2 or AMPKα/Nrf2 pathway in HaCaT cells. PMID:27722139

  9. Early diagnosis of gastric cancer with laser-induced fluorescence

    NASA Astrophysics Data System (ADS)

    Joffe, Alexander Y.; Sayenko, Valeriy F.; Denisov, Nikolay A.; Dets, Sergiy M.; Buryi, Alexander N.

    1999-02-01

    Optical biopsy of stomach mucosa was performed afterwards oral administration of encapsulated hyperflav (single dose was chosen to provide 0.1 - 0.15 mg/kg b.w.) A sufficient fluorescence contrast of suspicions versus normal tissue was obtained after incubation time from 4 to 10 hours. Fluorescence was induced by He - Cd laser coupled to fiber optic probe inserted into a biopsy channel of the endoscope. Fluorescent spectra were recorded in the range from 500 nm up to 700 nm with 2 nm resolution. We took two groups of patients with benign and malignant ulcer of the stomach and erosive gastritis. The first group consisted of 59 patients (male/female 36/23) was carried out with optical biopsy of stomach mucosa. The second group consisted of 60 patients (male/female 39/21) was carried out by routine method: gastroscopy and biopsy from 5 - 7 places of macroscopically changed mucosa.

  10. Vicenistatin induces early endosome-derived vacuole formation in mammalian cells.

    PubMed

    Nishiyama, Yuko; Ohmichi, Tomohiro; Kazami, Sayaka; Iwasaki, Hiroki; Mano, Kousuke; Nagumo, Yoko; Kudo, Fumitaka; Ichikawa, Sosaku; Iwabuchi, Yoshiharu; Kanoh, Naoki; Eguchi, Tadashi; Osada, Hiroyuki; Usui, Takeo

    2016-05-01

    Homotypic fusion of early endosomes is important for efficient protein trafficking and sorting. The key controller of this process is Rab5 which regulates several effectors and PtdInsPs levels, but whose mechanisms are largely unknown. Here, we report that vicenistatin, a natural product, enhanced homotypic fusion of early endosomes and induced the formation of large vacuole-like structures in mammalian cells. Unlike YM201636, another early endosome vacuolating compound, vicenistatin did not inhibit PIKfyve activity in vitro but activated Rab5-PAS pathway in cells. Furthermore, vicenistatin increased the membrane surface fluidity of cholesterol-containing liposomes in vitro, and cholesterol deprivation from the plasma membrane stimulated vicenistatin-induced vacuolation in cells. These results suggest that vicenistatin is a novel compound that induces the formation of vacuole-like structures by activating Rab5-PAS pathway and increasing membrane fluidity. PMID:27104762

  11. Early attentional bias for negative words when competition is induced.

    PubMed

    Ho, Ming-Chou; Li, Shuo-Heng; Yeh, Su-Ling

    2016-05-01

    Previous research (Zeelenberg, Wagenmakers, & Rotteveel, 2006) revealed that emotionally meaningful words were identified significantly better than neutral words, with no difference between positive and negative words. Since in that study only a single target word was displayed at a time, we hypothesized that the equivalent performances for positive and negative words were due to a lack of competition. To test this, in our Experiment 1, we replicated Zeelenberg and colleagues' finding, using emotion-laden Chinese words and the identical data-limited method, which measured the accuracy of a briefly shown target. We then introduced competition in Experiment 2 by simultaneously presenting two words during the target frame, and found evidence for an early attentional bias to negative words. In Experiment 3, we confirmed that the bias in Experiment 2 was not due to the inevitable repetition of stimuli. Taken together, these results support our hypothesis that, in the presence of competition, negative words receive attentional priority and consequently have enhanced perceptual representations.

  12. AMP-18 Targets p21 to Maintain Epithelial Homeostasis.

    PubMed

    Chen, Peili; Li, Yan Chun; Toback, F Gary

    2015-01-01

    Dysregulated homeostasis of epithelial cells resulting in disruption of mucosal barrier function is an important pathogenic mechanism in inflammatory bowel diseases (IBD). We have characterized a novel gastric protein, Antrum Mucosal Protein (AMP)-18, that has pleiotropic properties; it is mitogenic, anti-apoptotic and can stimulate formation of tight junctions. A 21-mer synthetic peptide derived from AMP-18 exhibits the same biological functions as the full-length protein and is an effective therapeutic agent in mouse models of IBD. In this study we set out to characterize therapeutic mechanisms and identify molecular targets by which AMP-18 maintains and restores disrupted epithelial homeostasis in cultured intestinal epithelial cells and a mouse model of IBD. Tumor necrosis factor (TNF)-α, a pro-inflammatory cytokine known to mediate gastrointestinal (GI) mucosal injury in IBD, was used to induce intestinal epithelial cell injury, and study the effects of AMP-18 on apoptosis and the cell cycle. An apoptosis array used to search for targets of AMP-18 in cells exposed to TNF-α identified the cyclin-dependent kinase inhibitor p21 WAF1/CIP1. Treatment with AMP-18 blunted increases in p21 expression and apoptosis, while reversing disturbed cell cycle kinetics induced by TNF-α. AMP-18 appears to act through PI3K/AKT pathways to increase p21 phosphorylation, thereby reducing its nuclear accumulation to overcome the antiproliferative effects of TNF-α. In vitamin D receptor-deficient mice with TNBS-induced IBD, the observed increase in p21 expression in colonic epithelial cells was suppressed by treatment with AMP peptide. The results indicate that AMP-18 can maintain and/or restore the homeostatic balance between proliferation and apoptosis in intestinal epithelial cells to protect and repair mucosal barrier homeostasis and function, suggesting a therapeutic role in IBD.

  13. Coupling signalling pathways to transcriptional control: nuclear factors responsive to cAMP.

    PubMed

    Tamai, K T; Monaco, L; Nantel, F; Zazopoulos, E; Sassone-Corsi, P

    1997-01-01

    Several endocrine and neuronal functions are governed by the cAMP-dependent signalling pathway. In eukaryotes, transcriptional regulation upon stimulation of the adenylyl cyclase signalling pathway is mediated by a family of cAMP-responsive nuclear factors. This family consists of a large number of members that may act as activators or repressors. These factors contain the basic domain/ leucine zipper motifs and bind as dimers to cAMP-response elements (CRE). The function of CRE-binding proteins (CREBs) is modulated by phosphorylation by several kinases. Direct activation of gene expression by CREB requires phosphorylation by the cAMP-dependent protein kinase A to the serine-133 residue. Among the repressors, ICER (Inducible cAMP Early Repressor) deserves special mention. ICER is generated from an alternative CREM promoter and constitutes the only inducible cAMP-responsive element binding protein. Furthermore, ICER negatively autoregulates the alternative promoter, thus generating a feedback loop. In contrast to the other members of the CRE-binding protein family, ICER expression is tissue specific and developmentally regulated. The kinetics of ICER expression are characteristic of an early response gene. Our results indicate that CREM plays a key physiological and developmental role within the hypothalamic-pituitary-gonadal axis. We have previously shown that the transcriptional activator CREM is highly expressed in postmeiotic cells. Spermiogenesis is a complex process by which postmeiotic male germ cells differentiate into mature spermatozoa. This process involves remarkable structural and biochemical changes that are under the hormonal control of the hypothalamic-pituitary axis. We have addressed the specific role of CREM in spermiogenesis using CREM-mutant mice generated by homologous recombination. Analysis of the seminiferous epithelium from mutant male mice reveals that spermatogenesis stops at the first step of spermiogenesis. Late spermatids are

  14. The Popeye Domain Containing Genes and cAMP Signaling

    PubMed Central

    Brand, Thomas; Poon, Kar Lai; Simrick, Subreena; Schindler, Roland F.R.

    2016-01-01

    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. PMID:27500161

  15. Early immature neuronal death initiates cerebral ischemia-induced neurogenesis in the dentate gyrus.

    PubMed

    Kim, D H; Lee, H E; Kwon, K J; Park, S J; Heo, H; Lee, Y; Choi, J W; Shin, C Y; Ryu, J H

    2015-01-22

    Throughout adulthood, neurons are continuously replaced by new cells in the dentate gyrus (DG) of the hippocampus, and this neurogenesis is increased by various neuronal injuries including ischemic stroke and seizure. While several mechanisms of this injury-induced neurogenesis have been elucidated, the initiation factor remains unclear. Here, we investigated which signal(s) trigger(s) ischemia-induced cell proliferation and neurogenesis in the hippocampal DG region. We found that early apoptotic cell death of the immature neurons occurred in the DG region following transient forebrain ischemia/reperfusion in mice. Moreover, early immature neuronal death in the DG initiated transient forebrain ischemia/reperfusion-induced neurogenesis through glycogen synthase kinase-3β/β-catenin signaling, which was mediated by microglia-derived insulin-like growth factor-1 (IGF-1). Additionally, we observed that the blockade of immature neuronal cell death, early microglial activation, or IGF-1 signaling attenuated ischemia-induced neurogenesis. These results suggest that early immature neuronal cell death initiates ischemia-induced neurogenesis through microglial IGF-1 in mice.

  16. Activation of the cAMP-PKA signaling pathway in rat dorsal root ganglion and spinal cord contributes toward induction and maintenance of bone cancer pain.

    PubMed

    Zhu, Gui-Qin; Liu, Su; He, Duan-Duan; Liu, Yue-Peng; Song, Xue-Jun

    2014-08-01

    The objective of this study was to explore the role of cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) signaling in the development of bone cancer pain in rats. Female Sprague-Dawley rats (N=48) were divided randomly into four groups: sham (n=8), tumor cell implantation (TCI) (n=16), TCI+saline (n=8), and TCI+PKA inhibitor (n=16). Bone cancer-induced pain behaviors - thermal hyperalgesia and mechanical allodynia - were tested at postoperative days -3, -1, 1, 3, 5, 7, 10, and 14. A PKA inhibitor, Rp-cAMPS (1 mmol/l/20 μl), was injected intrathecally on postoperative days 3, 4, and 5 (early phase) or 7, 8, and 9 postoperative days (late phase). The expression of PKA mRNA in dorsal root ganglia (DRG) was detected by reverse transcription-PCR. The concentration of cAMP and activity of PKA in DRG and spinal cord were measured by enzyme-linked immunosorbent assay. TCI treatment induced significant pain behaviors, manifested as thermal hyperalgesia and mechanical allodynia. Spinal administration of the PKA inhibitor Rp-cAMPS during the early phase and late phase significantly delayed or reversed, respectively, TCI-induced thermal hyperalgesia and mechanical allodynia. TCI treatment also led to obvious tumor growth and bone destruction. The level of PKA mRNA in the DRG, as well as the concentration of cAMP and the activity of PKA, in both the DRG and spinal cord were significantly increased after TCI treatment (P<0.01). We conclude that the inhibition of the cAMP-PKA signaling pathway may reduce bone cancer pain. PMID:24978483

  17. The cAMP response element-binding protein in the bed nucleus of the stria terminalis modulates the formalin-induced pain behavior in the female rat.

    PubMed

    Hagiwara, Hiroko; Ishida, Maho; Arita, Jun; Mitsushima, Dai; Takahashi, Takuya; Kimura, Fukuko; Funabashi, Toshiya

    2009-12-01

    Abstract Differences in male and female responses to pain are widely recognized in many species, including humans, but the cerebral mechanisms that generate these responses are unknown. Using the formalin test, we confirmed that proestrus female rats showed nociceptive behavior, modulated by estrogen that was distinct from male rats, particularly during the interphase period. We then explored the brain areas, which were involved in the female pattern of nociceptive behavior. We found that, after a formalin injection and at the time corresponding to the behavioral interphase, the number of phosphorylated cAMP response element-binding protein (pCREB)-immunoreactive neurons observed by immunocytochemistry increased in the dorsolateral division of the bed nucleus of the stria terminalis (BSTLD) in female but not male rats. There were no significant sex differences in pCREB expression following formalin in any region other than the BSTLD. The increased pCREB in female rats was eliminated after an ovariectomy and restored with 17beta-estradiol treatment. Neither an orchidectomy nor 17beta-estradiol treatment affected the pCREB response in male rats. The increase in pCREB expression in the BSTLD in female rats after formalin injection was confirmed with immunoblotting. To determine the role of CREB in the BSTLD, adenovirus-mediated expression of a dominant-negative form of CREB (mCREB) was carried out. The nociceptive behavior during interphase was significantly attenuated by injection of virus carrying mCREB into the BSTLD in female rats but not in male rats. These results suggest a novel role for CREB in the BSTLD as a modulator of the pain response in a female-specific, estrogen-dependent manner.

  18. Histone modifications induced by MDV infection at early cytolytic and latency phases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Marek’s disease (MD) is a highly contagious, lymphomatous disease of chickens induced by a herpesvirus, Marek’s disease virus (MDV) that is the cause of major annual losses to the poultry industry. MD pathogenesis involves multiple stages including an early cytolytic phase and latency, a...

  19. Cyclic AMP inhibits secretion from electroporated human neutrophils.

    PubMed

    Smolen, J E; Stoehr, S J; Kuczynski, B

    1991-02-01

    It has long been known that intracellular cAMP inhibits and cGMP enhances intact neutrophil function. However, these effects are modest and require relatively high concentrations of the cyclic nucleotides. We decided to re-examine the effects of cyclic nucleotides on Ca2(+)-induced secretion by electroporated cells. This system allowed us to bypass normal cell surface receptor-ligand interactions as well as to directly expose the intracellular space to native cyclic nucleotides. We found that concentrations of cAMP as low as 3 microM inhibited Ca2(+)-induced secretion; 30-300 microM cAMP was maximally inhibitory. cAMP was actually slightly more potent than dibutyryl cAMP, a membrane-permeant derivative. In contrast, cGMP was only slightly stimulatory at 3 microM and modestly inhibitory at 300 microM; dibutyryl cGMP was ineffective. A more detailed investigation of the effects of cAMP showed that inhibition was only obtained in the presence of Mg2+. Half-maximal inhibition by cAMP occurred at 10-30 microM. Inhibition by cAMP was achieved by shifting the Ca2+ dose-response curve for secretion to the right; this was observed for the release of both specific granules (vitamin B12 binding protein) and azurophil granules (B-glucuronidase). We previously showed that ATP could enhance Ca2(+)-induced secretion in the presence of Mg2+, apparently by interacting with a cell surface purine receptor. However, increasing concentrations of ATP could not overcome inhibition by cAMP; this suggested that cAMP acted at some site other than the purine receptor. Inhibition by cAMP was also less apparent in the presence of the protein kinase C agonist phorbol myristate acetate (PMA), suggesting that the cyclic nucleotide did not produce systemic desensitization of the neutrophils. In summary, these results demonstrate that low, physiologically relevant concentrations of cAMP can modulate neutrophil responsiveness. PMID:1846904

  20. Lung Cancer Workshop XI: Tobacco-Induced Disease: Advances in Policy, Early Detection and Management.

    PubMed

    Mulshine, James L; Avila, Rick; Yankelevitz, David; Baer, Thomas M; Estépar, Raul San Jose; Ambrose, Laurie Fenton; Aldigé, Carolyn R

    2015-05-01

    The Prevent Cancer Foundation Lung Cancer Workshop XI: Tobacco-Induced Disease: Advances in Policy, Early Detection and Management was held in New York, NY on May 16 and 17, 2014. The two goals of the Workshop were to define strategies to drive innovation in precompetitive quantitative research on the use of imaging to assess new therapies for management of early lung cancer and to discuss a process to implement a national program to provide high quality computed tomography imaging for lung cancer and other tobacco-induced disease. With the central importance of computed tomography imaging for both early detection and volumetric lung cancer assessment, strategic issues around the development of imaging and ensuring its quality are critical to ensure continued progress against this most lethal cancer. PMID:25898957

  1. Lung Cancer Workshop XI: Tobacco-Induced Disease: Advances in Policy, Early Detection and Management.

    PubMed

    Mulshine, James L; Avila, Rick; Yankelevitz, David; Baer, Thomas M; Estépar, Raul San Jose; Ambrose, Laurie Fenton; Aldigé, Carolyn R

    2015-05-01

    The Prevent Cancer Foundation Lung Cancer Workshop XI: Tobacco-Induced Disease: Advances in Policy, Early Detection and Management was held in New York, NY on May 16 and 17, 2014. The two goals of the Workshop were to define strategies to drive innovation in precompetitive quantitative research on the use of imaging to assess new therapies for management of early lung cancer and to discuss a process to implement a national program to provide high quality computed tomography imaging for lung cancer and other tobacco-induced disease. With the central importance of computed tomography imaging for both early detection and volumetric lung cancer assessment, strategic issues around the development of imaging and ensuring its quality are critical to ensure continued progress against this most lethal cancer.

  2. cAMP enhances BMP2-signaling through PKA and MKP1-dependent mechanisms

    SciTech Connect

    Ghayor, Chafik; Ehrbar, Martin; Miguel, Blanca San; Graetz, Klaus W.; Weber, Franz E.

    2009-04-03

    Recent studies suggest that the elevation of intracellular cyclic adenosine monophosphate (cAMP) and the activation of the protein kinase A regulate BMP-induced osteogenesis. However, the precise mechanisms underlying the enhancing effect of cAMP on BMP2 signaling were not completely revealed. In this study we investigated the effect of elevated cAMP level and PKA activation on the BMP2-induced osteoblastic differentiation in pluripotent C2C12 cells. Alkaline phosphatase activity and its mRNA were consistently induced by BMP2 treatment. The pretreatment of C2C12 cells with Forskolin, a cAMP generating agent, dbcAMP, an analogue of cAMP, or IBMX (3-isobutyl 1-methyl xanthine), and a nonspecific inhibitor of phosphodiesterases elicited further activation of alkaline phosphatase. Furthermore, elevated intracellular cAMP level increased BMP2-induced MKP1. On the other hand, BMP2-induced Erk phosphorylation (p44/p42) and cell proliferation were suppressed in the presence of cAMP. Thus, cAMP might enhance BMP2-induced osteoblastic differentiation by a MKP1-Erk-dependent mechanism.

  3. Phorbol esters modulate cyclic AMP accumulation in porcine thyroid cells

    SciTech Connect

    Emoto, T.; Kasai, K.; Hiraiwa, M.; Shimoda, S.

    1988-01-01

    In cultured porcine thyroid cells, during 60 min incubation phorbol 12-myristate 13-acetate (PMA) had no effect on basal cyclic AMP accumulation and slightly stimulated cyclic AMP accumulation evoked by thyroid stimulating hormone (TSH) or forskolin. Cholera toxin-induced cyclic AMP accumulation was significantly stimulated by PMA. On the other hand, cyclic AMP accumulation evoked by prostaglandin E/sub 1/ or E/sub 2/ (PGE/sub 1/ and PGE/sub 2/) was markedly depressed by simultaneous addition of PMA. These opposing effects of PMA on cyclic AMP accumulation evoked by PGE and cholera toxin were observed in a dose-related fashion, with half-maximal effect of around 10/sup -9/ M in either case. The almost same effects of PMA on cyclic AMP accumulation in basal and stimulated conditions were also observed in freshly prepared thyroid cells. The present study was performed in the presence of phosphodiesterase inhibitor, 3-iso-butyl-1-methylxanthine (IBMX), indicating that PMA affected adenylate cyclase activity. Therefore, it is suggested that PMA may modulate the production of cyclic AMP in response to different stimuli, possibly by affecting several sites in the adenylate cyclase complex in thyroid cells.

  4. Activation of reactive oxygen species/AMP activated protein kinase signaling mediates fisetin-induced apoptosis in multiple myeloma U266 cells.

    PubMed

    Jang, Ki Young; Jeong, Soo-Jin; Kim, Sun-Hee; Jung, Ji Hoon; Kim, Ji-Hyun; Koh, Wonil; Chen, Chang-Yan; Kim, Sung-Hoon

    2012-06-28

    We investigated the molecular mechanisms responsible for fisetin-induced apoptosis in U266 cells. Fisetin elicited the cytotoxicity in U266 cells, manifested as an increased fraction of the cells with sub-G1 content or stained positively with TUNEL labeling. Fisetin enhanced caspase-3 activation, downregulation of Bcl-2 and Mcl-1(L), and upregulation of Bax, Bim and Bad. Fisetin activated AMPK as well as its substrate acetyl-CoA carboxylase (ACC), along with a decreased phosphorylation of AKT and mTOR. Fisetin also stimulated generation of ROS in U266 cells. Conversely, compound C or N-acetyl-L-cystein blocked fisetin-induced apoptosis. Our data suggest that fisetin-induced apoptosis in U266 cells is through ROS and AMPK pathways.

  5. AMP-activated protein kinase inhibits TGF-β-induced fibrogenic responses of hepatic stellate cells by targeting transcriptional coactivator p300.

    PubMed

    Lim, Joong-Yeon; Oh, Min-A; Kim, Won Ho; Sohn, Hee-Young; Park, Sang Ick

    2012-03-01

    Liver fibrosis is a common consequence of various chronic liver injuries, including virus infection and ethanol. Activated hepatic stellate cells (HSCs) contribute to liver fibrosis through the accumulation of extracellular matrix proteins, including type I alpha collagen (COL1A). The activation of adenosine monophosphate-activated protein kinase (AMPK) modulates HSCs activation, but its underlying mechanism remains unclear. Here, we report that AMPK inhibits transforming growth factor (TGF)-β-induced fibrogenic property of HSCs by regulating transcriptional coactivator p300. We treated human (LX-2) and rat (CFSC-2G) HSC lines with TGF-β to induce fibrogenic activation of HSCs. Pharmacological activation of AMPK by treatment with 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), metformin, or adiponectin lowered TGF-β-induced expression of COL1A and myofibroblast marker alpha-smooth muscle actin (α-SMA). Transient transduction of constitutively active AMPKα (caAMPKα) was sufficient to attenuate COL1A and α-SMA expression, whereas an AMPK inhibitor considerably abrogated the inhibitory effect of AICAR on fibrogenic gene expression. Although AMPK significantly suppressed Smad-dependent transcription, it did not affect TGF-β-stimulated phosphorylation, nuclear localization, or DNA-binding activity of Smad2/3. AICAR rather attenuated TGF-β-induced Smad3 interaction with transcriptional coactivator p300 accompanying with reduction of Smad3 acetylation. Moreover, AICAR induced not only physical interaction between AMPK and p300 but also proteasomal degradation of p300 protein. Our data provide substantial evidence that AMPK could be a novel therapeutic target for treatment of liver fibrosis, by demonstrating the underlying mechanism of AMPK-induced antifibrotic function in HSCs.

  6. Early role of the κ opioid receptor in ethanol-induced reinforcement.

    PubMed

    Pautassi, Ricardo Marcos; Nizhnikov, Michael E; Acevedo, Ma Belén; Spear, Norman E

    2012-03-20

    Effects of early ethanol exposure on later ethanol intake emphasize the importance of understanding the neurobiology of ethanol-induced reinforcement early in life. Infant rats exhibit ethanol-induced appetitive conditioning and ethanol-induced locomotor activation, which have been linked in theory and may have mechanisms in common. The appetitive effects of ethanol are significantly modulated by μ and δ opioid receptors, whereas μ but not δ receptors are involved in the motor stimulant effects of ethanol during early development. The involvement of the κ opioid receptor (KOR) system in the motivational effects of ethanol has been much less explored. The present study assessed, in preweanling (infant) rats, the modulatory role of the KOR system in several paradigms sensitive to ethanol-induced reinforcement. Kappa opioid activation and blockade were examined in second-order conditioned place preference with varied timing before conditioning and with varied ethanol doses. The role of KOR on ethanol-induced locomotion and ethanol-induced taste conditioning was also explored. The experiments were based on the assumption that ethanol concurrently induces appetitive and aversive effects and that the latter may be mediated by activation of kappa receptors. The main result was that blockade of kappa function facilitated the expression of appetitive ethanol reinforcement in terms of tactile and taste conditioning. The effects of kappa activation on ethanol conditioning seemed to be independent from ethanol's stimulant effects. Kappa opioid activation potentiated the motor depressing effects of ethanol but enhanced motor activity in control subjects. Overall, the results support the hypothesis that a reduced function of the KOR system in nondependent subjects should attenuate the aversive consequences of ethanol.

  7. Early Activation of STAT3 Regulates Reactive Astrogliosis Induced by Diverse Forms of Neurotoxicity

    PubMed Central

    O'Callaghan, James P.; Kelly, Kimberly A.; VanGilder, Reyna L.; Sofroniew, Michael V.; Miller, Diane B.

    2014-01-01

    Astrogliosis, a cellular response characterized by astrocytic hypertrophy and accumulation of GFAP, is a hallmark of all types of central nervous system (CNS) injuries. Potential signaling mechanisms driving the conversion of astrocytes into “reactive” phenotypes differ with respect to the injury models employed and can be complicated by factors such as disruption of the blood-brain barrier (BBB). As denervation tools, neurotoxicants have the advantage of selective targeting of brain regions and cell types, often with sparing of the BBB. Previously, we found that neuroinflammation and activation of the JAK2-STAT3 pathway in astrocytes precedes up regulation of GFAP in the MPTP mouse model of dopaminergic neurotoxicity. Here we show that multiple mechanistically distinct mouse models of neurotoxicity (MPTP, AMP, METH, MDA, MDMA, KA, TMT) engender the same neuroinflammatory and STAT3 activation responses in specific regions of the brain targeted by each neurotoxicant. The STAT3 effects seen for TMT in the mouse could be generalized to the rat, demonstrating cross-species validity for STAT3 activation. Pharmacological antagonists of the neurotoxic effects blocked neuroinflammatory responses, pSTAT3tyr705 and GFAP induction, indicating that damage to neuronal targets instigated astrogliosis. Selective deletion of STAT3 from astrocytes in STAT3 conditional knockout mice markedly attenuated MPTP-induced astrogliosis. Monitoring STAT3 translocation in GFAP-positive cells indicated that effects of MPTP, METH and KA on pSTAT3tyr705 were localized to astrocytes. These findings strongly implicate the STAT3 pathway in astrocytes as a broadly triggered signaling pathway for astrogliosis. We also observed, however, that the acute neuroinflammatory response to the known inflammogen, LPS, can activate STAT3 in CNS tissue without inducing classical signs of astrogliosis. Thus, acute phase neuroinflammatory responses and neurotoxicity-induced astrogliosis both signal through

  8. Alkaline Phosphatase Protects Lipopolysaccharide-Induced Early Pregnancy Defects in Mice

    PubMed Central

    Lei, Wei; Ni, Hua; Herington, Jennifer; Reese, Jeff; Paria, Bibhash C.

    2015-01-01

    Excessive cytokine inflammatory response due to chronic or superphysiological level of microbial infection during pregnancy leads to pregnancy complications such as early pregnancy defects/loss and preterm birth. Bacterial toxin lipopolysaccharide (LPS), long recognized as a potent proinflammatory mediator, has been identified as a risk factor for pregnancy complications. Alkaline phosphatase (AP) isozymes have been shown to detoxify LPS by dephosphorylation. In this study, we examined the role of alkaline phosphatase (AP) in mitigating LPS-induced early pregnancy complications in mice. We found that 1) the uterus prior to implantation and implantation sites following embryo implantation produce LPS recognition and dephosphorylation molecules TLR4 and tissue non-specific AP (TNAP) isozyme, respectively; 2) uterine TNAP isozyme dephosphorylates LPS at its sites of production; 3) while LPS administration following embryo implantation elicits proinflammatory cytokine mRNA levels at the embryo implantation sites (EISs) and causes early pregnancy loss, dephosphorylated LPS neither triggers proinflammatory cytokine mRNA levels at the EISs nor induces pregnancy complications; 4) AP isozyme supplementation to accelerate LPS detoxification attenuates LPS-induced pregnancy complications following embryo implantation. These findings suggest that a LPS dephosphorylation strategy using AP isozyme may have a unique therapeutic potential to mitigate LPS- or Gram-negative bacteria-induced pregnancy complications in at-risk women. PMID:25910276

  9. Loss of AMP-Activated Protein Kinase Induces Mitochondrial Dysfunction and Proinflammatory Response in Unstimulated Abcd1-Knockout Mice Mixed Glial Cells

    PubMed Central

    Suhail, Hamid; Giri, Shailendra

    2015-01-01

    X-linked adrenoleukodystrophy (X-ALD) is caused by mutations and/or deletions in the ABCD1 gene. Similar mutations/deletions can give rise to variable phenotypes ranging from mild adrenomyeloneuropathy (AMN) to inflammatory fatal cerebral adrenoleukodystrophy (ALD) via unknown mechanisms. We recently reported the loss of the anti-inflammatory protein adenosine monophosphate activated protein kinase (AMPKα1) exclusively in ALD patient-derived cells. X-ALD mouse model (Abcd1-knockout (KO) mice) mimics the human AMN phenotype and does not develop the cerebral inflammation characteristic of human ALD. In this study we document that AMPKα1 levels in vivo (in brain cortex and spinal cord) and in vitro in Abcd1-KO mixed glial cells are similar to that of wild type mice. Deletion of AMPKα1 in the mixed glial cells of Abcd1-KO mice induced spontaneous mitochondrial dysfunction (lower oxygen consumption rate and ATP levels). Mitochondrial dysfunction in ALD patient-derived cells and in AMPKα1-deleted Abcd1-KO mice mixed glial cells was accompanied by lower levels of mitochondrial complex (1-V) subunits. More importantly, AMPKα1 deletion induced proinflammatory inducible nitric oxide synthase levels in the unstimulated Abcd1-KO mice mixed glial cells. Taken together, this study provides novel direct evidence for a causal role for AMPK loss in the development of mitochondrial dysfunction and proinflammatory response in X-ALD. PMID:25861159

  10. Regulation of Mct1 by cAMP-dependent internalization in rat brain endothelial cells.

    PubMed

    Smith, Jeffrey P; Uhernik, Amy L; Li, Lun; Liu, Zejian; Drewes, Lester R

    2012-10-22

    In the cerebrovascular endothelium, monocarboxylic acid transporter 1 (Mct1) controls blood-brain transport of short chain monocarboxylic and keto acids, including pyruvate and lactate, to support brain energy metabolism. Mct1 function is acutely decreased in rat brain cerebrovascular endothelial cells by β-adrenergic signaling through cyclic adenosine monophosphate (cAMP); however, the mechanism for this acute reduction in transport capacity is unknown. In this report, we demonstrate that cAMP induces the dephosphorylation and internalization of Mct1 from the plasma membrane into caveolae and early endosomes in the RBE4 rat brain cerebrovascular endothelial cell line. Additionally, we provide evidence that Mct1 constitutively cycles through clathrin vesicles and recycling endosomes in a pathway that is not dependent upon cAMP signaling in these cells. Our results are important because they show for the first time the regulated and unregulated vesicular trafficking of Mct1 in cerebrovascular endothelial cells; processes which have significance for better understanding normal brain energy metabolism, and the etiology and potential therapeutic approaches to treating brain diseases, such as stroke, in which lactic acidosis is a key component.

  11. Early growth response-1 is a regulator of DR5-induced apoptosis in colon cancer cells

    PubMed Central

    Mahalingam, D; Natoni, A; Keane, M; Samali, A; Szegezdi, E

    2010-01-01

    Background: Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) induces tumour cell apoptosis by binding to death receptor 4 (DR4) and DR5. DR4 and DR5 activation however can also induce inflammatory and pro-survival signalling. It is not known how these different cellular responses are regulated and what the individual role of DR4 vs DR5 is in these processes. Methods: DNA microarray study was carried out to identify genes differentially expressed after DR4 and DR5 activation. RT–PCR and western blotting was used to examine the expression of early growth response gene-1 (Egr-1) and the proteins of the TRAIL signalling pathway. The function of Egr-1 was studied by siRNA-mediated knockdown and overexpression of a dominant-negative version of Egr-1. Results: We show that the immediate early gene, Egr-1, regulates TRAIL sensitivity. Egr-1 is constitutively expressed in colon cancer cells and further induced upon activation of DR4 or DR5. Our results also show that DR4 mediates a type II, mitochondrion-dependent apoptotic pathway, whereas DR5 induces a mitochondrion-independent, type I apoptosis in HCT15 colon carcinoma cells. Egr-1 drives c-FLIP expression and the short splice variant of c-FLIP (c-FLIPS) specifically inhibits DR5 activation. Conclusion: Selective knockdown of c-FLIPS sensitises cells to DR5-induced but not DR4-induced apoptosis and Egr-1 exerts an effect as an inhibitor of the DR5-induced apoptotic pathway, possibly by regulating the expression of c-FLIPS. PMID:20087343

  12. Exendin-4 protects bone marrow-derived mesenchymal stem cells against oxygen/glucose and serum deprivation-induced apoptosis through the activation of the cAMP/PKA signaling pathway and the attenuation of ER stress.

    PubMed

    He, Jieqiong; Wang, Chao; Sun, Yunpeng; Lu, Bo; Cui, Jinjin; Dong, Nana; Zhang, Maomao; Liu, Youbing; Yu, Bo

    2016-04-01

    Exendin-4 (ex-4) is a long-acting glucagon-like peptide-1 receptor (GLP-1R) agonist which exerts beneficial effects on glycemic control and promotes cell viability. In the present study, we investigated the anti-apoptotic effects of ex-4, as well as the potential mechanisms responsible for these effects in rat bone marrow-derived mesenchymal stem cells (BM-MSCs) under conditions of oxygen, glucose and serum deprivation (OGD). The apoptosis of the MSCs was induced by subjecting the cells to OGD conditions for 4 h and was detected by Annexin V/PI and Hoechst 33258 staining. The MSCs were pre-conditioned with ex-4 for 12 h prior to being subjected to OGD conditions, and the expression levels of an apoptotic marker (cleaved caspase-3), endoplasmic reticulum (ER) stress markers [phosphorylated (p-)protein kinase RNA-like endoplasmic reticulum kinase (PERK), PERK, binding immunoglobulin protein (BIP), activating transcription factor 4 (ATF-4) and C/EBP homologous protein (CHOP)], as well as those of a survival marker (Bcl-2) were measured by western blot analysis. Furthermore, the mRNA levels of ATF-4 and CHOP were determined by RT-qPCR. ELISA was used to examine the activity of intracellular cAMP. Moreover, the GLP-1R antagonist, exendin9-39 (ex9-39), the protein kinase A (PKA) inhibitor, H89, and small interfering RNA (siRNA) targeting rat ATF-4 and CHOP were co-incubated with the MSCs. The apoptotic rate was markedly diminished following pre-conditioning with ex-4 in a dose‑dependent manner (P<0.05). The ER stress markers, p-PERK, BIP, ATF-4 and CHOP, were upregulated in the cells subjected to OGD conditions. Ex-4 pre-conditioning significantly decreased the mRNA and protein levels of ATF-4 and CHOP (P<0.05), and increased the activity of intracellular cAMP (P<0.05). Furthermore, the anti-apoptotic effects of ex-4 were almost reversed by treatment with either H89 or ex9-39 (P<0.05); transfection with siRNA-CHOP significantly reduced the apoptotic rate of the MSCs

  13. Exendin-4 protects bone marrow-derived mesenchymal stem cells against oxygen/glucose and serum deprivation-induced apoptosis through the activation of the cAMP/PKA signaling pathway and the attenuation of ER stress

    PubMed Central

    HE, JIEQIONG; WANG, CHAO; SUN, YUNPENG; LU, BO; CUI, JINJIN; DONG, NANA; ZHANG, MAOMAO; LIU, YOUBING; YU, BO

    2016-01-01

    Exendin-4 (ex-4) is a long-acting glucagon-like peptide-1 receptor (GLP-1R) agonist which exerts beneficial effects on glycemic control and promotes cell viability. In the present study, we investigated the anti-apoptotic effects of ex-4, as well as the potential mechanisms responsible for these effects in rat bone marrow-derived mesenchymal stem cells (BM-MSCs) under conditions of oxygen, glucose and serum deprivation (OGD). The apoptosis of the MSCs was induced by subjecting the cells to OGD conditions for 4 h and was detected by Annexin V/PI and Hoechst 33258 staining. The MSCs were pre-conditioned with ex-4 for 12 h prior to being subjected to OGD conditions, and the expression levels of an apoptotic marker (cleaved caspase-3), endoplasmic reticulum (ER) stress markers [phosphorylated (p-)protein kinase RNA-like endoplasmic reticulum kinase (PERK), PERK, binding immunoglobulin protein (BIP), activating transcription factor 4 (ATF-4) and C/EBP homologous protein (CHOP)], as well as those of a survival marker (Bcl-2) were measured by western blot analysis. Furthermore, the mRNA levels of ATF-4 and CHOP were determined by RT-qPCR. ELISA was used to examine the activity of intracellular cAMP. Moreover, the GLP-1R antagonist, exendin9-39 (ex9-39), the protein kinase A (PKA) inhibitor, H89, and small interfering RNA (siRNA) targeting rat ATF-4 and CHOP were co-incubated with the MSCs. The apoptotic rate was markedly diminished following pre-conditioning with ex-4 in a dose-dependent manner (P<0.05). The ER stress markers, p-PERK, BIP, ATF-4 and CHOP, were upregulated in the cells subjected to OGD conditions. Ex-4 pre-conditioning significantly decreased the mRNA and protein levels of ATF-4 and CHOP (P<0.05), and increased the activity of intracellular cAMP (P<0.05). Furthermore, the anti-apoptotic effects of ex-4 were almost reversed by treatment with either H89 or ex9-39 (P<0.05); transfection with siRNA-CHOP significantly reduced the apoptotic rate of the MSCs and

  14. Induction of functional CD154 (CD40 ligand) in neonatal T cells by cAMP-elevating agents

    PubMed Central

    Suárez, A; Mozo, L; Gayo, A; Simó, A; Gutiérrez, C

    2000-01-01

    A deficiency of neonatal T lymphocytes to express CD154 antigen in response to ionomycin and phorbol 12-myrsistate 13-acetate (PMA) stimulation or after CD3 cross-linking has been described. In the present report we describe that CD45RA+ newborn cells are able to synthesize and express CD154 at similar or even higher levels than adult cells in response to ionomycin and cAMP-elevating agents which trigger the protein kinase A (PKA) -mediated metabolic pathway. Peak CD154 protein concentrations in newborn cells were found between 4 and 8 hr after stimulation with ionomycin and dibutyryl cAMP. These agents, however, did not induce expression of the early activation antigen CD69. Surface levels of CD154 did not correlate with specific mRNA concentration, indicating that dibutyryl cAMP up-regulates CD154 by acting at a post-transcriptional stage. The CD154 antigen induced by PKA activation of newborn cells was functional, since upon binding to CD40 on B lymphocytes in the presence of interleukin-4 (IL-4), it promoted immunoglobulin heavy-class switching to IgE. We also found a different pattern of cytokine production between neonatal and adult CD4+ T cells. In response to ionomycin and dibutyryl cAMP, cord blood cells were more prone than adult lymphocytes to secrete the T helper type 2-derived immunosuppressive cytokines IL-4 and IL-10. Taking into account that the feto–maternal environment is rich in cAMP-elevating agents, the reduced risk of graft versus host disease associated with cord blood trasplantation, as compared with the risk with adult bone marrow cell transplants, may be due to the bias of neonatal cells to differentiate towards the T helper type 2 functional cell subset. PMID:10929069

  15. Amp Synthesis in Aqueous Solution of Adenosine and Phosphorus Pentoxide

    NASA Astrophysics Data System (ADS)

    Yamagata, Y.; Kojima, H.; Ejiri, K.; Inomata, K.

    1982-12-01

    Possible formation of a P4O10 molecule in magma, the stability of the molecule in hydrous volcanic gas at high temperatures and a possible prebiotic phosphate cycle were discussed in relation to chemical evolution. To demonstrate the utility of phosphorus pentoxide as a phosphorylating agent, aqueous solutions of adenosine (0.02M) and phosphorus pentoxide (0.2M) were incubated at 37°C for 5 months. The pH of the solutions was adjusted every day or every few days to each fixed value (9.0, 10.5, 11.5, 12.5) with 10 N NaOH. The HPLC analysis showed the formation of 2'-AMP, 3'-AMP, 5'-AMP, cyclic (2' 3')-AMP and cyclic (3' 5')-AMP. The main components of the products were 2'- and 3'-AMP, though cyclic (2' 3')-AMP was the main component in the early period of the incubation at pH 9.0. The yields (conversion rate of adenosine to AMPs) were increased almost linearly with the incubation time for 5 months in the case of pH 9.0. The final yields were about 3% (pH 9.0), 6% (pH 9.0, 1 M NaCl), 5% (pH 9.0, 0.01 M CaCl2, 0.01 M MgCl2), 7% (pH 9.0, 0.5 M NaCl, 0.01 M CaCl2, 0.01 M MgCl2), 9% (pH 9.0, 1 M NaCl, 0.01 M CaCl2, 0.01 M MgCl2), 32% (pH 10.5), 43% (pH 11.5), 35% (pH 12.5).

  16. Activation of AMP-Activated Protein Kinase α and Extracelluar Signal-Regulated Kinase Mediates CB-PIC-Induced Apoptosis in Hypoxic SW620 Colorectal Cancer Cells.

    PubMed

    Cho, Sung-Yun; Lee, Hyo-Jeong; Lee, Hyo-Jung; Jung, Deok-Beom; Kim, Hyunseok; Sohn, Eun Jung; Kim, Bonglee; Jung, Ji Hoon; Kwon, Byoung-Mog; Kim, Sung-Hoon

    2013-01-01

    Here, antitumor mechanism of cinnamaldehyde derivative CB-PIC was elucidated in human SW620 colon cancer cells. CB-PIC significantly exerted cytotoxicity, increased sub-G1 accumulation, and cleaved PARP with apoptotic features, while it enhanced the phosphorylation of AMPK alpha and ACC as well as activated the ERK in hypoxic SW620 cells. Furthermore, CB-PIC suppressed the expression of HIF1 alpha, Akt, and mTOR and activated the AMPK phosphorylation in hypoxic SW620 cells. Conversely, silencing of AMPK α blocked PARP cleavage and ERK activation induced by CB-PIC, while ERK inhibitor PD 98059 attenuated the phosphorylation of AMPK α in hypoxic SW620 cells, implying cross-talk between ERK and AMPK α . Furthermore, cotreatment of CB-PIC and metformin enhanced the inhibition of HIF1 α and Akt/mTOR and the activation of AMPK α and pACC in hypoxic SW620 cells. In addition, CB-PIC suppressed the growth of SW620 cells inoculated in BALB/c athymic nude mice, and immunohistochemistry revealed that CB-PIC treatment attenuated the expression of Ki-67, CD34, and CAIX and increased the expression of pAMPK α in CB-PIC-treated group. Interestingly, CP-PIC showed better antitumor activity in SW620 colon cancer cells under hypoxia than under normoxia, since it may be applied to chemoresistance. Overall, our findings suggest that activation of AMPK α and ERK mediates CB-PIC-induced apoptosis in hypoxic SW620 colon cancer cells.

  17. Nesfatin-1 Stimulates Fatty-Acid Oxidation by Activating AMP-Activated Protein Kinase in STZ-Induced Type 2 Diabetic Mice

    PubMed Central

    Xu, Huan; Wang, Peng-fei; Cai, Gui-ju; Song, Hai-feng; Wang, Chang-chen; Dong, Zhao-tong; Ju, Yan-jiao; Jiang, Zheng-yao

    2013-01-01

    Nesfatin-1 is an anorexigenic peptide involved in energy homeostasis. Recently, nesfatin-1 was reported to decrease blood glucose level and improve insulin sensitivity in high-fat diet-fed rats. However, little information is known about the influence of nesfatin-1 on lipid metabolism either in physiological or diabetic condition. This study undertook whether nesfatin-1 was involved in the pathophysiology in Streptozotocin-induced type 2 diabetic mice (T2DM), which was induced by a combination of high-calorie diet and two low-doses Streptozotocin. We observed that plasma nesfatin-1 was significantly increased while expression of nesfatin-1 neurons were decreased in hypothalamus in diabetes group compared to only high-calorie diet control group; intravenous injection of nesfatin-1 decreased 0–1h, 0–2h, 0–3h cumulative food intake in T2DM, but 0–24h total food intake had no difference between groups. Body weight and plasma FFA were normalized after nesfatin-1(10 µg/Kg) administration for 6 days. These results suggested that nesfatin-1 improved lipid disorder in T2DM. It was found that blood glucose and insulin resistance coefficient decreased with treatment of nesfatin-1 (both in 1 µg/Kg and 10 µg/Kg doses) in diabetes mice. For further understanding the role of nesfatin-1 on lipid metabolism, we detected p-AMPK and p-ACC of skeletal muscle in T2DM using western blotting. The expression of p-AMPK and p-ACC increased when nesfatin-1 was given with doses 1 µg/Kg but not in doses 10 µg/Kg. Taken together, nesfatin-1 participated in the development of T2DM and stimulated free fatty acid utilization via AMPK-ACC pathway in skeletal muscle in T2DM. PMID:24391760

  18. Proinflammatory adipokine leptin mediates disinfection byproduct bromodichloromethane-induced early steatohepatitic injury in obesity

    SciTech Connect

    Das, Suvarthi; Kumar, Ashutosh; Seth, Ratanesh Kumar; Tokar, Erik J.; Kadiiska, Maria B.; Waalkes, Michael P.; Mason, Ronald P.; Chatterjee, Saurabh

    2013-06-15

    Today's developed world faces a major public health challenge in the rise in the obese population and the increased incidence in fatty liver disease. There is a strong association among diet induced obesity, fatty liver disease and development of nonalcoholic steatohepatitis but the environmental link to disease progression remains unclear. Here we demonstrate that in obesity, early steatohepatitic lesions induced by the water disinfection byproduct bromodichloromethane are mediated by increased oxidative stress and leptin which act in synchrony to potentiate disease progression. Low acute exposure to bromodichloromethane (BDCM), in diet-induced obesity produced oxidative stress as shown by increased lipid peroxidation, protein free radical and nitrotyrosine formation and elevated leptin levels. Exposed obese mice showed histopathological signs of early steatohepatitic injury and necrosis. Spontaneous knockout mice for leptin or systemic leptin receptor knockout mice had significantly decreased oxidative stress and TNF-α levels. Co-incubation of leptin and BDCM caused Kupffer cell activation as shown by increased MCP-1 release and NADPH oxidase membrane assembly, a phenomenon that was decreased in Kupffer cells isolated from leptin receptor knockout mice. In obese mice that were BDCM-exposed, livers showed a significant increase in Kupffer cell activation marker CD68 and, increased necrosis as assessed by levels of isocitrate dehydrogenase, events that were decreased in the absence of leptin or its receptor. In conclusion, our results show that exposure to the disinfection byproduct BDCM in diet-induced obesity augments steatohepatitic injury by potentiating the effects of leptin on oxidative stress, Kupffer cell activation and cell death in the liver. - Highlights: ► BDCM acute exposure sensitizes liver to increased free radical stress in obesity. ► BDCM-induced higher leptin contributes to early steatohepatitic lesions. ► Increased leptin mediates protein

  19. Experiment definition studies for AMPS Spacelab

    NASA Technical Reports Server (NTRS)

    Liemohn, H.

    1975-01-01

    The electrical charging of the space shuttle orbiter is discussed in relation to the AMPS Spacelab payload along with an operations research technique for the selection of AMPS Spacelab experiments. Experiments proposed for AMPS include: hydromagnetic wave experiments; bistatic sounder of AMPS wake; and an artificial meteor gun. Experiment objectives and instrument functions are given for all experiments.

  20. Membrane remodeling, an early event in benzo[alpha]pyrene-induced apoptosis

    SciTech Connect

    Tekpli, Xavier; Rissel, Mary; Huc, Laurence; Catheline, Daniel; Sergent, Odile; Rioux, Vincent; Legrand, Philippe; Holme, Jorn A.; Dimanche-Boitrel, Marie-Therese; Lagadic-Gossmann, Dominique

    2010-02-15

    Benzo[alpha]pyrene (B[alpha]P) often serves as a model for mutagenic and carcinogenic polycyclic aromatic hydrocarbons (PAHs). Our previous work suggested a role of membrane fluidity in B[alpha]P-induced apoptotic process. In this study, we report that B[alpha]P modifies the composition of cholesterol-rich microdomains (lipid rafts) in rat liver F258 epithelial cells. The cellular distribution of the ganglioside-GM1 was markedly changed following B[alpha]P exposure. B[alpha]P also modified fatty acid composition and decreased the cholesterol content of cholesterol-rich microdomains. B[alpha]P-induced depletion of cholesterol in lipid rafts was linked to a reduced expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase). Aryl hydrocarbon receptor (AhR) and B[alpha]P-related H{sub 2}O{sub 2} formation were involved in the reduced expression of HMG-CoA reductase and in the remodeling of membrane microdomains. The B[alpha]P-induced membrane remodeling resulted in an intracellular alkalinization observed during the early phase of apoptosis. In conclusion, B[alpha]P altered the composition of plasma membrane microstructures through AhR and H{sub 2}O{sub 2} dependent-regulation of lipid biosynthesis. In F258 cells, the B[alpha]P-induced membrane remodeling was identified as an early apoptotic event leading to an intracellular alkalinization.

  1. Is a decrease in cyclic AMP a necessary and sufficient signal for maturation of amphibian oocytes

    SciTech Connect

    Gelerstein, S.; Shapira, H.; Dascal, N.; Yekuel, R.; Oron, Y.

    1988-05-01

    Acetylcholine rapidly lowered the intracellular levels of cyclic AMP in stage 5 and 6 Xenopus laevis oocytes. Acetylcholine alone did not induce oocyte maturation, though it did accelerate maturation induced by progesterone. The effect of acetylcholine on oocyte maturation was independent of extracellular calcium concentration. Adenosine increased cyclic AMP and abolished the progesterone-induced decrease in cyclic AMP levels in follicles and in denuded oocytes. This effect of adenosine was blocked by the Ra purinergic receptor antagonist, theophylline. Despite those effects, adenosine alone induced maturation in stage 6 oocytes and accelerated progesterone-induced maturation in both stage 5 and 6 cells. Adenosine also induced a significant increase in the rate of /sup 45/Ca efflux from oocytes in the presence and the absence of external calcium. We suggest that the activation of cell surface receptors involved in the release of calcium from cellular stores may induce or accelerate oocyte maturation independently of small changes in intracellular cyclic AMP concentration.

  2. Early onset Morganella morganii sepsis in a newborn infant with emergence of cephalosporin resistance caused by depression of AMPC beta-lactamase production..

    PubMed

    Sinha, Ajay K; Kempley, Stephen T; Price, Elizabeth; Sharma, Bal K; Livermore, David M

    2006-04-01

    A preterm infant with early onset Morganella morganii sepsis was treated with cefotaxime and gentamicin after confirmation of antimicrobial susceptibility. The infant developed persistent ventriculitis caused by the emergence of a cefotaxime-resistant Morganella variant with derepression of its AmpC beta-lactamase. When choosing antibiotic therapy, the risk of development of resistance to cephalosporins should be considered in infections caused by M. morganii and other Gram-negative organisms with inducible AmpC beta-lactamases.

  3. LdrP, a cAMP receptor protein/FNR family transcriptional regulator, serves as a positive regulator for the light-inducible gene cluster in the megaplasmid of Thermus thermophilus.

    PubMed

    Takano, Hideaki; Agari, Yoshihiro; Hagiwara, Kenta; Watanabe, Ren; Yamazaki, Ryuta; Beppu, Teruhiko; Shinkai, Akeo; Ueda, Kenji

    2014-12-01

    LdrP (TT_P0055) (LitR-dependent regulatory protein) is one of the four cAMP receptor protein (CRP)/FNR family transcriptional regulators retained by the extremely thermophilic bacterium Thermus thermophilus. Previously, we reported that LdrP served as a positive regulator for the light-induced transcription of crtB, a carotenoid biosynthesis gene encoded on the megaplasmid of this organism. Here, we showed that LdrP also functions as an activator of the expression of genes clustered around the crtB gene under the control of LitR, an adenosyl B12-bound light-sensitive regulator. Transcriptome analysis revealed the existence of 19 LitR-dependent genes on the megaplasmid. S1 nuclease protection assay confirmed that the promoters preceding TT_P0044 (P44), TT_P0049 (P49) and TT_P0070 (P70) were activated upon illumination in the WT strain. An ldrP mutant lost the ability to activate P44, P49 and P70, whilst disruption of litR resulted in constitutive transcription from these promoters irrespective of illumination, indicating that these genes were photo-dependently regulated by LdrP and LitR. An in vitro transcription experiment demonstrated that LdrP directly activated mRNA synthesis from P44 and P70 by the Thermus RNA polymerase holocomplex. The present evidence indicated that LdrP was the positive regulator essential for the transcription of the T. thermophilus light-inducible cluster encoded on the megaplasmid. PMID:25294106

  4. cAMP-responsive element binding protein mediates a cGMP/protein kinase G-dependent anti-apoptotic signal induced by nitric oxide in retinal neuro-glial progenitor cells.

    PubMed

    Nagai-Kusuhara, Azusa; Nakamura, Makoto; Mukuno, Hirokazu; Kanamori, Akiyasu; Negi, Akira; Seigel, Gail M

    2007-01-01

    Nitric oxide (NO) is cytoprotective to certain types of neuronal cells. The neuroprotective ability of NO in the retina was reportedly mediated by the cyclic GMP (cGMP) to protein kinase G (PKG) pathway. Cyclic AMP-responsive element binding protein (CREB) plays an essential role in the NO/cGMP/PKG-mediated survival of rat cerebellar granule cells. We tested whether CREB transduces the NO/cGMP/PKG anti-apoptotic cascade in R28 neuro-glial progenitor cells. Apoptosis was induced in R28 cells by serum deprivation for 24 h. Varying concentrations of two NO donors, sodium nitroprusside (SNP) and nipradilol, were added to medium with or without an NO scavenger, a soluble guanylyl cyclase inhibitor, or a PKG inhibitor. The cells were immunostained against activated caspase-3 and counterstained with Hoechst 33258. Apoptosis was quantified by counting activated caspase-3 positive or pyknotic cells. SNP and nipradilol rescued R28 cells from apoptosis in a dose-dependent manner, at an optimal concentration of 1.0 microM and 10 microM, respectively. Higher concentrations were cytotoxic. The NO scavenger and the inhibitors decreased the anti-apoptotic effect of the NO donors. Intracellular cGMP levels were increased after exposure to SNP and nipradilol. Western blotting showed that both NO donors increased CREB phosphorylation, which was blocked when pre-exposed to the inhibitors. Transfection with a dominant negative CREB construct defective of phosphorylation at Ser-133 interfered with the anti-apoptotic activity of SNP. These results indicate that CREB at least in part mediates the cGMP/PKG-dependent anti-apoptotic signal induced by NO in R28 cells. PMID:17081519

  5. MEK Inhibitors Reverse cAMP-Mediated Anxiety in Zebrafish

    PubMed Central

    Lundegaard, Pia R.; Anastasaki, Corina; Grant, Nicola J.; Sillito, Rowland R.; Zich, Judith; Zeng, Zhiqiang; Paranthaman, Karthika; Larsen, Anders Peter; Armstrong, J. Douglas; Porteous, David J.; Patton, E. Elizabeth

    2015-01-01

    Summary 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 the repositioning of MEK inhibitors as behavior stabilizers in the context of increased cAMP. PMID:26388333

  6. Mitochondrial Respiratory Defect Causes Dysfunctional Lactate Turnover via AMP-activated Protein Kinase Activation in Human-induced Pluripotent Stem Cell-derived Hepatocytes.

    PubMed

    Im, Ilkyun; Jang, Mi-Jin; Park, Seung Ju; Lee, Sang-Hee; Choi, Jin-Ho; Yoo, Han-Wook; Kim, Seyun; Han, Yong-Mahn

    2015-12-01

    A defective mitochondrial respiratory chain complex (DMRC) causes various metabolic disorders in humans. However, the pathophysiology of DMRC in the liver remains unclear. To understand DMRC pathophysiology in vitro, DMRC-induced pluripotent stem cells were generated from dermal fibroblasts of a DMRC patient who had a homoplasmic mutation (m.3398T→C) in the mitochondrion-encoded NADH dehydrogenase 1 (MTND1) gene and that differentiated into hepatocytes (DMRC hepatocytes) in vitro. DMRC hepatocytes showed abnormalities in mitochondrial characteristics, the NAD(+)/NADH ratio, the glycogen storage level, the lactate turnover rate, and AMPK activity. Intriguingly, low glycogen storage and transcription of lactate turnover-related genes in DMRC hepatocytes were recovered by inhibition of AMPK activity. Thus, AMPK activation led to metabolic changes in terms of glycogen storage and lactate turnover in DMRC hepatocytes. These data demonstrate for the first time that energy depletion may lead to lactic acidosis in the DMRC patient by reduction of lactate uptake via AMPK in liver. PMID:26491018

  7. Effect of ozone oxidative preconditioning in preventing early radiation-induced lung injury in rats.

    PubMed

    Bakkal, B H; Gultekin, F A; Guven, B; Turkcu, U O; Bektas, S; Can, M

    2013-09-01

    Ionizing radiation causes its biological effects mainly through oxidative damage induced by reactive oxygen species. Previous studies showed that ozone oxidative preconditioning attenuated pathophysiological events mediated by reactive oxygen species. As inhalation of ozone induces lung injury, the aim of this study was to examine whether ozone oxidative preconditioning potentiates or attenuates the effects of irradiation on the lung. Rats were subjected to total body irradiation, with or without treatment with ozone oxidative preconditioning (0.72 mg/kg). Serum proinflammatory cytokine levels, oxidative damage markers, and histopathological analysis were compared at 6 and 72 h after total body irradiation. Irradiation significantly increased lung malondialdehyde levels as an end-product of lipoperoxidation. Irradiation also significantly decreased lung superoxide dismutase activity, which is an indicator of the generation of oxidative stress and an early protective response to oxidative damage. Ozone oxidative preconditioning plus irradiation significantly decreased malondialdehyde levels and increased the activity of superoxide dismutase, which might indicate protection of the lung from radiation-induced lung injury. Serum tumor necrosis factor alpha and interleukin-1 beta levels, which increased significantly following total body irradiation, were decreased with ozone oxidative preconditioning. Moreover, ozone oxidative preconditioning was able to ameliorate radiation-induced lung injury assessed by histopathological evaluation. In conclusion, ozone oxidative preconditioning, repeated low-dose intraperitoneal administration of ozone, did not exacerbate radiation-induced lung injury, and, on the contrary, it provided protection against radiation-induced lung damage.

  8. Tocopherols inhibit oxidative and nitrosative stress in estrogen-induced early mammary hyperplasia in ACI rats.

    PubMed

    Das Gupta, Soumyasri; So, Jae Young; Wall, Brian; Wahler, Joseph; Smolarek, Amanda K; Sae-Tan, Sudathip; Soewono, Kelvin Y; Yu, Haixiang; Lee, Mao-Jung; Thomas, Paul E; Yang, Chung S; Suh, Nanjoo

    2015-09-01

    Oxidative stress is known to play a key role in estrogen-induced breast cancer. This study assessed the chemopreventive activity of the naturally occurring γ-tocopherol-rich mixture of tocopherols (γ-TmT) in early stages of estrogen-induced mammary hyperplasia in ACI rats. ACI rats provide an established model of rodent mammary carcinogenesis due to their high sensitivity to estrogen. Female rats were implanted with 9 mg of 17β-estradiol (E2) in silastic tubings and fed with control or 0.3% γ-TmT diet for 1, 3, 7, and 14 d. γ-TmT increased the levels of tocopherols and their metabolites in the serum and mammary glands of the rats. Histological analysis revealed mammary hyperplasia in the E2 treated rats fed with control or γ-TmT diet. γ-TmT decreased the levels of E2-induced nitrosative and oxidative stress markers, nitrotyrosine, and 8-oxo-dG, respectively, in the hyperplastic mammary tissues. 8-Isoprostane, a marker of oxidative stress in the serum, was also reduced by γ-TmT. Noticeably, γ-TmT stimulated Nrf2-dependent antioxidant response in the mammary glands of E2 treated rats, evident from the induced mRNA levels of Nrf2 and its downstream antioxidant enzymes, superoxide dismutase, catalase, and glutathione peroxidase. Therefore, inhibition of nitrosative/oxidative stress through induction of antioxidant response is the primary effect of γ-TmT in early stages of E2-induced mammary hyperplasia. Due to its cytoprotective activity, γ-TmT could be a potential natural agent for the chemoprevention of estrogen-induced breast cancer.

  9. Improved Long-Term Memory via Enhancing cGMP-PKG Signaling Requires cAMP-PKA Signaling

    PubMed Central

    Bollen, Eva; Puzzo, Daniela; Rutten, Kris; Privitera, Lucia; De Vry, Jochen; Vanmierlo, Tim; Kenis, Gunter; Palmeri, Agostino; D'Hooge, Rudi; Balschun, Detlef; Steinbusch, Harry MW; Blokland, Arjan; Prickaerts, Jos

    2014-01-01

    Memory consolidation is defined by the stabilization of a memory trace after acquisition, and consists of numerous molecular cascades that mediate synaptic plasticity. Commonly, a distinction is made between an early and a late consolidation phase, in which early refers to the first hours in which labile synaptic changes occur, whereas late consolidation relates to stable and long-lasting synaptic changes induced by de novo protein synthesis. How these phases are linked at a molecular level is not yet clear. Here we studied the interaction of the cyclic nucleotide-mediated pathways during the different phases of memory consolidation in rodents. In addition, the same pathways were studied in a model of neuronal plasticity, long-term potentiation (LTP). We demonstrated that cGMP/protein kinase G (PKG) signaling mediates early memory consolidation as well as early-phase LTP, whereas cAMP/protein kinase A (PKA) signaling mediates late consolidation and late-phase-like LTP. In addition, we show for the first time that early-phase cGMP/PKG signaling requires late-phase cAMP/PKA-signaling in both LTP and long-term memory formation. PMID:24813825

  10. Investigation of the early stages in laser-induced ignition by Schlieren photography and laser-induced fluorescence spectroscopy.

    PubMed

    Lackner, Maximilian; Charareh, S; Winter, F; Iskra, K; Rüdisser, D; Neger, T; Kopecek, H; Wintner, E

    2004-09-20

    Laser ignition has been discussed widely as a potentially superior ignition source for technical appliances such as internal combustion engines. Ignition strongly affects overall combustion, and its early stages in particular have strong implications on subsequent pollutant formation, flame quenching, and extinction. Our research here is devoted to the experimental investigation of the early stages of laser-induced ignition of CH4/air mixtures up to high pressures. Tests were performed in a 0.9-l combustion cell with initial pressures of up to 25 bar with stoichiometric to fuel-lean mixtures using a 5-ns 50-mJ 1064-nm Nd:YAG laser. Laserinduced fluorescence (LIF) was used to obtain two dimensionally resolved images of the OH radical distribution after the ignition event. These images were used to produce an animation of laser ignition and early flame kernel development. Schlieren photography was used to investigate the laserinduced shock wave, hot core gas, and developing flame ball. We extend existing knowledge to high-pressure regimes relevant for internal combustion engines.

  11. Investigation of the early stages in laser-induced ignition by Schlieren photography and laser-induced fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Lackner, Maximilian; Charareh, S.; Winter, F.; Iskra, K. F.; Rüdisser, D.; Neger, T.; Kopecek, H.; Wintner, E.

    2004-09-01

    Laser ignition has been discussed widely as a potentially superior ignition source for technical appliances such as internal combustion engines. Ignition strongly affects overall combustion, and its early stages in particular have strong implications on subsequent pollutant formation, flame quenching, and extinction. Our research here is devoted to the experimental investigation of the early stages of laser-induced ignition of CH4/air mixtures up to high pressures. Tests were performed in a 0.9-l combustion cell with initial pressures of up to 25 bar with stoichiometric to fuel-lean mixtures using a 5-ns 50-mJ 1064-nm Nd:YAG laser. Laserinduced fluorescence (LIF) was used to obtain two dimensionally resolved images of the OH radical distribution after the ignition event. These images were used to produce an animation of laser ignition and early flame kernel development. Schlieren photography was used to investigate the laserinduced shock wave, hot core gas, and developing flame ball. We extend existing knowledge to high-pressure regimes relevant for internal combustion engines.

  12. Regulation of the Dictyostelium glycogen phosphorylase 2 gene by cyclic AMP.

    PubMed

    Sucic, J F; Selmin, O; Rutherford, C L

    1993-01-01

    A crucial developmental event in the cellular slime mold, Dictyostelium discoideum, is glycogen degradation. The enzyme that catalyzes this degradation, glycogen phosphorylase 2 (gp-2), is developmentally regulated and cAMP appears to be involved in this regulation. We have examined several aspects of the cAMP regulation of gp-2. We show that addition of exogenous cAMP to aggregation competent amoebae induced the appearance of gp-2 mRNA. The induction of gp-2 mRNA occurred within 1 and 1.5 h after the initial exposure to cAMP. Exposure to exogenous cAMP concentrations as low as 1.0 microM could induce gp-2 mRNA. We also examined the molecular mechanism through which cAMP induction of gp-2 occurs. Induction of gp-2 appears to result from a mechanism that does not require intracellular cAMP signaling, and may occur directly through a cAMP binding protein without the requirement of any intracellular signalling. We also examined the promoter region of the gp-2 gene for cis-acting elements that are involved in the cAMP regulation of gp-2. A series of deletions of the promoter were fused to a luciferase reporter gene and then analyzed for cAMP responsiveness. The results indicated that a region from -258 nucleotides to the transcriptional start site is sufficient for essentially full activity and appears to carry all necessary cis-acting sites for cAMP induction. Further deletion of 58 nucleotides from the 5' end, results in fivefold less activity in the presence of cAMP. Deletion of the next 104 nucleotides eliminates the cAMP response entirely. PMID:8222346

  13. The proteasome inhibitor bortezomib induces testicular toxicity by upregulation of oxidative stress, AMP-activated protein kinase (AMPK) activation and deregulation of germ cell development in adult murine testis

    SciTech Connect

    Li, Wei; Fu, Jianfang; Zhang, Shun; Zhao, Jie; Xie, Nianlin; Cai, Guoqing

    2015-06-01

    Understanding how chemotherapeutic agents mediate testicular toxicity is crucial in light of compelling evidence that male infertility, one of the severe late side effects of intensive cancer treatment, occurs more often than they are expected to. Previous study demonstrated that bortezomib (BTZ), a 26S proteasome inhibitor used to treat refractory multiple myeloma (MM), exerts deleterious impacts on spermatogenesis in pubertal mice via unknown mechanisms. Here, we showed that intermittent treatment with BTZ resulted in fertility impairment in adult mice, evidenced by testicular atrophy, desquamation of immature germ cells and reduced caudal sperm storage. These deleterious effects may originate from the elevated apoptosis in distinct germ cells during the acute phase and the subsequent disruption of Sertoli–germ cell anchoring junctions (AJs) during the late recovery. Mechanistically, balance between AMP-activated protein kinase (AMPK) activation and Akt/ERK pathway appeared to be indispensable for AJ integrity during the late testicular recovery. Of particular interest, the upregulated testicular apoptosis and the following disturbance of Sertoli–germ cell interaction may both stem from the excessive oxidative stress elicited by BTZ exposure. We also provided the in vitro evidence that AMPK-dependent mechanisms counteract follicle-stimulating hormone (FSH) proliferative effects in BTZ-exposed Sertoli cells. Collectively, BTZ appeared to efficiently prevent germ cells from normal development via multiple mechanisms in adult mice. Employment of antioxidants and/or AMPK inhibitor may represent an attractive strategy of fertility preservation in male MM patients exposed to conventional BTZ therapy and warrants further investigation. - Highlights: • Intermittent treatment with BTZ caused fertility impairment in adult mice. • BTZ treatment elicited apoptosis during early phase of testicular recovery. • Up-regulation of oxidative stress by BTZ treatment

  14. Early-onset osteoarthritis of mouse temporomandibular joint induced by partial discectomy

    PubMed Central

    Xu, L.; Polur, I.; Lim, C.; Servais, J. M.; Dobeck, J.; Li, Y.; Olsen, B. R.

    2010-01-01

    Summary Objective The objective of this study is to characterize mouse temporomandibular joint (TMJ) following partial discectomy, since there is no documentation of whether or not partial discectomy can induce early-onset osteoarthritis (OA) in mouse TMJ. Methods Partial discs of TMJ in mice were removed by microsurgery. Histology was performed to characterize articular cartilages from the TMJ of mice. The morphology of the articular cartilages was evaluated using a modified Mankin scoring system. Immunohistostaining was carried out to examine the expression of discoidin domain receptor 2 (Ddr2), a type II collagen receptor, matrix metalloproteinase 13 (Mmp-13), and Mmp-derived type II collagen fragments in the articular cartilage of condyles from the mouse TMJ. Results Articular cartilage degeneration was seen in the mouse TMJ post discectomy, including increased proteoglycan staining in the extracellular matrix at 4 weeks, the appearance of chondrocyte clusters at 8 weeks, reduced proteoglycan staining and fibrillation at 12 weeks and the loss of articular cartilage at 16 weeks. Increased immunostaining for Ddr2, Mmp-13, and Mmp-derived type II collagen fragments was detected. Conclusion Results indicate that partial discectomy induces early-onset OA in mouse TMJ and that increased expression of Mmp-13, likely due to the elevated expression of Ddr2, may be one of the factors responsible for the early-onset OA in mouse TMJ. PMID:19230720

  15. Early recognition and management of fabricated or induced illness in children.

    PubMed

    Bass, Christopher; Glaser, Danya

    2014-04-19

    Fabricated or induced illness (previously known as Munchausen syndrome by proxy) takes place when a caregiver elicits health care on the child's behalf in an unjustified way. Although the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders specifies deception as a perpetrator characteristic, a far wider range is encountered clinically and is included in this Review. We describe the features of fabricated or induced illness, its effect on the child, and the psychosocial characteristics of caregivers and their possible motives. Present evidence suggests that somatoform and factitious disorders are over-represented in caregivers, with possible intergenerational transmission of abnormal illness behaviour from the caregiver to the child. Paediatricians' early recognition of perplexing presentations preceding fabricated or induced illness and their management might obviate the development of this disorder. In cases of fully developed fabricated or induced illness, as well as protection, the child will need help to return to healthy functioning and understand the fabricated or induced illness experience. Management of the perpetrator is largely dependent on their capacity to acknowledge the abusive behaviour and collaborate with helping agencies. If separation is necessary, reunification of mother and child is rare, but can be achieved in selected cases. More collaborative research is needed in this specialty, especially regarding close study of the characteristics of women with somatoform and factitious disorders who involve their children in abnormal illness behaviour. We recommend that general hospitals establish proactive networks including multidisciplinary cooperation between designated staff from both paediatric and adult mental health services. PMID:24612863

  16. Schwann Cells Metabolize Extracellular 2',3'-cAMP to 2'-AMP.

    PubMed

    Verrier, Jonathan D; Kochanek, Patrick M; Jackson, Edwin K

    2015-08-01

    The 3',5'-cAMP-adenosine pathway (3',5'-cAMP→5'-AMP→adenosine) and the 2',3'-cAMP-adenosine pathway (2',3'-cAMP→2'-AMP/3'-AMP→adenosine) are active in the brain. Oligodendrocytes participate in the brain 2',3'-cAMP-adenosine pathway via their robust expression of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase; converts 2',3'-cAMP to 2'-AMP). Because Schwann cells also express CNPase, it is conceivable that the 2',3'-cAMP-adenosine pathway exists in the peripheral nervous system. To test this and to compare the 2',3'-cAMP-adenosine pathway to the 3',5'-cAMP-adenosine pathway in Schwann cells, we examined the metabolism of 2',3'-cAMP, 2'-AMP, 3'-AMP, 3',5'-cAMP, and 5'-AMP in primary rat Schwann cells in culture. Addition of 2',3'-cAMP (3, 10, and 30 µM) to Schwann cells increased levels of 2'-AMP in the medium from 0.006 ± 0.002 to 21 ± 2, 70 ± 3, and 187 ± 10 nM/µg protein, respectively; in contrast, Schwann cells had little ability to convert 2',3'-cAMP to 3'-AMP or 3',5'-cAMP to either 3'-AMP or 5'-AMP. Although Schwann cells slightly converted 2',3'-cAMP and 2'-AMP to adenosine, they did so at very modest rates (e.g., 5- and 3-fold, respectively, more slowly compared with our previously reported studies in oligodendrocytes). Using transected myelinated rat sciatic nerves in culture medium, we observed a time-related increase in endogenous intracellular 2',3'-cAMP and extracellular 2'-AMP. These findings indicate that Schwann cells do not have a robust 3',5'-cAMP-adenosine pathway but do have a 2',3'-cAMP-adenosine pathway; however, because the pathway mostly involves 2'-AMP formation rather than 3'-AMP, and because the conversion of 2'-AMP to adenosine is slow, metabolism of 2',3'-cAMP mostly results in the accumulation of 2'-AMP. Accumulation of 2'-AMP in peripheral nerves postinjury could have pathophysiological consequences. PMID:25998049

  17. Zscan4 transiently reactivates early embryonic genes during the generation of induced pluripotent stem cells.

    PubMed

    Hirata, Tetsuya; Amano, Tomokazu; Nakatake, Yuhki; Amano, Misa; Piao, Yulan; Hoang, Hien G; Ko, Minoru S H

    2012-01-01

    The generation of induced pluripotent stem cells (iPSCs) by the forced expression of defined transcription factors in somatic cells holds great promise for the future of regenerative medicine. However, the initial reprogramming mechanism is still poorly understood. Here we show that Zscan4, expressed transiently in2-cell embryos and embryonic stem cells (ESCs), efficiently produces iPSCs from mouse embryo fibroblasts when coexpressed with Klf4, Oct4, and Sox2. Interestingly, the forced expression of Zscan4 is required onlyfor the first few days of iPSC formation. Microarray analysis revealed transient and early induction of preimplantation-specific genes in a Zscan4-dependent manner. Our work indicates that Zscan4 is a previously unidentified potent natural factor that facilitates the reprogramming process and reactivates early embryonic genes.

  18. Short-term second language and music training induces lasting functional brain changes in early childhood.

    PubMed

    Moreno, Sylvain; Lee, Yunjo; Janus, Monika; Bialystok, Ellen

    2015-01-01

    Immediate and lasting effects of music or second-language training were examined in early childhood using event-related potentials. Event-related potentials were recorded for French vowels and musical notes in a passive oddball paradigm in thirty-six 4- to 6-year-old children who received either French or music training. Following training, both groups showed enhanced late discriminative negativity (LDN) in their trained condition (music group-musical notes; French group-French vowels) and reduced LDN in the untrained condition. These changes reflect improved processing of relevant (trained) sounds, and an increased capacity to suppress irrelevant (untrained) sounds. After 1 year, training-induced brain changes persisted and new hemispheric changes appeared. Such results provide evidence for the lasting benefit of early intervention in young children. PMID:25346534

  19. Radiation-induced breast cancer: the question of early breast cancer screening in Hodgkin's lymphoma survivors.

    PubMed

    Hilal, Talal; Rudy, David W

    2016-02-01

    Chest irradiation is associated with numerous early and late complications that arise from ionizing radiation-induced damage to cellular structures within the field of therapy. In patients exposed to chest irradiation at an early age as part of the treatment of childhood cancer, specifically Hodgkin's lymphoma, the increased risk of breast cancer in the long run should be considered. A case of a 35-year-old woman who exposed to chest irradiation as part of the treatment of Hodgkin's lymphoma at the age of 20 years is presented here and serves as a reminder of this somewhat overlooked complication. The article presents the evidence available for and against breast cancer screening in this particular patient population. PMID:26949536

  20. Short-term second language and music training induces lasting functional brain changes in early childhood.

    PubMed

    Moreno, Sylvain; Lee, Yunjo; Janus, Monika; Bialystok, Ellen

    2015-01-01

    Immediate and lasting effects of music or second-language training were examined in early childhood using event-related potentials. Event-related potentials were recorded for French vowels and musical notes in a passive oddball paradigm in thirty-six 4- to 6-year-old children who received either French or music training. Following training, both groups showed enhanced late discriminative negativity (LDN) in their trained condition (music group-musical notes; French group-French vowels) and reduced LDN in the untrained condition. These changes reflect improved processing of relevant (trained) sounds, and an increased capacity to suppress irrelevant (untrained) sounds. After 1 year, training-induced brain changes persisted and new hemispheric changes appeared. Such results provide evidence for the lasting benefit of early intervention in young children.

  1. Short-term Second Language and Music Training Induces Lasting Functional Brain Changes in Early Childhood

    PubMed Central

    Moreno, Sylvain; Lee, Yunjo

    2014-01-01

    Immediate and lasting effects of music or second-language training were examined in early childhood using event-related potentials (ERPs). ERPs were recorded for French vowels and musical notes in a passive oddball paradigm in 36 four- to six-year-old children who received either French or music training. Following training, both groups showed enhanced late discriminative negativity (LDN) in their trained condition (music group–musical notes; French group–French vowels) and reduced LDN in the untrained condition. These changes reflect improved processing of relevant (trained) sounds, and an increased capacity to suppress irrelevant (untrained) sounds. After one year, training-induced brain changes persisted and new hemispheric changes appeared. Such results provide evidence for the lasting benefit of early intervention in young children. PMID:25346534

  2. Association with positive outcome induces early effects in event-related brain potentials.

    PubMed

    Schacht, Annekathrin; Adler, Nele; Chen, Peiyao; Guo, Taomei; Sommer, Werner

    2012-01-01

    Emotional pictures, faces, or words elicit an early posterior negativity (EPN) in the event-related potential, starting around 200-400 ms, followed by a late positive complex (LPC). Occasionally, also very early effects of emotion (VEEEs) are seen prior to 200 ms. The present study examined whether VEEEs can be due to direct links established by reinforcement learning. In the learning session, participants learned to associate previously unknown Chinese words with monetary gain, loss, or neither. In the test session, they were required to distinguish the learned stimuli from novel distracters. Specific to stimuli associated with positive outcome a VEEE, consisting of a posterior positivity, appeared around 150 ms and an LPC between 550 and 700 ms, whereas an EPN was absent. These results show that previous association with reward can induce VEEEs, indicating that emotion effects in ERPs may arise in the absence of biologically preparedness and semantic meaning. PMID:22027086

  3. Aortic VCAM-1: an early marker of vascular inflammation in collagen-induced arthritis.

    PubMed

    Denys, Anne; Clavel, Gaëlle; Lemeiter, Delphine; Schischmanoff, Olivier; Boissier, Marie-Christophe; Semerano, Luca

    2016-05-01

    Cardiovascular disease (CVD) is a major cause of morbidity and mortality in rheumatoid arthritis (RA). There are limited experimental data on vascular involvement in arthritis models. To study the link between CVD and inflammation in RA, we developed a model of vascular dysfunction and articular inflammation by collagen-induced arthritis (CIA) in C57Bl/6 (B6) mice. We studied the expression of vascular inflammatory markers in CIA with and without concomitant hyperlipidic diet (HD). Collagen-induced arthritis was induced with intradermal injection of chicken type-II collagen followed by a boost 21 days later. Mice with and without CIA were fed a standard diet or an HD for 12 weeks starting from the day of the boost. Arthritis severity was evaluated with a validated clinical score. Aortic mRNA levels of vascular cell adhesion molecule-1 (VCAM-1), inducible nitric oxide synthase (iNOS) and interleukin-17 were analysed by quantitative RT-PCR. Vascular cell adhesion molecule-1 localization in the aortic sinus was determined by immunohistochemistry. Atherosclerotic plaque presence was assessed in aortas. Collagen-induced arthritis was associated with increased expression of VCAM-1, independent of diet. VCAM-1 overexpression was detectable as early as 4 weeks after collagen immunization and persisted after 15 weeks. The HD induced atheroma plaque formation and aortic iNOS expression regardless of CIA. Concomitant CIA and HD had no additive effect on atheroma or VCAM-1 or iNOS expression. CIA and an HD diet induced a distinct and independent expression of large-vessel inflammation markers in B6 mice. This model may be relevant for the study of CVD in RA. PMID:26859834

  4. cAMP Signaling Prevents Podocyte Apoptosis via Activation of Protein Kinase A and Mitochondrial Fusion

    PubMed Central

    Xie, Kewei; Ni, Zhaohui; Yan, Yucheng; Wei, Kai; Chuang, Peter Y.; He, John Cijiang; Gu, Leyi

    2014-01-01

    Our previous in vitro studies suggested that cyclic AMP (cAMP) signaling prevents adriamycin (ADR) and puromycin aminonucleoside (PAN)-induced apoptosis in podocytes. As cAMP is an important second messenger and plays a key role in cell proliferation, differentiation and cytoskeleton formation via protein kinase A (PKA) or exchange protein directly activated by cAMP (Epac) pathways, we sought to determine the role of PKA or Epac signaling in cAMP-mediated protection of podocytes. In the ADR nephrosis model, we found that forskolin, a selective activator of adenylate cyclase, attenuated albuminuria and improved the expression of podocyte marker WT-1. When podocytes were treated with pCPT-cAMP (a selective cAMP/PKA activator), PKA activation was increased in a time-dependent manner and prevented PAN-induced podocyte loss and caspase 3 activation, as well as a reduction in mitochondrial membrane potential. We found that PAN and ADR resulted in a decrease in Mfn1 expression and mitochondrial fission in podocytes. pCPT-cAMP restored Mfn1 expression in puromycin or ADR-treated podocytes and induced Drp1 phosphorylation, as well as mitochondrial fusion. Treating podocytes with arachidonic acid resulted in mitochondrial fission, podocyte loss and cleaved caspase 3 production. Arachidonic acid abolished the protective effects of pCPT-cAMP on PAN-treated podocytes. Mdivi, a mitochondrial division inhibitor, prevented PAN-induced cleaved caspase 3 production in podocytes. We conclude that activation of cAMP alleviated murine podocyte caused by ADR. PKA signaling resulted in mitochondrial fusion in podocytes, which at least partially mediated the effects of cAMP. PMID:24642777

  5. Tormentic acid, a major component of suspension cells of Eriobotrya japonica, suppresses high-fat diet-induced diabetes and hyperlipidemia by glucose transporter 4 and AMP-activated protein kinase phosphorylation.

    PubMed

    Wu, Jin-Bin; Kuo, Yueh-Hsiung; Lin, Cheng-Hsiu; Ho, Hui-Ya; Shih, Chun-Ching

    2014-11-01

    This study was designed to evaluate the effects and mechanism of tormentic acid (PTA) on diabetes and dyslipidemia in high-fat (HF)-fed mice. Feeding C57BL/6J mice with a HF diet for 12 weeks induced type 2 diabetes and hyperlipidemia. During the last 4 weeks, the mice were given orally PTA (at two dosages) or rosiglitazone (Rosi) or water. In this study, the HF diet increased glucose, triglyceride, insulin, and leptin levels, whereas PTA effectively prevented these phenomena and ameliorated insulin resistance. PTA reduced visceral fat mass and hepatic triacylglycerol contents; moreover, PTA significantly decreased both the area of adipocytes and ballooning degeneration of hepatocytes. PTA caused increased skeletal muscular AMP-activated protein kinase (AMPK) phosphorylation and Akt phosphorylation and glucose transporter 4 (GLUT4) proteins, but reduced the hepatic expressions of phosphenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6 Pase) genes. PTA enhanced skeletal muscular Akt phosphorylation and increased insulin sensitivity. PTA also enhanced phospho-AMPK in the liver. Therefore, it is possible that the activation of AMPK by PTA results in decreasing hepatic glucose production while increasing skeletal muscular GLUT4 contents, thus contributing to attenuating the diabetic state. Moreover, PTA exhibits an antihyperlipidemic effect by down-regulations of the hepatic sterol regulatory element binding protein-1c (SREBP-1c) and apolipoprotein C-III (apo C-III) and an increased peroxisome proliferator activated receptor (PPAR)-α expression, thus resulting in decreases in blood triglycerides. These findings demonstrated that PTA was effective for the treatment of diabetes and hyperlipidemia in HF-fed mice.

  6. Pheromone-induced expression of immediate early genes in the mouse vomeronasal sensory system.

    PubMed

    Haga-Yamanaka, Sachiko; Touhara, Kazushige

    2013-01-01

    Immediate early genes (IEGs) are powerful tools for visualizing activated neurons and extended circuits that are stimulated by sensory input. Several kinds of IEGs (e.g., c-fos, egr-1) have been utilized for detecting activated receptor neurons in the pheromone sensory organ called the vomeronasal organ (VNO), as well as for mapping the neurons within the central nervous system (CNS) excited by pheromones.In this chapter, we describe the procedure for the detection of pheromone-induced neural activation in the VNO and CNS using the c-Fos immunostaining technique.

  7. Optical-fiber-based laser-induced breakdown spectroscopy for detection of early caries

    NASA Astrophysics Data System (ADS)

    Sasazawa, Shuhei; Kakino, Satoko; Matsuura, Yuji

    2015-06-01

    A laser-induced breakdown spectroscopy (LIBS) system targeting for the in vivo analysis of tooth enamel is described. The system is planned to enable real-time analysis of teeth during laser dental treatment by utilizing a hollow optical fiber that transmits both Q-switched Nd:YAG laser light for LIBS and infrared Er:YAG laser light for tooth ablation. The sensitivity of caries detection was substantially improved by expanding the spectral region under analysis to ultraviolet (UV) light and by focusing on emission peaks of Zn in the UV region. Subsequently, early caries were distinguished from healthy teeth with accuracy rates above 80% in vitro.

  8. [Modern methods of early screening for preeclampsia and pregnancy-induced hypertension--a review].

    PubMed

    Poprawski, Grzegorz; Wender-Ozegowska, Ewa; Zawiejska, Agnieszka; Brazert, Jacek

    2012-09-01

    Preeclampsia remains to be a serious perinatal complication and early screening for this disease to identify the high risk population before the first symptoms develop constitutes a considerable clinical challenge. Modern methods of screening for preeclampsia and pregnancy-induced hypertension include patients history biochemical serum markers and foetal DNA and RNA in maternal serum. They aid the process of developing an optimal protocol to initiate treatment in early pregnancy and to reduce the rate of complications. Our review presents an overview of the novel methods and techniques used for early screening for preeclampsia and pregnancy-induced hypertension. Most of the research focuses on 11-13 weeks of gestation due to the fact that the first prenatal examination is performed at that time. The most important information seems to be: weight, mass, mean blood pressure, history of pregnancy-induced hypertension or preeclampsia at previous pregnancies as well as the ethnic origin. During an ultrasound scan, pulsatility index of the uterine arteries is measured. Blood samples are obtained during the last part of the examination. At the moment only a few markers seem to be strong predictors of hypertensive disorders during pregnancy: pregnancy-associated plasma protein-A (PAPP-A), placental growth factor (PIGF) and soluble fms-like tyrosine kinase-1 (sFlt-1). Also, fetal DNA and RNA in maternal plasma are helpful in the prediction of preeclampsia as they are markers of the trophoblast apoptosis. Researchers aim at identifying the population at high risk of pregnancy-induced hypertension and preeclampsia in order to offer appropriate antenatal care to these women. At the moment many drugs and diet supplements are investigated to reduce the prevalence of hypertensive disorders in pregnancy. These medications are usually administrated in early gestation (up to 16 week of gestation) before the first clinical symptoms present. Low doses of aspirin were found to decrease

  9. Photoactivated adenylyl cyclase (PAC) reveals novel mechanisms underlying cAMP-dependent axonal morphogenesis

    PubMed Central

    Zhou, Zhiwen; Tanaka, Kenji F.; Matsunaga, Shigeru; Iseki, Mineo; Watanabe, Masakatsu; Matsuki, Norio; Ikegaya, Yuji; Koyama, Ryuta

    2016-01-01

    Spatiotemporal regulation of axonal branching and elongation is essential in the development of refined neural circuits. cAMP is a key regulator of axonal growth; however, whether and how intracellular cAMP regulates axonal branching and elongation remain unclear, mainly because tools to spatiotemporally manipulate intracellular cAMP levels have been lacking. To overcome this issue, we utilized photoactivated adenylyl cyclase (PAC), which produces cAMP in response to blue-light exposure. In primary cultures of dentate granule cells transfected with PAC, short-term elevation of intracellular cAMP levels induced axonal branching but not elongation, whereas long-term cAMP elevation induced both axonal branching and elongation. The temporal dynamics of intracellular cAMP levels regulated axonal branching and elongation through the activation of protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac), respectively. Thus, using PAC, our study for the first time reveals that temporal cAMP dynamics could regulate axonal branching and elongation via different signaling pathways. PMID:26795422

  10. Photoactivated adenylyl cyclase (PAC) reveals novel mechanisms underlying cAMP-dependent axonal morphogenesis.

    PubMed

    Zhou, Zhiwen; Tanaka, Kenji F; Matsunaga, Shigeru; Iseki, Mineo; Watanabe, Masakatsu; Matsuki, Norio; Ikegaya, Yuji; Koyama, Ryuta

    2016-01-01

    Spatiotemporal regulation of axonal branching and elongation is essential in the development of refined neural circuits. cAMP is a key regulator of axonal growth; however, whether and how intracellular cAMP regulates axonal branching and elongation remain unclear, mainly because tools to spatiotemporally manipulate intracellular cAMP levels have been lacking. To overcome this issue, we utilized photoactivated adenylyl cyclase (PAC), which produces cAMP in response to blue-light exposure. In primary cultures of dentate granule cells transfected with PAC, short-term elevation of intracellular cAMP levels induced axonal branching but not elongation, whereas long-term cAMP elevation induced both axonal branching and elongation. The temporal dynamics of intracellular cAMP levels regulated axonal branching and elongation through the activation of protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac), respectively. Thus, using PAC, our study for the first time reveals that temporal cAMP dynamics could regulate axonal branching and elongation via different signaling pathways. PMID:26795422

  11. Molecular characterization and epidemiology of cefoxitin resistance among Enterobacteriaceae lacking inducible chromosomal ampC genes from hospitalized and non-hospitalized patients in Algeria: description of new sequence type in Klebsiella pneumoniae isolates.

    PubMed

    Gharout-Sait, Alima; Touati, Abdelaziz; Guillard, Thomas; Brasme, Lucien; de Champs, Christophe

    2015-01-01

    In this study, 922 consecutive non-duplicate clinical isolates of Enterobacteriaceae obtained from hospitalized and non-hospitalized patients at Bejaia, Algeria were analyzed for AmpC-type β-lactamases production. The ampC genes and their genetic environment were characterized using polymerase chain reaction (PCR) and sequencing. Plasmid incompatibility groups were determined by using PCR-based replicon typing. Phylogenetic grouping and multilocus sequence typing were determined for molecular typing of the plasmid-mediated AmpC (pAmpC) isolates. Of the isolates, 15 (1.6%) were identified as AmpC producers including 14 CMY-4-producing isolates and one DHA-1-producing Klebsiella pneumoniae. All AmpC-producing isolates co-expressed the broad-spectrum TEM-1 β-lactamase and three of them co-produced CTX-M and/or SHV-12 ESBL. Phylogenetic grouping and virulence genotyping of the E. coli isolates revealed that most of them belonged to groups D and B1. Multilocus sequence typing analysis of K. pneumoniae isolates identified four different sequence types (STs) with two new sequences: ST1617 and ST1618. Plasmid replicon typing indicates that blaCMY-4 gene was located on broad host range A/C plasmid, while LVPK replicon was associated with blaDHA-1. All isolates carrying blaCMY-4 displayed the transposon-like structures ISEcp1/ΔISEcp1-blaCMY-blc-sugE. Our study showed that CMY-4 was the main pAmpC in the Enterobacteriaceae isolates in Algeria.

  12. 17β-estradiol ameliorates oxygen-induced retinopathy in the early hyperoxic phase

    PubMed Central

    Zhang, Hongbing; Wang, Xiaodong; Xu, Kun; Wang, Yao; Wang, Yani; Liu, Xianning; Zhang, Xianjiao; Wang, Liang; Li, Xiaogang

    2016-01-01

    Retinopathy of prematurity (ROP) is a major and leading cause of blindness in premature infants. It has been realized that early treatment for ROP is important. However, all the early treatments of ROP are focusing on peripheral retinal ablation which does not surmount the limit of extinguishing retinal neovascularization and protecting the retinas of children with ROP from the injury of ablation. In this study, we investigated the morphological changes of retina and oxidative stress alterations in the early phase of oxygen-induced retinopathy (OIR) and tested the effects of 17β-estradiol (17β-E2) , a nonselective estrogen receptor (ER) agonist, on early phase OIR development. We found that large central capillary-free areas were induced in the retinas of pups exposed to hyperoxia on postnatal day 9 (P9) , whereas vascularization was almost complete in the retinas of pups exposed to normoxia at the same age. The concentrations of malondiadehyde (MDA) , an end-product of oxidative stress, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a major enzyme producing free radicals, as well as the activity of NADPH oxidase were significantly elevated in the retinas of pups exposed to hyperoxia on P9 and postnatal day 13 (P13) compared to those in age matched pups exposed to normoxia. Treatment with 17β-E2 decreased not only the percentage of the central capillary-free area to total retina area but also the concentrations of MDA and NADPH oxidase as well as the activity of NADPH oxidase in a dose-dependent manner in pups exposed to hyperoxia on p9 and P13. The concentration of VEGF was significantly decreased on P9 but increased on P14 in the retinas of pups exposed to hyperoxia, whereas it was significantly elevated on P9 but decreased on P14 in the retinas of pups treated with 17β-E2. The effect of 17β-E2 could be reversed by the co-treatment with ICI182780, a high affinity estrogen receptor antagonist, which suggested that 17β-E2 might exert its

  13. Alarm pheromone induces immediate-early gene expression and slow behavioral response in honey bees.

    PubMed

    Alaux, Cédric; Robinson, Gene E

    2007-07-01

    Primer and releaser pheromones are molecules used for communication that induce species-specific responses. In contrast to primer pheromones, it is not known whether the quicker-acting releaser pheromones can affect brain gene expression. We show here that isopentyl acetate (IPA), a releaser pheromone that communicates alarm in honey bees, not only provokes a quick defensive response but also influences behavior for a longer period of time and affects brain gene expression. Exposure to IPA affected behavioral responsiveness to subsequent exposures to IPA and induced the expression of the immediate early gene and transcription factor c-Jun in the antennal lobes. Our findings blur the long-standing distinction between primer and releaser pheromone and highlight the pervasiveness of environmental regulation of brain gene expression. PMID:17505874

  14. A change in liver metabolism but not in brown adipose tissue thermogenesis is an early event in ovariectomy-induced obesity in rats.

    PubMed

    Nigro, Mariana; Santos, Anderson T; Barthem, Clarissa S; Louzada, Ruy A N; Fortunato, Rodrigo S; Ketzer, Luisa A; Carvalho, Denise P; de Meis, Leopoldo

    2014-08-01

    Menopause is associated with increased visceral adiposity and disrupted glucose homeostasis, but the underlying molecular mechanisms related to these metabolic changes are still elusive. Brown adipose tissue (BAT) plays a key role in energy expenditure that may be regulated by sexual steroids, and alterations in glucose homeostasis could precede increased weight gain after ovariectomy. Thus, the aim of this work was to evaluate the metabolic pathways in both the BAT and the liver that may be disrupted early after ovariectomy. Ovariectomized (OVX) rats had increased food efficiency as early as 12 days after ovariectomy, which could not be explained by differences in feces content. Analysis of isolated BAT mitochondria function revealed no differences in citrate synthase activity, uncoupling protein 1 expression, oxygen consumption, ATP synthesis, or heat production in OVX rats. The addition of GDP and BSA to inhibit uncoupling protein 1 decreased oxygen consumption in BAT mitochondria equally in both groups. Liver analysis revealed increased triglyceride content accompanied by decreased levels of phosphorylated AMP-activated protein kinase and phosphorylated acetyl-CoA carboxylase in OVX animals. The elevated expression of gluconeogenic enzymes in OVX and OVX + estradiol rats was not associated with alterations in glucose tolerance test or in serum insulin but was coincident with higher glucose disposal during the pyruvate tolerance test. Although estradiol treatment prevented the ovariectomy-induced increase in body weight and hepatic triglyceride and cholesterol accumulation, it was not able to prevent increased gluconeogenesis. In conclusion, the disrupted liver glucose homeostasis after ovariectomy is neither caused by estradiol deficiency nor is related to increased body mass. PMID:24914935

  15. Modulation of iridovirus-induced apoptosis by endocytosis, early expression, JNK, and apical caspase

    SciTech Connect

    Chitnis, Nilesh S.; D'Costa, Susan M.; Paul, Eric R.; Bilimoria, Shaen L.

    2008-01-20

    Chilo iridescent virus (CIV) is the type species for the family Iridoviridae, which are large, isometric, cytoplasmic dsDNA viruses. We examined the mechanism of apoptosis induction by CIV. High CIV doses (CIV{sub XS}; 400 {mu}g/ml), UV-irradiated virus (CIV{sub UV}; 10 {mu}g/ml) and CVPE (CIV protein extract; 10 {mu}g/ml) induced apoptosis in 60% of treated Choristoneura fumiferana (IPRI-CF-124T) cells. Normal doses of infectious CIV (10 {mu}g/ml) induced apoptosis in only 10% of C. fumiferana (CF) cells. Apoptosis was inhibited by Z-IETD-FMK, an apical caspase inhibitor, indicating that CIV-induced apoptosis requires caspase activity. The putative caspase in CF cells was designated Cf-caspase-i. CIV{sub UV} or CVPE enhanced Cf-caspase-i activity by 80% at 24 h relative to mock-treated cells. Since the MAP kinase pathway induces or inhibits apoptosis depending on the context, we used JNK inhibitor SP600125 and demonstrated drastic suppression of CVPE-induced apoptosis. Thus, the JNK signaling pathway is significant for apoptosis in this system. Virus interaction with the cell surface was not sufficient for apoptosis since CIV{sub UV} particles bound to polysterene beads failed to induce apoptosis. Endocytosis inhibitors (bafilomycin or ammonium chloride) negated apoptosis induction by CIV{sub UV}, CIV{sub XS} or CVPE indicating that entry through this mode is required. Given the weak apoptotic response to infectious CIV, we postulated that viral gene expression inhibited apoptosis. CIV infection of cells pretreated with cycloheximide induced apoptosis in 69% of the cells compared to 10% in normal infections. Furthermore, blocking viral DNA replication with aphidicolin or phosphonoacetic acid suppressed apoptosis and Cf-caspase-i activity, indicating that early viral expression is necessary for inhibition of apoptosis, and de novo synthesis of viral proteins is not required for induction. We show for the first time that, in a member of the family Iridoviridae

  16. Clinical features and drug induced side effects in early versus late antidepressant responders.

    PubMed

    Fabbri, Chiara; Marsano, Agnese; Balestri, Martina; De Ronchi, Diana; Serretti, Alessandro

    2013-10-01

    Early antidepressant response (2nd week) has been reported as the result of a true antidepressant effect and a predictor of subsequent stable response. With the purpose to study the clinical profile of early response/remission (2nd week) compared to late response/remission (4th-6th weeks), two independent major depressive disorder (MDD) samples (the Sequenced Treatment Alternatives to Relieve Depression or STAR*D n=1922 and an Italian sample n=171) were investigated. Patients were treated with citalopram in the STAR*D while in a naturalistic setting in the Italian sample. Depressive symptomatology was assessed by the Hamilton Depressive Rating Scale weekly in the Italian sample and biweekly by the Quick Inventory of Depressive Symptomatology Clinician Rated in the STAR*D. Logistic regression was used to investigate possible predictors of early response and the Bonferroni correction was applied. In the STAR*D, higher levels of baseline core depressive symptoms (Bech subscale) were associated with early response (p=0.00017), as well as lower baseline insomnia (p=0.003) and higher work and social functioning (p=0.001). In the Italian sample none of these variables were associated with the phenotype, but a non significant trend of lower baseline quality of life (p=0.078) was observed in late remitters. In the STAR*D late responders reported higher levels of antidepressant induced side effects, especially difficulty in sleeping (p=5.68e-13), with a non significant trend in the same direction in the Italian sample (p=0.09). The identification of late versus early antidepressant responders at the beginning of the treatment may be useful to guide therapeutic choices in clinical settings. PMID:23800418

  17. [Early and late complications in induced abortions of primigravidae (including suggested measures)].

    PubMed

    Kreibich, H; Ludwig, A

    1980-04-01

    Of 7823 induced abortions which were performed between 1970 and 1978, 798 were performed on 13-18 year old primigravidae. About 88% of these women were 16-18 years of age. The 2 most frequent early complications were fever (4.27%) and palpable evidence of infection (parametritis/salpingitis) (6.4%). Most complications were observed after the use of Hegar dilation and vacuum aspiration. 390 of the patients who underwent abortion between 1976 and 1978 were examined 14 days and 3 months after the abortion. 1.79% reported residual bleeding at both examinations, even though placental residue was not found. The incidence of infection fell from 4.62% to 3.58%. 1.79% reported menstrual irregularities 3 months after the operation. The rate of spontaneous abortion following a previous induced abortion among all patients increased from 1.68% in 1970 to 17.54% in 1978. 652 of the 796 adolescents were interviewed in 1977: 418 were married. Of these, 10.29% had had spontaneous abortions, 11% premature births, 64.11% had had normal births, and 5.5% secondary sterility. Of the 234 single women, 78.63% used oral contraceptives, 6.84% had undergone subsequent induced abortion(s), and 5.98% had undergone spontaneous abortion. Better sex education is needed to help avoid an increase in induced abortion and its consequences among adolescents.

  18. Enriched Environment Protects the Optic Nerve from Early Diabetes-Induced Damage in Adult Rats

    PubMed Central

    Dorfman, Damián; Aranda, Marcos L.; Rosenstein, Ruth E.

    2015-01-01

    Diabetic retinopathy is a leading cause of reduced visual acuity and acquired blindness. Axoglial alterations of the distal (close to the chiasm) optic nerve (ON) could be the first structural change of the visual pathway in streptozotocin (STZ)-induced diabetes in rats. We analyzed the effect of environmental enrichment on axoglial alterations of the ON provoked by experimental diabetes. For this purpose, three days after vehicle or STZ injection, animals were housed in enriched environment (EE) or remained in a standard environment (SE) for 6 weeks. Anterograde transport, retinal morphology, optic nerve axons (toluidine blue staining and phosphorylated neurofilament heavy immunoreactivity), microglia/macrophages (ionized calcium binding adaptor molecule 1 (Iba-1) immunoreactivity), astrocyte reactivity (glial fibrillary acid protein-immunostaining), myelin (myelin basic protein immunoreactivity), ultrastructure, and brain derived neurotrophic factor (BDNF) levels were assessed in non-diabetic and diabetic animals housed in SE or EE. No differences in retinal morphology or retinal ganglion cell number were observed among groups. EE housing which did not affect the STZ-induced weight loss and hyperglycemia, prevented a decrease in the anterograde transport from the retina to the superior colliculus, ON axon number, and phosphorylated neurofilament heavy immunoreactivity. Moreover, EE housing prevented an increase in Iba-1 immunoreactivity, and astrocyte reactivity, as well as ultrastructural myelin alterations in the ON distal portion at early stages of diabetes. In addition, EE housing avoided a decrease in BDNF levels induced by experimental diabetes. These results suggest that EE induced neuroprotection in the diabetic visual pathway. PMID:26312758

  19. Early LPS-induced ERK activation in retinal pigment epithelium cells is dependent on PIP 2 -PLC.

    PubMed

    Mateos, Melina V; Kamerbeek, Constanza B; Giusto, Norma M; Salvador, Gabriela A

    2016-06-01

    This article presents additional data regarding the study "The phospholipase D pathway mediates the inflammatory response of the retinal pigment epithelium" [1]. The new data presented here show that short exposure of RPE cells to lipopolysaccharide (LPS) induces an early and transient activation of the extracellular signal-regulated kinase (ERK1/2). This early ERK1/2 activation is dependent on phosphatidylinositol bisphosphate-phospholipase C (PIP2-PLC). On the contrary, neither the phospholipase D 1 (PLD1) nor the PLD2 inhibition is able to modulate the early ERK1/2 activation induced by LPS in RPE cells.

  20. Early-stage development of human induced pluripotent stem cell-derived neurons.

    PubMed

    Ohara, Yuki; Koganezawa, Noriko; Yamazaki, Hiroyuki; Roppongi, Reiko T; Sato, Kaoru; Sekino, Yuko; Shirao, Tomoaki

    2015-12-01

    Recent advances in human induced pluripotent stem cells (hiPSCs) offer new possibilities for biomedical research and clinical applications. Differentiated neurons from hiPSCs are expected to be useful for developing novel methods of treatment for various neurological diseases. However, the detailed process of functional maturation of hiPSC-derived neurons (hiPS neurons) remains poorly understood. This study analyzes development of hiPS neurons, focusing specifically on early developmental stages through 48 hr after cell seeding; development was compared with that of primary cultured neurons derived from the rat hippocampus. At 5 hr after cell seeding, neurite formation occurs in a similar manner in both neuronal populations. However, very few neurons with axonal polarization were observed in the hiPS neurons even after 48 hr, indicating that hiPS neurons differentiate more slowly than rat neurons. We further investigated the elongation speed of axons and found that hiPS neuronal axons were slower. In addition, we characterized the growth cones. The localization patterns of skeletal proteins F-actin, microtubule, and drebrin were similar to those of rat neurons, and actin depolymerization by cytochalasin D induced similar changes in cytoskeletal distribution in the growth cones between hiPS neurons and rat neurons. These results indicate that, during the very early developmental stage, hiPS neurons develop comparably to rat hippocampal neurons with regard to axonal differentiation, but the growth of axons is slower.

  1. Early detection of oil-induced stress in crops using spectral and thermal responses

    NASA Astrophysics Data System (ADS)

    Emengini, Ebele Josephine; Blackburn, George Alan; Theobald, Julian Charles

    2013-01-01

    Oil pollution is a major source of environmental degradation, and requires accurate monitoring and timely detection for an effective control of its occurrence. This paper examines the potential of a remote sensing approach using the spectral and thermal responses of crops for the early detection of stress caused by oil pollution. In a glasshouse, pot-grown maize was treated with oil at sublethal and lethal applications. Thereafter, leaf thermal, spectral and physiological measurements were taken every two to three days to monitor the development of stress responses. Our results indicate that absolute leaf temperature was a poor indicator of developing stress. However, a derived thermal index (IG) responded consistently in the early stages of physiological damage. Various spectral reflectance features were highly sensitive to oil-induced stress. A narrow-band index using wavelengths in the near-infrared and red-edge region, (R755-R716)/(R755+R716), was optimal for previsual detection of oil-induced stress. This index had a strong linear relationship with photosynthetic rate. This indicates that by detecting vegetation stress, thermal and hyperspectral remote sensing has considerable potential for the timely detection of oil pollution in the environment.

  2. Early maternal deprivation in rats induces gender-dependent effects on developing hippocampal and cerebellar cells.

    PubMed

    Llorente, Ricardo; Gallardo, Meritxell López; Berzal, Alvaro Llorente; Prada, Carmen; Garcia-Segura, Luis Miguel; Viveros, María-Paz

    2009-05-01

    Adult animals submitted to a single prolonged episode of maternal deprivation [24h, postnatal day 9-10] show behavioral alterations that resemble specific symptoms of schizophrenia. According to the neurodevelopmental theory, these behavioral deficits might be mediated by detrimental neurodevelopmental processes that might be associated, at least partially, with stress-induced corticosterone responses. In order to address this hypothesis, we have focused on the hippocampus and cerebellar cortex, two brain regions that show high density of glucocorticoid receptors, and analyzed possible neuronal and glial alterations by immunohistochemical techniques. To evaluate the presence of degenerated neurons we used Fluoro-Jade-C (FJ-C) staining and for the study of astrocytes we employed glial fibrillary acidic protein (GFAP). Within control animals, females showed significantly more GFAP positive cells than males and a trend towards more FJ-C positive cells. Maternal deprivation induced neuronal degeneration and astroglial changes in the hippocampus and cerebellar cortex of neonatal rats that, in general, were more marked in males. This differential effect may be attributable to a greater vulnerability of males to this kind of early environmental insult and/or to sex-dependent differences in the onset and/or progression of the effects. The present experimental procedure may be instrumental in elucidating sex-dependent mechanisms of neurodevelopmental psychiatric disorders with a basis in early environmental insults.

  3. Expression of immediate-early genes in the dorsal cochlear nucleus in salicylate-induced tinnitus.

    PubMed

    Hu, Shou-Sen; Mei, Ling; Chen, Jian-Yong; Huang, Zhi-Wu; Wu, Hao

    2016-02-01

    Spontaneous neuronal activity in dorsal cochlear nucleus (DCN) may be involved in the physiological processes underlying salicylate-induced tinnitus. As a neuronal activity marker, immediate-early gene (IEG) expression, especially activity-dependent cytoskeletal protein (Arc/Arg3.1) and the early growth response gene-1 (Egr-1), appears to be highly correlated with sensory-evoked neuronal activity. However, their relationships with tinnitus induced by salicylate have rarely been reported in the DCN. In this study, we assessed the effect of acute and chronic salicylate treatment on the expression of N-methyl D-aspartate receptor subunit 2B (NR2B), Arg3.1, and Egr-1. We also observed ultrastructural alterations in the DCN synapses in an animal model of tinnitus. Levels of mRNA and protein expression of NR2B and Arg3.1 were increased in rats that were chronically administered salicylate (200 mg/kg, twice daily for 3, 7, or 14 days). These levels returned to baseline 14 days after cessation of treatment. However, no significant changes were observed in Egr-1 gene expression in any groups. Furthermore, rats subjected to long-term salicylate administration showed more presynaptic vesicles, thicker and longer postsynaptic densities, and increased synaptic interface curvature. Alterations of Arg3.1 and NR2B may be responsible for the changes in the synaptic ultrastructure. These changes confirm that salicylate can cause neural plasticity changes at the DCN level. PMID:25636249

  4. Early Detection of Drug-Induced Renal Hemodynamic Dysfunction Using Sonographic Technology in Rats

    PubMed Central

    Fisch, Sudeshna; Liao, Ronglih; Hsiao, Li-Li; Lu, Tzongshi

    2016-01-01

    The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is estimated to contribute to 19- 25% of all clinical cases of acute kidney injury (AKI) in critically ill patients. AKI detection has historically relied on metrics such as serum creatinine (sCr) or blood urea nitrogen (BUN) which are demonstrably inadequate in full assessment of nephrotoxicity in the early phase of renal dysfunction. Currently, there is no robust diagnostic method to accurately detect hemodynamic alteration in the early phase of AKI while such alterations might actually precede the rise in serum biomarker levels. Such early detection can help clinicians make an accurate diagnosis and help in in decision making for therapeutic strategy. Rats were treated with Cisplatin to induce AKI. Nephrotoxicity was assessed for six days using high-frequency sonography, sCr measurement and upon histopathology of kidney. Hemodynamic evaluation using 2D and Color-Doppler images were used to serially study nephrotoxicity in rats, using the sonography. Our data showed successful drug-induced kidney injury in adult rats by histological examination. Color-Doppler based sonographic assessment of AKI indicated that resistive-index (RI) and pulsatile-index (PI) were increased in the treatment group; and peak-systolic velocity (mm/s), end-diastolic velocity (mm/s) and velocity-time integral (VTI, mm) were decreased in renal arteries in the same group. Importantly, these hemodynamic changes evaluated by sonography preceded the rise of sCr levels. Sonography-based indices such as RI or PI can thus be useful predictive markers of declining renal function in rodents. From our sonography-based observations in the kidneys of rats that underwent AKI, we showed that these noninvasive hemodynamic measurements may consider as an accurate, sensitive and robust method in detecting early stage kidney dysfunction. This study also

  5. Increase in Endogenous and Exogenous Cyclic AMP Levels Inhibits Sclerotial Development in Sclerotinia sclerotiorum

    PubMed Central

    Rollins, Jeffrey A.; Dickman, Martin B.

    1998-01-01

    Growth and development of a wild-type Sclerotinia sclerotiorum isolate were examined in the presence of various pharmacological compounds to investigate signal transduction pathways that influence the development of sclerotia. Compounds known to increase endogenous cyclic AMP (cAMP) levels in other organisms by inhibiting phosphodiesterase activity (caffeine and 3-isobutyl-1-methyl xanthine) or by activating adenylate cyclase (NaF) reduced or eliminated sclerotial development in S. sclerotiorum. Growth in the presence of 5 mM caffeine correlated with increased levels of endogenous cAMP in mycelia. In addition, incorporation of cAMP into the growth medium decreased or eliminated the production of sclerotia in a concentration-dependent manner and increased the accumulation of oxalic acid. Inhibition of sclerotial development was cAMP specific, as exogenous cyclic GMP, AMP, and ATP did not influence sclerotial development. Transfer of developing cultures to cAMP-containing medium at successive time points demonstrated that cAMP inhibits development prior to or during sclerotial initiation. Together, these results indicate that cAMP plays a role in the early transition between mycelial growth and sclerotial development. PMID:9647827

  6. Laser microbeam-induced DNA damage inhibits cell division in fertilized eggs and early embryos.

    PubMed

    Wang, Zhong-Wei; Ma, Xue-Shan; Ma, Jun-Yu; Luo, Yi-Bo; Lin, Fei; Wang, Zhen-Bo; Fan, Heng-Yu; Schatten, Heide; Sun, Qing-Yuan

    2013-10-15

    DNA double-strand breaks are caused by both intracellular physiological processes and environmental stress. In this study, we used laser microbeam cut (abbreviated microcut or cut), which allows specific DNA damage in the pronucleus of a fertilized egg and in individual blastomere(s) of an early embryo, to investigate the response of early embryos to DNA double-strand breaks. Line type γH2AX foci were detected in the cut region, while Chk2 phosphorylation staining was observed in the whole nuclear region of the cut pronuclei or blastomeres. Zygotes with cut male or female pronucleus showed poor developmental capability: the percentage of cleavage embryos was significantly decreased, and the embryos failed to complete further development to blastocysts. The cut blastomeres in 2-cell, 4-cell, and 8-cell embryos ceased cleavage, and they failed to incorporate into compacted morulae, but instead underwent apoptosis and cell death at the blastocyst stage; the uncut part of embryos could develop to blastocysts, with a reduced percentage or decreased cell number. When both blastomeres of the 2-cell embryos were cut by laser microbeam, cell death occurred 24 h earlier, suggesting important functions of the uncut blastomere in delaying cell death of the cut blastomere. Taken together, we conclude that microbeam-induced DNA damage in early embryos causes compromised development, and that embryos may have their own mechanisms to exclude DNA-damaged blastomeres from participating in further development. PMID:24036543

  7. Fisetin induces Sirt1 expression while inhibiting early adipogenesis in 3T3-L1 cells.

    PubMed

    Kim, Sang Chon; Kim, Yoo Hoon; Son, Sung Wook; Moon, Eun-Yi; Pyo, Suhkneung; Um, Sung Hee

    2015-11-27

    Fisetin (3,7,3',4'-tetrahydroxyflavone) is a naturally found flavonol in many fruits and vegetables and is known to have anti-aging, anti-cancer and anti-viral effects. However, the effects of fisetin on early adipocyte differentiation and the epigenetic regulator controlling adipogenic transcription factors remain unclear. Here, we show that fisetin inhibits lipid accumulation and suppresses the expression of PPARγ in 3T3-L1 cells. Fisetin suppressed early stages of preadipocyte differentiation, and induced expression of Sirt1. Depletion of Sirt1 abolished the inhibitory effects of fisetin on intracellular lipid accumulation and on PPARγ expression. Mechanistically, fisetin facilitated Sirt1-mediated deacetylation of PPARγ and FoxO1, and enhanced the association of Sirt1 with the PPARγ promoter, leading to suppression of PPARγ transcriptional activity, thereby repressing adipogenesis. Lowering Sirt1 levels reversed the effects of fisetin on deacetylation of PPARγ and increased PPARγ transactivation. Collectively, our results suggest the effects of fisetin in increasing Sirt1 expression and in epigenetic control of early adipogenesis.

  8. Wnt Signaling Inhibits Osteoclast Differentiation by Activating Canonical and Noncanonical cAMP/PKA Pathways

    PubMed Central

    Weivoda, Megan M; Ruan, Ming; Hachfeld, Christine M; Pederson, Larry; Howe, Alan; Davey, Rachel A; Zajac, Jeffrey D; Kobayashi, Yasuhiro; Williams, Bart O; Westendorf, Jennifer J; Khosla, Sundeep; Oursler, Merry Jo

    2016-01-01

    Although there has been extensive characterization of the Wnt signaling pathway in the osteoblast lineage, the effects of Wnt proteins on the osteoclast lineage are less well studied. We found that osteoclast lineage cells express canonical Wnt receptors. Wnt3a reduced osteoclast formation when applied to early bone-marrow macrophage (BMM) osteoclast differentiation cultures, whereas late addition did not suppress osteoclast formation. Early Wnt3a treatment inactivated the crucial transcription factor NFATc1 in osteoclast progenitors. Wnt3a led to the accumulation of nuclear β-catenin, confirming activation of canonical Wnt signaling. Reducing low-density lipoprotein receptor-related proteins (Lrp) 5 and Lrp6 protein expression prevented Wnt3a-induced inactivation of NFATc1; however, deletion of β-catenin did not block Wnt3a inactivation of NFATc1, suggesting that this effect was mediated by a noncanonical pathway. Wnt3a rapidly activated the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway and pharmacological stimulation of cAMP/PKA signaling suppressed osteoclast differentiation; Wnt3a-induced NFATc1 phosphorylation was blocked by inhibiting interactions between PKA and A-kinase anchoring proteins (AKAPs). These data indicate that Wnt3a directly suppresses osteoclast differentiation through both canonical (β-catenin) and noncanonical (cAMP/PKA) pathways in osteoclast precursors. In vivo reduction of Lrp5 and Lrp6 expressions in the early osteoclast lineage via Rank promoter Cre recombination reduced trabecular bone mass, whereas disruption of Lrp5/6 expression in late osteoclast precursors via cathepsin K (Ctsk) promoter Cre recombination did not alter the skeletal phenotype. Surprisingly, reduction of Lrp5/6 in the early osteoclast lineage decreased osteoclast numbers, as well as osteoblast numbers. Published studies have previously noted that β-catenin signaling is required for osteoclast progenitor proliferation. Our in vivo data

  9. Protective effects of dexamethasone on early acute lung injury induced by oleic acid in rats

    PubMed Central

    Huang, Bin; Wang, Dao-Xin; Deng, Wang

    2014-01-01

    Objective: Whether alveolar edema could be cleared by alveolar epithelial is a key to the treatment and prognosis of ALI (acute lung injury). In this study, oleic acid(OA)-induced ALI model was established, the expression of α1 Na+/K+-ATPase (NKA) and β1 Na+/K+-ATPase were performed in vivo to investigate the mechanism of alveolar fluid clearance (AFC) in ALI and the effect of early low doses of dexamethasone on alveolar fluid clearance. Methods: In this study, Male rats were challenged by OA with or without dexamethasone (1 mg/kg, iv) post-treatment. Lung histopathology, blood gas, pulmonary vascular permeability, BALF IL-6, MPO and NKA activity of lung were examined. α1NKA and β1NKA mRNA and protein expression were detected. Results: The results indicated that compared with sham operated group, NKA activity, mRNA and protein expression of α1NKA and β1NKA were decreased in OA treated group, while wet/dry ratio, lung index, IL-6, and MPO activity were increased significantly. Pulmonary edema was obviously seen under light microscope. Those indexes were improved in dexamethasone treated group compared to OA treated group. Conclusion: The expression of NKA to decline for the lung injury is one important mechanism of pulmonary edema. Early low dose of dexamethasone treatment could suppress the expression of inflammatory mediators, improved lung epithelial-endothelial barrier permeability, increased the expressions of α1 NKA and β1 NKA mRNA, α1 NKA and β1 NKA protein level, stimulated NKA activity and decreased pulmonary edema. In conclusion, these observations suggest that early low dose of dexamethasone treatment has a protective effect on OA induced ALI. PMID:25663967

  10. Sucrose-induced analgesia during early life modulates adulthood learning and memory formation.

    PubMed

    Nuseir, Khawla Q; Alzoubi, Karem H; Alabwaini, Jehad; Khabour, Omar F; Kassab, Manal I

    2015-06-01

    This study is aimed at examining the long-term effects of chronic pain during early life (postnatal day 0 to 8weeks), and intervention using sucrose, on cognitive functions during adulthood in rats. Pain was induced in rat pups via needle pricks of the paws. Sucrose solution or paracetamol was administered for analgesia before the paw prick. Control groups include tactile stimulation to account for handling and touching the paws, and sucrose alone was used. All treatments were started on day one of birth and continued for 8weeks. At the end of the treatments, behavioral studies were conducted to test the spatial learning and memory using radial arm water maze (RAWM), as well as pain threshold via foot-withdrawal response to a hot plate apparatus. Additionally, the hippocampus was dissected, and blood was collected. Levels of neurotrophins (BDNF, IGF-1 and NT-3) and endorphins were assessed using ELISA. The results show that chronic noxious stimulation resulted in comparable foot-withdrawal latency between noxious and tactile groups. On the other hand, pretreatment with sucrose or paracetamol increased pain threshold significantly both in naive rats and noxiously stimulated rats (P<0.05). Chronic pain during early life impaired short-term memory, and sucrose treatment prevented such impairment (P<0.05). Sucrose significantly increased serum levels of endorphin and enkephalin. Chronic pain decreased levels of BDNF in the hippocampus and this decrease was prevented by sucrose and paracetamol treatments. Hippocampal levels of NT-3 and IGF-1 were not affected by any treatment. In conclusion, chronic pain induction during early life induced short memory impairment, and pretreatment with sucrose prevented this impairment via mechanisms that seem to involve BDNF. As evident in the results, sucrose, whether alone or in the presence of pre-noxious stimulation, increases pain threshold in such circumstances; most likely via a mechanism that involves an increase in endogenous

  11. Recharge of the early atmosphere of Mars by impact-induced release of CO2

    USGS Publications Warehouse

    Carr, Michael H.

    1989-01-01

    Channels on the Martian surface suggest that Mars had an early, relatively thick atmosphere. If the atmosphere was thick enough for water to be stable at the surface, CO2 in the atmosphere would have been fixed as carbonates on a relatively short time scale, previously estimated to be 1 bar every 107 years. This loss must have been offset by some replenishment mechanism to account for the numerous valley networks in the oldest surviving terrains. Impacts could have released CO2 into the atmosphere by burial, by shock-induced release during impact events, and by addition of carbon to Mars from the impacting bolides. Depending on the relationship between the transient cavity diameter and the diameter of the resulting crater, burial rates as a result of impact gardening at the end of heavy bombardment are estimated to range from 20 to 45 m/106 years, on the assumption that cratering rates in Mars were similar to those of the Nectarian Period on the Moon. At these rates 0.1-0.2 bar of CO2 could have been released every 107 years as a result of burial to depths where dissociation temperatures of carbonates were reached. Modeling of large impacts suggests that an additional 0.01 to 0.02 bar of CO2 could have been released every 107 years during the actual impacts. In the unlikely event that all the impacting material was composed of carbonaceous chondrites, a further 0.3 bar of CO2 could have been added to the atmosphere every 107 years by oxidation of meteoritic carbon. Even when supplemented by the volcanically induced release of CO2, these release rates are barely sufficient to sustain an early atmosphere if water were continuously present at the surface. The results suggest that water may have been only intermittently present on the surface early in the planet's history.

  12. Sucrose-induced analgesia during early life modulates adulthood learning and memory formation.

    PubMed

    Nuseir, Khawla Q; Alzoubi, Karem H; Alabwaini, Jehad; Khabour, Omar F; Kassab, Manal I

    2015-06-01

    This study is aimed at examining the long-term effects of chronic pain during early life (postnatal day 0 to 8weeks), and intervention using sucrose, on cognitive functions during adulthood in rats. Pain was induced in rat pups via needle pricks of the paws. Sucrose solution or paracetamol was administered for analgesia before the paw prick. Control groups include tactile stimulation to account for handling and touching the paws, and sucrose alone was used. All treatments were started on day one of birth and continued for 8weeks. At the end of the treatments, behavioral studies were conducted to test the spatial learning and memory using radial arm water maze (RAWM), as well as pain threshold via foot-withdrawal response to a hot plate apparatus. Additionally, the hippocampus was dissected, and blood was collected. Levels of neurotrophins (BDNF, IGF-1 and NT-3) and endorphins were assessed using ELISA. The results show that chronic noxious stimulation resulted in comparable foot-withdrawal latency between noxious and tactile groups. On the other hand, pretreatment with sucrose or paracetamol increased pain threshold significantly both in naive rats and noxiously stimulated rats (P<0.05). Chronic pain during early life impaired short-term memory, and sucrose treatment prevented such impairment (P<0.05). Sucrose significantly increased serum levels of endorphin and enkephalin. Chronic pain decreased levels of BDNF in the hippocampus and this decrease was prevented by sucrose and paracetamol treatments. Hippocampal levels of NT-3 and IGF-1 were not affected by any treatment. In conclusion, chronic pain induction during early life induced short memory impairment, and pretreatment with sucrose prevented this impairment via mechanisms that seem to involve BDNF. As evident in the results, sucrose, whether alone or in the presence of pre-noxious stimulation, increases pain threshold in such circumstances; most likely via a mechanism that involves an increase in endogenous

  13. Histamine reverses a memory deficit induced in rats by early postnatal maternal deprivation.

    PubMed

    Benetti, Fernando; da Silveira, Clarice Kras Borges; da Silva, Weber Cláudio; Cammarota, Martín; Izquierdo, Iván

    2012-01-01

    Early partial maternal deprivation causes long-lasting neurochemical, behavioral and brain structural effects. In rats, it causes a deficit in memory consolidation visible in adult life. Some of these deficits can be reversed by donepezil and galantamine, which suggests that they may result from an impairment of brain cholinergic transmission. One such deficit, representative of all others, is an impairment of memory consolidation, clearly observable in a one-trial inhibitory avoidance task. Recent data suggest a role of brain histaminergic systems in the regulation of behavior, particularly inhibitory avoidance learning. Here we investigate whether histamine itself, its analog SKF-91844, or various receptor-selective histamine agonists and antagonists given into the CA1 region of the hippocampus immediately post-training can affect retention of one-trial inhibitory avoidance in rats submitted to early postnatal maternal deprivation. We found that histamine, SKF-91844 and the H2 receptor agonist, dimaprit enhance consolidation on their own and reverse the consolidation deficit induced by maternal deprivation. The enhancing effect of histamine was blocked by the H2 receptor antagonist, ranitidine, but not by the H1 receptor antagonist pyrilamine or by the H3 antagonist thioperamide given into CA1 at doses known to have other behavioral actions, without altering locomotor and exploratory activity or the anxiety state of the animals. The present results suggest that the memory deficit induced by early postnatal maternal deprivation in rats may in part be due to an impairment of histamine mediated mechanisms in the CA1 region of the rat hippocampus.

  14. Spectroscopic Analysis of Stimulated Raman Scattering in the Early Stage of Laser-Induced Breakdown in Water

    NASA Astrophysics Data System (ADS)

    Yui, Hiroharu; Yoneda, Yoshinori; Kitamori, Takehiko; Sawada, Tsuguo

    1999-05-01

    We have investigated laser-induced breakdown (LIB) in water by measuring stimulated Raman scattering (SRS) induced in the early stage of the LIB phenomenon. The SRS is emitted before the breakdown plasma continuum emission becomes dominant and lasts about 80 picoseconds. The time-resolved spectra of the SRS indicate that ice VII structure is formed partially and inhomogeneously in the focal volume of the exciting beam on a picosecond time scale. The mechanism of the SRS inducement is also discussed.

  15. Early responses of tobacco suspension cells to rhizobacterial elicitors of induced systemic resistance.

    PubMed

    van Loon, Leendert C; Bakker, Peter A H M; van der Heijdt, Walter H W; Wendehenne, David; Pugin, Alain

    2008-12-01

    Colonization of roots by selected strains of fluorescent Pseudomonas spp. can trigger induced systemic resistance (ISR) against foliar pathogens in a plant species-specific manner. It has been suggested that early responses in cell suspension cultures in response to rhizobacterial elicitors, such as generation of active oxygen species (AOS) and extracellular medium alkalinization (MA), are linked to the development of ISR in whole plants. Perception of flagellin was demonstrated to elicit ISR in Arabidopsis, and bacterial lipopolysaccharides (LPS) have been shown to elicit several defense responses and to act as bacterial determinants of ISR in various plant species. In the present study, the LPS-containing cell walls, the pyoverdine siderophores, and the flagella of Pseudomonas putida WCS358, P. fluorescens WCS374, and P. fluorescens WCS417, which are all known to act as elicitors of ISR in selected plant species, were tested for their effects on the production of AOS, MA, elevation of cytoplasmic Ca(2+) ([Ca(2+)](cyt)), and defense-related gene expression in tobacco suspension cells. The LPS of all three strains, the siderophore of WCS374, and the flagella of WCS358 induced a single, transient, early burst of AOS, whereas the siderophores of WCS358 and WCS417 and the flagella of WCS374 and WCS417 did not. None of the compounds caused cell death. Once stimulated by the active compounds, the cells became refractory to further stimulation by any of the active elicitors, but not to the elicitor cryptogein from the oomycete Phytophthora cryptogea, indicating that signaling upon perception of the different rhizobacterial compounds rapidly converges into a common response pathway. Of all compounds tested, only the siderophores of WCS358 and WCS417 did not induce MA; the flagella of WCS374 and WCS417, although not active as elicitors of AOS, did induce MA. These results were corroborated by using preparations from relevant bacterial mutants. The active rhizobacterial

  16. Activation of calpains mediates early lung neutrophilic inflammation in ventilator-induced lung injury

    PubMed Central

    Liu, Dejie; Yan, Zhibo; Minshall, Richard D.; Schwartz, David E.; Chen, Yuguo

    2012-01-01

    Lung inflammatory responses in the absence of infection are considered to be one of primary mechanisms of ventilator-induced lung injury. Here, we determined the role of calpain in the pathogenesis of lung inflammation attributable to mechanical ventilation. Male C57BL/6J mice were subjected to high (28 ml/kg) tidal volume ventilation for 2 h in the absence and presence of calpain inhibitor I (10 mg/kg). To address the isoform-specific functions of calpain 1 and calpain 2 during mechanical ventilation, we utilized a liposome-based delivery system to introduce small interfering RNAs targeting each isoform in pulmonary vasculature in vivo. Mechanical ventilation with high tidal volume induced rapid (within minutes) and persistent calpain activation and lung inflammation as evidenced by neutrophil recruitment, production of TNF-α and IL-6, pulmonary vascular hyperpermeability, and lung edema formation. Pharmaceutical calpain inhibition significantly attenuated these inflammatory responses caused by lung hyperinflation. Depletion of calpain 1 or calpain 2 had a protective effect against ventilator-induced lung inflammatory responses. Inhibition of calpain activity by means of siRNA silencing or pharmacological inhibition also reduced endothelial nitric oxide (NO) synthase (NOS-3)-mediated NO production and subsequent ICAM-1 phosphorylation following high tidal volume ventilation. These results suggest that calpain activation mediates early lung inflammation during ventilator-induced lung injury via NOS-3/NO-dependent ICAM-1 phosphorylation and neutrophil recruitment. Inhibition of calpain activation may therefore provide a novel and promising strategy for the prevention and treatment of ventilator-induced lung injury. PMID:22140070

  17. Hypermutator Salmonella Heidelberg induces an early cell death in epithelial cells.

    PubMed

    Le Gall-David, Sandrine; Zenbaa, Neila; Bouchard, Damien; Lavault, Marie-Thérèse; Bonnaure-Mallet, Martine; Jolivet-Gougeon, Anne; Bousarghin, Latifa

    2015-10-22

    We have previously described that a strain of Salmonella Heidelberg with a hypermutator phenotype, B182, adhered strongly to HeLa cells. In this work, we showed that this hypermutator Salmonella strain invaded HeLa epithelial cells and induced cytoskeleton alteration. Those changes lead to HeLa cell death which was characteristic of apoptosis. For the first time, we showed that this hypermutator strain induced apoptosis associated with the activation of caspases 2, 9 and 3. Complementation of B182 strain showed a decrease in cells death induction. In the presence of other Salmonella Heidelberg with a normomutator phenotype, such as WT and SL486, cell death and caspase 3 were undetectable. These results suggested that early apoptosis and caspase 3 activation were specific to B182. Besides, B182 induced LDH release and caspase 3 activation in CaCo-2 and HCT116 cells. Heat-treated B182 and diffusible products failed to induce this phenotype. Epithelial cells treatment with cytochalasin D caused the inhibition of B182 internalisation and caspase 3 activation. These results showed that this cell death required active S. Heidelberg B182 protein synthesis and bacterial internalisation. However sipB and sopB, usually involved in apoptosis induced by Salmonella were not overexpressed in B182, contrary to fimA and fliC. Comparative genome analysis showed numerous mutations as in rpoS which would be more investigated. The role of the hypermutator phenotype might be suspected to be implicated in these specific features. This result expands our knowledge about strong mutators frequently found in bacterial organisms isolated from clinical specimens.

  18. Early ice retreat and ocean warming may induce copepod biogeographic boundary shifts in the Arctic Ocean

    NASA Astrophysics Data System (ADS)

    Feng, Zhixuan; Ji, Rubao; Campbell, Robert G.; Ashjian, Carin J.; Zhang, Jinlun

    2016-08-01

    Early ice retreat and ocean warming are changing various facets of the Arctic marine ecosystem, including the biogeographic distribution of marine organisms. Here an endemic copepod species, Calanus glacialis, was used as a model organism, to understand how and why Arctic marine environmental changes may induce biogeographic boundary shifts. A copepod individual-based model was coupled to an ice-ocean-ecosystem model to simulate temperature- and food-dependent copepod life history development. Numerical experiments were conducted for two contrasting years: a relatively cold and normal sea ice year (2001) and a well-known warm year with early ice retreat (2007). Model results agreed with commonly known biogeographic distributions of C. glacialis, which is a shelf/slope species and cannot colonize the vast majority of the central Arctic basins. Individuals along the northern boundaries of this species' distribution were most susceptible to reproduction timing and early food availability (released sea ice algae). In the Beaufort, Chukchi, East Siberian, and Laptev Seas where severe ocean warming and loss of sea ice occurred in summer 2007, relatively early ice retreat, elevated ocean temperature (about 1-2°C higher than 2001), increased phytoplankton food, and prolonged growth season created favorable conditions for C. glacialis development and caused a remarkable poleward expansion of its distribution. From a pan-Arctic perspective, despite the great heterogeneity in the temperature and food regimes, common biogeographic zones were identified from model simulations, thus allowing a better characterization of habitats and prediction of potential future biogeographic boundary shifts.

  19. Dendrobium officinale Prevents Early Complications in Streptozotocin-Induced Diabetic Rats.

    PubMed

    Hou, Shao-Zhen; Liang, Chu-Yan; Liu, Hua-Zhen; Zhu, Dong-Mei; Wu, Ya-Yun; Liang, Jian; Zhao, Ya; Guo, Jian-Ru; Huang, Song; Lai, Xiao-Ping

    2016-01-01

    Background. Dendrobium officinale (DO) Kimura et Migo is a precious Chinese herb that is considered beneficial for health due to its antioxidant and antidiabetes properties, and so on. In this research, we try to determine the preventive effect of DO on the early complications of STZ-induced diabetic rats. Methods. Type 1 diabetic rats were produced with a single intraperitoneal injection of STZ (50 mg/kg). DO (1 g/kg/day) was then orally administered for 5 weeks. Blood glucose, TC, TG, BUN, CREA, and GSH-PX levels were determined, and electroretinographic activity and hypoalgesia were investigated. Pathological sections of the eyes, hearts, aortas, kidneys, and livers were analyzed. Results. Treatment with DO significantly attenuated the serum levels of TC, TG, BUN, and CREA, markedly increased the amplitudes of ERG a- and b-waves and Ops, and reduced the hypoalgesia and histopathological changes of vital organs induced by hyperglycemia. The protective effect of DO in diabetic rats may be associated with its antioxidant activity, as evidenced by the marked increase in the serum level of glutathione peroxidase. However, DO had no significant effect on blood glucose levels and bodyweight of diabetic rats. Conclusions. DO supplementation is an effective treatment to prevent STZ-induced diabetic complications. PMID:27034693

  20. Neutrophil Extracellular Traps Enhance Early Inflammatory Response in Sendai Virus-Induced Asthma Phenotype.

    PubMed

    Akk, Antonina; Springer, Luke E; Pham, Christine T N

    2016-01-01

    Paramyxoviral infection in childhood has been linked to a significant increased rate of asthma development. In mice, paramyxoviral infection with the mouse parainfluenza virus type I, Sendai virus (Sev), causes a limited bronchiolitis followed by persistent asthma traits. We have previously shown that the absence of cysteine protease dipeptidyl peptidase I (DPPI) dampened the acute lung inflammatory response and the subsequent asthma phenotype induced by Sev. Adoptive transfer of wild-type neutrophils into DPPI-deficient mice restored leukocyte influx, the acute cytokine response, and the subsequent mucous cell metaplasia that accompanied Sev-induced asthma phenotype. However, the exact mechanism by which DPPI-sufficient neutrophils promote asthma development following Sev infection is still unknown. We hypothesize that neutrophils recruited to the alveolar space following Sev infection elaborate neutrophil extracellular traps (NETs) that propagate the inflammatory cascade, culminating in the eventual asthma phenotype. Indeed, we found that Sev infection was associated with NET formation in the lung and release of cell-free DNA complexed to myeloperoxidase in the alveolar space and plasma that peaked on day 2 post infection. Absence of DPPI significantly attenuated Sev-induced NET formation in vivo and in vitro. Furthermore, concomitant administration of DNase 1, which dismantled NETs, or inhibition of peptidylarginine deiminase 4 (PAD4), an essential mediator of NET formation, suppressed the early inflammatory responses to Sev infection. Lastly, NETs primed bone marrow-derived cells to release cytokines that can amplify the inflammatory cascade. PMID:27617014

  1. Ethylene signalling is mediating the early cadmium-induced oxidative challenge in Arabidopsis thaliana.

    PubMed

    Schellingen, Kerim; Van Der Straeten, Dominique; Remans, Tony; Vangronsveld, Jaco; Keunen, Els; Cuypers, Ann

    2015-10-01

    Cadmium (Cd) induces the generation of reactive oxygen species (ROS) and stimulates ethylene biosynthesis. The phytohormone ethylene is a regulator of many developmental and physiological plant processes as well as stress responses. Previous research indicated various links between ethylene signalling and oxidative stress. Our results support a correlation between the Cd-induced oxidative challenge and ethylene signalling in Arabidopsis thaliana leaves. The effects of 24 or 72 h exposure to 5 μM Cd on plant growth and several oxidative stress-related parameters were compared between wild-type (WT) and ethylene insensitive mutants (etr1-1, ein2-1, ein3-1). Cadmium-induced responses observed in WT plants were mainly affected in etr1-1 and ein2-1 mutants, of which the growth was less inhibited by Cd exposure as compared to WT and ein3-1 mutants. Both etr1-1 and ein2-1 showed a delayed response in the glutathione (GSH) metabolism, including GSH levels and transcript levels of GSH synthesising and recycling enzymes. Furthermore, the expression of different oxidative stress marker genes was significantly lower in Cd-exposed ein2-1 mutants, evidencing that ethylene signalling is involved in early responses to Cd stress. A model for the cross-talk between ethylene signalling and oxidative stress is proposed. PMID:26398798

  2. Early Amiodarone-Induced Pulmonary Toxicity after Endovascular Aneurysm Repair: A Case Report.

    PubMed

    Yoon, Uzung; Marinelli, Laura; Ali, Sayed; Huberfeld, Seymour; Barrera, Rafael; Chang, John B

    2016-09-01

    Amiodarone is an antiarrhythmic drug that has been commonly used to treat supraventricular and ventricular arrhythmias. This drug is an iodine-containing compound that tends to accumulate in several organs, including the lungs. Especially, its main metabolically active metabolite desethylamiodarone can adversely affect many organs. A very well-known severe complication of amiodarone therapy is the amiodarone-induced pulmonary toxicity. This article presents the case study of an 82-year-old male patient with acute amiodarone-induced pulmonary toxicity. The patient underwent endovascular aneurysm repair for rapidly increasing abdominal aortic aneurysm. During the postoperative period the patient developed rapid atrial fibrillation and amiodarone therapy was initiated. Subsequently, the patient went into acute respiratory failure and was requiring high supplemental oxygen support and a chest X-ray revealed bilateral pulmonary infiltrates. During the hospital course the patient required mechanical ventilator support. With discontinuation of amiodarone, supportive therapy and steroid treatment patient symptoms significantly improved. Amiodarone-induced pulmonary toxicity must be considered in the differential diagnosis of all patients on the medication with progressive or acute respiratory symptoms. Early discontinuation of amiodarone and aggressive corticosteroid therapy should be considered as a viable treatment strategy. PMID:27574388

  3. Environmental enrichment rescues the degraded auditory temporal resolution of cortical neurons induced by early noise exposure.

    PubMed

    Jiang, Cuiping; Xu, Xiaoxiao; Yu, Liping; Xu, Jinghong; Zhang, Jiping

    2015-09-01

    The accurate processing of sound temporal information is crucial to human speech perception and other species-specific communication. During postnatal development, the auditory cortex shows environmental and experience-dependent plasticity. However, how the postnatal environment affects cortical processing of sound temporal information is not fully understood. The aim of the present study was to determine whether postnatal noise exposure impairs neural temporal resolution in the auditory cortex, and, if so, whether environmental enrichment can rescue this degraded neural temporal acuity. Using the neural gap detection threshold determined in anesthetized rats as an index of temporal acuity, we found that exposure of juvenile rats to moderate-level noise induced much higher neural gap detection thresholds in adulthood than exposure of adult rats to the same noise. Environmental enrichment did not affect cortical neural gap detection thresholds in normally developing rats. However, rearing of rats with early noise exposure in an enriched environment promoted recovery from the noise-induced degraded neural temporal resolution. In addition, the tonal stimuli in the enriched environment contributed to only a portion of the recovery. These results provide evidence for noise-induced developmental impairment in neural gap detection thresholds in the auditory cortex, and suggest a therapeutic potential for environmental enrichment as a non-invasive approach to rescue developmentally degraded auditory temporal processing.

  4. Early Detection of T cell Transfer-induced Autoimmune Colitis by In Vivo Imaging System

    PubMed Central

    Chen, Yu-Ling; Chen, Yi-Ting; Lo, Cheng-Feng; Hsieh, Ching-I; Chiu, Shang-Yi; Wu, Chang-Yen; Yeh, Yu-Shan; Hung, Shu-Hsuan; Cheng, Po-Hao; Su, Yu-Hsuan; Jiang, Si-Tse; Chin, Hsian-Jean; Su, Yu-Chia

    2016-01-01

    Inflammatory bowel disease is a chronic and progressive inflammatory intestinal disease that includes two major types, namely ulcerative colitis and Crohn’s disease (CD). CD is characterized by intestinal epithelial hyperplasia and inflammatory cell infiltration. Transfer of CD25−CD45RBhiCD4+ (naïve) T cells into immunodeficiency mice induces autoimmune colitis with pathological lesions similar to CD and loss of body weight 4 weeks after cell transfer. However, weight loss neither has sufficient sensitivity nor totally matches the pathological findings of CD. To establish an early and sensitive indicator of autoimmune colitis model, the transferred T cell-induced colitis mouse model was modified by transferring luciferase-expressing donor T cells and determining the colitis by in vivo imaging system (IVIS). Colitis was detected with IVIS 7–10 days before the onset of body weight loss and diarrhea. IVIS was also applied in the dexamethasone treatment trial, and was a more sensitive indicator than body weight changes. All IVIS signals were parallel to the pathological abnormalities of the gut and immunological analysis results. In summary, IVIS provides both sensitive and objective means to monitor the disease course of transferred T cell-induced CD and fulfills the 3Rs principle of humane care of laboratory animals. PMID:27762297

  5. Neutrophil Extracellular Traps Enhance Early Inflammatory Response in Sendai Virus-Induced Asthma Phenotype

    PubMed Central

    Akk, Antonina; Springer, Luke E.; Pham, Christine T. N.

    2016-01-01

    Paramyxoviral infection in childhood has been linked to a significant increased rate of asthma development. In mice, paramyxoviral infection with the mouse parainfluenza virus type I, Sendai virus (Sev), causes a limited bronchiolitis followed by persistent asthma traits. We have previously shown that the absence of cysteine protease dipeptidyl peptidase I (DPPI) dampened the acute lung inflammatory response and the subsequent asthma phenotype induced by Sev. Adoptive transfer of wild-type neutrophils into DPPI-deficient mice restored leukocyte influx, the acute cytokine response, and the subsequent mucous cell metaplasia that accompanied Sev-induced asthma phenotype. However, the exact mechanism by which DPPI-sufficient neutrophils promote asthma development following Sev infection is still unknown. We hypothesize that neutrophils recruited to the alveolar space following Sev infection elaborate neutrophil extracellular traps (NETs) that propagate the inflammatory cascade, culminating in the eventual asthma phenotype. Indeed, we found that Sev infection was associated with NET formation in the lung and release of cell-free DNA complexed to myeloperoxidase in the alveolar space and plasma that peaked on day 2 post infection. Absence of DPPI significantly attenuated Sev-induced NET formation in vivo and in vitro. Furthermore, concomitant administration of DNase 1, which dismantled NETs, or inhibition of peptidylarginine deiminase 4 (PAD4), an essential mediator of NET formation, suppressed the early inflammatory responses to Sev infection. Lastly, NETs primed bone marrow-derived cells to release cytokines that can amplify the inflammatory cascade.

  6. Ethylene signalling is mediating the early cadmium-induced oxidative challenge in Arabidopsis thaliana.

    PubMed

    Schellingen, Kerim; Van Der Straeten, Dominique; Remans, Tony; Vangronsveld, Jaco; Keunen, Els; Cuypers, Ann

    2015-10-01

    Cadmium (Cd) induces the generation of reactive oxygen species (ROS) and stimulates ethylene biosynthesis. The phytohormone ethylene is a regulator of many developmental and physiological plant processes as well as stress responses. Previous research indicated various links between ethylene signalling and oxidative stress. Our results support a correlation between the Cd-induced oxidative challenge and ethylene signalling in Arabidopsis thaliana leaves. The effects of 24 or 72 h exposure to 5 μM Cd on plant growth and several oxidative stress-related parameters were compared between wild-type (WT) and ethylene insensitive mutants (etr1-1, ein2-1, ein3-1). Cadmium-induced responses observed in WT plants were mainly affected in etr1-1 and ein2-1 mutants, of which the growth was less inhibited by Cd exposure as compared to WT and ein3-1 mutants. Both etr1-1 and ein2-1 showed a delayed response in the glutathione (GSH) metabolism, including GSH levels and transcript levels of GSH synthesising and recycling enzymes. Furthermore, the expression of different oxidative stress marker genes was significantly lower in Cd-exposed ein2-1 mutants, evidencing that ethylene signalling is involved in early responses to Cd stress. A model for the cross-talk between ethylene signalling and oxidative stress is proposed.

  7. Neutrophil Extracellular Traps Enhance Early Inflammatory Response in Sendai Virus-Induced Asthma Phenotype

    PubMed Central

    Akk, Antonina; Springer, Luke E.; Pham, Christine T. N.

    2016-01-01

    Paramyxoviral infection in childhood has been linked to a significant increased rate of asthma development. In mice, paramyxoviral infection with the mouse parainfluenza virus type I, Sendai virus (Sev), causes a limited bronchiolitis followed by persistent asthma traits. We have previously shown that the absence of cysteine protease dipeptidyl peptidase I (DPPI) dampened the acute lung inflammatory response and the subsequent asthma phenotype induced by Sev. Adoptive transfer of wild-type neutrophils into DPPI-deficient mice restored leukocyte influx, the acute cytokine response, and the subsequent mucous cell metaplasia that accompanied Sev-induced asthma phenotype. However, the exact mechanism by which DPPI-sufficient neutrophils promote asthma development following Sev infection is still unknown. We hypothesize that neutrophils recruited to the alveolar space following Sev infection elaborate neutrophil extracellular traps (NETs) that propagate the inflammatory cascade, culminating in the eventual asthma phenotype. Indeed, we found that Sev infection was associated with NET formation in the lung and release of cell-free DNA complexed to myeloperoxidase in the alveolar space and plasma that peaked on day 2 post infection. Absence of DPPI significantly attenuated Sev-induced NET formation in vivo and in vitro. Furthermore, concomitant administration of DNase 1, which dismantled NETs, or inhibition of peptidylarginine deiminase 4 (PAD4), an essential mediator of NET formation, suppressed the early inflammatory responses to Sev infection. Lastly, NETs primed bone marrow-derived cells to release cytokines that can amplify the inflammatory cascade. PMID:27617014

  8. Identification of a cyclic-AMP-responsive element within the rat somatostatin gene.

    PubMed Central

    Montminy, M R; Sevarino, K A; Wagner, J A; Mandel, G; Goodman, R H

    1986-01-01

    We have examined the regulation of somatostatin gene expression by cAMP in PC12 rat pheochromocytoma cells transfected with the rat somatostatin gene. Forskolin at 10 microM caused a 4-fold increase in somatostatin mRNA levels within 4 hr of treatment in stably transfected cells. Chimeric genes containing the somatostatin gene promoter fused to the bacterial reporter gene encoding chloramphenicol acetyltransferase were also induced by cAMP in PC12 cells. To delineate the sequences required for response to cAMP, we constructed a series of promoter deletion mutants. Our studies defined a region between 60 and 29 base pairs upstream from the transcriptional initiation site that conferred cAMP responsiveness when placed adjacent to the simian virus 40 promoter. Within the cAMP-responsive element of the somatostatin gene, we observed an 8-base palindrome, 5'-TGACGTCA-3', which is highly conserved in many other genes whose expression is regulated by cAMP. cAMP responsiveness was greatly reduced when the somatostatin fusion genes were transfected into the mutant PC12 line A126-1B2, which is deficient in cAMP-dependent protein kinase 2. Our studies indicate that transcriptional regulation of the somatostatin gene by cAMP requires protein kinase 2 activity and may depend upon a highly conserved promoter element. Images PMID:2875459

  9. Impact of AmpC Derepression on Fitness and Virulence: the Mechanism or the Pathway?

    PubMed Central

    Pérez-Gallego, Marcelo; Torrens, Gabriel; Castillo-Vera, Jane; Moya, Bartolomé; Zamorano, Laura; Hultenby, Kjell; Albertí, Sebastián; Mellroth, Peter; Henriques-Normark, Birgitta; Normark, Staffan

    2016-01-01

    ABSTRACT Understanding the interplay between antibiotic resistance and bacterial fitness and virulence is essential to guide individual treatments and improve global antibiotic policies. A paradigmatic example of a resistance mechanism is the intrinsic inducible chromosomal β-lactamase AmpC from multiple Gram-negative bacteria, including Pseudomonas aeruginosa, a major nosocomial pathogen. The regulation of ampC expression is intimately linked to peptidoglycan recycling, and AmpC-mediated β-lactam resistance is frequently mediated by inactivating mutations in ampD, encoding an N-acetyl-anhydromuramyl-l-alanine amidase, affecting the levels of ampC-activating muropeptides. Here we dissect the impact of the multiple pathways causing AmpC hyperproduction on P. aeruginosa fitness and virulence. Through a detailed analysis, we demonstrate that the lack of all three P. aeruginosa AmpD amidases causes a dramatic effect in fitness and pathogenicity, severely compromising growth rates, motility, and cytotoxicity; the latter effect is likely achieved by repressing key virulence factors, such as protease LasA, phospholipase C, or type III secretion system components. We also show that ampC overexpression is required but not sufficient to confer the growth-motility-cytotoxicity impaired phenotype and that alternative pathways leading to similar levels of ampC hyperexpression and resistance, such as those involving PBP4, had no fitness-virulence cost. Further analysis indicated that fitness-virulence impairment is caused by overexpressing ampC in the absence of cell wall recycling, as reproduced by expressing ampC from a plasmid in an AmpG (muropeptide permease)-deficient background. Thus, our findings represent a major step in the understanding of β-lactam resistance biology and its interplay with fitness and pathogenesis. PMID:27795406

  10. Amps particle accelerator definition study

    NASA Technical Reports Server (NTRS)

    Sellen, J. M., Jr.

    1975-01-01

    The Particle Accelerator System of the AMPS (Atmospheric, Magnetospheric, and Plasmas in Space) payload is a series of charged particle accelerators to be flown with the Space Transportation System Shuttle on Spacelab missions. In the configuration presented, the total particle accelerator system consists of an energetic electron beam, an energetic ion accelerator, and both low voltage and high voltage plasma acceleration devices. The Orbiter is illustrated with such a particle accelerator system.

  11. Farnesoid X receptor activation by chenodeoxycholic acid induces detoxifying enzymes through AMP-activated protein kinase and extracellular signal-regulated kinase 1/2-mediated phosphorylation of CCAAT/enhancer binding protein β.

    PubMed

    Noh, Kyoung; Kim, Young Mi; Kim, Young Woo; Kim, Sang Geon

    2011-08-01

    Farnesoid X receptor (FXR) regulates redox homeostasis and elicits a cytoprotective effect. CCAAT/enhancer binding protein-β (C/EBPβ) plays a role in regulating the expression of hepatocyte-specific genes and contributes to hepatocyte protection and liver regeneration. In view of the role of FXR in xenobiotic metabolism and hepatocyte survival, this study investigated the potential of FXR to activate C/EBPβ for the induction of detoxifying enzymes and the responsible regulatory pathway. Chenodeoxycholic acid (CDCA), a major component in bile acids, activates FXR. In HepG2 cells, CDCA treatment activated C/EBPβ, as shown by increases in its phosphorylation, nuclear accumulation, and expression. 3-(2,6-Dichlorophenyl)-4-(3'-carboxy-2-chlorostilben-4-yl-)oxymethyl-5-isopropyl-isoxazole (GW4064), a synthetic FXR ligand, had similar effects. In addition, CDCA enhanced luciferase gene transcription from the construct containing -1.65-kb GSTA2 promoter, which contained C/EBP response element (pGL-1651). Moreover, CDCA treatment activated AMP-activated protein kinase (AMPK), which led to extracellular signal-regulated kinase 1/2 (ERK1/2) activation, as evidenced by the results of experiments using a dominant-negative mutant of AMPKα and chemical inhibitor. The activation of ERK1/2 was responsible for the activating phosphorylation of C/EBPβ. FXR knockdown attenuated the ability of CDCA to activate AMPK and ERK1/2 and phosphorylate C/EBPβ. Consistently, enforced expression of FXR promoted the phosphorylation of AMPKα, ERK1/2, and C/EBPβ, verifying that C/EBPβ phosphorylation elicited by CDCA results from the activation of AMPK and ERK1/2 by FXR. In mice, CDCA treatment activated C/EBPβ with the induction of detoxifying enzymes in the liver. Our results demonstrate that CDCA induces antioxidant and xenobiotic-metabolizing enzymes by activating C/EBPβ through AMPK-dependent ERK1/2 pathway downstream of FXR.

  12. Sitagliptin inhibits endothelin-1 expression in the aortic endothelium of rats with streptozotocin-induced diabetes by suppressing the nuclear factor-κB/IκBα system through the activation of AMP-activated protein kinase

    PubMed Central

    TANG, SONG-TAO; SU, HUAN; ZHANG, QIU; TANG, HAI-QIN; WANG, CHANG-JIANG; ZHOU, QING; WEI, WEI; ZHU, HUA-QING; WANG, YUAN

    2016-01-01

    Emerging evidence suggests that dipeptidyl peptidase-4 (DPP-4) inhibitors, including sitagliptin, exert favourable effects on the vascular endothelium. DPP-4 inhibitors suppress the degradation of glucagon-like peptide-1 (GLP-1), which has been reported to enhance nitric oxide (NO) production. However, the effects of DPP-4 inhibitors on endothelin-1 (ET-1) expression in the aorta, as well as the underlying mechanisms responsible for these effects, have yet to be investigated in animal models of diabetes mellitus (DM). In the present study, the rats were randomly divided into the following four groups: i) control; ii) DM; iii) DM + low-dose sitagliptin (10 mg/kg); and iv) DM + high-dose sitagliptin (30 mg/kg). Apart from the control group, all the rats received a high-fat diet for 8 weeks prior to the induction of diabetes with an intraperitoneal injection of streptozotocin. The treatments were then administered for 12 weeks. The serum levels of ET-1, NO, GLP-1 and insulin were measured as well as endothelial function. The expression of ET-1, AMP-activated protein kinase (AMPK) and nuclear factor (NF)-κB/IκBα were determined. After 12 weeks of treatment, the diabetic rats receiving sitagliptin showed significantly elevated serum levels of GLP-1 and NO, and reduced levels of ET-1. Moreover, sitagliptin significantly attenuated endothelial dysfunction as well as the remodeling of the aortic wall. Notably, sitagliptin inhibited ET-1 expression at the transcriptional and translational level in the aorta, which may have been mediated by the suppression of the NF-κB/IκBα system induced by AMPK activation. The majority of the above-mentioned effects were dose dependent. Taken together, the findings of the present study indicate that sitagliptin inhibits ET-1 expression in the aortic endothelium by suppressing the NF-κB/IκBα system through the activation of the AMPK pathway in diabetic rats. These findings further demonstrate some of the vasoprotective properties

  13. Sitagliptin inhibits endothelin-1 expression in the aortic endothelium of rats with streptozotocin-induced diabetes by suppressing the nuclear factor-κB/IκBα system through the activation of AMP-activated protein kinase.

    PubMed

    Tang, Song-Tao; Su, Huan; Zhang, Qiu; Tang, Hai-Qin; Wang, Chang-Jiang; Zhou, Qing; Wei, Wei; Zhu, Hua-Qing; Wang, Yuan

    2016-06-01

    Emerging evidence suggests that dipeptidyl peptidase-4 (DPP-4) inhibitors, including sitagliptin, exert favourable effects on the vascular endothelium. DPP-4 inhibitors suppress the degradation of glucagon-like peptide-1 (GLP‑1), which has been reported to enhance nitric oxide (NO) production. However, the effects of DPP-4 inhibitors on endothelin-1 (ET-1) expression in the aorta, as well as the underlying mechanisms responsible for these effects, have yet to be investigated in animal models of diabetes mellitus (DM). In the present study, the rats were randomly divided into the following four groups: i) control; ii) DM; iii) DM + low‑dose sitagliptin (10 mg/kg); and iv) DM + high‑dose sitagliptin (30 mg/kg). Apart from the control group, all the rats received a high-fat diet for 8 weeks prior to the induction of diabetes with an intraperitoneal injection of streptozotocin. The treatments were then administered for 12 weeks. The serum levels of ET-1, NO, GLP-1 and insulin were measured as well as endothelial function. The expression of ET-1, AMP-activated protein kinase (AMPK) and nuclear factor (NF)-κB/IκBα were determined. After 12 weeks of treatment, the diabetic rats receiving sitagliptin showed significantly elevated serum levels of GLP-1 and NO, and reduced levels of ET-1. Moreover, sitagliptin significantly attenuated endothelial dysfunction as well as the remodeling of the aortic wall. Notably, sitagliptin inhibited ET-1 expression at the transcriptional and translational level in the aorta, which may have been mediated by the suppression of the NF-κB/IκBα system induced by AMPK activation. The majority of the above-mentioned effects were dose dependent. Taken together, the findings of the present study indicate that sitagliptin inhibits ET-1 expression in the aortic endothelium by suppressing the NF-κB/IκBα system through the activation of the AMPK pathway in diabetic rats. These findings further demonstrate some of the

  14. Agile manufacturing prototyping system (AMPS)

    SciTech Connect

    Garcia, P.

    1998-05-09

    The Agile Manufacturing Prototyping System (AMPS) is being integrated at Sandia National Laboratories. AMPS consists of state of the industry flexible manufacturing hardware and software enhanced with Sandia advancements in sensor and model based control; automated programming, assembly and task planning; flexible fixturing; and automated reconfiguration technology. AMPS is focused on the agile production of complex electromechanical parts. It currently includes 7 robots (4 Adept One, 2 Adept 505, 1 Staubli RX90), conveyance equipment, and a collection of process equipment to form a flexible production line capable of assembling a wide range of electromechanical products. This system became operational in September 1995. Additional smart manufacturing processes will be integrated in the future. An automated spray cleaning workcell capable of handling alcohol and similar solvents was added in 1996 as well as parts cleaning and encapsulation equipment, automated deburring, and automated vision inspection stations. Plans for 1997 and out years include adding manufacturing processes for the rapid prototyping of electronic components such as soldering, paste dispensing and pick-and-place hardware.

  15. Changes in the cyclic AMP content during growth and development of Acetabularia.

    PubMed

    Minder, C; Vanden Driessche, T

    1978-05-26

    The 3',5'-adenosine monophosphate (cyclic-AMP) content of the unicellular alga Acetabularia has been examined at various developmental stages. It has been found that very young algae, less than 10mm in length, have a high cAMP content [more than 7 pmoles per 100 mg wet weight (WW)], but that with the growth of the algae, the cAMP content decreases rapidly, reaching the low level of 0.5--1.0 pmoles per 100mg WW. The cAMP content remains at this level until cap differentiation, after which an increase in cAMP content accompanies cap enlargement. It has been shown that these results are unlikely to be affected by changes in the cAMP content induced by variations in circadian rhythm. Treatment with theophylline (2.10(-3) M), a phosphodieterase inhibitor, results in an increase in the cAMP content and delays growth and cap formation. Experiments on the effects of theophylline upon the circadian rhythm of oxygen evolution have shown that the continuous presence of theophylline in the culture medium does not induce a phase shift in the rhythm. The cAMP content of anucleate Acetabularia shows development stage variations parallel to that of the whole algae.

  16. Ovarian insufficiency and early pregnancy loss induced by activation of the innate immune system

    PubMed Central

    Erlebacher, Adrian; Zhang, Dorothy; Parlow, Albert F.; Glimcher, Laurie H.

    2004-01-01

    We describe a murine model of early pregnancy failure induced by systemic activation of the CD40 immune costimulatory pathway. Although fetal loss involved an NK cell intermediate, it was not due to lymphocyte-mediated destruction of the fetus and placenta. Rather, pregnancy failure resulted from impaired progesterone synthesis by the corpus luteum of the ovary, an endocrine defect in turn associated with ovarian resistance to the gonadotropic effects of prolactin. Pregnancy failure also required the proinflammatory cytokine TNF-α and correlated with the luteal induction of the prolactin receptor signaling inhibitors suppressor of cytokine signaling 1 (Socs1) and Socs3. Such links between immune activation and reproductive endocrine dysfunction may be relevant to pregnancy loss and other clinical disorders of reproduction. PMID:15232610

  17. Early detection of dysplasia in colon and bladder tissue using laser-induced fluorescence

    NASA Astrophysics Data System (ADS)

    Rava, Richard P.; Richards-Kortum, Rebecca R.; Fitzmaurice, Maryann; Cothren, Robert M., Jr.; Petras, Robert; Sivak, Michael J., Jr.; Levine, Howard H.

    1991-06-01

    Laser induced fluorescence has been explored as an early detection scheme for two clinically important examples of neoplasia: colorectal dysplasia and transitional cell carcinoma in the urinary bladder. In both, it is desirable to detect microscopic and biochemical changes of pre-cancer in order to identify patients at risk for developing invasive carcinoma. This paper will compare the fluorescence obtained from these two pre-cancerous conditions, and discuss the connection between the fluorescence and the morphological/molecular changes occurring in the tissue. The similarities and differences in the fluorescence will be compared to determine the general features of pre-cancerous changes that might be utilized for detection of the disease.

  18. Recharge of the early atmosphere of Mars by impact-induced release of CO2

    NASA Technical Reports Server (NTRS)

    Carr, Michael H.

    1989-01-01

    The question as to whether high impact rates early in the history of Mars could have aided in maintaining a relatively thick CO2 atmosphere is discussed. Such impacts could have released CO2 into the atmosphere by burial, by shock-induced release during impact events, and by the addition of carbon to Mars from the impacting bolides. On the assumption that cratering rates on Mars were comparable to those of the moon's Nectarial period, burial rates are a result of 'impact gardening' at the end of heavy bombardment are estimated to have ranged from 20 to 45 m/million years; at these rates, 0.1-0.2 bar of CO2 would have been released every 10 million years as a result of burial to depths at which carbonate dissociation temperatures are encountered.

  19. Early inflammatory damage to intestinal neurons occurs via inducible nitric oxide synthase.

    PubMed

    Venkataramana, Shriram; Lourenssen, S; Miller, K G; Blennerhassett, M G

    2015-03-01

    Intestinal inflammation affects the enteric nervous system (ENS) that lies adjacent to the smooth muscle layers. Previously, we showed that the loss of ENS neurons in animal models such as tri-nitrobenzene sulphonic acid (TNBS)-induced colitis was a limited and early event despite progressive worsening of inflammation. Here, we demonstrated that the rapid appearance of activated immune cells in the intestinal wall is selectively neurotoxic via iNOS-derived NO, using TNBS-induced colitis in both rats and mice, and a co-culture model of ENS neurons and smooth muscle. An influx of neutrophils and macrophages occurred within hours of initiation of rat colitis, correlating with iNOS expression, acutely elevated NO and neuronal death. In vitro, chemical donors of NO selectively caused axonal damage and neuronal death. These outcomes were similar to those seen with combined culture with either activated peritoneal immune cells or the immune cell lines RAW-264 and RBL-2H3. Immune cell-mediated neurotoxicity was blocked by the iNOS inhibitor L-NIL, and neuronal death was inhibited by the RIP-1 kinase inhibitor necrostatin. In a mouse model, the stereotypic loss of myenteric neurons by Day 4 post-TNBS was abrogated by the selective iNOS inhibitors L-NIL or 1400W without effect on other parameters of intestinal inflammation. Preservation of ENS neurons also ameliorated the hyperplasia of smooth muscle that is characteristic of intestinal inflammation, in line with prior work showing neural regulation of smooth muscle phenotype. This identifies a predominant pathway of immune cell damage to the ENS, where early, acute elevation of NO from iNOS can be cytotoxic to myenteric neurons.

  20. Early or late antibiotic intervention prevents Helicobacter pylori-induced gastric cancer in a mouse model.

    PubMed

    Zhang, Songhua; Lee, Dong Soo; Morrissey, Rhiannon; Aponte-Pieras, Jose R; Rogers, Arlin B; Moss, Steven F

    2014-12-01

    H. pylori infection causes gastritis, peptic ulcers and gastric cancer. Eradicating H. pylori prevents ulcers, but to what extent this prevents cancer remains unknown, especially if given after intestinal metaplasia has developed. H. pylori infected wild-type (WT) mice do not develop cancer, but mice lacking the tumor suppressor p27 do so, thus providing an experimental model of H. pylori-induced cancer. We infected p27-deficient mice with H. pylori strain SS1 at 6-8 weeks of age. Persistently H. pylori-infected WT C57BL/6 mice served as controls. Mice in the eradication arms received antimicrobial therapy (omeprazole, metronidazole and clarithromycin) either "early" (at 15 weeks post infection, WPI) or "late" at 45 WPI. At 70 WPI, mice were euthanized for H. pylori determination, histopathology and cytokine/chemokine expression. Persistently infected mice developed premalignant lesions including high-grade dysplasia, whereas those given antibiotics did not. Histologic activity scores in the eradication groups were similar to each other, and were significantly decreased compared with controls for inflammation, epithelial defects, hyperplasia, metaplasia, atrophy and dysplasia. IP-10 and MIG levels in groups that received antibiotics were significantly lower than controls. There were no significant differences in expression of IFN-γ, TNF-α, IL-1β, RANTES, MCP-1, MIP-1α or MIP-1β among the three groups. Thus, H. pylori eradication given either early or late after infection significantly attenuated gastric inflammation, gastric atrophy, hyperplasia, and dysplasia in the p27-deficient mice model of H. pylori-induced gastric cancer, irrespective of the timing of antibiotic administration. This was associated with reduced expression of IP-10 and MIG.

  1. Role of phosphodiesterases in the shaping of sub-plasma-membrane cAMP oscillations and pulsatile insulin secretion.

    PubMed

    Tian, Geng; Sågetorp, Jenny; Xu, Yunjian; Shuai, Hongyan; Degerman, Eva; Tengholm, Anders

    2012-11-01

    Specificity and versatility in cyclic AMP (cAMP) signalling are governed by the spatial localisation and temporal dynamics of the signal. Phosphodiesterases (PDEs) are important for shaping cAMP signals by hydrolyzing the nucleotide. In pancreatic β-cells, glucose triggers sub-plasma-membrane cAMP oscillations, which are important for insulin secretion, but the mechanisms underlying the oscillations are poorly understood. Here, we investigated the role of different PDEs in the generation of cAMP oscillations by monitoring the concentration of cAMP in the sub-plasma-membrane space ([cAMP](pm)) with ratiometric evanescent wave microscopy in MIN6 cells or mouse pancreatic β-cells expressing a fluorescent translocation biosensor. The general PDE inhibitor IBMX increased [cAMP](pm), and whereas oscillations were frequently observed at 50 µM IBMX, 300 µM-1 mM of the inhibitor caused a stable increase in [cAMP](pm). The [cAMP](pm) was nevertheless markedly suppressed by the adenylyl cyclase inhibitor 2',5'-dideoxyadenosine, indicating IBMX-insensitive cAMP degradation. Among IBMX-sensitive PDEs, PDE3 was most important for maintaining a low basal level of [cAMP](pm) in unstimulated cells. After glucose induction of [cAMP](pm) oscillations, inhibitors of PDE1, PDE3 and PDE4 inhibitors the average cAMP level, often without disturbing the [cAMP](pm) rhythmicity. Knockdown of the IBMX-insensitive PDE8B by shRNA in MIN6 cells increased the basal level of [cAMP](pm) and prevented the [cAMP](pm)-lowering effect of 2',5'-dideoxyadenosine after exposure to IBMX. Moreover, PDE8B-knockdown cells showed reduced glucose-induced [cAMP](pm) oscillations and loss of the normal pulsatile pattern of insulin secretion. It is concluded that [cAMP](pm) oscillations in β-cells are caused by periodic variations in cAMP generation, and that several PDEs, including PDE1, PDE3 and the IBMX-insensitive PDE8B, are required for shaping the sub-membrane cAMP signals and pulsatile insulin release.

  2. Early transcriptional responses to mercury: a role for ethylene in mercury-induced stress.

    PubMed

    Montero-Palmero, M Belén; Martín-Barranco, Amanda; Escobar, Carolina; Hernández, Luis E

    2014-01-01

    Understanding the cellular mechanisms of plant tolerance to mercury (Hg) is important for developing phytoremediation strategies of Hg-contaminated soils. The early responses of alfalfa (Medicago sativa) seedlings to Hg were studied using transcriptomics analysis. A Medicago truncatula microarray was hybridized with high-quality root RNA from M. sativa treated with 3 μM Hg for 3, 6 and 24 h. The transcriptional pattern data were complementary to the measurements of root growth inhibition, lipid peroxidation, hydrogen peroxide (H2 O2 ) accumulation and NADPH-oxidase activity as stress indexes. Of 559 differentially expressed genes (DEGs), 91% were up-regulated. The majority of DEGs were shared between the 3 and 6 h (60%) time points, including the 'stress', 'secondary metabolism' and 'hormone metabolism' functional categories. Genes from ethylene metabolism and signalling were highly represented, suggesting that this phytohormone may be relevant for metal perception and homeostasis. Ethylene-insensitive alfalfa seedlings preincubated with the ethylene signalling inhibitor 1-methylcyclopronene and Arabidopsis thaliana ein2-5 mutants confirmed that ethylene participates in the early perception of Hg stress. It modulates root growth inhibition, NADPH-oxidase activity and Hg-induced apoplastic H2 O2 accumulation. Therefore, ethylene signalling attenuation could be useful in future phytotechnological applications to ameliorate stress symptoms in Hg-polluted plants. PMID:24033367

  3. Cerebrospinal fluid control of neurogenesis induced by retinoic acid during early brain development.

    PubMed

    Alonso, M I; Martín, C; Carnicero, E; Bueno, D; Gato, A

    2011-07-01

    Embryonic-cerebrospinal fluid (E-CSF) plays crucial roles in early brain development including the control of neurogenesis. Although FGF2 and lipoproteins present in the E-CSF have previously been shown to be involved in neurogenesis, the main factor triggering this process remains unknown. E-CSF contains all-trans-retinol and retinol-binding protein involved in the synthesis of retinoic acid (RA), a neurogenesis inducer. In early chick embryo brain, only the mesencephalic-rombencephalic isthmus (IsO) is able to synthesize RA. Here we show that in chick embryo brain development: (1) E-CSF helps to control RA synthesis in the IsO by means of the RBP and all-trans-retinol it contains; (2) E-CSF has retinoic acid activity, which suggests it may act as a diffusion pathway for RA; and (3) the influence of E-CSF on embryonic brain neurogenesis is to a large extent due to its involvement in RA synthesis. These data help to understand neurogenesis from neural progenitor cells. PMID:21594951

  4. Proton and Fe Ion-Induced Early and Late Chromosome Aberrations in Different Cell Types

    NASA Technical Reports Server (NTRS)

    Wu, Honglu; Lu, Tao; Yeshitla, Samrawit; Zhang, Ye; Kadhim, Munira

    2016-01-01

    An early stage of cancer development is believed to be genomic instability (GI) which accelerates the mutation rate in the descendants of the cells surviving radiation exposure. To investigate GI induced by charged particles, we exposed human lymphocytes, human fibroblast cells, and human mammary epithelial cells to high energy protons and Fe ions. In addition, we also investigated GI in bone marrow cells isolated from CBA/CaH (CBA) and C57BL/6 (C57) mice, by analyzing cell survival and chromosome aberrations in the cells after multiple cell divisions. Results analyzed so far from the experiments indicated different sensitivities to charged particles between CBA/CaH (CBA) and C57BL/6 (C57) mouse strains, suggesting that there are two main types of response to irradiation: 1) responses associated with survival of damaged cells and 2) responses associated with the induction of non-clonal chromosomal instability in the surviving progeny of stem cells. Previously, we reported that the RBE for initial chromosome damages was high in human lymphocytes exposed to Fe ions. Our results with different cell types demonstrated different RBE values between different cell types and between early and late chromosomal damages. This study also attempts to offer an explanation for the varying RBE values for different cancer types.

  5. Social Preference Deficits in Juvenile Zebrafish Induced by Early Chronic Exposure to Sodium Valproate

    PubMed Central

    Liu, Xiuyun; Zhang, Yinglan; Lin, Jia; Xia, Qiaoxi; Guo, Ning; Li, Qiang

    2016-01-01

    Prenatal exposure to sodium valproate (VPA), a widely used anti-epileptic drug, is related to a series of dysfunctions, such as deficits in language and communication. Clinical and animal studies have indicated that the effects of VPA are related to the concentration and to the exposure window, while the neurobehavioral effects of VPA have received limited research attention. In the current study, to analyze the neurobehavioral effects of VPA, zebrafish at 24 h post-fertilization (hpf) were treated with early chronic exposure to 20 μM VPA for 7 h per day for 6 days or with early acute exposure to 100 μM VPA for 7 h. A battery of behavioral screenings was conducted at 1 month of age to investigate social preference, locomotor activity, anxiety, and behavioral response to light change. A social preference deficit was only observed in animals with chronic VPA exposure. Acute VPA exposure induced a change in the locomotor activity, while chronic VPA exposure did not affect locomotor activity. Neither exposure procedure influenced anxiety or the behavioral response to light change. These results suggested that VPA has the potential to affect some behaviors in zebrafish, such as social behavior and the locomotor activity, and that the effects were closely related to the concentration and the exposure window. Additionally, social preference seemed to be independent from other simple behaviors. PMID:27812327

  6. Early transcriptional responses to mercury: a role for ethylene in mercury-induced stress.

    PubMed

    Montero-Palmero, M Belén; Martín-Barranco, Amanda; Escobar, Carolina; Hernández, Luis E

    2014-01-01

    Understanding the cellular mechanisms of plant tolerance to mercury (Hg) is important for developing phytoremediation strategies of Hg-contaminated soils. The early responses of alfalfa (Medicago sativa) seedlings to Hg were studied using transcriptomics analysis. A Medicago truncatula microarray was hybridized with high-quality root RNA from M. sativa treated with 3 μM Hg for 3, 6 and 24 h. The transcriptional pattern data were complementary to the measurements of root growth inhibition, lipid peroxidation, hydrogen peroxide (H2 O2 ) accumulation and NADPH-oxidase activity as stress indexes. Of 559 differentially expressed genes (DEGs), 91% were up-regulated. The majority of DEGs were shared between the 3 and 6 h (60%) time points, including the 'stress', 'secondary metabolism' and 'hormone metabolism' functional categories. Genes from ethylene metabolism and signalling were highly represented, suggesting that this phytohormone may be relevant for metal perception and homeostasis. Ethylene-insensitive alfalfa seedlings preincubated with the ethylene signalling inhibitor 1-methylcyclopronene and Arabidopsis thaliana ein2-5 mutants confirmed that ethylene participates in the early perception of Hg stress. It modulates root growth inhibition, NADPH-oxidase activity and Hg-induced apoplastic H2 O2 accumulation. Therefore, ethylene signalling attenuation could be useful in future phytotechnological applications to ameliorate stress symptoms in Hg-polluted plants.

  7. Early β-Amyloid-induced Synaptic Dysfunction Is Counteracted by Estrogen in Organotypic Hippocampal Cultures.

    PubMed

    Merlo, Sara; Spampinato, Simona Federica; Capani, Francisco; Sortino, Maria Angela

    2016-01-01

    In the present study we set up a model of slow progression of neuronal injury by exposing organotypic hippocampal cultures to a low concentration of Amyloid β (25-35) peptide (Aβ, 2 μM) to analyze the time-related effects of 17-β estradiol (17β-E2, 10 nM). Neuronal death occurs after 7 d and is prevented by addition of 17β-E2 24 h prior to, together with or 48 h after exposure to Aβ. This effect is mimicked by selective ERα agonist PPT (100 nM). Treatment with Aβ leads to early and transient (16-72 h) increase of pre- and post-synaptic proteins synaptophysin and PSD95, followed by a decrease coincident with neuronal death (7d), all prevented by 17β-E2. At 72 h of Aβ exposure, synaptic activity is increased, as by higher levels of glutamate and increased loading and unloading of FM 1-43-labeled synaptic vesicles. All these effects are also prevented by 17β-E2. These data point out beneficial effects of estrogen on early Aβ-induced synaptic disruption.

  8. Salicylate-induced changes in immediate-early genes in the hippocampal CA1 area.

    PubMed

    Wu, Hao; Xu, Feng-Lei; Yin, Yong; Da, Peng; You, Xiao-Dong; Xu, Hui-Min; Tang, Yan

    2015-08-01

    Studies have suggested that salicylate affects neuronal function via interactions with specific membrane channels/receptors. However, the effect of salicylate on activity and synaptic morphology of the hippocampal Cornu Ammonis (CA) 1 area remains to be elucidated. The activation of immediate-early genes (IEGs) was reported to correlate with neuronal activity, in particular activity-regulated cytoskeleton-associated protein and early growth response gene 1. The aim of the present study was to evaluate the expression of these IEGs, as well that of N-methyl D-aspartate (NMDA) receptor subunit 2B in rats following acute and chronic salicylate treatment. Protein and messenger RNA levels of all three genes were increased in rats following chronic administration of salicylate (300 mg/kg for 10 days), returning to baseline levels 14 days post-cessation of treatment. The transient upregulation of gene expression following treatment was accompanied by ultrastructural alterations in hippocampal CA1 area synapses. An increase in synaptic interface curvature was observed as well as an increased number of presynaptic vesicles; in addition, postsynaptic densities thickened and lengthened. In conclusion, the results of the present study indicated that chronic exposure to salicylate may lead to structural alteration of hippocampal CA1 neurons, and it was suggested that this process occurs through induced expression of IEGs via NMDA receptor activation. PMID:25873216

  9. Water permeability and TCDD-induced edema in zebrafish early-life stages.

    PubMed

    Hill, Adrian J; Bello, Susan M; Prasch, Amy L; Peterson, Richard E; Heideman, Warren

    2004-03-01

    A common response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in teleost embryos is blue-sac disease, characterized by pericardial and yolk-sac edema. The cellular and extracellular fluids of freshwater fish are hyperosmotic compared to the surrounding water. In order to be in osmotic balance, freshwater fish must maintain a barrier to minimize water entry and excrete excess water that passes the barrier. We hypothesized that edema observed in TCDD-exposed zebrafish was caused by a failure of a barrier to incoming water. As a test of this hypothesis, we removed the osmotic gradient that drives water entry by increasing the osmolarity of the surrounding water with mannitol. Abolishing the osmotic gradient between the interior body fluids and the water environment of the developing zebrafish significantly reduced both pericardial and yolk-sac edema. When added after edema formation had already started, mannitol only partially reversed pre-existing edema. An alternate hypothesis is that TCDD impairs water excretion, allowing water to accumulate as edema fluid. However, we were unable to demonstrate an alteration in kidney function: expression of early markers for kidney development appeared normal, and we did not observe TCDD-induced changes in kidney filtration. An alteration in the overall shape of the kidney was observed, but this may be a consequence of compression by edema. In conclusion, TCDD exposure may inhibit the function of a permeability barrier to water, which is critical for maintaining osmotic balance in early development.

  10. Cerebrospinal fluid control of neurogenesis induced by retinoic acid during early brain development.

    PubMed

    Alonso, M I; Martín, C; Carnicero, E; Bueno, D; Gato, A

    2011-07-01

    Embryonic-cerebrospinal fluid (E-CSF) plays crucial roles in early brain development including the control of neurogenesis. Although FGF2 and lipoproteins present in the E-CSF have previously been shown to be involved in neurogenesis, the main factor triggering this process remains unknown. E-CSF contains all-trans-retinol and retinol-binding protein involved in the synthesis of retinoic acid (RA), a neurogenesis inducer. In early chick embryo brain, only the mesencephalic-rombencephalic isthmus (IsO) is able to synthesize RA. Here we show that in chick embryo brain development: (1) E-CSF helps to control RA synthesis in the IsO by means of the RBP and all-trans-retinol it contains; (2) E-CSF has retinoic acid activity, which suggests it may act as a diffusion pathway for RA; and (3) the influence of E-CSF on embryonic brain neurogenesis is to a large extent due to its involvement in RA synthesis. These data help to understand neurogenesis from neural progenitor cells.

  11. Salicylate-induced changes in immediate-early genes in the hippocampal CA1 area

    PubMed Central

    WU, HAO; XU, FENG-LEI; YIN, YONG; DA, PENG; YOU, XIAO-DONG; XU, HUI-MIN; TANG, YAN

    2015-01-01

    Studies have suggested that salicylate affects neuronal function via interactions with specific membrane channels/receptors. However, the effect of salicylate on activity and synaptic morphology of the hippocampal Cornu Ammonis (CA) 1 area remains to be elucidated. The activation of immediate-early genes (IEGs) was reported to correlate with neuronal activity, in particular activity-regulated cytoskeleton-associated protein and early growth response gene 1. The aim of the present study was to evaluate the expression of these IEGs, as well that of N-methyl D-aspartate (NMDA) receptor subunit 2B in rats following acute and chronic salicylate treatment. Protein and messenger RNA levels of all three genes were increased in rats following chronic administration of salicylate (300 mg/kg for 10 days), returning to baseline levels 14 days post-cessation of treatment. The transient upregulation of gene expression following treatment was accompanied by ultrastructural alterations in hippocampal CA1 area synapses. An increase in synaptic interface curvature was observed as well as an increased number of presynaptic vesicles; in addition, postsynaptic densities thickened and lengthened. In conclusion, the results of the present study indicated that chronic exposure to salicylate may lead to structural alteration of hippocampal CA1 neurons, and it was suggested that this process occurs through induced expression of IEGs via NMDA receptor activation. PMID:25873216

  12. BRODIFACOUM INDUCES EARLY HEMOGLOBINURIA AND LATE HEMATURIA IN RATS: NOVEL RAPID BIOMARKERS OF POISONING

    PubMed Central

    Ware, Kyle M; Feinstein, Douglas L; Rubinstein, Israel; Weinberg, Guy; Rovin, Brad H; Hebert, Lee; Muni, Navin; Cianciolo, Rachel E.; Satoskar, Anjali A; Nadasdy, Tibor; Brodsky, Sergey V

    2015-01-01

    Introduction Brodifacoum (BDF) is a superwarfarin that is used primarily as a rodenticide. There have been increasing number of reports of human cases of accidental or intentional BDF ingestion with high mortality rate. Its broad availability and high lethality suggest that BDF should be considered a potential chemical threat. Currently, there is no biomarker for early detection of BDF ingestion in humans; patients typically present with severe coagulopathy. Since we demonstrated earlier that warfarin can induce acute kidney injury with hematuria, we tested whether BDF would also lead to change in urinary biomarkers. Material and methods BDF was administered to Sprague Dawley rats via oral gavage. N-acetylcysteine (NAC) was given per os in drinking water 24 hours prior to BDF. Urinalysis was performed at different times after BDF administration. Anticoagulation and serum creatinine levels were analyzed in the blood. Results We observed that within a few hours the animals developed BDF-dose-dependent transient hemoglobinuria, which ceased within 24 hours. This was accompanied by a transient decrease in hematocrit, gross hemolysis and an increase in free hemoglobin in the serum. At later times, animals developed true hematuria with red blood cells in the urine, which was associated with BDF anticoagulation. NAC prevented early hemoglobinuria, but not late hematuria associated with BDF. Conclusions We propose that transient early hemoglobinuria (associated with oxidative stress) with consecutive late hematuria (associated with anticoagulation) are novel biomarkers of BDF poisoning and they can be used in clinical setting or in mass-casualty with BDF to identify poisoned patients. PMID:26111556

  13. Cyclic AMP Affects Oocyte Maturation and Embryo Development in Prepubertal and Adult Cattle

    PubMed Central

    Bernal-Ulloa, Sandra Milena; Heinzmann, Julia; Herrmann, Doris; Hadeler, Klaus-Gerd; Aldag, Patrick; Winkler, Sylke; Pache, Dorit; Baulain, Ulrich; Lucas-Hahn, Andrea; Niemann, Heiner

    2016-01-01

    High cAMP levels during in vitro maturation (IVM) have been related to improved blastocyst yields. Here, we employed the cAMP/cGMP modulators, forskolin, IBMX, and cilostamide, during IVM to unravel the role of high cAMP in early embryonic development produced from prepubertal and adult bovine oocytes. Oocytes were collected via transvaginal aspiration and randomly assigned to three experimental groups: TCM24 (24h IVM/control), cAMP30 (2h pre-IVM (forskolin-IBMX), 30h IVM-cilostamide), and DMSO30 (Dimethyl Sulfoxide/vehicle control). After IVM, oocytes were fertilized in vitro and zygotes were cultured in vitro to blastocysts. Meiotic progression, cAMP levels, mRNA abundance of selected genes and DNA methylation were evaluated in oocytes. Blastocysts were used for gene expression or DNA methylation analyses. Blastocysts from the cAMP30 groups were transferred to recipients. The cAMP elevation delayed meiotic progression, but developmental rates were not increased. In immature oocytes, mRNA abundance of PRKACA was higher for cAMP30 protocol and no differences were found for PDE3A, SMAD2, ZAR1, PRDX1 and SLC2A8. EGR1 gene was up-regulated in prepubertal cAMP30 immature oocytes and down-regulated in blastocysts from all in vitro treatments. A similar gene expression profile was observed for DNMT3b, BCL2L1, PRDX1 and SLC2A8 in blastocysts. Satellite DNA methylation profiles were different between prepubertal and adult oocytes and blastocysts derived from the TCM24 and DMSO30 groups. Blastocysts obtained from prepubertal and adult oocytes in the cAMP30 treatment displayed normal methylation profiles and produced offspring. These data indicate that cAMP regulates IVM in prepubertal and adult oocytes in a similar manner, with impact on the establishment of epigenetic marks and acquisition of full developmental competency. PMID:26926596

  14. Transcriptomic analysis of cyclic AMP response in bovine cumulus cells.

    PubMed

    Khan, D R; Guillemette, C; Sirard, M A; Richard, F J

    2015-09-01

    Acquisition of oocyte developmental competence needs to be understood to improve clinical outcomes of assisted reproduction. The stimulation of cumulus cell concentration of cyclic adenosine 3'5'-monophosphate (cAMP) by pharmacological agents during in vitro maturation (IVM) participates in improvement of oocyte quality. However, precise coordination and downstream targets of cAMP signaling in cumulus cells are largely unknown. We have previously demonstrated better embryo development after cAMP stimulation for first 6 h during IVM. Using this model, we investigated cAMP signaling in cumulus cells through in vitro culture of cumulus-oocyte complexes (COCs) in the presence of cAMP raising agents: forskolin, IBMX, and dipyridamole (here called FID treatment). Transcriptomic analysis of cumulus cells indicated that FID-induced differentially expressed transcripts were implicated in cumulus expansion, steroidogenesis, cell metabolism, and oocyte competence. Functional genomic analysis revealed that protein kinase-A (PKA), extracellular signal regulated kinases (ERK1/2), and calcium (Ca(2+)) pathways as key regulators of FID signaling. Inhibition of PKA (H89) in FID-supplemented COCs or substitution of FID with calcium ionophore (A23187) demonstrated that FID activated primarily the PKA pathway which inhibited ERK1/2 phosphorylation and was upstream of calcium signaling. Furthermore, inhibition of ERK1/2 phosphorylation by FID supported a regulation by dual specific phosphatase (DUSP1) via PKA. Our findings imply that cAMP (FID) regulates cell metabolism, steroidogenesis, intracellular signaling and cumulus expansion through PKA which modulates these functions through optimization of ERK1/2 phosphorylation and coordination of calcium signaling. These findings have implications for development of new strategies for improving oocyte in vitro maturation leading to better developmental competence.

  15. Transcriptomic analysis of cyclic AMP response in bovine cumulus cells

    PubMed Central

    Khan, D. R.; Guillemette, C.; Sirard, M. A.

    2015-01-01

    Acquisition of oocyte developmental competence needs to be understood to improve clinical outcomes of assisted reproduction. The stimulation of cumulus cell concentration of cyclic adenosine 3′5′-monophosphate (cAMP) by pharmacological agents during in vitro maturation (IVM) participates in improvement of oocyte quality. However, precise coordination and downstream targets of cAMP signaling in cumulus cells are largely unknown. We have previously demonstrated better embryo development after cAMP stimulation for first 6 h during IVM. Using this model, we investigated cAMP signaling in cumulus cells through in vitro culture of cumulus-oocyte complexes (COCs) in the presence of cAMP raising agents: forskolin, IBMX, and dipyridamole (here called FID treatment). Transcriptomic analysis of cumulus cells indicated that FID-induced differentially expressed transcripts were implicated in cumulus expansion, steroidogenesis, cell metabolism, and oocyte competence. Functional genomic analysis revealed that protein kinase-A (PKA), extracellular signal regulated kinases (ERK1/2), and calcium (Ca2+) pathways as key regulators of FID signaling. Inhibition of PKA (H89) in FID-supplemented COCs or substitution of FID with calcium ionophore (A23187) demonstrated that FID activated primarily the PKA pathway which inhibited ERK1/2 phosphorylation and was upstream of calcium signaling. Furthermore, inhibition of ERK1/2 phosphorylation by FID supported a regulation by dual specific phosphatase (DUSP1) via PKA. Our findings imply that cAMP (FID) regulates cell metabolism, steroidogenesis, intracellular signaling and cumulus expansion through PKA which modulates these functions through optimization of ERK1/2 phosphorylation and coordination of calcium signaling. These findings have implications for development of new strategies for improving oocyte in vitro maturation leading to better developmental competence. PMID:26082143

  16. Early treatment of chlorine-induced airway hyperresponsiveness and inflammation with corticosteroids

    SciTech Connect

    Jonasson, Sofia; Wigenstam, Elisabeth; Koch, Bo; Bucht, Anders

    2013-09-01

    Chlorine (Cl{sub 2}) is an industrial gas that is highly toxic and irritating when inhaled causing tissue damage and an acute inflammatory response in the airways followed by a long-term airway dysfunction. The aim of this study was to evaluate whether early anti-inflammatory treatment can protect against the delayed symptoms in Cl{sub 2}-exposed mice. BALB/c mice were exposed by nose-only inhalation using 200 ppm Cl{sub 2} during 15 min. Assessment of airway hyperresponsiveness (AHR), inflammatory cell counts in bronchoalveolar lavage, occurrence of lung edema and lung fibrosis were analyzed 24 h or 14 days post-exposure. A single dose of the corticosteroid dexamethasone (10 or 100 mg/kg) was administered intraperitoneally 1, 3, 6, or 12 h following Cl{sub 2} exposure. High-dose of dexamethasone reduced the acute inflammation if administered within 6 h after exposure but treated animals still displayed a significant lung injury. The effect of dexamethasone administered within 1 h was dose-dependent; high-dose significantly reduced acute airway inflammation (100 mg/kg) but not treatment with the relatively low-dose (10 mg/kg). Both doses reduced AHR 14 days later, while lung fibrosis measured as collagen deposition was not significantly reduced. The results point out that the acute inflammation in the lungs due to Cl{sub 2} exposure only partly is associated with the long-term AHR. We hypothesize that additional pathogenic mechanisms apart from the inflammatory reactions contribute to the development of long-term airway dysfunction. By using this mouse model, we have validated early administration of corticosteroids in terms of efficacy to prevent acute lung injury and delayed symptoms induced by Cl{sub 2} exposure. - Highlights: • Inhalation of Cl{sub 2} may lead to a long-standing airway hyperresponsiveness. • The symptoms in Cl{sub 2}-exposed mice are similar to those described for RADS in humans. • Corticosteroids prevent delayed symptoms such as AHR in

  17. Early transcriptomic events in microdissected Arabidopsis nematode-induced giant cells.

    PubMed

    Barcala, Marta; García, Alejandra; Cabrera, Javier; Casson, Stuart; Lindsey, Keith; Favery, Bruno; García-Casado, Gloria; Solano, Roberto; Fenoll, Carmen; Escobar, Carolina

    2010-02-01

    Root-knot nematodes differentiate highly specialized feeding cells in roots (giant cells, GCs), through poorly characterized mechanisms that include extensive transcriptional changes. While global transcriptome analyses have used galls, which are complex root structures that include GCs and surrounding tissues, no global gene expression changes specific to GCs have been described. We report on the differential transcriptome of GCs versus root vascular cells, induced in Arabidopsis by Meloidogyne javanica at a very early stage of their development, 3 days after infection (d.p.i.). Laser microdissection was used to capture GCs and root vascular cells for microarray analysis, which was validated through qPCR and by a promoter-GUS fusion study. Results show that by 3 d.p.i., GCs exhibit major gene repression. Although some genes showed similar regulation in both galls and GCs, the majority had different expression patterns, confirming the molecular distinctiveness of the GCs within the gall. Most of the differentially regulated genes in GCs have no previously assigned function. Comparisons with other transcriptome analyses revealed similarities between GCs and cell suspensions differentiating into xylem cells. This suggests a molecular link between GCs and developing vascular cells, which represent putative GC stem cells. Gene expression in GCs at 3 d.p.i. was also found to be similar to crown galls induced by Agrobacterium tumefaciens, a specialized root biotroph. PMID:20003167

  18. Early ultrastructural changes of apoptosis induced by fumonisin B1 in rat liver.

    PubMed

    Moon, W S; Kim, J H; Kang, M J; Lee, D G

    2000-04-01

    A time sequential study was performed to investigate the histological and ultrastructural findings of fumonisin B1-induced apoptosis in the male Sprague-Dawley rat liver. Six hours after administration of FB1, marked morphologic changes of hepatocytes included the appearance of small vacuoles along the margin of cell membrane. Twelve hours after injection of FB1, acidophilic degeneration of cells occurred, but no fragmented nucleus was evident around the centrilobular area, with few apoptotic cells. By electron microscope, the degenerated acidophilic cells revealed following changes: characteristic formation of cytoplasmic vacuoles, condensed cytoplasm, detachment from neighboring cells, and as well as margination of nuclear chromatin and swollen mitochondria with amorphous matrical deposit. The number of apoptotic cells or bodies was further enhanced at 24 hours in the vicinity of dense acidophilic cells, resulting in a marked increase over the values of control rats. Serum analysis revealed the elevation of cholesterol levels from the beginning to the end of this experiment. Morphologic data and serum findings in this study support the theory that FB1-induced alteration of membrane lipid constituents of the hepatocytes are likely to be early key events in explaining the FB1 apoptotic effect.

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

    SciTech Connect

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

    2005-06-01

    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.

  20. Early biomarkers of doxorubicin-induced heart injury in a mouse model

    SciTech Connect

    Desai, Varsha G.; Kwekel, Joshua C.; Vijay, Vikrant; Moland, Carrie L.; Herman, Eugene H.; Lee, Taewon; Han, Tao; Lewis, Sherry M.; Davis, Kelly J.; Muskhelishvili, Levan; Kerr, Susan; Fuscoe, James C.

    2014-12-01

    Cardiac troponins, which are used as myocardial injury markers, are released in plasma only after tissue damage has occurred. Therefore, there is a need for identification of biomarkers of earlier events in cardiac injury to limit the extent of damage. To accomplish this, expression profiling of 1179 unique microRNAs (miRNAs) was performed in a chronic cardiotoxicity mouse model developed in our laboratory. Male B6C3F{sub 1} mice were injected intravenously with 3 mg/kg doxorubicin (DOX; an anti-cancer drug), or saline once a week for 2, 3, 4, 6, and 8 weeks, resulting in cumulative DOX doses of 6, 9, 12, 18, and 24 mg/kg, respectively. Mice were euthanized a week after the last dose. Cardiac injury was evidenced in mice exposed to 18 mg/kg and higher cumulative DOX dose whereas examination of hearts by light microscopy revealed cardiac lesions at 24 mg/kg DOX. Also, 24 miRNAs were differentially expressed in mouse hearts, with the expression of 1, 1, 2, 8, and 21 miRNAs altered at 6, 9, 12, 18, and 24 mg/kg DOX, respectively. A pro-apoptotic miR-34a was the only miRNA that was up-regulated at all cumulative DOX doses and showed a significant dose-related response. Up-regulation of miR-34a at 6 mg/kg DOX may suggest apoptosis as an early molecular change in the hearts of DOX-treated mice. At 12 mg/kg DOX, up-regulation of miR-34a was associated with down-regulation of hypertrophy-related miR-150; changes observed before cardiac injury. These findings may lead to the development of biomarkers of earlier events in DOX-induced cardiotoxicity that occur before the release of cardiac troponins. - Highlights: • Upregulation of miR-34a before doxorubicin-induced cardiac tissue injury • Apoptosis might be an early event in mouse heart during doxorubicin treatment. • Expression of miR-150 declined before doxorubicin-induced cardiac tissue injury.

  1. Subtoxic Alterations in Hepatocyte-Derived Exosomes: An Early Step in Drug-Induced Liver Injury?

    PubMed

    Holman, Natalie S; Mosedale, Merrie; Wolf, Kristina K; LeCluyse, Edward L; Watkins, Paul B

    2016-06-01

    Drug-induced liver injury (DILI) is a significant clinical and economic problem in the United States, yet the mechanisms that underlie DILI remain poorly understood. Recent evidence suggests that signaling molecules released by stressed hepatocytes can trigger immune responses that may be common across DILI mechanisms. Extracellular vesicles released by hepatocytes, principally hepatocyte-derived exosomes (HDEs), may constitute one such signal. To examine HDE alterations as a function of drug-induced stress, this work utilized prototypical hepatotoxicant acetaminophen (APAP) in male Sprague-Dawley (SD) rats, SD rat hepatocytes, and primary human hepatocytes. HDE were isolated using ExoQuick precipitation reagent and analyzed by quantification of the liver-specific RNAs albumin and microRNA-122 (miR-122). In vivo, significant elevations in circulating exosomal albumin mRNA were observed at subtoxic APAP exposures. Significant increases in exosomal albumin mRNA were also observed in primary rat hepatocytes at subtoxic APAP concentrations. In primary human hepatocytes, APAP elicited increases in both exosomal albumin mRNA and exosomal miR-122 without overt cytotoxicity. However, the number of HDE produced in vitro in response to APAP did not increase with exosomal RNA quantity. We conclude that significant drug-induced alterations in the liver-specific RNA content of HDE occur at subtoxic APAP exposures in vivo and in vitro, and that these changes appear to reflect selective packaging rather than changes in exosome number. The current findings demonstrate that translationally relevant HDE alterations occur in the absence of overt hepatocellular toxicity, and support the hypothesis that HDE released by stressed hepatocytes may mediate early immune responses in DILI. PMID:26962055

  2. Arthritis Induces Early Bone High Turnover, Structural Degradation and Mechanical Weakness

    PubMed Central

    Vidal, Bruno; Cascão, Rita; Vale, Ana Catarina; Cavaleiro, Inês; Vaz, Maria Fátima; Brito, José Américo Almeida; Canhão, Helena; Fonseca, João Eurico

    2015-01-01

    Background We have previously found in the chronic SKG mouse model of arthritis that long standing (5 and 8 months) inflammation directly leads to high collagen bone turnover, disorganization of the collagen network, disturbed bone microstructure and degradation of bone biomechanical properties. The main goal of the present work was to study the effects of the first days of the inflammatory process on the microarchitecture and mechanical properties of bone. Methods Twenty eight Wistar adjuvant-induced arthritis (AIA) rats were monitored during 22 days after disease induction for the inflammatory score, ankle perimeter and body weight. Healthy non-arthritic rats were used as controls for compar-ison. After 22 days of disease progression rats were sacrificed and bone samples were collected for histomorphometrical, energy dispersive X-ray spectroscopical analysis and 3-point bending. Blood samples were also collected for bone turnover markers. Results AIA rats had an increased bone turnover (as inferred from increased P1NP and CTX1, p = 0.0010 and p = 0.0002, respectively) and this was paralleled by a decreased mineral content (calcium p = 0.0046 and phos-phorus p = 0.0046). Histomorphometry showed a lower trabecular thickness (p = 0.0002) and bone volume (p = 0.0003) and higher trabecular sepa-ration (p = 0.0009) in the arthritic group as compared with controls. In addition, bone mechanical tests showed evidence of fragility as depicted by diminished values of yield stress and ultimate fracture point (p = 0.0061 and p = 0.0279, re-spectively) in the arthritic group. Conclusions We have shown in an AIA rat model that arthritis induc-es early bone high turnover, structural degradation, mineral loss and mechanical weak-ness. PMID:25617902

  3. Endothelial dysfunction and inflammation induced by iron oxide nanoparticle exposure: Risk factors for early atherosclerosis.

    PubMed

    Zhu, Mo-Tao; Wang, Bing; Wang, Yun; Yuan, Lan; Wang, Hua-Jian; Wang, Meng; Ouyang, Hong; Chai, Zhi-Fang; Feng, Wei-Yue; Zhao, Yu-Liang

    2011-06-10

    More recently, the correlation between exposure to nanoparticles and cardiovascular diseases is of particular concern in nanotoxicology related fields. Nanoparticle-triggered endothelial dysfunction is hypothesized to be a dominant mechanism in the development of the diseases. To test this hypothesis, iron oxide nanoparticles (Fe₂O₃ and Fe₃O₄), as two widely used nanomaterials and the main metallic components in particulate matter, were selected to assess their potential risks on human endothelial system. The direct effects of iron oxide nanoparticles on human aortic endothelial cells (HAECs) and the possible effects mediated by monocyte (U937 cells) phagocytosis and activation were investigated. In the study, HAECs and U937 cells were exposed to 2, 20, 100 μg/mL of 22-nm-Fe₂O₃ and 43-nm-Fe₃O₄ particles. Our results indicate that cytoplasmic vacuolation, mitochondrial swelling and cell death were induced in HAEC. A significant increase in nitric oxide (NO) production was induced which coincided with the elevation of nitric oxide synthase (NOS) activity in HAECs. Adhesion of monocytes to the HAECs was significantly enhanced as a consequence of the up-regulation of intracellular cell adhesion molecule-1 (ICAM-1) and interleukin-8 (IL-8) expression, all of which are considered as early steps of atheroscelerosis. Phagocytosis and dissolution of nanoparticles by monocytes were found to simultaneously provoke oxidative stress and mediate severe endothelial toxicity. We conclude that intravascular iron oxide nanoparticles may induce endothelial system inflammation and dysfunction by three ways: (1) nanoparticles may escape from phagocytosis that interact directly with the endothelial monolayer; (2) nanoparticles are phagocytized by monocytes and then dissolved, thus impact the endothelial cells as free iron ions; or (3) nanoparticles are phagocytized by monocytes to provoke oxidative stress responses.

  4. Atmospheric, Magnetospheric, and Plasmas in Space (AMPS) spacelab payload definition study, technical summary document

    NASA Technical Reports Server (NTRS)

    Keeley, J. T.

    1976-01-01

    Some 60 instrument candidates and 80 possible science investigations were evaluated. The early analysis emphasized the science aspect in terms of the functional requirements for each of the potential experiments identified by the AMPS science working group. These requirements were then used for the grouping of instruments into practical payloads which would fit the capabilities of the Shuttle/Spacelab. This analysis resulted in the definition of eleven different AMPS configurations. The data were then used to define a typical set of requirements for a flexible AMPS laboratory. The data gathered to this point showed that a planned sequential buildup of the laboratory would be necessary to meet both physical and funding limitations. This led to the definition of five strawman payloads by the science working group, which were used to establish a conceptual laboratory and to define preliminary design of a configuration which could satisfy AMPS needs during the early program period.

  5. Complex Regulation Pathways of AmpC-Mediated β-Lactam Resistance in Enterobacter cloacae Complex.

    PubMed

    Guérin, François; Isnard, Christophe; Cattoir, Vincent; Giard, Jean Christophe

    2015-12-01

    Enterobacter cloacae complex (ECC), an opportunistic pathogen causing numerous infections in hospitalized patients worldwide, is able to resist β-lactams mainly by producing the AmpC β-lactamase enzyme. AmpC expression is highly inducible in the presence of some β-lactams, but the underlying genetic regulation, which is intricately linked to peptidoglycan recycling, is still poorly understood. In this study, we constructed different mutant strains that were affected in genes encoding enzymes suspected to be involved in this pathway. As expected, the inactivation of ampC, ampR (which encodes the regulator protein of ampC), and ampG (encoding a permease) abolished β-lactam resistance. Reverse transcription-quantitative PCR (qRT-PCR) experiments combined with phenotypic studies showed that cefotaxime (at high concentrations) and cefoxitin induced the expression of ampC in different ways: one involving NagZ (a N-acetyl-β-D-glucosaminidase) and another independent of NagZ. Unlike the model established for Pseudomonas aeruginosa, inactivation of DacB (also known as PBP4) was not responsible for a constitutive ampC overexpression in ECC, whereas it caused AmpC-mediated high-level β-lactam resistance, suggesting a post-transcriptional regulation mechanism. Global transcriptomic analysis by transcriptome sequencing (RNA-seq) of a dacB deletion mutant confirmed these results. Lastly, analysis of 37 ECC clinical isolates showed that amino acid changes in the AmpD sequence were likely the most crucial event involved in the development of high-level β-lactam resistance in vivo as opposed to P. aeruginosa where dacB mutations have been commonly found. These findings bring new elements for a better understanding of β-lactam resistance in ECC, which is essential for the identification of novel potential drug targets. PMID:26438498

  6. Complex Regulation Pathways of AmpC-Mediated β-Lactam Resistance in Enterobacter cloacae Complex

    PubMed Central

    Guérin, François; Isnard, Christophe; Giard, Jean Christophe

    2015-01-01

    Enterobacter cloacae complex (ECC), an opportunistic pathogen causing numerous infections in hospitalized patients worldwide, is able to resist β-lactams mainly by producing the AmpC β-lactamase enzyme. AmpC expression is highly inducible in the presence of some β-lactams, but the underlying genetic regulation, which is intricately linked to peptidoglycan recycling, is still poorly understood. In this study, we constructed different mutant strains that were affected in genes encoding enzymes suspected to be involved in this pathway. As expected, the inactivation of ampC, ampR (which encodes the regulator protein of ampC), and ampG (encoding a permease) abolished β-lactam resistance. Reverse transcription-quantitative PCR (qRT-PCR) experiments combined with phenotypic studies showed that cefotaxime (at high concentrations) and cefoxitin induced the expression of ampC in different ways: one involving NagZ (a N-acetyl-β-d-glucosaminidase) and another independent of NagZ. Unlike the model established for Pseudomonas aeruginosa, inactivation of DacB (also known as PBP4) was not responsible for a constitutive ampC overexpression in ECC, whereas it caused AmpC-mediated high-level β-lactam resistance, suggesting a post-transcriptional regulation mechanism. Global transcriptomic analysis by transcriptome sequencing (RNA-seq) of a dacB deletion mutant confirmed these results. Lastly, analysis of 37 ECC clinical isolates showed that amino acid changes in the AmpD sequence were likely the most crucial event involved in the development of high-level β-lactam resistance in vivo as opposed to P. aeruginosa where dacB mutations have been commonly found. These findings bring new elements for a better understanding of β-lactam resistance in ECC, which is essential for the identification of novel potential drug targets. PMID:26438498

  7. AMP-18 protects barrier function of colonic epithelial cells: role of tight junction proteins

    PubMed Central

    Walsh-Reitz, Margaret M.; Huang, Erick F.; Musch, Mark W.; Chang, Eugene B.; Martin, Terence E.; Kartha, Sreedharan; Toback, F. Gary

    2005-01-01

    AMP-18, a novel gastric antrum mucosal protein, and a synthetic peptide of amino acids 77-97, have mitogenic and motogenic properties for epithelial cells. The possibility that AMP-18 is also protective was evaluated in the colonic mucosa of mice and monolayer cultures of human colonic epithelial Caco2/bbe (C2) cells. Administration of AMP peptide to mice with dextran sulfate sodium (DSS)-induced colonic injury delayed the onset of bloody diarrhea, and reduced weight loss. Treatment of C2 cells with AMP peptide protected monolayers against decreases in transepithelial electrical resistance (TER) induced by the oxidant monochloramine, indomethacin, or DSS. A molecular mechanism for these barrier-protective effects was sought by asking if AMP peptide acted on specific tight junction (TJ) proteins. Immunoblots of detergent-insoluble fractions of C2 cells treated with AMP peptide exhibited increased accumulation of specific TJ proteins. Occludin immunoreactivity was also increased in detergent-insoluble fractions obtained from colonic mucosal cells of mice injected with AMP peptide. Laser scanning confocal microscopy (CF) supported the capacity of AMP peptide to enhance accumulation of occludin and ZO-1 in TJ domains of C2 cell monolayers, and together with immunoblot analysis showed that the peptide protected against loss of these TJ proteins following oxidant injury. AMP peptide also protected against a fall in TER during disruption of actin filaments by cytochalasin D, and stabilized perijunctional actin during oxidant injury when assessed by CF. These findings suggest that AMP-18 could protect the intestinal mucosal barrier by acting on specific TJ proteins and stabilizing perijunctional actin. PMID:15961882

  8. Effect of cholera toxin on cAMP levels and Na/sup +/ influx in isolated intestinal epithelial cells

    SciTech Connect

    Hyun, C.S.; Kimmich, G.A.

    1982-09-01

    Freshly isolated chicken intestinal cells contain approximately 20 pmol adenosine 3',5'-cyclic monophosphate (cAMP)/mg cellular protein. Incubation with 3 ..mu..g/ml cholera toxin (CT) at 37/sup 0/C induces an elevation of cellular cAMP beginning 10-15 min after initial exposure. The response is linear with time for 40-50 min and causes a six- to eightfold increase over control levels at steady state. Dibutyryl cAMP and agents that increase cAMP production inhibit Na/sup +/ influx into the isolated enterocytes. Chlorpromazine completely abolishes the toxin-induced elevation of cAMP in the isolated cells and also reverses the effect on Na/sup +/ entry. The data provide evidence for a cAMP-mediated control of intestinal cell Na/sup +/ uptake, which may represent the mechanistic basis for the antiabsorptive effect of CT on Na/sup +/ during induction of intestinal secretory activity. Studies on the time-dependent effects of chlorpromazine on both intracellular cAMP concentration and Na/sup +/ influx suggest that the reactivation of the Na/sup +/ transport system after cAMP-induced inhibition is slow relative to the disappearance of cAMP.

  9. [beta]1-Adrenoceptor or [alpha]1-Adrenoceptor Activation Initiates Early Odor Preference Learning in Rat Pups: Support for the Mitral Cell/cAMP Model of Odor Preference Learning

    ERIC Educational Resources Information Center

    Harley, Carolyn W.; Darby-King, Andrea; McCann, Jennifer; McLean, John H.

    2006-01-01

    We proposed that mitral cell [beta]1-adrenoceptor activation mediates rat pup odor preference learning. Here we evaluate [beta]1-, [beta]2-, [alpha]1-, and [alpha]2-adrenoceptor agonists in such learning. The [beta]1-adrenoceptor agonist, dobutamine, and the [alpha]1-adrenoceptor agonist, phenylephrine, induced learning, and both exhibited an…

  10. A Cell-Autonomous Molecular Cascade Initiated by AMP-Activated Protein Kinase Represses Steroidogenesis

    PubMed Central

    Abdou, Houssein S.; Bergeron, Francis

    2014-01-01

    Steroid hormones regulate essential physiological processes, and inadequate levels are associated with various pathological conditions. In testosterone-producing Leydig cells, steroidogenesis is strongly stimulated by luteinizing hormone (LH) via its receptor leading to increased cyclic AMP (cAMP) production and expression of the steroidogenic acute regulatory (STAR) protein, which is essential for the initiation of steroidogenesis. Steroidogenesis then passively decreases with the degradation of cAMP into AMP by phosphodiesterases. In this study, we show that AMP-activated protein kinase (AMPK) is activated following cAMP-to-AMP breakdown in MA-10 and MLTC-1 Leydig cells. Activated AMPK then actively inhibits cAMP-induced steroidogenesis by repressing the expression of key regulators of steroidogenesis, including Star and Nr4a1. Similar results were obtained in Y-1 adrenal cells and in the constitutively steroidogenic R2C cells. We have also determined that maximum AMPK activation following stimulation of steroidogenesis in MA-10 Leydig cells occurs when steroid hormone production has reached a plateau. Our data identify AMPK as a molecular rheostat that actively represses steroid hormone biosynthesis to preserve cellular energy homeostasis and prevent excess steroid production. PMID:25225331

  11. AmpG is required for BlaXc beta-lactamase expression in Xanthomonas campestris pv. campestris str. 17.

    PubMed

    Yang, Tsuey-Ching; Chen, Tzu-Fan; Tsai, Jeffrey J P; Hu, Rouh-Mei

    2013-03-01

    The chromosomal ampR(Xc) -bla(Xc) module is essential for the β-lactam resistance of Xanthomonas campestris pv. campestris. Bla(Xc) β-lactamase is expressed at a high basal level in the absence of an inducer and its expression can be further induced by β-lactam. In enterobacteria, ampG encodes an inner membrane facilitator involved in the recycling of murein degradation compounds. An isogenic ampG mutant (XcampG) of X. campestris pv. campestris str. 17 (Xc17) was constructed to investigate the link between murein recycling and bla(Xc) expression. Our data demonstrate that (1) XcampG is susceptible to β-lactam antibiotics; (2) AmpG(Xc) is essential for expression of bla(Xc) ; (3) AmpGs of Xc17, Stenotrophomonas maltophilia KJ (SmKJ) and Escherichia coli DH5α can complement the defect of XcampG; (4) overexpression of AmpG(X) (c) significantly increased bla(Xc) expression; and (5) AmpG(Xc) from Xc17 is able to restore β-lactamase induction of the ampN(Xc) -ampG(Xc) double mutant of SmKJ. In Xc17, ampG(Xc) can be expressed from the promoter residing in the intergenic region of ampN(Xc) -ampG(Xc) and the expression is independent of β-lactam induction. AmpN, which is required for β-lactamases induction in SmKJ, is not required for the β-lactam antibiotic resistance of Xc17.

  12. Paternal Diet-Induced Obesity Retards Early Mouse Embryo Development, Mitochondrial Activity and Pregnancy Health

    PubMed Central

    Binder, Natalie K.; Hannan, Natalie J.; Gardner, David K.

    2012-01-01

    Worldwide, 48% of adult males are overweight or obese. An association between infertility and excessive body weight is now accepted, although focus remains primarily on females. It has been shown that parental obesity results in compromised embryo development, disproportionate changes in embryo metabolism and reduced blastocyst cell number. The aim of this study was to determine whether paternal obesity has negative effects on the resultant embryo. Specifically, using in vitro fertilisation (IVF), we wanted to isolate the functional effects of obesity on sperm by examining the subsequent embryo both pre- and post-implantation. Epididymal sperm was collected from age matched normal and obese C57BL/6 mice and cryopreserved for subsequent IVF with oocytes collected from Swiss females (normal diet/weight). Obesity was induced in male mice by feeding a high fat diet of 22% fat for 10 weeks. Resultant embryos were cultured individually and development monitored using time-lapse microscopy. Paternal obesity resulted in a significant delay in preimplantation embryo development as early as syngamy (P<0.05). Metabolic parameters were measured across key developmental stages, demonstrating significant reduction in mitochondrial membrane potential (P<0.01). Blastocysts were stained to determine trophectoderm (TE) and inner cell mass (ICM) cell numbers, revealing significant differences in the ratio of cell allocation to TE and ICM lineages (P<0.01). Functional studies examining blastocyst attachment, growth and implantation demonstrated that blastocysts derived from sperm of obese males displayed significantly reduced outgrowth on fibronectin in vitro (P<0.05) and retarded fetal development in vivo following embryo transfer (P<0.05). Taken together, these data clearly demonstrate that paternal obesity has significant negative effects on the embryo at a variety of key early developmental stages, resulting in delayed development, reduced placental size and smaller offspring

  13. An improved method of early diagnosis of smoking-induced respiratory changes using machine learning algorithms.

    PubMed

    Amaral, Jorge L M; Lopes, Agnaldo J; Jansen, José M; Faria, Alvaro C D; Melo, Pedro L

    2013-12-01

    The purpose of this study was to develop an automatic classifier to increase the accuracy of the forced oscillation technique (FOT) for diagnosing early respiratory abnormalities in smoking patients. The data consisted of FOT parameters obtained from 56 volunteers, 28 healthy and 28 smokers with low tobacco consumption. Many supervised learning techniques were investigated, including logistic linear classifiers, k nearest neighbor (KNN), neural networks and support vector machines (SVM). To evaluate performance, the ROC curve of the most accurate parameter was established as baseline. To determine the best input features and classifier parameters, we used genetic algorithms and a 10-fold cross-validation using the average area under the ROC curve (AUC). In the first experiment, the original FOT parameters were used as input. We observed a significant improvement in accuracy (KNN=0.89 and SVM=0.87) compared with the baseline (0.77). The second experiment performed a feature selection on the original FOT parameters. This selection did not cause any significant improvement in accuracy, but it was useful in identifying more adequate FOT parameters. In the third experiment, we performed a feature selection on the cross products of the FOT parameters. This selection resulted in a further increase in AUC (KNN=SVM=0.91), which allows for high diagnostic accuracy. In conclusion, machine learning classifiers can help identify early smoking-induced respiratory alterations. The use of FOT cross products and the search for the best features and classifier parameters can markedly improve the performance of machine learning classifiers. PMID:24001924

  14. Early impairment of skeletal muscle endothelial glycocalyx barrier properties in diet‐induced obesity in mice

    PubMed Central

    Eskens, Bart J. M.; Leurgans, Thomas M.; Vink, Hans; VanTeeffelen, Jurgen W. G. E.

    2014-01-01

    Abstract While previous studies have indicated an important role for the endothelial glycocalyx in regulation of microvascular function, it was recently shown that acute enzymatic glycocalyx degradation in rats was associated with an impaired insulin‐mediated glucose disposal. The aim of this study was to determine whether glycocalyx damage in skeletal muscle occurs at an early stage of diet‐induced obesity (DIO). The microcirculation of the hindlimb muscle of anesthetized C57Bl/6 mice, fed chow (CON) or a high‐fat diet (HFD) for 6 and 18 weeks (w), respectively, was visualized with a Sidestream Dark‐Field camera, and glycocalyx barrier properties were derived from the calculated perfused boundary region (PBR). Subsequently, an intraperitoneal glucose tolerance test was performed and the area under the curve (AUC) of blood glucose was calculated. Impairment of glycocalyx barrier properties was already apparent after 6 weeks of HFD and remained after 18 weeks of HFD (PBR [in μm]: 0.81 ± 0.03 in CON_6w vs. 0.97 ± 0.04 in HFD_6w and 1.02 ± 0.07 in HFD_18w [both P < 0.05]). Glucose intolerance appeared to develop more slowly (AUC [in mmol/L × 120 min]: 989 ± 61 in CON_6w vs. 1204 ± 89 in HFD_6w [P = 0.11] and 1468 ± 84 in HFD_18w [P < 0.05]) than the impairment of glycocalyx barrier properties. The data indicate that damage to the endothelial glycocalyx is an early event in DIO. It is suggested that glycocalyx damage may contribute to the development of insulin resistance in obesity. PMID:24744873

  15. Environmental tobacco smoke in the early postnatal period induces impairment in brain myelination.

    PubMed

    Torres, Larissa H; Annoni, Raquel; Balestrin, Natalia T; Coleto, Priscila L; Duro, Stephanie O; Garcia, Raphael C T; Pacheco-Neto, Maurílio; Mauad, Thais; Camarini, Rosana; Britto, Luiz R G; Marcourakis, Tania

    2015-11-01

    Environmental tobacco smoke (ETS) is associated with high morbidity and mortality, mainly in children. However, few studies focus on the brain development effects of ETS exposure. Myelination mainly occurs in the early years of life in humans and the first three postnatal weeks in rodents and is sensitive to xenobiotics exposure. This study investigated the effects of early postnatal ETS exposure on myelination. BALB/c mice were exposed to ETS generated from 3R4F reference research cigarettes from the third to the fourteenth days of life. The myelination of nerve fibers in the optic nerve by morphometric analysis and the levels of Olig1 and myelin basic protein (MBP) were evaluated in the cerebellum, diencephalon, telencephalon, and brainstem in infancy, adolescence, and adulthood. Infant mice exposed to ETS showed a decrease in the percentage of myelinated fibers in the optic nerve, compared with controls. ETS induced a decrease in Olig1 protein levels in the cerebellum and brainstem and an increase in MBP levels in the cerebellum at infant. It was also found a decrease in MBP levels in the telencephalon and brainstem at adolescence and in the cerebellum and diencephalon at adulthood. The present study demonstrates that exposure to ETS, in a critical phase of development, affects the percentage of myelinated fibers and myelin-specific proteins in infant mice. Although we did not observe differences in the morphological analysis in adolescence and adulthood, there was a decrease in MBP levels in distinctive brain regions suggesting a delayed effect in adolescence and adulthood.

  16. Early Intervention for Spinal Cord Injury with Human Induced Pluripotent Stem Cells Oligodendrocyte Progenitors

    PubMed Central

    All, Angelo H.; Gharibani, Payam; Gupta, Siddharth; Bazley, Faith A.; Pashai, Nikta; Chou, Bin-Kuan; Shah, Sandeep; Resar, Linda M.; Cheng, Linzhao; Gearhart, John D.; Kerr, Candace L.

    2015-01-01

    Induced pluripotent stem (iPS) cells are at the forefront of research in regenerative medicine and are envisaged as a source for personalized tissue repair and cell replacement therapy. Here, we demonstrate for the first time that oligodendrocyte progenitors (OPs) can be derived from iPS cells generated using either an episomal, non-integrating plasmid approach or standard integrating retroviruses that survive and differentiate into mature oligodendrocytes after early transplantation into the injured spinal cord. The efficiency of OP differentiation in all 3 lines tested ranged from 40% to 60% of total cells, comparable to those derived from human embryonic stem cells. iPS cell lines derived using episomal vectors or retroviruses generated a similar number of early neural progenitors and glial progenitors while the episomal plasmid-derived iPS line generated more OPs expressing late markers O1 and RIP. Moreover, we discovered that iPS-derived OPs (iPS-OPs) engrafted 24 hours following a moderate contusive spinal cord injury (SCI) in rats survived for approximately two months and that more than 70% of the transplanted cells differentiated into mature oligodendrocytes that expressed myelin associated proteins. Transplanted OPs resulted in a significant increase in the number of myelinated axons in animals that received a transplantation 24 h after injury. In addition, nearly a 5-fold reduction in cavity size and reduced glial scarring was seen in iPS-treated groups compared to the control group, which was injected with heat-killed iPS-OPs. Although further investigation is needed to understand the mechanisms involved, these results provide evidence that patient-specific, iPS-derived OPs can survive for three months and improve behavioral assessment (BBB) after acute transplantation into SCI. This is significant as determining the time in which stem cells are injected after SCI may influence their survival and differentiation capacity. PMID:25635918

  17. Endogenous hydrogen sulfide mediates the cardioprotection induced by ischemic postconditioning in the early reperfusion phase

    PubMed Central

    HUANG, YI-E; TANG, ZHI-HAN; XIE, WEI; SHEN, XIN-TIAN; LIU, MI-HUA; PENG, XIANG-PING; ZHAO, ZHAN-ZHI; NIE, DE-BO; LIU, LU-SHAN; JIANG, ZHI-SHENG

    2012-01-01

    Hydrogen sulfide (H2S), produced by cystanthionine-γ-lysase (CSE) in the cardiovascular system, has been suggested to be the third gasotransmitter in addition to nitric oxide (NO) and carbon monoxide (CO). The present study aimed to investigate the role of H2S in ischemic postconditioning (IPO) during the early period of reperfusion. IPO with 6 episodes of 10 sec reperfusion followed by 6 episodes of 10 sec ischemia (IPO 2’) was administered when reperfusion was initiated. Cardiodynamics and the concentration of H2S were measured at 1, 2, 3, 4, 5, 10, 20, 30, 60, 90 and 120 min of reperfusion. Lactate dehydrogenase (LDH) levels and infarct size were determined at the end of the reperfusion. The concentration of H2S was stable during the whole experiment in the control group, whereas it reached a peak at the first minute of reperfusion in the ischemia-reperfusion (IR) group. The concentration of H2S at the first minute of reperfusion in the IPO 2’ group was higher compared to that of the IR group, which correlated with cardioprotection including improved heart contractile function and reduced infarct size and LDH levels. However, the above effects of IPO 2’ were attenuated by pre-treatment with blockade of endogenous H2S production with DL-propargylglycine for 20 min prior to global ischemia. Furthermore, we found that other forms of IPO, IPO commencing at 1 min after reperfusion (delayed IPO) or lasting only for 1 min (IPO 1’), failed to increase the concentration of H2S and protect the myocardium. We conclude that the peak of endogenous H2S in the early reperfusion phase is the key to cardioprotection induced by IPO. PMID:23226785

  18. Early constraint-induced movement therapy promotes functional recovery and neuronal plasticity in a subcortical hemorrhage model rat.

    PubMed

    Ishida, Akimasa; Misumi, Sachiyo; Ueda, Yoshitomo; Shimizu, Yuko; Cha-Gyun, Jung; Tamakoshi, Keigo; Ishida, Kazuto; Hida, Hideki

    2015-05-01

    Constraint-induced movement therapy (CIMT) promotes functional recovery of impaired forelimbs after hemiplegic strokes, including intracerebral hemorrhage (ICH). We used a rat model of subcortical hemorrhage to compare the effects of delivering early or late CIMT after ICH. The rat model was made by injecting collagenase into the globus pallidus near the internal capsule, and then forcing rats to use the affected forelimb for 7 days starting either 1 day (early CIMT) or 17 days (late CIMT) after the lesion. Recovery of forelimb function in the skilled reaching test and the ladder stepping test was found after early-CIMT, while no significant recovery was shown after late CIMT or in the non-CIMT controls. Early CIMT was associated with greater numbers of ΔFosB-positive cells in the ipsi-lesional sensorimotor cortex layers II-III and V. Additionally, we found expression of the growth-related genes brain-derived neurotrophic factor (BDNF) and growth-related protein 43 (GAP-43), and abundant dendritic arborization of pyramidal neurons in the sensorimotor area. Similar results were not detected in the contra-lesional cortex. In contrast to early CIMT, late CIMT failed to induce any changes in plasticity. We conclude that CIMT induces molecular and morphological plasticity in the ipsi-lesional sensorimotor cortex and facilitates better functional recovery when initiated immediately after hemorrhage.

  19. Psychiatric sequelae to term birth and induced early and late abortion: a longitudinal study.

    PubMed

    Athanasiou, R; Oppel, W; Michelson, L; Unger, T; Yager, M

    1973-01-01

    Three obstetric groups matched for parity, age, color, marital and socioeconomic status, were compared for the psychosocial antecedents to abortion or term delivery and sequelae that did exist. One group had induced abortion by suction curettage, the second had induced abortion by saline injection, and the third had term delivery. 373 women were interviewed during pregnancy; 211 were included in follow-up after an average of 16 months for abortion patients and 13 months for the term delivery group. The 373 women received a structured 90-minute interview about family relationships, consort relationships, living arrangements, income, education, religious behavior, sexual behavior, contraceptive knowledge and use, pregnancy history and desired family size. The Srole Anomia scale and the Rosenberg Self-Esteem scale were also used. At the followup interview, similar questions were asked and the two attitude scales were again presented. The MMPI (Minnesota Multiphasic Personality Inventory) and the SCL (Symptom Check List) were also used. 12% of the 211 follow-up scored higher than 70 on the MMPI for mania and psychopathy scales; these women were evenly divided among the three groups. SCL scales showed the three groups of patients to be similar after abortion or birth, with the only statistically significant difference being the suction curettage group, which had fewer complaints after abortion than either of the other two groups. There was no difference on the anomia or self-esteem scale between pre- or post-procedure for any of the groups. The data suggest neither great harm nor great benefit with regard to these variables due to either early or late abortion or term birth. Early abortion by suction curettage was possibly more therapeutic than carrying a pregnancy to term. There were striking improvements with regard to contraceptive use after the procedure. 71% of each group used effective contraception for more than 12 months after abortion or birth: a 20% increase

  20. Effects of glucagon-like peptide-1 on advanced glycation endproduct-induced aortic endothelial dysfunction in streptozotocin-induced diabetic rats: possible roles of Rho kinase- and AMP kinase-mediated nuclear factor κB signaling pathways.

    PubMed

    Tang, Song-Tao; Zhang, Qiu; Tang, Hai-Qin; Wang, Chang-Jiang; Su, Huan; Zhou, Qing; Wei, Wei; Zhu, Hua-Qing; Wang, Yuan

    2016-07-01

    Interaction between advanced glycation endproducts (AGEs) and receptor for AGEs (RAGE) as well as downstream pathways leads to vascular endothelial dysfunction in diabetes. Glucagon-like peptide-1 (GLP-1) has been reported to attenuate endothelial dysfunction in the models of atherosclerosis. However, whether GLP-1 exerts protective effects on aortic endothelium in diabetic animal model and the underlying mechanisms are still not well defined. Experimental diabetes was induced through administration with combination of high-fat diet and intraperitoneal injection of streptozotocin. Rats were randomly divided into four groups, including controls, diabetes, diabetes + sitagliptin (30 mg/kg/day), diabetes + exenatide (3 μg/kg/12 h). Eventually, endothelial damage, markers of inflammation and oxidative stress, were measured. After 12 weeks administration, diabetic rats received sitagliptin and exenatide showed significant elevation of serum NO level and reduction of ET-1 as well as inflammatory cytokines levels. Moreover, sitagliptin and exenatide significantly inhibited aortic oxidative stress level and improved aortic endothelial function in diabetic rats. Importantly, these drugs inhibited the protein expression level in AGE/RAGE-induced RhoA/ROCK/NF-κB/IκBα signaling pathways and activated AMPK in diabetic aorta. Finally, the target proteins of p-eNOS, iNOS, and ET-1, which reflect endothelial function, were also changed by these drugs. Our present study indicates that sitagliptin and exenatide administrations can improve endothelial function in diabetic aorta. Of note, RAGE/RhoA/ROCK and AMPK mediated NF-κB signaling pathways may be the intervention targets of these drugs to protect aortic endothelium. PMID:26758998

  1. Arsenic-induced phosphate limitation under experimental Early Proterozoic oceanic conditions

    NASA Astrophysics Data System (ADS)

    Chi Fru, Ernest; Hemmingsson, Christoffer; Holm, Mikaela; Chiu, Beverly; Iñiguez, Enrique

    2016-01-01

    Comparison of phosphorus concentrations associated with modern hydrothermal Fe(III)(oxyhydr)oxides and ancient Fe(III) oxide-rich iron formations, is used to estimate bioavailable Precambrian marine phosphorus (P) concentrations. This led to the proposition of a low dissolved P budget of ˜10-25% of present-day levels, before ˜1.9 billion years ago. Estimates incorporating ancient marine Si levels ≥ 0.67 mM instead suggested global dissolved P levels greater than today. Here we unite current experimental models that have considered NaCl solutions containing elevated dissolved Fe(II), Si, Ca2+ and Mg2+ ions in the incorporation of P in Precambrian marine Fe(III)(oxyhydr)oxides, in addition to arsenic as a hydrothermal proxy. We show that the coprecipitation of dissolved P and Fe(III)(oxyhydr)oxides from arsenic-rich marine waters produces an average P distribution coefficient of ˜0.072 (± 0.01) μM-1. This is comparable to the ˜ 0.07 μM-1 predicted for Fe(III)(oxyhydr)oxides in modern arsenic-rich, submarine hydrothermal settings, from which the lower Early Proterozoic dissolved marine P concentrations were predicted. As/P molar ratios below modern seawater ratios removed the negative feedback effect high Si impose on P scavenging by Fe(III)(oxyhydr)oxides. The binding of As(III) to Fe(III)(oxyhydr)oxides exhibits a lower competitive influence on P fixation. As(V) that likely became prominent in the surficially oxidized Early Proterozoic oceans induced dissolved P limitation because of preferential P sequestration at the expense of dissolved As(V) enrichment. The control of As on P scavenging by the precipitating Fe(III)(oxyhydr)oxides is strong regardless of common seawater cations (Mg2+ and Ca2+). The data suggest that the application of Si and Fe(III)(oxyhydr)oxides as an ancient seawater P proxy should consider chemical variability between depositional basins, taking into account the rather strong role hydrothermal arsenic has on the distribution of P in

  2. Neutrino-Induced Nucleosynthesis in Helium Shells of Early Core-Collapse Supernovae

    NASA Astrophysics Data System (ADS)

    Banerjee, Projjwal; Qian, Yong-Zhong; Heger, Alexander; Haxton, Wick

    2016-02-01

    We summarize our studies on neutrino-driven nucleosynthesis in He shells of early core-collapse supernovae with metallicities of Z ≲ 10-3 Z⊙. We find that for progenitors of ˜ 11-15 M⊙, the neutrons released by 4He(ν¯ee, e+n)3H in He shells can be captured to produce nuclei with mass numbers up to A ˜ 200. This mechanism is sensitive to neutrino emission spectra and flavor oscillations. In addition, we find two new primary mechanisms for neutrino-induced production of 9Be in He shells. The first mechanism produces 9Be via 7Li(n,γ)8Li(n,γ)9Li(e- ν¯ee)9Be and relies on a low explosion energy for its survival. The second mechanism operates in progenitors of ˜ 8 M⊙, where 9Be can be produced directly via 7Li(3H, n0)9Be during the rapid expansion of the shocked Heshell material. The light nuclei 7Li and 3H involved in these mechanisms are produced by neutrino interactions with 4He. We discuss the implications of neutrino-induced nucleosynthesis in He shells for interpreting the elemental abundances in metal-poor stars.

  3. Early stages of oxidative stress-induced membrane permeabilization: a neutron reflectometry study.

    PubMed

    Smith, Hillary L; Howland, Michael C; Szmodis, Alan W; Li, Qijuan; Daemen, Luke L; Parikh, Atul N; Majewski, Jaroslaw

    2009-03-18

    Neutron reflectometry was used to probe in situ the structure of supported lipid bilayers at the solid-liquid interface during the early stages of UV-induced oxidative degradation. Single-component supported lipid bilayers composed of gel phase, dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and fluid phase, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), phospholipids were exposed to low-dose oxidative stress generated by UV light and their structures were examined by neutron reflectometry. An interrupted illumination mode, involving exposures in 15 min increments with 2 h intervals between subsequent exposures, and a continuous mode involving a single 60 (or 90) min exposure period were employed. In both cases, pronounced differences in the structure of the lipid bilayer after exposure were observed. Interrupted exposure led to a substantial decrease in membrane coverage but preserved its total thickness at reduced scattering length densities. These results indicate that the initial phase during UV-induced membrane degradation involves the formation of hydrophilic channels within the membrane. This is consistent with the loss of some lipid molecules we observe and attendant reorganization of residual lipids forming hemimicellar edges of the hydrophilic channels. In contrast, continuous illumination produced a graded interface of continuously varied scattering length density (and hence hydrocarbon density) extending 100-150 A into the liquid phase. Exposure of a DPPC bilayer to UV light in the presence of a reservoir of unfused vesicles showed low net membrane disintegration during oxidative stress, presumably because of surface back-filling from the bulk reservoir. Chemical evidence for membrane degradation was obtained by mass spectrometry and Fourier transform infrared spectroscopy. Further evidence for the formation of hydrophilic channels was furnished by fluorescence microscopy and imaging ellipsometry data.

  4. Early Stages of Oxidative Stress-Induced Membrane Permeabilization: A Neutron Reflectometry Study

    PubMed Central

    Smith, Hillary L.; Howland, Michael C.; Szmodis, Alan W.; Li, Qijuan; Daemen, Luke L.; Parikh, Atul N.; Majewski, Jaroslaw

    2009-01-01

    Neutron reflectometry was used to probe in situ the structure of supported lipid bilayers at the solid–liquid interface during the early stages of UV-induced oxidative degradation. Single-component supported lipid bilayers composed of gel phase, dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and fluid phase, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), phospholipids were exposed to low-dose oxidative stress generated by UV light and their structures were examined by neutron reflectometry. An interrupted illumination mode, involving exposures in 15 min increments with 2 h intervals between subsequent exposures, and a continuous mode involving a single 60 (or 90) min exposure period were employed. In both cases, pronounced differences in the structure of the lipid bilayer after exposure were observed. Interrupted exposure led to a substantial decrease in membrane coverage but preserved its total thickness at reduced scattering length densities. These results indicate that the initial phase during UV-induced membrane degradation involves the formation of hydrophilic channels within the membrane. This is consistent with the loss of some lipid molecules we observe and attendant reorganization of residual lipids forming hemimicellar edges of the hydrophilic channels. In contrast, continuous illumination produced a graded interface of continuously varied scattering length density (and hence hydrocarbon density) extending 100–150 Å into the liquid phase. Exposure of a DPPC bilayer to UV light in the presence of a reservoir of unfused vesicles showed low net membrane disintegration during oxidative stress, presumably because of surface back-filling from the bulk reservoir. Chemical evidence for membrane degradation was obtained by mass spectrometry and Fourier transform infrared spectroscopy. Further evidence for the formation of hydrophilic channels was furnished by fluorescence microscopy and imaging ellipsometry data. PMID:19275260

  5. Serum microRNAs are early indicators of survival after radiation-induced hematopoietic injury

    PubMed Central

    Acharya, Sanket S.; Fendler, Wojciech; Watson, Jacqueline; Hamilton, Abigail; Pan, Yunfeng; Gaudiano, Emily; Moskwa, Patryk; Bhanja, Payel; Saha, Subhrajit; Guha, Chandan; Parmar, Kalindi; Chowdhury, Dipanjan

    2015-01-01

    Accidental radiation exposure is a threat to human health that necessitates effective clinical planning and diagnosis. Minimally invasive biomarkers that can predict long-term radiation injury are urgently needed for optimal management after a radiation accident. We have identified serum microRNA (miRNA) signatures that indicate long-term impact of total body irradiation (TBI) in mice when measured within 24 hours of exposure. Impact of TBI on the hematopoietic system was systematically assessed to determine a correlation of residual hematopoietic stem cells (HSCs) with increasing doses of radiation. Serum miRNA signatures distinguished untreated mice from animals exposed to radiation and correlated with the impact of radiation on HSCs. Mice exposed to sublethal (6.5 Gy) and lethal (8 Gy) doses of radiation were indistinguishable for 3 to 4 weeks after exposure. A serum miRNA signature detectable 24 hours after radiation exposure consistently segregated these two cohorts. Furthermore, using either a radioprotective agent before, or radiation mitigation after, lethal radiation, we determined that the serum miRNA signature correlated with the impact of radiation on animal health rather than the radiation dose. Last, using humanized mice that had been engrafted with human CD34+ HSCs, we determined that the serum miRNA signature indicated radiation-induced injury to the human bone marrow cells. Our data suggest that serum miRNAs can serve as functional dosimeters of radiation, representing a potential breakthrough in early assessment of radiation-induced hematopoietic damage and timely use of medical countermeasures to mitigate the long-term impact of radiation. PMID:25972001

  6. Exploring the early stages of the pH-induced conformational change of influenza hemagglutinin.

    PubMed

    Zhou, Yu; Wu, Chao; Zhao, Lifeng; Huang, Niu

    2014-10-01

    Hemagglutinin (HA) mediates the membrane fusion process of influenza virus through its pH-induced conformational change. However, it remains challenging to study its structure reorganization pathways in atomic details. Here, we first applied continuous constant pH molecular dynamics approach to predict the pK(a) values of titratable residues in H2 subtype HA. The calculated net-charges in HA1 globular heads increase from 0e (pH 7.5) to +14e (pH 4.5), indicating that the charge repulsion drives the detrimerization of HA globular domains. In HA2 stem regions, critical pH sensors, such as Glu103(2), His18(1), and Glu89(1), are identified to facilitate the essential structural reorganizations in the fusing pathways, including fusion peptide release and interhelical loop transition. To probe the contribution of identified pH sensors and unveil the early steps of pH-induced conformational change, we carried out conventional molecular dynamics simulations in explicit water with determined protonation state for each titratable residue in different environmental pH conditions. Particularly, energy barriers involving previously uncharacterized hydrogen bonds and hydrophobic interactions are identified in the fusion peptide release pathway. Nevertheless, comprehensive comparisons across HA family members indicate that different HA subtypes might employ diverse pH sensor groups along with different fusion pathways. Finally, we explored the fusion inhibition mechanism of antibody CR6261 and small molecular inhibitor TBHQ, and discovered a novel druggable pocket in H2 and H5 subtypes. Our results provide the underlying mechanism for the pH-driven conformational changes and also novel insight for anti-flu drug development.

  7. DBcAMP stimulates vesicle transport and HRP excretion in isolated perfused rat liver.

    PubMed

    Hayakawa, T; Bruck, R; Ng, O C; Boyer, J L

    1990-11-01

    To clarify the effect of adenosine 3',5'-cyclic monophosphate (cAMP) on the transcytotic vesicle pathway, we measured the biliary excretion of bile acid, phospholipid, and horseradish peroxidase (HRP) in the isolated perfused rat liver (IPRL) with or without infusion of N6,2'-O-dibutyryl-cAMP (DBcAMP). A linear relationship between bile flow and bile acid excretion was observed in both control and DBcAMP-infused livers. DBcAMP increased the y-axis intercept from 1.10 +/- 0.16 to 1.48 +/- 0.19 microliters.min-1.g liver-1 (P less than 0.01) and the slope from 6.5 +/- 1.99 to 10.77 +/- 1.71 microliters/mumol bile acid (P less than 0.01). DBcAMP also increased the biliary excretion of bile acid and phospholipid during a 1.0 mumol/min infusion of taurocholate. When HRP was pulse loaded for 1 min, HRP appeared in bile in early (4-6 min) and late (20-25 min) peaks. DBcAMP markedly increased the late peak of HRP from 0.33 +/- 0.08 to 1.15 +/- 0.32 ng.min-1.g liver-1 (P less than 0.01), a phenomenon blocked by colchicine. An electron-microscopic morphometric analysis indicated that DBcAMP increased both the density and %area of HRP-containing vesicles in the pericanalicular area, compared with controls, 18 min after a 1-min pulse of HRP. DBcAMP had no effect on the uptake rate of HRP in 4-h primary hepatocyte cultures but stimulated biliary excretion of HRP when preloaded in the IPRL. These findings indicate that cAMP regulates excretory function in part by stimulating the microtubule-dependent transcytotic vesicle transport system.

  8. From drought sensing to developmental control: evolution of cyclic AMP signaling in social amoebas.

    PubMed

    Ritchie, Allyson V; van Es, Saskia; Fouquet, Celine; Schaap, Pauline

    2008-10-01

    Amoebas and other protists commonly encyst when faced with environmental stress. Although little is known of the signaling pathways that mediate encystation, the analogous process of spore formation in dictyostelid social amoebas is better understood. In Dictyostelium discoideum, secreted cyclic AMP (cAMP) mediates the aggregation of starving amoebas and induces the differentiation of prespore cells. Intracellular cAMP acting on cAMP-dependent protein kinase (PKA) triggers the maturation of spores and prevents their germination under the prevalent conditions of high osmolality in the spore head. The osmolyte-activated adenylate cyclase, ACG, produces cAMP for prespore differentiation and inhibition of spore germination. To retrace the origin of ACG function, we investigated ACG gene conservation and function in species that span the dictyostelid phylogeny. ACG genes, osmolyte-activated ACG activity, and osmoregulation of spore germination were detected in species that represent the 4 major groups of Dictyostelia. Unlike the derived species D. discoideum, many basal Dictyostelia have retained the ancestral mechanism of encystation from solitary amoebas. In these species and in solitary amoebas, encystation is independently triggered by starvation or by high osmolality. Osmolyte-induced encystation was accompanied by an increase in cAMP and prevented by inhibition of PKA, indicating that ACG and PKA activation mediate this response. We propose that high osmolality signals drought in soil amoebas and that developmental cAMP signaling in the Dictyostelia has evolved from this stress response.

  9. Partial dopaminergic denervation-induced impairment in stimulus discrimination acquisition in parkinsonian rats: a model for early Parkinson's disease.

    PubMed

    Eagle, Andrew L; Olumolade, Oluyemi O; Otani, Hajime

    2015-03-01

    Parkinson's disease (PD) produces progressive nigrostriatal dopamine (DA) denervation resulting in cognitive and motor impairment. However, it is unknown whether cognitive impairments, such as instrumental learning deficits, are associated with the early stage PD-induced mild DA denervation. The current study sought to model early PD-induced instrumental learning impairments by assessing the effects of low dose (5.5μg), bilateral 6OHDA-induced striatal DA denervation on acquisition of instrumental stimulus discrimination in rats. 6OHDA (n=20) or sham (n=10) lesioned rats were tested for stimulus discrimination acquisition either 1 or 2 weeks post surgical lesion. Stimulus discrimination acquisition across 10 daily sessions was used to assess discriminative accuracy, or a probability measure of the shift toward reinforced responding under one stimulus condition (Sd) away from extinction, when reinforcement was withheld, under another (S(d) phase). Striatal DA denervation was assayed by tyrosine hydroxylase (TH) staining intensity. Results indicated that 6OHDA lesions produced significant loss of dorsal striatal TH staining intensity and marked impairment in discrimination acquisition, without inducing akinetic motor deficits. Rather 6OHDA-induced impairment was associated with perseveration during extinction (S(Δ) phase). These findings suggest that partial, bilateral striatal DA denervation produces instrumental learning deficits, prior to the onset of gross motor impairment, and suggest that the current model is useful for investigating mild nigrostriatal DA denervation associated with early stage clinical PD.

  10. Butyrate activates the cAMP-protein kinase A-cAMP response element-binding protein signaling pathway in Caco-2 cells.

    PubMed

    Wang, Aihua; Si, Hongwei; Liu, Dongmin; Jiang, Honglin

    2012-01-01

    Butyrate is a major SCFA produced by microbial fermentation of dietary fiber in the gastrointestinal tract. Butyrate is widely thought to mediate the benefits of fiber and resistant starch consumption to colon health in humans. Besides serving as a substrate for energy production, butyrate has many regulatory effects in animals. Little is known about the signaling mechanisms underlying the regulatory effects of butyrate and other SCFA. In this study, we determined whether butyrate can activate cAMP-protein kinase A (PKA)- cAMP response element (CRE)-binding protein (CREB) signaling in Caco-2 cells, a model of intestinal epithelial cells. Butyrate promoted luciferase expression from a CRE-reporter construct, induced phosphorylation of CREB, increased the activity of PKA, and elevated the levels of cAMP in Caco-2 cells. These data suggest that butyrate activates cAMP-PKA-CREB signaling in Caco-2 cells. Butyrate, however, had no effect on the activities of adenylyl cyclase (AC) and phosphodiesterase (PDE), two enzymes that determine the production and degradation of intracellular cAMP, respectively. Because the activities of AC and PDE are primarily regulated by G protein-coupled receptor (GPR)-mediated intracellular signaling, lack of an effect of butyrate on these two enzymes suggests that butyrate does not activate cAMP-PKA-CREB signaling through GPR. Butyrate-treated Caco-2 cells had greater concentrations of ATP than untreated cells. Because ATP is the substrate for cAMP production, this difference suggests that butyrate may activate cAMP-PKA-CREB signaling in Caco-2 cells through increased ATP production. Overall, this study raises the possibility that some of the regulatory effects of butyrate in animals, including those on the colonocytes, may be mediated by the cAMP-PKA-CREB signaling pathway at the cellular level.

  11. Autophagic degradation of aquaporin-2 is an early event in hypokalemia-induced nephrogenic diabetes insipidus.

    PubMed

    Khositseth, Sookkasem; Uawithya, Panapat; Somparn, Poorichaya; Charngkaew, Komgrid; Thippamom, Nattakan; Hoffert, Jason D; Saeed, Fahad; Michael Payne, D; Chen, Shu-Hui; Fenton, Robert A; Pisitkun, Trairak

    2015-12-17

    Hypokalemia (low serum potassium level) is a common electrolyte imbalance that can cause a defect in urinary concentrating ability, i.e., nephrogenic diabetes insipidus (NDI), but the molecular mechanism is unknown. We employed proteomic analysis of inner medullary collecting ducts (IMCD) from rats fed with a potassium-free diet for 1 day. IMCD protein quantification was performed by mass spectrometry using a label-free methodology. A total of 131 proteins, including the water channel AQP2, exhibited significant changes in abundance, most of which were decreased. Bioinformatic analysis revealed that many of the down-regulated proteins were associated with the biological processes of generation of precursor metabolites and energy, actin cytoskeleton organization, and cell-cell adhesion. Targeted LC-MS/MS and immunoblotting studies further confirmed the down regulation of 18 selected proteins. Electron microscopy showed autophagosomes/autophagolysosomes in the IMCD cells of rats deprived of potassium for only 1 day. An increased number of autophagosomes was also confirmed by immunofluorescence, demonstrating co-localization of LC3 and Lamp1 with AQP2 and several other down-regulated proteins in IMCD cells. AQP2 was also detected in autophagosomes in IMCD cells of potassium-deprived rats by immunogold electron microscopy. Thus, enhanced autophagic degradation of proteins, most notably including AQP2, is an early event in hypokalemia-induced NDI.

  12. Heat shock during early somitogenesis induces caudal vertebral column defects in Atlantic salmon (Salmo salar).

    PubMed

    Wargelius, Anna; Fjelldal, Per Gunnar; Hansen, Tom

    2005-07-01

    In several terrestrial vertebrates, heat shock (HS) during somitogenesis causes vertebral deformities. To determine if vertebral deformities can occur due to sudden temperature changes during early development in fish, Atlantic salmon embryos were HS treated during somitogenesis. Ten months later these individuals displayed a high prevalence of caudal vertebral column condensations (27-34%). The defects were located caudally of the abdominal cavity, displaying an even distribution in this region independent of time of HS. To determine if HS disturbed vertebral development during somitogenesis, two genes coding for markers of skeletal development were identified, namely, the secreted protein Shh (Sashh) and the transcription factor Twist (Satwist). These proteins are involved in the proliferation and specification of presumptive skeletal cells (sclerotome) in vertebrates. The spatial expression pattern of sashh and satwist in salmon indicated a functional conservation of these proteins. Furthermore, HS embryos displayed expressional disturbance in both sashh and satwist, indicating an effect of HS on sclerotomal cell patterning. However, the HS-protecting ability in embryos seems to be individually regulated because reduction in gene expression was not detected at all stages; in addition, HS did not induce somitic disturbance and vertebral deformity in all embryos.

  13. ALD1 Regulates Basal Immune Components and Early Inducible Defense Responses in Arabidopsis.

    PubMed

    Cecchini, Nicolás M; Jung, Ho Won; Engle, Nancy L; Tschaplinski, Timothy J; Greenberg, Jean T

    2015-04-01

    Robust immunity requires basal defense machinery to mediate timely responses and feedback cycles to amplify defenses against potentially spreading infections. AGD2-LIKE DEFENSE RESPONSE PROTEIN 1 (ALD1) is needed for the accumulation of the plant defense signal salicylic acid (SA) during the first hours after infection with the pathogen Pseudomonas syringae and is also upregulated by infection and SA. ALD1 is an aminotransferase with multiple substrates and products in vitro. Pipecolic acid (Pip) is an ALD1-dependent bioactive product induced by P. syringae. Here, we addressed roles of ALD1 in mediating defense amplification as well as the levels and responses of basal defense machinery. ALD1 needs immune components PAD4 and ICS1 (an SA synthesis enzyme) to confer disease resistance, possibly through a transcriptional amplification loop between them. Furthermore, ALD1 affects basal defense by controlling microbial-associated molecular pattern (MAMP) receptor levels and responsiveness. Vascular exudates from uninfected ALD1-overexpressing plants confer local immunity to the wild type and ald1 mutants yet are not enriched for Pip. We infer that, in addition to affecting Pip accumulation, ALD1 produces non-Pip metabolites that play roles in immunity. Thus, distinct metabolite signals controlled by the same enzyme affect basal and early defenses versus later defense responses, respectively.

  14. The circadian clock gene regulatory module enantioselectively mediates imazethapyr-induced early flowering in Arabidopsis thaliana.

    PubMed

    Qian, Haifeng; Han, Xiao; Peng, Xiaofeng; Lu, Tao; Liu, Weiping; Fu, Zhengwei

    2014-03-01

    Plant growth and development are strongly affected by environmental pollutants, such as herbicides. Widely used herbicides can remain in soil or aquatic systems for long periods of time. Herbicide pollutants have been reported to heavily affect global plant growth and pose a significant challenge to agriculture. However, it is unclear whether herbicides affect plant flowering. Here, we demonstrated that imazethapyr (IM), a chiral herbicide, can enantioselectively promote flowering in Arabidopsis thaliana. We clarified the possible mechanism by which IM promotes flowering and found that the photoperiod pathway may play an important role in propagating the IM stress signal. IM enantiomers decreased the amplitude of core oscillators (CIRCADIAN CLOCK ASSOCIATED 1 and LATE ELONGATED HYPOCOTYL) and utilized the up-regulation of the GIGANTEA-(CONSTANS)-FLOWERING LOCUS T pathway to induce floral gene, APETALA1 over-expression enantioselectively; this treatment ultimately caused early flowering. Our findings provide new insight into the method by which plants control reproductive timing in response to herbicide stress. Flowering time is an important trait in crops and affects the life cycles of pollinator species. The persistence of herbicides in the biosphere will alter plant life cycles and diversity. PMID:24484962

  15. Social exclusion induces early-stage perceptual and behavioral changes in response to social cues.

    PubMed

    Kawamoto, Taishi; Nittono, Hiroshi; Ura, Mitsuhiro

    2014-01-01

    Social exclusion is so aversive that it causes broad cognitive and behavioral changes to regulate the individual's belonging status. The present study examined whether such changes also occur at early neural or automatic behavioral levels in response to social cues. Event-related brain potentials (ERPs) and facial electromyograms (EMGs) were recorded during a task in which participants viewed smiling, disgusted, and neutral faces after experiencing social exclusion or inclusion. Social exclusion was manipulated using a simple ball-tossing game (Cyberball), and need threat was assessed after the game. We found that zygomaticus major muscle activity, which reflects facial mimicry, was larger in response to smiling faces after exclusion than after inclusion. In addition, P1 amplitude, which reflects visual attention, was larger for disgusted faces than for neutral faces following social exclusion. N170 amplitude, which reflects structural encoding of the face, was correlated with heightened need threat. These findings demonstrate that social exclusion induces immediate and rapid changes in attention, perception, and automatic behavior. These findings reflect the rapid and primary regulation of belonging.

  16. Pharmacology at work for cardio-oncology: ranolazine to treat early cardiotoxicity induced by antitumor drugs.

    PubMed

    Minotti, Giorgio

    2013-09-01

    Antitumor drugs may cause asymptomatic diastolic dysfunction that introduces a lifetime risk of heart failure or myocardial infarction. Cardio-oncology is the discipline committed to the cardiac surveillance and management of cancer patients and survivors; however, cardio-oncology teams do not always attempt to treat early diastolic dysfunction. Common cardiovascular drugs, such as β blockers or angiotensin-converting enzyme inhibitors or others, would be of uncertain efficacy in diastolic dysfunction. This perspective describes the potential value of ranolazine, an antianginal drug that improves myocardial perfusion by relieving diastolic wall tension and dysfunction. Ranolazine acts by inhibiting the late inward sodium current, and pharmacological reasonings anticipate that antitumor anthracyclines and nonanthracycline chemotherapeutics might well induce anomalous activation of this current. These notions formed the rationale for a clinical study of the efficacy and safety of ranolazine in cancer patients. This study was not designed to demonstrate that ranolazine reduced the lifetime risk of cardiac events; it was designed as a short term proof-of-concept study that probed the following hypotheses: 1) asymptomatic diastolic dysfunction could be detected a few days after patients completed antitumor therapy, and 2) ranolazine was active and safe in relieving echocardiographic and/or biohumoral indices of diastolic dysfunction, measured at 5 weeks or 6 months of ranolazine administration. These facts illustrate the translational value of pharmacology, which goes from identifying therapeutic opportunities to validating hypotheses in clinical settings. Pharmacology is a key to the success of cardio-oncology. PMID:23818683

  17. Autophagic degradation of aquaporin-2 is an early event in hypokalemia-induced nephrogenic diabetes insipidus

    PubMed Central

    Khositseth, Sookkasem; Uawithya, Panapat; Somparn, Poorichaya; Charngkaew, Komgrid; Thippamom, Nattakan; Hoffert, Jason D.; Saeed, Fahad; Michael Payne, D.; Chen, Shu-Hui; Fenton, Robert A.; Pisitkun, Trairak

    2015-01-01

    Hypokalemia (low serum potassium level) is a common electrolyte imbalance that can cause a defect in urinary concentrating ability, i.e., nephrogenic diabetes insipidus (NDI), but the molecular mechanism is unknown. We employed proteomic analysis of inner medullary collecting ducts (IMCD) from rats fed with a potassium-free diet for 1 day. IMCD protein quantification was performed by mass spectrometry using a label-free methodology. A total of 131 proteins, including the water channel AQP2, exhibited significant changes in abundance, most of which were decreased. Bioinformatic analysis revealed that many of the down-regulated proteins were associated with the biological processes of generation of precursor metabolites and energy, actin cytoskeleton organization, and cell-cell adhesion. Targeted LC-MS/MS and immunoblotting studies further confirmed the down regulation of 18 selected proteins. Electron microscopy showed autophagosomes/autophagolysosomes in the IMCD cells of rats deprived of potassium for only 1 day. An increased number of autophagosomes was also confirmed by immunofluorescence, demonstrating co-localization of LC3 and Lamp1 with AQP2 and several other down-regulated proteins in IMCD cells. AQP2 was also detected in autophagosomes in IMCD cells of potassium-deprived rats by immunogold electron microscopy. Thus, enhanced autophagic degradation of proteins, most notably including AQP2, is an early event in hypokalemia-induced NDI. PMID:26674602

  18. Right ventricular angiogenesis is an early adaptive response to chronic hypoxia-induced pulmonary hypertension

    PubMed Central

    Kolb, Todd M.; Peabody, Jacelyn; Baddoura, Philip; Fallica, Jon; Mock, Jason R.; Singer, Benjamin D.; D’Alessio, Franco R.; Damarla, Mahendra; Damico, Rachel L.; Hassoun, Paul M.

    2015-01-01

    Objective Myocardial angiogenesis is presumed to play a role in right ventricular (RV) adaptation to pulmonary hypertension (PH), though definitive evidence and functional correlations are lacking. We aimed to use definitive methods to correlate RV angiogenesis, hypertrophy, and function in a murine PH model. Methods Mice were exposed to chronic hypoxia for 21 days to induce PH (CH-PH) and RV remodeling. We used unbiased stereology and flow cytometry to quantify angiogenesis and myocyte hypertrophy, and pressure-volume loops to measure RV function. Results Within 7 days, RV-specific increases in total capillary length (10576±2574 cm vs. 6822±1379 cm; P = 0.02), surface area (10±3.3 cm2 vs. 4.9±1.5 cm2; P = 0.01), and volume (0.0013±0.0005 cm3 vs. 0.0006±0.0001 cm3; P = 0.02) were observed, and RV endothelial cell proliferation increased nearly 10-fold. Continued exposure led to progressive RV hypertrophy without additional angiogenesis. RV function was preserved, but activation of hypoxia-dependent gene expression was observed in both ventricles after 21 days. Conclusions Early RV remodeling in CH-PH is associated with RV angiogenesis and preserved RV function. Continued CH-PH is associated with RV hypertrophy but not angiogenesis, leading to biventricular activation of hypoxia-dependent gene expression. PMID:26352923

  19. The fungicide imazalil induces developmental abnormalities and alters locomotor activity during early developmental stages in zebrafish.

    PubMed

    Jin, Yuanxiang; Zhu, Zhihong; Wang, Yueyi; Yang, Enlu; Feng, Xiayan; Fu, Zhengwei

    2016-06-01

    The fungicide imazalil (IMZ) is used extensively to protect vegetable fields, fruit plantations and post-harvest crops from rot. Likely due to its wide-spread use, IMZ is frequently detected in vegetable, fruit, soil and even surface water samples. Even though several previous studies have reported on the neurotoxicity of IMZ, its effects on the neurobehavior of zebrafish have received little attention to date. In this study, we show that the heartbeat and hatchability of zebrafish were significantly influenced by IMZ concentrations of 300 μg L(-1) or higher. Moreover, in zebrafish larvae, locomotor behaviors such as average swimming speed and swimming distance were significantly decreased after exposure to 300 μg L(-1) IMZ for 96 h, and acetylcholinesterase (AChE) expression and activity were consistently inhibited in IMZ-treated fish. Our results further suggest that IMZ could act as a neuroendocrine disruptor by decreasing the expression of neurotoxicity-related genes such as Glial fibrillary acidic protein (Gfap), Myelin basic protein (Mbp) and Sonic hedgehog a (Shha) during early developmental stages of zebrafish. In conclusion, we show that exposure to IMZ has the potential to induce developmental toxicity and locomotor behavior abnormalities during zebrafish development. PMID:27035382

  20. Early changes in the liver-soluble proteome from mice fed a nonalcoholic steatohepatitis inducing diet.

    PubMed

    Thomas, Anja; Stevens, Axel P; Klein, Matthias S; Hellerbrand, Claus; Dettmer, Katja; Gronwald, Wolfram; Oefner, Peter J; Reinders, Jörg

    2012-05-01

    Despite the increasing incidence of nonalcoholic steatohepatitis (NASH) with the rise in lifestyle-related diseases such as the metabolic syndrome, little is known about the changes in the liver proteome that precede the onset of inflammation and fibrosis. Here, we investigated early changes in the liver-soluble proteome of female C57BL/6N mice fed an NASH-inducing diet by 2D-DIGE and nano-HPLC-MS/MS. In parallel, histology and measurements of hepatic content of triglycerides, cholesterol and intermediates of the methionine cycle were performed. Hepatic steatosis manifested itself after 2 days of feeding, albeit significant changes in the liver-soluble proteome were not evident before day 10 in the absence of inflammatory or fibrotic signs. Proteomic alterations affected mainly energy and amino acid metabolism, detoxification processes, urea cycle, and the one-carbon/S-adenosylmethionine pathways. Additionally, intermediates of relevant affected pathways were quantified from liver tissue, confirming the findings from the proteomic analysis.

  1. Early iron-deficiency-induced transcriptional changes in Arabidopsis roots as revealed by microarray analyses

    PubMed Central

    Buckhout, Thomas J; Yang, Thomas JW; Schmidt, Wolfgang

    2009-01-01

    Background Iron (Fe) is an essential nutrient in plants and animals, and Fe deficiency results in decreased vitality and performance. Due to limited bio-availability of Fe, plants have evolved sophisticated adaptive alterations in development, biochemistry and metabolism that are mainly regulated at the transcriptional level. We have investigated the early transcriptional response to Fe deficiency in roots of the model plant Arabidopsis, using a hydroponic system that permitted removal of Fe from the nutrient solution within seconds and transferring large numbers of plants with little or no mechanical damage to the root systems. We feel that this experimental approach offers significant advantages over previous and recent DNA microarray investigations of the Fe-deficiency response by increasing the resolution of the temporal response and by decreasing non-Fe deficiency-induced transcriptional changes, which are common in microarray analyses. Results The expression of sixty genes were changed after 6 h of Fe deficiency and 65% of these were found to overlap with a group of seventy-nine genes that were altered after 24 h. A disproportionally high number of transcripts encoding ion transport proteins were found, which function to increase the Fe concentration and decrease the zinc (Zn) concentration in the cytosol. Analysis of global changes in gene expression revealed that changes in Fe availability were associated with the differential expression of genes that encode transporters with presumed function in uptake and distribution of transition metals other than Fe. It appeared that under conditions of Fe deficiency, the capacity for Zn uptake increased, most probably the result of low specificity of the Fe transporter IRT1 that was induced upon Fe deficiency. The transcriptional regulation of several Zn transports under Fe deficiency led presumably to the homeostatic regulation of the cytosolic concentration of Zn and of other transition metal ions such as Mn to

  2. Cyclic AMP regulates the biosynthesis of cellobiohydrolase in Cellulomonas flavigena growing in sugar cane bagasse.

    PubMed

    Herrera-Herrera, Jesús Antonio; Pérez-Avalos, Odilia; Salgado, Luis M; Ponce-Noyola, Teresa

    2009-10-01

    Cellulomonas flavigena produces a battery of cellulase components that act concertedly to degrade cellulose. The addition of cAMP to repressed C. flavigena cultures released catabolic repression, while addition of cAMP to induced C. flavigena cultures led to a cellobiohydrolase hyperproduction. Exogenous cAMP showed positive regulation on cellobiohydrolase production in C. flavigena grown on sugar cane bagasse. A C. flavigena cellobiohydrolase gene was cloned (named celA), which coded for a 71- kDa enzyme. Upstream, a repressor celR1, identified as a 38 kDa protein, was monitored by use of polyclonal antibodies.

  3. Sodium butyrate improves locomotor impairment and early mortality in a rotenone-induced Drosophila model of Parkinson's disease.

    PubMed

    St Laurent, R; O'Brien, L M; Ahmad, S T

    2013-08-29

    Parkinson's disease (PD) is a neurodegenerative disorder primarily affecting the dopaminergic neurons in the nigrastriatal pathway resulting in debilitating motor impairment in both familial and sporadic cases. Histone deacetylase (HDAC) inhibitors have been recently implicated as a therapeutic candidate because of their ability to correct the disrupted HDAC activity in PD and other neurodegenerative diseases. Sodium butyrate (SB), an HDAC inhibitor, reduces degeneration of dopaminergic neurons in a mutant alpha-synuclein Drosophila transgenic model of familial PD. Chronic exposure to the pesticide rotenone also causes selective degeneration of dopaminergic neurons and causes locomotor impairment and early mortality in a Drosophila model of chemically induced PD. This study investigated the effects of sodium butyrate on locomotor impairment and early mortality in a rotenone-induced PD model. We show that treatment with 10mM SB-supplemented food rescued the rotenone-induced locomotor impairment and early mortality in flies. Additionally, flies with the genetic knockdown of HDAC activity through Sin3A loss-of-function mutation (Sin3A(lof)) were resistant to rotenone-induced locomotor impairment and early mortality. Furthermore, SB-supplemented Sin3A(lof) flies had a modest additive effect for improving locomotor impairment. We also show SB-mediated improvement of rotenone-induced locomotor impairment was associated with elevated dopamine levels in the brain. However, the possibility of SB-mediated protective role through mechanisms independent from dopamine system is also discussed. These findings demonstrate that HDAC inhibitors like SB can ameliorate locomotor impairment in a rotenone-induced PD model.

  4. The plasma cyclic AMP response to catecholamines as potentiated by phentolamine in rats.

    PubMed

    Kunitada, S; Ui, M

    1978-05-15

    Norepinephrine failed to increase plasma cyclic AMP when injected alone into fasted rats, in contrast with sharp increases elicited by isoproterenol, epinephrine or tyramine. In rats pretreated with 6-hydroxydopamine or cocain, however, there was significant increase in plasma cyclic AMP after norepinephrine injection, suggesting that the rapid neuronal catecholamine uptake was at least partly responsible for the lack of norepinephrine action. Phentolamine was very effective in enhancing the epinephrine-, norepinephrine- or tyramine-induced increase in plasma cyclic AMP but without effect on the isoproterenol-induced increase. Blockade of postsynaptic alpha-adrenoceptors, rather than of presynaptic receptors, is likely to be involved in the phentolamine potentiation, since it was even observed in rats treated with 6-hydroxydopamine or cocaine. A discussion is presented regarding the mechanism by which cyclic AMP generation is influenced by the alpha- and beta-adrenoceptor interaction on effector cell membranes.

  5. Hydrogen peroxide induces murine macrophage chemokine gene transcription via extracellular signal-regulated kinase- and cyclic adenosine 5'-monophosphate (cAMP)-dependent pathways: involvement of NF-kappa B, activator protein 1, and cAMP response element binding protein.

    PubMed

    Jaramillo, Maritza; Olivier, Martin

    2002-12-15

    Hydrogen peroxide (H(2)O(2)) has been shown to act as a second messenger that activates chemokine expression. In the present study, we investigated the mechanisms underlying this cellular regulation in the murine macrophage cell line B10R. We report that H(2)O(2) increases mRNA expression of various chemokines, macrophage-inflammatory protein (MIP)-1alpha/CC chemokine ligand (CCL)3, MIP-1beta/CCL4, MIP-2/CXC chemokine ligand 2, and monocyte chemoattractant protein-1/CCL2, by activating the extracellular signal-regulated kinase (ERK) pathway and the nuclear translocation of the transcription factors NF-kappaB, AP-1, and CREB. Blockage of the ERK pathway with specific inhibitors against mitogen-activated protein kinase kinase 1/2 and ERK1/ERK2 completely abolished both the H(2)O(2)-mediated chemokine up-regulation and the activation of all NF studied. Similarly, selective inhibition of cAMP and NF-kappaB strongly down-regulated the induction of all chemokine transcripts as well as CREB and NF-kappaB activation, respectively. Of interest, we detected a significant decrease of NF-kappaB, AP-1, and CREB DNA binding activities by reciprocal competition for these binding sites when either specific cold oligonucleotides (NF-kappaB, AP-1, and CREB) or Abs against various transcription factor subunits (p50, p65, c-Fos, Jun B, c-Jun, and CREB-1) were added. These findings indicate that cooperation between ERK- and cAMP-dependent pathways seems to be required to achieve the formation of an essential transcriptional factor complex for maximal H(2)O(2)-dependent chemokine modulation. Finally, experiments performed with actinomycin D suggest that H(2)O(2)-mediated MIP-1beta mRNA up-regulation results from transcriptional control, whereas that of MIP-1alpha, MIP-2, and monocyte chemoattractant protein-1 is due to both gene transcription activation and mRNA posttranscriptional stabilization.

  6. The growth factor-inducible immediate-early gene 3CH134 encodes a protein-tyrosine-phosphatase.

    PubMed Central

    Charles, C H; Sun, H; Lau, L F; Tonks, N K

    1993-01-01

    Stimulation of fibroblasts with serum growth factors results in the rapid activation of a set of immediate-early genes, among them 3CH134. We have purified a bacterially expressed form of the 3CH134-encoded polypeptide and demonstrated that it has intrinsic protein-tyrosine-phosphatase (PTPase; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) activity in vitro. This activity is optimal at pH 7.5, is sensitive to vanadate and cysteinyl modifying agents, and is insensitive to a panel of serine/threonine phosphatase inhibitors. Purified 3CH134 protein displays a high degree of selectivity among the tyrosine-phosphorylated polypeptide substrates tested. Under our assay conditions, the rates of dephosphorylation are in the order EDNDYINASL peptide < myelin basic protein < reduced, carboxyamidomethylated, and maleylated lysozyme (RCML) < p42mapk. There is a 200-fold range in rates for these substrates, with p42mapk dephosphorylated 15-fold more rapidly than RCML. Although 3CH134 is most closely related to the tyrosine/serine dual-specificity phosphatase VH1, we failed to detect any 3CH134-directed activity on casein or RCML phosphorylated on serine/threonine residues by cAMP-dependent protein kinase. Since 3CH134 expression is controlled transcriptionally and posttranscriptionally, it may represent a class of PTPases whose activity is regulated at the level of protein synthesis and degradation. Images Fig. 1 Fig. 4 Fig. 5 Fig. 6 PMID:8389479

  7. 5′-AMP impacts lymphocyte recirculation through activation of A2B receptors

    PubMed Central

    Bouma, Hjalmar R.; Mandl, Judith N.; Strijkstra, Arjen M.; Boerema, Ate S.; Kok, Jan-Willem; van Dam, Annie; IJzerman, Ad; Kroese, Frans G. M.; Henning, Robert H.

    2013-01-01

    Natural hibernation consists of torpid phases with metabolic suppression alternating with euthermic periods. Induction of torpor holds substantial promise in various medical conditions, including trauma, major surgery, and transplantation. Torpor in mice can be induced pharmacologically by 5′-AMP. Previously, we showed that during natural torpor, the reduction in body temperature results in lymphopenia via a reduction in plasma S1P. Here, we show that during torpor induced by 5′-AMP, there is a similar reduction in the number of circulating lymphocytes that is a result of their retention in secondary lymphoid organs. This lymphopenia could be mimicked by engagement of A2BRs by a selective A2BR agonist (LUF6210) in the absence of changes in temperature and prevented by A2BR antagonists during 5′-AMP-induced torpor. In addition, forced cooling of mice led to peripheral blood lymphopenia, independent of A2BR signaling. The induction of torpor using 5′-AMP impacted the migration of lymphocytes within and between secondary lymphoid organs. During torpor, the homing into LNs was impaired, and two-photon intravital microscopy revealed that cell motility was decreased significantly and rapidly upon 5′-AMP administration. Furthermore, the S1P plasma concentration was reduced by 5′-AMP but not by LUF6210. S1P plasma levels restored upon arousal. Likely, the reduced migration in LNs combined with the reduced S1P plasma level substantially reduces lymphocyte egress after injection of 5′-AMP. In conclusion, 5′-AMP induces a state of pharmacological torpor in mice, during which, lymphopenia is governed primarily by body temperature-independent suppression of lymphocyte egress from LNs. PMID:23682128

  8. Atrazine Acts as an Endocrine Disrupter by Inhibiting cAMP-specific Phosphodiesterase-4

    PubMed Central

    Kucka, Marek; Pogrmic-Majkic, Kristina; Fa, Svetlana; Stojilkovic, Stanko S.; Kovacevic, Radmila

    2014-01-01

    Atrazine, one of the most commonly used herbicides worldwide, acts as an endocrine disruptor, but the mechanism of its action has not been characterized. In this study, we show that atrazine rapidly increases cAMP levels in cultured rat pituitary and testicular Leydig cells in a concentration-dependent manner, but less effectively than 3-isobutyl-1-methylxanthine, a competitive non-specific inhibitor of phosphodiesterases (PDEs). In forskolin (an activator of adenylyl cyclase)- and probenecid (an inhibitor of cyclic nucleotide transporters)-treated cells, but not in 3-isobutyl-1-methylxanthine-treated cells, atrazine further increased cAMP levels, indicating that inhibition of PDEs accounts for accumulation of cAMP. In contrast to cAMP, atrazine did not alter cGMP levels, further indicating that it inhibits cAMP-specific PDEs. Atrazine-induced changes in cAMP levels were sufficient to stimulate prolactin release in pituitary cells and androgen production in Leydig cells, indicating that it acts as an endocrine disrupter both in cells that secrete by exocytosis of prestored hormones and in cells that secrete by de novo hormone synthesis. Rolipram abolished the stimulatory effect of atrazine on cAMP release in both cell types, suggesting that it acts as an inhibitor of PDE4s, isoforms whose mRNA transcripts dominate in pituitary and Leydig cells together with mRNA for PDE8A. In contrast, immortalized lacto-somatotrophs showed low expression of these mRNA transcripts and several fold higher cAMP levels compared to normal pituitary cells, and atrazine was unable to further increase cAMP levels. These results indicate that atrazine acts as a general endocrine disrupter by inhibiting cAMP-specific PDE4s. PMID:23022511

  9. Positive Effect of Carbon Sources on Natural Transformation in Escherichia coli: Role of Low-Level Cyclic AMP (cAMP)-cAMP Receptor Protein in the Derepression of rpoS

    PubMed Central

    Guo, Mengyue; Wang, Huanyu; Xie, Nengbin

    2015-01-01

    ABSTRACT Natural plasmid transformation of Escherichia coli is a complex process that occurs strictly on agar plates and requires the global stress response factor σS. Here, we showed that additional carbon sources could significantly enhance the transformability of E. coli. Inactivation of phosphotransferase system genes (ptsH, ptsG, and crr) caused an increase in the transformation frequency, and the addition of cyclic AMP (cAMP) neutralized the promotional effect of carbon sources. This implies a negative role of cAMP in natural transformation. Further study showed that crp and cyaA mutations conferred a higher transformation frequency, suggesting that the cAMP-cAMP receptor protein (CRP) complex has an inhibitory effect on transformation. Moreover, we observed that rpoS is negatively regulated by cAMP-CRP in early log phase and that both crp and cyaA mutants show no transformation superiority when rpoS is knocked out. Therefore, it can be concluded that both the crp and cyaA mutations derepress rpoS expression in early log phase, whereby they aid in the promotion of natural transformation ability. We also showed that the accumulation of RpoS during early log phase can account for the enhanced transformation aroused by additional carbon sources. Our results thus demonstrated that the presence of additional carbon sources promotes competence development and natural transformation by reducing cAMP-CRP and, thus, derepressing rpoS expression during log phase. This finding could contribute to a better understanding of the relationship between nutrition state and competence, as well as the mechanism of natural plasmid transformation in E. coli. IMPORTANCE Escherichia coli, which is not usually considered to be naturally transformable, was found to spontaneously take up plasmid DNA on agar plates. Researching the mechanism of natural transformation is important for understanding the role of transformation in evolution, as well as in the transfer of pathogenicity and

  10. Efficacy of troxerutin on streptozotocin-induced rat model in the early stage of diabetic retinopathy.

    PubMed

    Chung, Han Kook; Choi, Seul Min; Ahn, Byoung Ok; Kwak, Hyun Hee; Kim, Jeong Hoon; Kim, Won Bae

    2005-01-01

    The vascular changes associated with early diabetic retinopathy, which include the formation of microaneurysms and acellular capillaries, vessel dilation, vascular endothelial growth factor expression, were investigated experimentally in streptozotocin-induced diabetic rats treated with antioxidants: troxerutin (trihydroxy-ethylrutoside, CAS 7085-55-4), Vaccinium myrtillus, and calcium dobesilate (hydroquinone calcium sulfonate, CAS 20123-80-2). The development and progression of retinopathy was followed using fundus photography. After 3 months, the rats were sacrificed and half of the eyes were prepared for neovascularization analysis, and the other half were used for VEGF (vascular endothelial growth factor) analysis. The results from fundus photography and ADPase (adenosine diphosphatase) staining were quantified by the percentage area of the retinal vasculature using a commercial image analyzer. The VEGF protein in the retinal homogenates was assessed using an ELISA (enzyme linked immunosorbent assay) kit and VEGF-mRNA by RT-PCR (reverse transcription polymerase chain reaction). In the ADPase stain, the retinal vascular percent area increased significantly in the diabetic control. Neovascularization and aneurysms were observed in the diabetic control and were attenuated by 50 mg/kg troxerutin, but the retinal vascular percentage area was not significantly different from the diabetic control. The VEGF protein concentration was higher in diabetic rats than in the nondiabetic rats (21.5 +/- 2.1 vs 27.7 +/- 5.8 pg/mg, p < 0.05), and this increase was attenuated by 10 mg/kg troxerutin (24.5 +/- 3.8 pg/mg, p < 0.05) and prevented by 50 mg/kg troxerutin (19.5 +/- 2.2 pg/mg, p < 0.05). However, there were no significant differences between the groups. The VEGF-mRNA density showed a increasing tendency by 20% in the diabetic rats compared with the non-diabetic rats (1.0 +/- 0.1 vs 1.2 +/- 0.1 VEGF/beta-actin), and this increase was corrected by 10 mg/kg troxerutin (1

  11. Neddylation is required for herpes simplex virus type I (HSV-1)-induced early phase interferon-beta production

    PubMed Central

    Zhang, Xueying; Ye, Zhenjie; Pei, Yujun; Qiu, Guihua; Wang, Qingyang; Xu, Yunlu; Shen, Beifen; Zhang, Jiyan

    2016-01-01

    Type I interferons such as interferon-beta (IFN-β) play essential roles in the host innate immune response to herpes simplex virus type I (HSV-1) infection. The transcription of type I interferon genes is controlled by nuclear factor-κB (NF-κB) and interferon regulatory factor (IRF) family members including IRF3. NF-κB activation depends on the phosphorylation of inhibitor of κB (IκB), which triggers its ubiqitination and degradation. It has been reported that neddylation inhibition by a pharmacological agent MLN4924 potently suppresses lipopolysaccharide (LPS)-induced proinflammatory cytokine production with the accumulation of phosphorylated IκBα. However, the role of neddylation in type I interferon expression remains unknown. Here, we report that neddylation inhibition with MLN4924 or upon UBA3 deficiency led to accumulation of phosphorylated IκBα, impaired IκBα degradation, and impaired NF-κB nuclear translocation in the early phase of HSV-1 infection even though phosphorylation and nuclear translocation of IRF3 were not affected. The blockade of NF-κB nuclear translocation by neddylation inhibition becomes less efficient at the later time points of HSV-1 infection. Consequently, HSV-1-induced early phase IFN-β production significantly decreased upon MLN4924 treatment and UBA3 deficiency. NF-κB inhibitor JSH-23 mimicked the effects of neddylation inhibition in the early phase of HSV-1 infection. Moreover, the effects of neddylation inhibition on HSV-1-induced early phase IFN-β production diminished in the presence of NF-κB inhibitor JSH-23. Thus, neddylation contributes to HSV-1-induced early phase IFN-β production through, at least partially, promoting NF-κB activation. PMID:27593482

  12. Neddylation is required for herpes simplex virus type I (HSV-1)-induced early phase interferon-beta production.

    PubMed

    Zhang, Xueying; Ye, Zhenjie; Pei, Yujun; Qiu, Guihua; Wang, Qingyang; Xu, Yunlu; Shen, Beifen; Zhang, Jiyan

    2016-09-01

    Type I interferons such as interferon-beta (IFN-β) play essential roles in the host innate immune response to herpes simplex virus type I (HSV-1) infection. The transcription of type I interferon genes is controlled by nuclear factor-κB (NF-κB) and interferon regulatory factor (IRF) family members including IRF3. NF-κB activation depends on the phosphorylation of inhibitor of κB (IκB), which triggers its ubiqitination and degradation. It has been reported that neddylation inhibition by a pharmacological agent MLN4924 potently suppresses lipopolysaccharide (LPS)-induced proinflammatory cytokine production with the accumulation of phosphorylated IκBα. However, the role of neddylation in type I interferon expression remains unknown. Here, we report that neddylation inhibition with MLN4924 or upon UBA3 deficiency led to accumulation of phosphorylated IκBα, impaired IκBα degradation, and impaired NF-κB nuclear translocation in the early phase of HSV-1 infection even though phosphorylation and nuclear translocation of IRF3 were not affected. The blockade of NF-κB nuclear translocation by neddylation inhibition becomes less efficient at the later time points of HSV-1 infection. Consequently, HSV-1-induced early phase IFN-β production significantly decreased upon MLN4924 treatment and UBA3 deficiency. NF-κB inhibitor JSH-23 mimicked the effects of neddylation inhibition in the early phase of HSV-1 infection. Moreover, the effects of neddylation inhibition on HSV-1-induced early phase IFN-β production diminished in the presence of NF-κB inhibitor JSH-23. Thus, neddylation contributes to HSV-1-induced early phase IFN-β production through, at least partially, promoting NF-κB activation. PMID:27593482

  13. The AzTEC Mathematics Project (AMP).

    ERIC Educational Resources Information Center

    Johnson, Gae R.

    The AzTEC Mathematics Project (AMP) is a statewide partnership among Arizona's Regents universities and state community colleges, partner school districts, and economic communities. AzTec is committed to preparing highly qualified K-12 mathematics and science teachers. AMP targeted Native American teachers and teachers of Native American students…