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Sample records for activates extracellular signal-regulated

  1. Brain-derived neurotrophic factor activation of extracellular signal-regulated kinase is autonomous from the dominant extrasynaptic NMDA receptor extracellular signal-regulated kinase shutoff pathway.

    Science.gov (United States)

    Mulholland, P J; Luong, N T; Woodward, J J; Chandler, L J

    2008-01-24

    NMDA receptors bidirectionally modulate extracellular signal-regulated kinase (ERK) through the coupling of synaptic NMDA receptors to an ERK activation pathway that is opposed by a dominant ERK shutoff pathway thought to be coupled to extrasynaptic NMDA receptors. In the present study, synaptic NMDA receptor activation of ERK in rat cortical cultures was partially inhibited by the highly selective NR2B antagonist Ro25-6981 (Ro) and the less selective NR2A antagonist NVP-AAM077 (NVP). When Ro and NVP were added together, inhibition appeared additive and equal to that observed with the NMDA open-channel blocker MK-801. Consistent with a selective coupling of extrasynaptic NMDA receptors to the dominant ERK shutoff pathway, pre-block of synaptic NMDA receptors with MK-801 did not alter the inhibitory effect of bath-applied NMDA on ERK activity. Lastly, in contrast to a complete block of synaptic NMDA receptor activation of ERK by extrasynaptic NMDA receptors, activation of extrasynaptic NMDA receptors had no effect upon ERK activation by brain-derived neurotrophic factor. These results suggest that the synaptic NMDA receptor ERK activation pathway is coupled to both NR2A and NR2B containing receptors, and that the extrasynaptic NMDA receptor ERK inhibitory pathway is not a non-selective global ERK shutoff.

  2. Extracellular signal-regulated kinases 1 and 2 activation in endothelial cells exposed to cyclic strain

    Science.gov (United States)

    Ikeda, M.; Takei, T.; Mills, I.; Kito, H.; Sumpio, B. E.

    1999-01-01

    The aim of this study was to determine whether extracellular signal-regulated kinases 1/2 (ERK1/ERK2) are activated and might play a role in enhanced proliferation and morphological change induced by strain. Bovine aortic endothelial cells (BAEC) were subjected to an average of 6 or 10% strain at a rate of 60 cycles/min for up to 4 h. Cyclic strain caused strain- and time-dependent phosphorylation and activation of ERK1/ERK2. Peak phosphorylation and activation of ERK1/ERK2 induced by 10% strain were at 10 min. A specific ERK1/ERK2 kinase inhibitor, PD-98059, inhibited phosphorylation and activation of ERK1/ERK2 but did not inhibit the increased cell proliferation and cell alignment induced by strain. Treatment of BAEC with 2,5-di-tert-butyl-1, 4-benzohydroquinone, to deplete inositol trisphosphate-sensitive calcium storage, and gadolinium chloride, a Ca2+ channel blocker, did not inhibit the activation of ERK1/ERK2. Strain-induced ERK1/ERK2 activation was partly inhibited by the protein kinase C inhibitor calphostin C and completely inhibited by the tyrosine kinase inhibitor genistein. These data suggest that 1) ERK1/ERK2 are not critically involved in the strain-induced cell proliferation and orientation, 2) strain-dependent activation of ERK1/ERK2 is independent of intracellular and extracellular calcium mobilization, and 3) protein kinase C activation and tyrosine kinase regulate strain-induced activation of ERK1/ERK2.

  3. Induction of interleukin-8 by Naegleria fowleri lysates requires activation of extracellular signal-regulated kinase in human astroglial cells.

    Science.gov (United States)

    Kim, Jong-Hyun; Sohn, Hae-Jin; Lee, Sang-Hee; Kwon, Daeho; Shin, Ho-Joon

    2012-08-01

    Naegleria fowleri is a pathogenic free-living amoeba which causes primary amoebic meningoencephalitis in humans and experimental animals. To investigate the mechanisms of such inflammatory diseases, potential chemokine gene activation in human astroglial cells was investigated following treatment with N. fowleri lysates. We demonstrated that N. fowleri are potent inducers for the expression of interleukin-8 (IL-8) genes in human astroglial cells which was preceded by activation of extracellular signal-regulated kinase (ERK). In addition, N. fowleri lysates induces the DNA binding activity of activator protein-1 (AP-1), an important transcription factor for IL-8 induction. The specific mitogen-activated protein kinase kinase/ERK inhibitor, U0126, blocks N. fowleri-mediated AP-1 activation and subsequent IL-8 induction. N. fowleri-induced IL-8 expression requires activation of ERK in human astroglial cells. These findings indicate that treatment of N. fowleri on human astroglial cells leads to the activation of AP-1 and subsequent expression of IL-8 which are dependent on ERK activation. These results may help understand the N. fowleri-mediated upregulation of chemokine and cytokine expression in the astroglial cells.

  4. Inhibition of host extracellular signal-regulated kinase (ERK) activation decreases new world alphavirus multiplication in infected cells

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    Voss, Kelsey; Amaya, Moushimi [National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, 10650 Pyramid Place, Manassas, VA (United States); Mueller, Claudius [Center for Applied Proteomics and Personalized Medicine, George Mason University, 10900 University Boulevard, Manassas, VA (United States); Roberts, Brian [Leidos Health Life Sciences, 5202 Presidents Court, Suite 110, Frederick, MD (United States); Kehn-Hall, Kylene; Bailey, Charles [National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, 10650 Pyramid Place, Manassas, VA (United States); Petricoin, Emanuel [Center for Applied Proteomics and Personalized Medicine, George Mason University, 10900 University Boulevard, Manassas, VA (United States); Narayanan, Aarthi, E-mail: anaraya1@gmu.edu [National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, 10650 Pyramid Place, Manassas, VA (United States)

    2014-11-15

    New World alphaviruses belonging to the family Togaviridae are classified as emerging infectious agents and Category B select agents. Our study is focused on the role of the host extracellular signal-regulated kinase (ERK) in the infectious process of New World alphaviruses. Infection of human cells by Venezuelan equine encephalitis virus (VEEV) results in the activation of the ERK-signaling cascade. Inhibition of ERK1/2 by the small molecule inhibitor Ag-126 results in inhibition of viral multiplication. Ag-126-mediated inhibition of VEEV was due to potential effects on early and late stages of the infectious process. While expression of viral proteins was down-regulated in Ag-126 treated cells, we did not observe any influence of Ag-126 on the nuclear distribution of capsid. Finally, Ag-126 exerted a broad-spectrum inhibitory effect on New World alphavirus multiplication, thus indicating that the host kinase, ERK, is a broad-spectrum candidate for development of novel therapeutics against New World alphaviruses. - Highlights: • VEEV infection activated multiple components of the ERK signaling cascade. • Inhibition of ERK activation using Ag-126 inhibited VEEV multiplication. • Activation of ERK by Ceramide C6 increased infectious titers of TC-83. • Ag-126 inhibited virulent strains of all New World alphaviruses. • Ag-126 treatment increased percent survival of infected cells.

  5. NPNT is Expressed by Osteoblasts and Mediates Angiogenesis via the Activation of Extracellular Signal-regulated Kinase

    Science.gov (United States)

    Kuek, Vincent; Yang, Zhifan; Chim, Shek Man; Zhu, Sipin; Xu, Huazi; Chow, Siu To; Tickner, Jennifer; Rosen, Vicki; Erber, Wendy; Li, Xiucheng; An, Qin; Qian, Yu; Xu, Jiake

    2016-01-01

    Angiogenesis plays an important role in bone development and remodeling and is mediated by a plethora of potential angiogenic factors. However, data regarding specific angiogenic factors that are secreted within the bone microenvironment to regulate osteoporosis is lacking. Here, we report that Nephronectin (NPNT), a member of the epidermal growth factor (EGF) repeat superfamily proteins and a homologue of EGFL6, is expressed in osteoblasts. Intriguingly, the gene expression of NPNT is reduced in the bone of C57BL/6J ovariectomised mice and in osteoporosis patients. In addition, the protein levels of NPNT and CD31 are also found to be reduced in the tibias of OVX mice. Exogenous addition of mouse recombinant NPNT on endothelial cells stimulates migration and tube-like structure formation in vitro. Furthermore, NPNT promotes angiogenesis in an ex vivo fetal mouse metatarsal angiogenesis assay. We show that NPNT stimulates the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated kinase (MAPK) in endothelial cells. Inhibition of ERK1/2 impaired NPNT-induced endothelial cell migration, tube-like structure formation and angiogenesis. Taken together, these results demonstrate that NPNT is a paracrine angiogenic factor and may play a role in pathological osteoporosis. This may lead to new targets for treatment of bone diseases and injuries. PMID:27782206

  6. α1A-adrenergic receptor induces activation of extracellular signal-regulated kinase 1/2 through endocytic pathway.

    Directory of Open Access Journals (Sweden)

    Fei Liu

    Full Text Available G protein-coupled receptors (GPCRs activate mitogen-activated protein kinases through a number of distinct pathways in cells. Increasing evidence has suggested that endosomal signaling has an important role in receptor signal transduction. Here we investigated the involvement of endocytosis in α(1A-adrenergic receptor (α(1A-AR-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2. Agonist-mediated endocytic traffic of α(1A-AR was assessed by real-time imaging of living, stably transfected human embryonic kidney 293A cells (HEK-293A. α(1A-AR was internalized dynamically in cells with agonist stimulation, and actin filaments regulated the initial trafficking of α(1A-AR. α(1A-AR-induced activation of ERK1/2 but not p38 MAPK was sensitive to disruption of endocytosis, as demonstrated by 4°C chilling, dynamin mutation and treatment with cytochalasin D (actin depolymerizing agent. Activation of protein kinase C (PKC and C-Raf by α(1A-AR was not affected by 4°C chilling or cytochalasin D treatment. U73122 (a phospholipase C [PLC] inhibitor and Ro 31-8220 (a PKC inhibitor inhibited α(1B-AR- but not α(1A-AR-induced ERK1/2 activation. These data suggest that the endocytic pathway is involved in α(1A-AR-induced ERK1/2 activation, which is independent of G(q/PLC/PKC signaling.

  7. Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors

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    Rössler, Oliver G.; Glatzel, Daniel; Thiel, Gerald, E-mail: gerald.thiel@uks.eu

    2015-03-01

    Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interrupts the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1. - Highlights: • The plant polyphenol resveratrol upregulates Egr-1 expression and activity. • The stimulation of Egr-1 requires the protein kinases ERK and Raf. • Resveratrol treatment upregulates the transcriptional activation potential of Elk-1. • Resveratrol-induced stimulation of Egr-1 requires ternary complex factors. • Two distinct resveratrol-responsive elements were identified.

  8. Cue-elicited reward-seeking requires extracellular signal-regulated kinase activation in the nucleus accumbens.

    Science.gov (United States)

    Shiflett, Michael W; Martini, Ross P; Mauna, Jocelyn C; Foster, Rebecca L; Peet, Eloise; Thiels, Edda

    2008-02-01

    The motivation to seek out rewards can come under the control of stimuli associated with reward delivery. The ability of cues to motivate reward-seeking behavior depends on the nucleus accumbens (NAcc). The molecular mechanisms in the NAcc that underlie the ability of a cue to motivate reward-seeking are not well understood. We examined whether extracellular signal-regulated kinase (ERK), an important intracellular signaling pathway in learning and memory, has a role in these motivational processes. We first examined p42 ERK (ERK2) activation in the NAcc after rats were trained to associate an auditory stimulus with food delivery and found that, as a consequence of training, presentation of the auditory cue itself was sufficient to increase ERK2 activation in the NAcc. To examine whether inhibition of ERK in the NAcc prevents cue-induced reward-seeking, we infused an inhibitor of ERK, U0126, into the NAcc before assessing rats' instrumental responding in the presence versus absence of the conditioned cue. We found that, whereas vehicle-infused rats showed increased instrumental responding during cue presentation, rats infused with U0126 showed a profound impairment in cue-induced instrumental responding. In contrast, intra-NAcc U0126 infusion had no effect on rats' food-reinforced instrumental responding or their ability to execute conditioned approach behavior. Our results demonstrate learning-related changes in ERK signaling in the NAcc, and that disruption of ERK activation in this structure interferes with the incentive-motivational effects of conditioned stimuli. The molecular mechanisms described here may have implications for cue-elicited drug craving after repeated exposure to drugs of abuse.

  9. Extracellular signal-regulated kinase 1/2 signalling in SLE T cells is influenced by oestrogen and disease activity.

    Science.gov (United States)

    Gorjestani, S; Rider, V; Kimler, B F; Greenwell, C; Abdou, N I

    2008-06-01

    Systemic lupus erythematosus (SLE) is an autoimmune disease that occurs primarily in women of reproductive age. The disease is characterized by exaggerated T-cell activity and abnormal T-cell signalling. The mitogen-activated protein kinase (MAPK) pathway is involved in the maintenance of T-cell tolerance that fails in patients with SLE. Oestrogen is a female sex hormone that binds to nuclear receptors and alters the rate of gene transcription. Oestrogen can also act through the plasma membrane and rapidly stimulate second messengers including calcium flux and kinase activation. In this study, we investigated whether oestrogen influences the activation of MAPK signalling through the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in activated SLE T cells. SLE and control T cells were cultured in serum-free medium without and with oestradiol (10(-7) M) for 18 h. The T cells were activated with phorbol 12 myristate 13-acetate and ionomycin for various time points (0-60 min), and the amount of phosphorylated ERK1/2 was measured by immunoblotting. There were no differences in ERK1/2 phosphorylation between SLE and control T cells at 5 and 15 min after the activation stimulus. However, comparison between the amount of phosphorylated ERK1/2 in SLE T cells from the same patients cultured without and with oestradiol showed a significant oestrogen-dependent suppression (P=0.48) of ERK1/2 in patients with inactive/mild systemic lupus erythematosus disease activity index (SLEDAI) (0-2) compared with patients with moderate (4-6) or active (8-12) SLEDAI scores. These results suggest that the suppression of MAPK through ERK1/2 phosphorylation is sensitive to oestradiol in patients with inactive or mild disease, but the sensitivity is not maintained when disease activity increases. Furthermore, studies are now necessary to understand the mechanisms by which oestrogen influences MAPK activation in SLE T cells. PMID:18539708

  10. Cytoplasmic vacuolation in cultured rat astrocytes induced by an organophosphorus agent requires extracellular signal-regulated kinase activation

    International Nuclear Information System (INIS)

    There are various toxic chemicals that cause cell death. However, in certain cases deleterious agents elicit various cellular responses prior to cell death. To determine the cellular mechanisms by which such cellular responses are induced is important, but sufficient attention has not been paid to this issue to date. In this study, we showed the characteristic effects of an organophosphorus (OP) agent, bis(pinacolyl methyl)phosphonate (BPMP), which we synthesized for the study of OP nerve agents, on cultured rat astrocytes. Morphologically, BPMP induced cytoplasmic vacuolation and stellation in the rat astrocytes. Cytoplasmic vacuolation is a cell pathological change observed, for example, in vacuolar degeneration, and stellation has been reported in astrocytic reactions against various stimuli. By pretreatment with cycloheximide, a protein synthesis inhibitor, stellation was inhibited, although vacuolation was not. Cell staining with a mitochondrion-selective dye indicated that the vacuolation probably occurs in the mitochondria that are swollen and vacuolatred in the center. Interestingly, the extracellular signal-regulated kinase (ERK) cascade inhibitor inhibited vacuolation and, to some extent, stellation. These results suggest that the ERK signaling cascade is important for the induction of mitochondrial vacuolation. We expect that a detailed study of these astrocytic reactions will provide us new perspectives regarding the variation and pathological significance of cell morphological changes, such as vacuolar degeneration, and also the mechanisms underlying various neurological disorders

  11. Localization of active, dually phosphorylated extracellular signal-regulated kinase 1 and 2 in colorectal cancer with or without activating BRAF and KRAS mutations

    DEFF Research Database (Denmark)

    Holck, Susanne; Bonde, Jesper; Pedersen, Helle;

    2016-01-01

    . The same applied to 4 of the 14 KRAS(m) tumors. A phophorylation-insensitive antibody demonstrated that lack of pERK staining did not reflect defect expression of ERK1/2 protein. Thus, increased staining for pERK does not correlate to BRAF or KRAS mutations even with a highly optimized procedure. Further......Colorectal cancers (CRC) often show activating mutations of the KRAS or BRAF genes, which stimulate the extracellular signal-regulated kinase (ERK) pathway, thus increasing cell proliferation and inhibiting apoptosis. However, immunohistochemical results on ERK activation in such tumors differ...... greatly. Recently, using a highly optimized immunohistochemical method, we obtained evidence that high levels of ERK activation in rectal adenocarcinomas were associated with resistance to radiochemotherapy. In order to determine whether KRAS and/or BRAF mutations correlate to immunohistochemically...

  12. Neuronal extracellular signal-regulated kinase (ERK activity as marker and mediator of alcohol and opioid dependence

    Directory of Open Access Journals (Sweden)

    Eva R. Zamora-Martinez

    2014-03-01

    Full Text Available Early pioneering work in the field of biochemistry identified phosphorylation as a crucial post-translational modification of proteins with the ability to both indicate and arbitrate complex physiological processes. More recent investigations have functionally linked phosphorylation of extracellular signal-regulated kinase (ERK to a variety of neurophysiological mechanisms ranging from acute neurotransmitter action to long-term gene expression. ERK phosphorylation serves as an intracellular bridging mechanism that facilitates neuronal communication and plasticity. Drugs of abuse, including alcohol and opioids, act as artificial yet powerful rewards that impinge upon natural reinforcement processes critical for survival. The graded progression from initial exposure to addiction (or substance dependence is believed to result from drug- and drug context-induced adaptations in neuronal signaling processes across brain reward and stress circuits following excessive drug use. In this regard, commonly abused drugs as well as drug-associated experiences are capable of modifying the phosphorylation of ERK within central reinforcement systems. In addition, chronic drug and alcohol exposure may drive ERK-regulated epigenetic and structural alterations that underlie a long-term propensity for escalating drug use. Under the influence of such a neurobiological vulnerability, encountering drug-associated cues and contexts can produce subsequent alterations in ERK signaling that drive relapse to drug and alcohol seeking. Current studies are determining precisely which molecular and regional ERK phosphorylation-associated events contribute to the addiction process, as well as which neuroadaptations need to be targeted in order to return dependent individuals to a healthy state.

  13. Organ culture of the trigeminal ganglion induces enhanced expression of calcitonin gene-related peptide via activation of extracellular signal-regulated protein kinase 1/2

    DEFF Research Database (Denmark)

    Tajti, János; Kuris, Anikó; Vécsei, László;

    2011-01-01

    , phosphorylated ERK1/2 (pERK1/2), total ERK1/2 (tERK1/2), phosphorylated p38 (pp38), phosphorylated C-Jun-N-terminal protein kinase (pJNK), pro-calcitonin (pro-CT), CGRP receptor activity modifying protein 1 (RAMP1), glutamine synthetase (GS) and pro-CT were used. To explore molecular mechanisms involved......BACKGROUND AND OBJECTIVE: Clinical and experimental studies have revealed a central role of calcitonin gene-related peptide (CGRP) in primary headaches. The role of extracellular signal-regulated kinase 1 and 2 (ERK1/2) in neuronal and glial cell expression of CGRP- immunoreactivity (-ir) in rat...

  14. The effect of active and passive intravenous cocaine administration on the extracellular signal-regulated kinase (ERK) activity in the rat brain.

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    Miszkiel, Joanna; Detka, Jan; Cholewa, Joanna; Frankowska, Małgorzata; Nowak, Ewa; Budziszewska, Bogusława; Przegaliński, Edmund; Filip, Małgorzata

    2014-08-01

    According to a current hypothesis of learning processes, recent papers pointed out to an important role of the extracellular signal-regulated kinase (ERK), in drug addiction. We employed the Western blotting techniques to examine the ERK activity immediately after cocaine iv self-administration and in different drug-free withdrawal periods in rats. To distinguish motivational vs. pharmacological effects of the psychostimulant intake, a "yoked" procedure was used. Animals were decapitated after 14 daily cocaine self-administration sessions or on the 1st, 3rd or 10th extinction days. At each time point the activity of the ERK was assessed in several brain structures, including the prefrontal cortex, hippocampus, dorsal striatum and nucleus accumbens. Passive, repeated iv cocaine administration resulted in a 45% increase in ERK phosphorylation in the hippocampus while cocaine self-administration did not change brain ERK activity. On the 1st day of extinction, the activity of the ERK in the prefrontal cortex was decreased in rats with a history of cocaine chronic intake: by 66% for "active" cocaine group and by 35% for "yoked" cocaine group. On the 3rd day the reduction in the ERK activity (25-34%) was observed in the hippocampus for both cocaine-treated groups, and also in the nucleus accumbens for "yoked" cocaine group (40%). On the 10th day of extinction there was no significant alteration in ERK activity in any group of rats. Our findings suggest that cortical ERK is involved in cocaine seeking behavior in rats. They also indicate the time and regional adaptations in this enzyme activity after cocaine withdrawal. PMID:24948065

  15. Pre-LTP requires extracellular signal-regulated kinase in the ACC

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    Yamanaka, Manabu; Tian, Zhen; Darvish-Ghane, Soroush

    2016-01-01

    The extracellular signal-regulated kinase is an important protein kinase for cortical plasticity. Long-term potentiation in the anterior cingulate cortex is believed to play important roles in chronic pain, fear, and anxiety. Previous studies of extracellular signal-regulated kinase are mainly focused on postsynaptic form of long-term potentiation (post-long-term potentiation). Little is known about the relationship between extracellular signal-regulated kinase and presynaptic long-term potentiation (pre-long-term potentiation) in cortical synapses. In this study, we examined whether pre-long-term potentiation in the anterior cingulate cortex requires the activation of presynaptic extracellular signal-regulated kinase. We found that p42/p44 mitogen-activated protein kinase inhibitors, PD98059 and U0126, suppressed the induction of pre-long-term potentiation. By contrast, these inhibitors did not affect the maintenance of pre-long-term potentiation. Using pharmacological inhibitors, we found that pre-long-term potentiation recorded for 1 h did not require transcriptional or translational processes. Our results strongly indicate that the activation of presynaptic extracellular signal-regulated kinase is required for the induction of pre-long-term potentiation, and this involvement may explain the contribution of extracellular signal-regulated kinase to mood disorders. PMID:27178245

  16. Agonist-induced activation of histamine H3 receptor signals to extracellular signal-regulated kinases 1 and 2 through PKC-, PLD-, and EGFR-dependent mechanisms.

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    Lai, Xiangru; Ye, Lingyan; Liao, Yuan; Jin, Lili; Ma, Qiang; Lu, Bing; Sun, Yi; Shi, Ying; Zhou, Naiming

    2016-04-01

    The histamine H3 receptor (H3R), abundantly expressed in the central and the peripheral nervous system, has been recognized as a promising target for the treatment of various important CNS diseases including narcolepsy, Alzheimer's disease, and attention deficit hyperactivity disorder. The H3R acts via Gi/o -proteins to inhibit adenylate cyclase activity and modulate MAPK activity. However, the underlying molecular mechanisms for H3R mediation of the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) remain to be elucidated. In this study, using HEK293 cells stably expressing human H3R and mouse primary cortical neurons endogenously expressing mouse H3R, we found that the H3R-mediated activation of ERK1/2 was significantly blocked by both the pertussis toxin and the MEK1/2 inhibitor U0126. Upon stimulation by H3R agonist histamine or imetit, H3R was shown to rapidly induce ERK1/2 phosphorylation via PLC/PKC-, PLDs-, and epidermal growth factor receptor (EGFR) transactivation-dependent pathways. Furthermore, it was also indicated that while the βγ-subunits play a key role in H3R-activated ERK1/2 phosphorylation, β-arrestins were not required for ERK1/2 activation. In addition, when the cultured mouse cortical neurons were exposed to oxygen and glucose deprivation conditions (OGD), imetit exhibited neuroprotective properties through the H3R. Treatment of cells with the inhibitor UO126 abolished these protective effects. This suggests a possible neuroprotective role of the H3R-mediated ERK1/2 pathway under hypoxia conditions. These observations may provide new insights into the pharmacological effects and the physiological functions modulated by the H3R-mediated activation of ERK1/2. Histamine H3 receptors are abundantly expressed in the brain and play important roles in various CNS physiological functions. However, the underlying mechanisms for H3R-induced activation of extracellular signal-regulated kinase (ERK)1/2 remain largely unknown. Here

  17. Differential extracellular signal-regulated kinases 1 and 2 activation by the angiotensin type 1 receptor supports distinct phenotypes of cardiac myocytes

    DEFF Research Database (Denmark)

    Aplin, Mark; Christensen, Gitte Lund; Schneider, Mikael;

    2007-01-01

    The angiotensin II (AngII) type 1 receptor (AT(1)R) is a seven-transmembrane receptor well established to activate extracellular signal-regulated kinases 1 and 2 (ERK1/2) by discrete G protein-dependent and beta-arrestin2-dependent pathways. The biological importance of this, however, remains...... obscure. Application of the modified analogue [Sar(1), Ile(4), Ile(8)]-AngII ([SII] AngII) allowed us to dissect the two pathways of ERK1/2 activation in native cardiac myocytes. Although cytosol-retained, the beta-arrestin2-bound pool of ERK1/2 represents an active signalling component...... that phosphorylates p90 Ribosomal S6 Kinase, a ubiquitous and versatile mediator of ERK1/2 signal transduction. Moreover, the beta-arrestin2-dependent ERK1/2 signal supports intact proliferation of cardiac myocytes. In contrast to G(q)-activated ERK1/2, and in keeping with its failure to translocate to the nucleus...

  18. Contribution of phospholipase D in endothelin-1-mediated extracellular signal-regulated kinase activation and proliferation in rat uterine leiomyoma cells.

    Science.gov (United States)

    Robin, Philippe; Chouayekh, Sondes; Bole-Feysot, Christine; Leiber, Denis; Tanfin, Zahra

    2005-01-01

    Endothelin (ET)-1 is a mitogenic factor in numerous cell types, including rat myometrial cells. In the present study, we investigated the potential role of ET-1 in the proliferation of tumoral uterine smooth muscle cells (ELT-3 cells). We found that ET-1 exerted a more potent mitogenic effect in ELT-3 cells than in normal myometrial cells, as indicated by the increase in [3H]thymidine incorporation, cell number, and bromodeoxyuridine incorporation. The ET-1 was more efficient than platelet-derived growth factor and epidermal growth factor to stimulate proliferation. The ET-1-mediated cell proliferation was inhibited in the presence of U0126, a specific inhibitor of (mitogen-activated protein kinase ERK kinase), indicating that extracellular signal-regulated kinase (ERK) activation is involved. Additionally, ET-1 induced the activation of phospholipase (PL) D, leading to the synthesis of phosphatidic acid (PA). The ET-1-induced activation of PLD was twofold higher in ELT-3 cells compared to that in normal cells. The two cell types expressed mRNA for PLD1a and PLD2, whereas PLD1b was expressed only in ELT-3 cells. The exposure of cells to butan-1-ol reduced ET-1-mediated production of PA by PLD and partially inhibited ERK activation and DNA synthesis. Addition of exogenous PLD or PA in the medium reproduced the effect of ET-1 on ERK activation and cell proliferation. Collectively, these data indicate that ET-1 is a potent mitogenic factor in ELT-3 cells via a signaling pathway involving a PLD-dependent activation of ERK. This highlights the potential role of ET-1 in the development of uterine leiomyoma, and it reinforces the role of PLD in tumor growth. PMID:15355882

  19. Inhibition of Adipocyte Differentiation by Phytoestrogen Genistein Through a Potential Downregulation of Extracellular Signal-Regulated Kinases 1/2 Activity

    Science.gov (United States)

    Liao, Qing-Chuan; Li, Ya-Lin; Qin, Yan-Fang; Quarles, L. Darryl; Xu, Kang-Kang; Li, Rong; Zhou, Hong-Hao; Xiao, Zhou-Sheng

    2016-01-01

    In the current study, we investigated the effects of genistein on adipogenic differentiation of mouse bone marrow-derived mesenchymal stem cell (BMSC) cultures and its potential signaling pathway. The terminal adipogenic differentiation was assessed by western-blotting analysis of adipogenic-specific proteins such as PPARγ, C/EBPα, and aP2 and the formation of adipocytes. Treatment of mouse BMSC cultures with adipogenic cocktail resulted in sustained activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), which are members of the mitogen-activated protein kinase (MAPK) family, at the early phase of adipogenesis (from days 3 to 9). Inhibition of ERK1/2 activation by PD98059, a specific MEK inhibitor, reversed the induced adipogenic differentiation. Genistein dose-dependently decreased the phosphorylation of ERK1/2 in mouse BMSC cultures. Genistein incubation for the entire culture period, as well as that applied during the early phase of the culture period, significantly inhibited the adipogenic differentiation of mouse BMSC cultures. While genistein was incubated at the late stage (after day 9), no inhibitory effect on adipogenic differentiation was observed. BMSC cultures treated with genistein in the presence of fibroblast growth factor-2 (FGF-2), an activator of the ERK1/2 signaling pathway, expressed normal levels of ERK1/2 activity, and, in so doing, are capable of undergoing adipogenesis. Our results suggest that activation of the ERK1/2 signaling pathway during the early phase of adipogenesis (from days 3 to 9) is essential to adipogenic differentiation of BMSC cultures, and that genistein inhibits the adipogenic differentiation through a potential downregulation of ERK1/2 activity at this early phase of adipogenesis. PMID:18384126

  20. Xenin-induced feeding suppression is not mediated through the activation of central extracellular signal-regulated kinase signaling in mice.

    Science.gov (United States)

    Kim, Eun Ran; Lew, Pei San; Spirkina, Alexandra; Mizuno, Tooru M

    2016-10-01

    Xenin is a gut hormone that reduces food intake by partly acting through the hypothalamus via neurotensin receptor 1 (Ntsr1). However, specific signaling pathways that mediate xenin-induced feeding suppression are not fully understood. Activation of Ntsr1 leads to the activation of the extracellular signal-regulated kinase (ERK). Hypothalamic ERK participates in the regulation of food intake by mediating the effect of hormonal signals. Therefore, we hypothesized that the anorectic effect of xenin is mediated by hypothalamic ERK signaling. To address this hypothesis, we compared levels of phosphorylation of ERK1/2 (pERK1/2) in the hypothalamus of both control and xenin-treated mice. The effect of xenin on ERK1/2 phosphorylation was also examined in mouse hypothalamic neuronal cell lines with or without Ntsr1. We also examined the effect of the blockade of central ERK signaling on xenin-induced feeding suppression in mice. The intraperitoneal (i.p.) injection of xenin caused a significant increase in the number of pERK1/2-immunoreactive cells in the hypothalamic arcuate nucleus. The majority of pERK1/2-positive cells expressed neuronal nuclei (NeuN), a marker for neurons. Xenin treatment increased pERK1/2 levels in one cell line expressing Ntsr1 but not another line without Ntsr1 expression. Both i.p. and intracerebroventricular (i.c.v.) injections of xenin reduced food intake in mice. The i.c.v. pre-treatment with U0126, a selective inhibitor of ERK1/2 upstream kinases, did not affect xenin-induced reduction in food intake. These findings suggest that although xenin activates ERK signaling in subpopulations of hypothalamic neurons, xenin does not require the activation of hypothalamic ERK signaling pathway to elicit feeding suppression. PMID:27316340

  1. Downregulation of Aquaporin 4 Expression through Extracellular Signal-regulated Kinases1/2 Activation in Cultured Astrocytes Following Scratch-injury

    Institute of Scientific and Technical Information of China (English)

    SHI Zhong Fang; ZHAO Wei Jiang; XU Li Xin; DONG Li Ping; YANG Shao Hua; YUAN Fang

    2015-01-01

    ObjectiveTo investigate the role of extracellular signal-regulated kinase1/2 (ERK1/2) pathway in the regulation of aquaporin 4 (AQP4) expression inculturedastrocytes after scratch-injury. MethodsThe scratch-injury model was produced in cultured astrocytes of rat by a 10-μL plastic pipette tip. The morphological changes of astrocytes and lactate dehydrogenase (LDH) leakages were observed to assess the degree of scratch-injury. AQP4 expressionwas detected by immunofluorescence staining and Western blot, and phosphorylated-ERK1/2 (p-ERK1/2) expression was determined by Western blot. To explore the effect of ERK1/2 pathway on AQP4 expression in scratch-injured astrocytes, 10 µmol/L U0126 (ERK1/2inhibitor) was incubated in the medium at 30 min before the scratch-injury in some groups. ResultsIncreases in LDH leakage were observed at 1, 12, and 24 h after scratch-injury, and AQP4 expression was reduced simultaneously. Decrease in AQP4 expressionwas associated with a significant increase in ERK1/2 activation. Furthermore, pretreatment with U0126 blocked both ERK1/2 activation and decrease in AQP4 expression induced by scratch-injury. ConclusionThese results indicate that ERK1/2 pathway down-regulates AQP4 expression in scratch-injured astrocytes, and ERK1/2 pathway might be a novel therapeutic target in reversing the effects of astrocytes that contribute to traumatic brain edema.

  2. Atorvastatin induces autophagy of mesenchymal stem cells under hypoxia and serum deprivation conditions by activating the mitogenactivated protein kinase/extracellular signal-regulated kinase pathway

    Institute of Scientific and Technical Information of China (English)

    Li Na; Zhang Qian; Qian Haiyan; Jin Chen; Yang Yuejin; Gao Runlin

    2014-01-01

    Background The survival ratio of implanted mesenchymal stem cells (MSCs) in the infarcted myocardium is low.Autophagy is a complex "self-eating" process and can be utilized for cell survival.We have found that atorvastatin (ATV) can effectively activate autophagy to enhance MSCs survival during hypoxia and serum deprivation (H/SD).The mitogenactivated protein kinase/extracellular signal-regulated kinase (MEK/ERK) pathway is a non-canonical autophagy pathway.We hypothesized that the MEK/ERK pathway mediated ATV-induced autophagy of MSCs under H/SD.Methods MSCs were pretreated with ATV (0.01-10 μmol/L) under H/SD for three hours.For inhibitor studies,the cells were pre-incubated with the MEK1/2 inhibitor U0126.Cell autophagy was assessed by acidic vesicular organelles (AVO)-positive cells using flow cytometry,autophagy related protein using Western blotting and autophagosome using transmission electron microscopy.Results Autophagy was elevated in the H/SD group compared with the normal group.ATV further enhanced the autophagic activity as well as the phosphorylation of ERK1/2 evidenced by more AVO-positive cells ((8.63±0.63)% vs.(5.77±0.44)%,P <0.05),higher LC3-Ⅱ/LC3-Ⅰ ratio (4.36±0.31 vs.2.52±0.18,P <0.05) and more autophagosomes.And treatment with U0126 downregulated the phosphorylation of ERK1/2 and attenuated ATV-induced autophagy.Conclusion The MEK/ERK pathway participates in ATV-induced autophagy in MSCs under H/SD,and modulation of the pathway could be a novel strategy to improve MSCs survival.

  3. Corticotrigeminal projections from the insular cortex to the trigeminal caudal subnucleus regulate orofacial pain after nerve injury via extracellular signal-regulated kinase activation in insular cortex neurons

    Directory of Open Access Journals (Sweden)

    Jian eWang

    2015-12-01

    Full Text Available Cortical neuroplasticity alterations are implicated in the pathophysiology of chronic orofacial pain. However, the relationship between critical cortex excitability and orofacial pain maintenance has not been fully elucidated. We recently demonstrated a top-down corticospinal descending pain modulation pathway from the anterior cingulate cortex (ACC to the spinal dorsal horn that could directly regulate nociceptive transmission. Thus, we aimed to investigate possible corticotrigeminal connections that directly influence orofacial nociception in rats. Infraorbital nerve chronic constriction injury (IoN-CCI induced significant orofacial nociceptive behaviors as well as pain-related negative emotions such as anxiety/depression in rats. By combining retrograde and anterograde tract tracing, we found powerful evidence that the trigeminal caudal subnucleus (Vc, especially the superficial laminae (I/II, received direct descending projections from granular and dysgranular parts of the insular cortex (IC. Extracellular signal-regulated kinase (ERK, an important signaling molecule involved in neuroplasticity, was significantly activated in the IC following IoN-CCI. Moreover, in IC slices from IoN-CCI rats, U0126, an inhibitor of ERK activation, decreased both the amplitude and the frequency of spontaneous excitatory postsynaptic currents (sEPSCs and reduced the paired-pulse ratio (PPR of Vc-projecting neurons. Additionally, U0126 also reduced the number of action potentials in the Vc-projecting neurons. Finally, intra-IC infusion of U0126 obviously decreased Fos expression in the Vc, accompanied by the alleviation of both nociceptive behavior and negative emotions. Thus, the corticotrigeminal descending pathway from the IC to the Vc could directly regulate orofacial pain, and ERK deactivation in the IC could effectively alleviate neuropathic pain as well as pain-related negative emotions in IoN-CCI rats, probably through this top-down pathway. These

  4. Calcitonin gene-related peptide induces proliferation and monocyte chemoattractant protein-1 expression via extracellular signal-regulated kinase activation in rat osteoblasts

    Institute of Scientific and Technical Information of China (English)

    HAN Na; ZHANG Dian-ying; WANG Tian-bing; ZHANG Pei-xun; JIANG Bao-guo

    2010-01-01

    Background Calcitonin gene-related peptide (CGRP), a sensory neuropeptide, affects osteoblast proliferation and bone formation. However, the mechanisms are not fully understood. Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that stimulates the migration of monocytes and plays important roles in regulating bone remolding during fracture repair, In this study, we investigated the effects of CGRP on proliferation and MCP-1 expression in cultured rat osteoblasts.Methods Primary rat osteoblasts were isolated from fetal rats calvariae. Cells were exposed to gradient concentrations (10-9 to 10-7 mol/L) of CGRP. Protein and mRNA levels of MCP-1 were quantified by Western blotting and semiquantitative reverse transcription-polymerase chain reaction, respectively. The protein level of MCP-1 was investigated and compared in cell culture media by enzyme linked immunosorbent assay (ELISA). Phospho-extracellular signal-regulated kinase (ERK) expression was detected by Western blotting. Cell proliferative activity was measured by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) and BrdU assay. The effects of MAPK/ERK kinase (MEK)-inhibitor U0126 on CGRP-induced MCP-1 expression in primary rat osteoblasts were examined.Results CGRP effectively enhanced primary rat osteoblast proliferation and led to significant increases in the expression of MCP-1 mRNA and protein in time- and dose-dependent manners. CGRP activated the ERK pathway.Pretreatment of cultured rat osteoblasts with MEK inhibitor U0126 resulted in dose-dependent inhibitions of CGRP-induced MCP-1 mRNA and protein levels. Thus, CGRP promoted cell proliferation and stimulated MCP-1 expression in cultured rat osteoblasts.Conclusion These studies document novel links between CGRP and MCP-1 and illuminate the effects of CGRP in regulating bone remodeling.

  5. Imbalanced expression of mitogen-activated protein kinase phosphatase-1 and phosphorylated extracellular signal-regulated kinases in lung squamous cell carcinoma

    Institute of Scientific and Technical Information of China (English)

    Kai WANG; Min ZHANG; Ying-ying QIAN; Zhe-yuan DING; Jun-huiLV; Hua-hao SHEN

    2011-01-01

    Objective:Mitogen-activated protein kinases (MAPKs) are correlated with a more malignant phenotype in many cancers.This study was designed to evaluate the predictive value of the expression of MAPK phosphatase-1 (MKP-1) and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2),as the key regulatory mechanism of the MAPKs,in lung squamous cell carcinoma (SCC).Methods:We assessed the expressions of MKP-1 and p-ERK1/2in twenty subjects at different differentiation degree of SCC and five normal lungs by immunohistochemistry and real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis.Results:Immunohistochemistry and real-time RT-PCR assay showed that the expression of MKP-1 was gradually decreased as tissue type went from normal lung tissues to increasingly undifferentiated carcinoma,and it was negatively correlated with tumor differentiation (P<0.01).However,the expression of p-ERK1/2 or ERK1/2 was gradually increased as tissue type went from normal lung tissues to increasingly undifferentiated carcinoma,and it was positively correlated with tumor differentiation (P<0.01).Conclusions:Our data indicates the relevance of MKP-1 and p-ERK1/2 in SCC as a potential positive and negative prognostic factor.The imbalanced expression of MKP-1 and p-ERK1/2 may play a role in the development of SCC and these two molecules may be new targets for the therapy and prognosis of SCC.

  6. Region- or state-related differences in expression and activation of extracellular signal-regulated kinases (ERKs in naïve and pain-experiencing rats

    Directory of Open Access Journals (Sweden)

    Cui Xiu-Yu

    2007-07-01

    Full Text Available Abstract Background Extracellular signal-regulated kinase (ERK, one member of the mitogen-activated protein kinase (MAPK family, has been suggested to regulate a diverse array of cellular functions, including cell growth, differentiation, survival, as well as neuronal plasticity. Recent evidence indicates a role for ERKs in nociceptive processing in both dorsal root ganglion and spinal cord. However, little literature has been reported to examine the differential distribution and activation of ERK isoforms, ERK1 and ERK2, at different levels of pain-related pathways under both normal and pain states. In the present study, quantitative blot immunolabeling technique was used to determine the spatial and temporal expression of ERK1 and ERK2, as well as their activated forms, in the spinal cord, primary somatosensory cortex (SI area of cortex, and hippocampus under normal, transient pain and persistent pain states. Results In naïve rats, we detected regional differences in total expression of ERK1 and ERK2 across different areas. In the spinal cord, ERK1 was expressed more abundantly than ERK2, while in the SI area of cortex and hippocampus, there was a larger amount of ERK2 than ERK1. Moreover, phosphorylated ERK2 (pERK2, not phosphorylated ERK1 (pERK1, was normally expressed with a high level in the SI area and hippocampus, but both pERK1 and pERK2 were barely detectable in normal spinal cord. Intraplantar saline or bee venom injection, mimicking transient or persistent pain respectively, can equally initiate an intense and long-lasting activation of ERKs in all three areas examined. However, isoform-dependent differences existed among these areas, that is, pERK2 exhibited stronger response than pERK1 in the spinal cord, whereas ERK1 was more remarkably activated than ERK2 in the S1 area and hippocampus. Conclusion Taken these results together, we conclude that: (1 under normal state, while ERK immunoreactivity is broadly distributed in the rat

  7. Involvement of extracellular signal-regulated kinase/mitogen activated protein kinase pathway in multidrug resistance induced by HBx in hepatoma cell line

    Institute of Scientific and Technical Information of China (English)

    Jian Guan; Xiao-Ping Chen; Hong Zhu; Shun-Feng Luo; Bin Cao; Lei Ding

    2004-01-01

    AIM: To investigate the molecular mechanism of the influence of HBx protein on multidrug resistance associated genes:multidrug resistance 1 (MDR-1), multidrug related protein (MRP-1), lung resistance related protein (LRP) in hepatoma cells and the potential role of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway in this process.METHODS: A cell model stably expressing the HBx protein was established by liposome-mediated transfection of HBx gene into HepG2 cell line. The expression of multidrug resistance associated genes and proteins was detected by RT-PCR and Western blot. AnnexinV-FITC/PI assay was used to confirm the multidrug resistance (MDR) phenotype of transfected cells by fluorescence cytometry (FACS). The ERK/MAPK pathway activation was measured by Western blot through comparing the ratio of phosphorylation of ERK/MAPK to total ERK/MAPK protein. After treated with the ERK/MAPK pathway inhibitor U0126, the HBx-expressing cells were harvested. Then RT-PCR, Western blot and FACS were used to analyze the alterations in the expression of multidrug resistance associated genes and the MDR phenotype after exposure.RESULTS: Compared with the control group, the transfected cells showed a higher expression of MDR associated genes and proteins. Marked elevations in MDR-1 (64.3%), MRP-1 (87.5%) and LRP (90.8%) were observed in the transfected cells (P<0.05). RT-PCR revealed that the over-expression of MDR associated proteins was due to amplification of such genes (MDR1 2.9 fold, MRP1 1.67 fold, LRP1.95 fold).Furthermore, we found that the ERK/MAPK activity was remarkably high in the HBx-expressing cells. The activation of ERK/MAPK, as measured by the ratio of phosphorylated ERK bands normalized to the total ERK bands, was increased by 2.3-fold in HBx-transfected cells compared with cells transfected with the empty vector. After treated with the ERK/MAPK pathway inhibitor, the level of MDR associated genes and proteins in the

  8. The angiotensin type 1 receptor activates extracellular signal-regulated kinases 1 and 2 by G protein-dependent and -independent pathways in cardiac myocytes and langendorff-perfused hearts

    DEFF Research Database (Denmark)

    Aplin, Mark; Christensen, Gitte Lund; Schneider, Mikael;

    2007-01-01

    The angiotensin II (AngII) type 1 receptor (AT(1)R) has been shown to activate extracellular signal-regulated kinases 1 and 2 (ERK1/2) through G proteins or G protein-independently through beta-arrestin2 in cellular expression systems. As activation mechanisms may greatly influence the biological...... effects of ERK1/2 activity, differential activation of the AT(1)R in its native cellular context could have important biological and pharmacological implications. To examine if AT(1)R activates ERK1/2 by G protein-independent mechanisms in the heart, we used the [Sar(1), Ile(4), Ile(8)]-AngII ([SII] Ang......II) analogue in native preparations of cardiac myocytes and beating hearts. We found that [SII] AngII does not activate G(q)-coupling, yet stimulates the beta-arrestin2-dependent ERK1/2. The G(q)-activated pool of ERK1/2 rapidly translocates to the nucleus, while the beta-arrestin2-scaffolded pool remains...

  9. Leptin stimulates pituitary prolactin release through an extracellular signal-regulated kinase-dependent pathway

    DEFF Research Database (Denmark)

    Tipsmark, Christian K; Strom, Christina N; Bailey, Sean T;

    2008-01-01

    of the actions of leptin are mediated by the activation of extracellular signal-regulated kinase (ERK1/2) but nothing is known about the cellular mechanisms by which leptin might regulate PRL secretion in vertebrates. We therefore tested whether ERK1/2 might be involved in the leptin PRL response and found...... that the ERK inhibitor, PD98059, hindered leptin-induced PRL release. We further analyzed leptin response by quantifying tyrosine and threonine phosphorylation of ERK1/2 using western blots. One hour incubation with leptin induced a concentration-dependent increase in phosphorylated, and thus active, ERK1/2....... Our data show that leptin is a powerful stimulator of in vitro PRL release and that its actions occur in part through stimulation of ERK1/2....

  10. Association between GRB2/Sos and insulin receptor substrate 1 is not sufficient for activation of extracellular signal-regulated kinases by interleukin-4: implications for Ras activation by insulin.

    Science.gov (United States)

    Pruett, W; Yuan, Y; Rose, E; Batzer, A G; Harada, N; Skolnik, E Y

    1995-03-01

    Insulin receptor substrate 1 (IRS-1) mediates the activation of a variety of signaling pathways by the insulin and insulin-like growth factor 1 receptors by serving as a docking protein for signaling molecules with SH2 domains. We and others have shown that in response to insulin stimulation IRS-1 binds GRB2/Sos and have proposed that this interaction is important in mediating Ras activation by the insulin receptor. Recently, it has been shown that the interleukin (IL)-4 receptor also phosphorylates IRS-1 and an IRS-1-related molecule, 4PS. Unlike insulin, however, IL-4 fails to activate Ras, extracellular signal-regulated kinases (ERKs), or mitogen-activated protein kinases. We have reconstituted the IL-4 receptor into an insulin-responsive L6 myoblast cell line and have shown that IRS-1 is tyrosine phosphorylated to similar degrees in response to insulin and IL-4 stimulation in this cell line. In agreement with previous findings, IL-4 failed to activate the ERKs in this cell line or to stimulate DNA synthesis, whereas the same responses were activated by insulin. Surprisingly, IL-4's failure to activate ERKs was not due to a failure to stimulate the association of tyrosine-phosphorylated IRS-1 with GRB2/Sos; the amounts of GRB2/Sos associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. Moreover, the amounts of phosphatidylinositol 3-kinase activity associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. In contrast to insulin, however, IL-4 failed to induce tyrosine phosphorylation of Shc or association of Shc with GRB2. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Previous studies have indicated that activation of ERks in this cell line is dependent upon Ras since a dominant-negative Ras (Asn-17) blocks ERK activation by insulin. Our findings, taken in the context

  11. Prorenin and renin-induced extracellular signal-regulated kinase 1/2 activation in monocytes is not blocked by aliskiren or the handle-region peptide

    NARCIS (Netherlands)

    S. Feldt (Sandra); W.W. Batenburg (Wendy); I. Mazak (Istvan); U. Maschke (Ulrike); M. Wellner (Maren); H. Kvakan (Heda); R. Dechend (Ralf); A. Fiebeler (Anette); C. Burckle (Celine); A. Contrepas (Aurelie); A.H.J. Danser (Jan); M. Bader (Michael); G. Nguyen (Genevieve); F.C. Luft (Friedrich); D. Müller (Dominik)

    2008-01-01

    textabstractThe recently cloned (pro)renin receptor [(P)RR] mediates renin-stimulated cellular effects by activating mitogen-activated protein kinases and promotes nonproteolytic prorenin activation. In vivo, (P)RR is said to be blocked with a peptide consisting of 10 amino acids from the prorenin p

  12. Regulation of extracellular signal-regulated kinase 1/2 inlfuences hippocampal neuronal survival in a rat model of diabetic cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    Yaning Zhao; Jianmin Li; Qiqun Tang; Pan Zhang; Liwei Jing; Changxiang Chen; Shuxing Li

    2014-01-01

    Activation of extracellular signal-regulated kinase 1/2 has been demonstrated in acute brain ischemia. We hypothesized that activated extracellular signal-regulated kinase 1/2 can protect hippocampal neurons from injury in a diabetic model after cerebral ischemia/reperfusion. In this study, transient whole-brain ischemia was induced by four-vessel occlusion in normal and diabetic rats, and extracellular signal-regulated kinase 1/2 inhibitor (U0126) was administered into diabetic rats 30 minutes before ischemia as a pretreatment. Results showed that the number of surviving neurons in the hippocampal CA1 region was reduced, extracellular signal-regulated kinase 1/2 phosphorylation and Ku70 activity were decreased, and pro-apoptotic Bax expression was upregulated after intervention using U0126. These ifndings demonstrate that inhibition of extracellular signal-regulated kinase 1/2 activity aggravated neuronal loss in the hippocampus in a diabetic rat after cerebral ischemia/reperfusion, further decreased DNA repairing ability and ac-celerated apoptosis in hippocampal neurons. Extracellular signal-regulated kinase 1/2 activation plays a neuroprotective role in hippocampal neurons in a diabetic rat after cerebral ischemia/reperfusion.

  13. Ras-mutant cancer cells display B-Raf binding to Ras that activates extracellular signal-regulated kinase and is inhibited by protein kinase A phosphorylation.

    Science.gov (United States)

    Li, Yanping; Takahashi, Maho; Stork, Philip J S

    2013-09-20

    The small G protein Ras regulates proliferation through activation of the mitogen-activated protein (MAP) kinase (ERK) cascade. The first step of Ras-dependent activation of ERK signaling is Ras binding to members of the Raf family of MAP kinase kinase kinases, C-Raf and B-Raf. Recently, it has been reported that in melanoma cells harboring oncogenic Ras mutations, B-Raf does not bind to Ras and does not contribute to basal ERK activation. For other types of Ras-mutant tumors, the relative contributions of C-Raf and B-Raf are not known. We examined non-melanoma cancer cell lines containing oncogenic Ras mutations and express both C-Raf and B-Raf isoforms, including the lung cancer cell line H1299 cells. Both B-Raf and C-Raf were constitutively bound to oncogenic Ras and contributed to Ras-dependent ERK activation. Ras binding to B-Raf and C-Raf were both subject to inhibition by the cAMP-dependent protein kinase PKA. cAMP inhibited the growth of H1299 cells and Ras-dependent ERK activation via PKA. PKA inhibited the binding of Ras to both C-Raf and B-Raf through phosphorylations of C-Raf at Ser-259 and B-Raf at Ser-365, respectively. These studies demonstrate that in non-melanocytic Ras-mutant cancer cells, Ras signaling to B-Raf is a significant contributor to ERK activation and that the B-Raf pathway, like that of C-Raf, is a target for inhibition by PKA. We suggest that cAMP and hormones coupled to cAMP may prove useful in dampening the effects of oncogenic Ras in non-melanocytic cancer cells through PKA-dependent actions on B-Raf as well as C-Raf.

  14. Constitutive activation of extracellular signal-regulated kinase predisposes diffuse large B-cell lymphoma cell lines to CD40-mediated cell death

    DEFF Research Database (Denmark)

    Hollmann, C Annette; Owens, Trevor; Nalbantoglu, Josephine;

    2006-01-01

    CD40 promotes survival, proliferation, and differentiation of normal B cells but can cause activation-induced cell death in malignant B lymphocytes. CD40 ligand and anti-CD40 antibodies have been used successfully to induce apoptosis in lymphoma lines both in vitro and in xenograft tumor models...... a specific cell line or tumor will undergo apoptosis when stimulated with CD40 and to identify targets downstream of CD40 that affect only the apoptotic arm of CD40 signaling. We have analyzed gene expression patterns in CD40-sensitive and CD40-resistant diffuse large B-cell lymphoma (DLBCL) cell lines...... and no increase in ERK activity in response to CD40 stimulation. Our results suggest that constitutive activation of ERK may be required for death signaling by CD40....

  15. Activated extracellular signal-regulated kinases are necessary and sufficient to initiate tubulogenesis in renal tubular MDCK strain I cell cysts.

    Science.gov (United States)

    Hellman, Nathan E; Greco, Andres J; Rogers, Katherine K; Kanchagar, Chitra; Balkovetz, Daniel F; Lipschutz, Joshua H

    2005-10-01

    A classic in vitro model of renal cyst and tubule formation utilizes the Madin-Darby canine kidney (MDCK) cell line, of which two strains exist. Most cyst and tubule formation studies that utilized MDCK cells have been performed with MDCK strain II cells. MDCK strain II cells form hollow cysts in a three-dimensional collagen matrix over 10 days and tubulate in response to hepatocyte growth factor, which increases levels of active (phosphorylated) ERK1/2. In this study, we demonstrate that MDCK strain I cells also form cysts when grown in a collagen matrix; however, MDCK strain I cell cysts spontaneously initiate the primary steps in tubulogenesis. Analysis of time-lapse microscopy of both MDCK strain I and strain II cell cysts during the initial stages of tubulogenesis demonstrates a highly dynamic process with cellular extensions and retractions occurring rapidly and continuously. MDCK strain I cell cysts can spontaneously initiate tubulogenesis mainly because of relatively higher levels of active ERK in MDCK strain I, compared with strain II, cells. The presence of either of two distinct inhibitors of ERK activation (UO126 and PD09059) prevents tubulogenesis from occurring spontaneously in MDCK strain I cell cysts and, in response to hepatocyte growth factor, in strain II cell cysts. The difference between MDCK strain I and strain II cell lines is likely explained by differing embryological origins, with strain I cells being of collecting duct, and hence ureteric bud, origin. Ureteric bud cells also have high levels of active ERK and spontaneously tubulate in our in vitro collagen gel system, with tubulogenesis inhibited by UO126 and PD09059. These results suggest that a seminal event in kidney development may be the activation of ERK in the mesonephric duct/ureteric bud cells destined to form the collecting tubules.

  16. Differential activation of p38 and extracellular signal-regulated kinase in spinal cord in a model of bee venom-induced inflammation and hyperalgesia

    Directory of Open Access Journals (Sweden)

    Kobayashi Kimiko

    2008-04-01

    Full Text Available Abstract Background Honeybee's sting on human skin can induce ongoing pain, hyperalgesia and inflammation. Injection of bee venom (BV into the intraplantar surface of the rat hindpaw induces an early onset of spontaneous pain followed by a lasting thermal and mechanical hypersensitivity in the affected paw. The underlying mechanisms of BV-induced thermal and mechanical hypersensitivity are, however, poorly understood. In the present study, we investigated the role of mitogen-activated protein kinase (MAPK in the generation of BV-induced pain hypersensitivity. Results We found that BV injection resulted in a quick activation of p38, predominantly in the L4/L5 spinal dorsal horn ipsilateral to the inflammation from 1 hr to 7 d post-injection. Phosphorylated p38 (p-p38 was expressed in both neurons and microglia, but not in astrocytes. Intrathecal administration of the p38 inhibitor, SB203580, prevented BV-induced thermal hypersensitivity from 1 hr to 3 d, but had no effect on mechanical hypersensitivity. Activated ERK1/2 was observed exclusively in neurons in the L4/L5 dorsal horn from 2 min to 1 d, peaking at 2 min after BV injection. Intrathecal administration of the MEK inhibitor, U0126, prevented both mechanical and thermal hypersensitivity from 1 hr to 2 d. p-ERK1/2 and p-p38 were expressed in neurons in distinct regions of the L4/L5 dorsal horn; p-ERK1/2 was mainly in lamina I, while p-p38 was mainly in lamina II of the dorsal horn. Conclusion The results indicate that differential activation of p38 and ERK1/2 in the dorsal horn may contribute to the generation and development of BV-induced pain hypersensitivity by different mechanisms.

  17. P2X7 receptor activates extracellular signal-regulated kinases ERK1 and ERK2 independently of Ca2+ influx

    DEFF Research Database (Denmark)

    Amstrup, Jan; Novak, Ivana

    2003-01-01

    P2X7 nucleotide receptors modulate a spectrum of cellular events in various cells including epithelia, such as exocrine pancreas. Although the pharmacology and channel properties of the P2X7 receptors have been studied intensively, signal transduction pathways are relatively unknown. In this study...... we applied a heterologous expression system of rat P2X7 receptors in HEK-293 cells. We followed the receptor expression and function using the enhanced green fluorescent protein (EGFP) tag, activation of intracellular proteins and increases in cellular Ca2+. EGFP-P2X7 receptors localized...

  18. Icariin inhibits oxidized low-density lipoprotein-induced proliferation of vascular smooth muscle cells by suppressing activation of extracellular signal-regulated kinase 1/2 and expression of proliferating cell nuclear antigen.

    Science.gov (United States)

    Hu, Yanwu; Liu, Kai; Yan, Mengtong; Zhang, Yang; Wang, Yadi; Ren, Liqun

    2016-03-01

    Icariin, a flavonoid isolated from the traditional Chinese herbal medicine Epimedium brevicornum Maxim, has been shown to possess anti-inflammatory, anti‑oxidant and anti-atherosclerotic activities in vivo and in vitro. The aim of the present study was to investigate the effects of icariin on oxidized low‑density lipoprotein (ox-LDL)-induced proliferation of vascular smooth muscle cells (VSMCs) and the possible underlying mechanism. VSMCs were cultured and pre‑treated with various concentrations of icariin (0, 10, 20 or 40 µm) prior to stimulation by ox‑LDL (50 µg/ml). Cell proliferation was evaluated by an MTT assay. Flow cytometry was used to study the influence of icariin on the cell cycle. Proliferating cell nuclear antigen (PCNA) expression and phosphorylation levels of extracellular signal-regulated kinase (ERK)1/2 were detected by western blot analysis. The results indicated that icariin significantly inhibited ox‑LDL‑induced proliferation of VSMCs and phosphorylation of ERK1/2. Furthermore, icariin also blocked the ox‑LDL‑induced cell‑cycle progression at G1/S‑interphase and downregulated the expression of PCNA in VSMCs. In conclusion, the present study indicated for the first time that icariin reduced the amount of ox‑LDL‑induced proliferation of VSMCs through suppression of PCNA expression and inactivation of ERK1/2.

  19. Curcumin exerts antinociceptive effects by inhibiting the activation of astrocytes in spinal dorsal horn and the intracellular extracellular signal-regulated kinase signaling pathway in rat model of chronic constriction injury

    Institute of Scientific and Technical Information of China (English)

    JI Feng-tao; LIANG Jiang-jun; LIU Ling; CAO Ming-hui; LI Feng

    2013-01-01

    Background Activation of glial cells and the extracellular signal-regulated kinase (ERK) signaling pathway play an important role in the development and maintenance of neuropathic pain.Curcumin can alleviate the symptom of inflammatory pain by inhibiting the production and release of interleukin and tumor necrosis factor.However,whether curcumin affects neuropathic pain induced by nerve injury and the possible mechanism involved are still unknown.This study investigated the effects of tolerable doses of curcumin on the activation of astrocytes and ERK signaling in the spinal dorsal horn in rat model of neuropathic pain.Methods Adult male Sprague-Dawley rats were randomly divided into three groups:a control (sham operated) group,and chronic constriction injury groups (to induce neuropathic pain) that were either untreated or treated with curcumin.Thermal and mechanical hyperalgesia thresholds were measured.The distribution and morphological changes of astrocytes were observed by immunofluorescence.Western blotting was used to detect changes in the expression of glial flbrillary acid protein (GFAP) and phosphorylated ERK.Results Injured rats showed obvious mechanical allodynia and thermal hyperalgesia.The number of GFAP-positive astrocytes,and the fluorescence intensity of GFAP were significantly increased in the spinal dorsal horn of injured compared with control rats.The soma of astrocytes also appeared hypertrophied in injured animals.Expression of GFAP and phosphorylated ERK was also significantly increased in the spinal dorsal hom of injured compared with control rats.Curcumin reduced the injury-induced thermal and mechanical hyperalgesia,the increase in the fluorescence intensity of GFAP and the hypertrophy of astrocytic soma,activation of GFAP and phosphorylation of ERK in the spinal dorsal horn.Conclusions Curcumin can markedly alleviate nerve injury-induced neuropathic pain in rats.The analgesic effect of curcumin may be attributed to its inhibition of

  20. Epstein-Barr virus-negative aggressive natural killer-cell leukaemia with high P-glycoprotein activity and phosphorylated extracellular signal-regulated protein kinases 1 and 2

    Directory of Open Access Journals (Sweden)

    Sanja Perkovic

    2012-09-01

    Full Text Available Aggressive natural killer-cell leukaemia (ANKL is a rare type of disease with fulminant course and poor outcome. The disease is more prevalent among Asians than in other ethnic groups and shows strong association with Epstein-Barr virus (EBV and P-glycoprotein (P-gp expression associated with multidrug resistance. Here we present a case of a 47 year old Caucasian female with a prior medical history of azathioprine treated ulcerative colitis who developed EBV-negative form of ANKL. The patient presented with hepatosplenomegaly, fever and nausea with peripheral blood and bone marrow infiltration with up to 70% of atypical lymphoid cells positive for cCD3, CD2, CD7, CD56, CD38, CD45, TIA1 and granzyme B, and negative for sCD3, CD4, CD5, CD8, CD34 and CD123 indicative of ANKL. Neoplastic CD56+ NK-cells showed high level of P-glycoprotein expression and activity, but also strong expression of phosphorylated extracellular signal-regulated protein kinases 1 and 2 (ERK1/2 MAP kinase. The patient was treated with an intensive polychemotherapy regimen designed for treatment of acute lymphoblastic leukaemia, but one month after admission developed sepsis, coma and died of cardiorespiratory arrest. We present additional evidence that, except for the immunophenotype, leukaemic NK-cells resemble normal NK-cells in terms of P-gp functional capacity and expression of phosphorylated ERK1/2 signalling molecule. In that sense drugs that block P-glycoprotein activity and activated signalling pathways might represent new means for targeted therapy.

  1. Nicotine shifts the temporal activation of hippocampal protein kinase A and extracellular signal-regulated kinase 1/2 to enhance long-term, but not short-term, hippocampus-dependent memory.

    Science.gov (United States)

    Gould, Thomas J; Wilkinson, Derek S; Yildirim, Emre; Poole, Rachel L F; Leach, Prescott T; Simmons, Steven J

    2014-03-01

    Acute nicotine enhances hippocampus-dependent learning through nicotine binding to β2-containing nicotinic acetylcholine receptors (nAChRs), but it is unclear if nicotine is targeting processes involved in short-term memory (STM) leading to a strong long-term memory (LTM) or directly targeting LTM. In addition, the molecular mechanisms involved in the effects of nicotine on learning are unknown. Previous research indicates that protein kinase A (PKA), extracellular signal-regulated kinase 1/2 (ERK1/2), and protein synthesis are crucial for LTM. Therefore, the present study examined the effects of nicotine on STM and LTM and the involvement of PKA, ERK1/2, and protein synthesis in the nicotine-induced enhancement of hippocampus-dependent contextual learning in C57BL/6J mice. The protein synthesis inhibitor anisomycin impaired contextual conditioning assessed at 4 h but not 2 h post-training, delineating time points for STM (2 h) and LTM (4 h and beyond). Nicotine enhanced contextual conditioning at 4, 8, and 24 h but not 2 h post-training, indicating nicotine specifically enhances LTM but not STM. Furthermore, nicotine did not rescue deficits in contextual conditioning produced by anisomycin, suggesting that the nicotine enhancement of contextual conditioning occurs through a protein synthesis-dependent mechanism. In addition, inhibition of dorsal hippocampal PKA activity blocked the effect of acute nicotine on learning, and nicotine shifted the timing of learning-related PKA and ERK1/2 activity in the dorsal and ventral hippocampus. Thus, the present results suggest that nicotine specifically enhances LTM through altering the timing of PKA and ERK1/2 signaling in the hippocampus, and suggests that the timing of PKA and ERK1/2 activity could contribute to the strength of memories.

  2. Post-exercise impact of ingested whey protein hydrolysate on gene expression profiles in rat skeletal muscle: activation of extracellular signal-regulated kinase 1/2 and hypoxia-inducible factor-1α.

    Science.gov (United States)

    Kanda, Atsushi; Ishijima, Tomoko; Shinozaki, Fumika; Nakayama, Kyosuke; Fukasawa, Tomoyuki; Nakai, Yuji; Abe, Keiko; Kawahata, Keiko; Ikegami, Shuji

    2014-06-28

    We have previously shown that whey protein hydrolysate (WPH) causes a greater increase in muscle protein synthesis than does a mixture of amino acids that is identical in amino acid composition. The present study was conducted to investigate the effect of WPH on gene expression. Male Sprague-Dawley rats subjected to a 2 h swimming exercise were administered either a carbohydrate-amino acid diet or a carbohydrate-WPH diet immediately after exercise. At 1 h after exercise, epitrochlearis muscle mRNA was sampled and subjected to DNA microarray analysis. We found that ingestion of WPH altered 189 genes after considering the false discovery rate. Among the up-regulated genes, eight Gene Ontology (GO) terms were enriched, which included key elements such as Cd24, Ccl2, Ccl7 and Cxcl1 involved in muscle repair after exercise. In contrast, nine GO terms were enriched in gene sets that were down-regulated by the ingestion of WPH, and these GO terms fell into two clusters, 'regulation of ATPase activity' and 'immune response'. Furthermore, we found that WPH activated two upstream proteins, extracellular signal-regulated kinase 1/2 (ERK1/2) and hypoxia-inducible factor-1α (HIF-1α), which might act as key factors for regulating gene expression. These results suggest that ingestion of WPH, compared with ingestion of a mixture of amino acids with an identical amino acid composition, induces greater changes in the post-exercise gene expression profile via activation of the proteins ERK1/2 and HIF-1α. PMID:24598469

  3. Intracellular transactivation of epidermal growth factor receptor by α1A-adrenoceptor is mediated by phosphatidylinositol 3-kinase independently of activation of extracellular signal regulated kinases 1/2 and serine-threonine kinases in Chinese hamster ovary cells.

    Science.gov (United States)

    Ulu, Nadir; Henning, Robert H; Guner, Sahika; Zoto, Teuta; Duman-Dalkilic, Basak; Duin, Marry; Gurdal, Hakan

    2013-10-01

    Transactivation of epidermal growth factor receptor (EGFR) by α1-adrenoceptor (α1-AR) is implicated in contraction and hypertrophy of vascular smooth muscle (VSM). We examine whether all α1-AR subtypes transactivate EGFR and explore the mechanism of transactivation. Chinese hamster ovary (CHO) cells stably expressing one subtype of α1-AR were transiently transfected with EGFR. The transactivation mechanism was examined both by coexpression of a chimeric erythropoietin (EPO)-EGFR with an extracellular EPO and intracellular EGFR domain, and by pharmacologic inhibition of external and internal signaling routes. All three α1-AR subtypes transactivated EGFR, which was dependent on the increase in intracellular calcium. The EGFR kinase inhibitor AG1478 [4-(3'-chloroanilino)-6,7-dimethoxyquinazoline] abrogated α1A-AR and α1D-AR induced phosphorylation of EGFR, but both the inhibition of matrix metalloproteinases by GM6001 [(R)-N4-hydroxy-N(1)-[(S)-2-(1H-indol-3-yl)-1-methylcarbamoyl-ethyl]-2-isobutyl-succinamide] or blockade of EGFR by cetuximab did not. Stimulation of α1A-AR and α1D-AR also induced phosphorylation of EPO-EGFR chimeric receptors. Moreover, α1A-AR stimulation enhanced phosphorylation of extracellular signal regulated kinase (ERK) 1/2 and serine-threonine kinases (Akt), which were both unaffected by AG1478, indicating that ERK1/2 and Akt phosphorylation is independent of EGFR transactivation. Accordingly, inhibitors of ERK1/2 or Akt did not influence the α1A-AR-mediated EGFR transactivation. Inhibition of calcium/calmodulin-dependent kinase II (CaMKII), phosphatidylinositol 3-kinase (PI3K), and Src, however, did block EGFR transactivation by α1A-AR and α1D-AR. These findings demonstrate that all α1-AR subtypes transactivate EGFR, which is dependent on an intracellular signaling route involving an increase in calcium and activation of CaMKII, PI3K, and Src, but not the of ERK1/2 and Akt pathways.

  4. Extracellular signal-regulated kinase-2 within the ventral tegmental area regulates responses to stress.

    Science.gov (United States)

    Iñiguez, Sergio D; Vialou, Vincent; Warren, Brandon L; Cao, Jun-Li; Alcantara, Lyonna F; Davis, Lindsey C; Manojlovic, Zarko; Neve, Rachael L; Russo, Scott J; Han, Ming-Hu; Nestler, Eric J; Bolaños-Guzmán, Carlos A

    2010-06-01

    Neurotrophic factors and their signaling pathways have been implicated in the neurobiological adaptations in response to stress and the regulation of mood-related behaviors. A candidate signaling molecule implicated in mediating these cellular responses is the extracellular signal-regulated kinase (ERK1/2), although its functional role in mood regulation remains to be fully elucidated. Here we show that acute (1 d) or chronic (4 weeks) exposure to unpredictable stress increases phosphorylation of ERK1/2 and of two downstream targets (ribosomal S6 kinase and mitogen- and stress-activated protein kinase 1) within the ventral tegmental area (VTA), an important substrate for motivated behavior and mood regulation. Using herpes simplex virus-mediated gene transfer to assess the functional significance of this ERK induction, we show that overexpressing ERK2 within the VTA increases susceptibility to stress as measured in the forced swim test, responses to unconditioned nociceptive stimuli, and elevated plus maze in Sprague Dawley male rats, and in the tail suspension test and chronic social defeat stress procedure in C57BL/6 male mice. In contrast, blocking ERK2 activity in the VTA produces stress-resistant behavioral responses in these same assays and also blocks a chronic stress-induced reduction in sucrose preference. The effects induced by ERK2 blockade were accompanied by decreases in the firing frequency of VTA dopamine neurons, an important electrophysiological hallmark of resilient-like behavior. Together, these results strongly implicate a role for ERK2 signaling in the VTA as a key modulator of responsiveness to stress and mood-related behaviors. PMID:20519540

  5. Advanced Glycation End Products Affect Osteoblast Proliferation and Function by Modulating Autophagy Via the Receptor of Advanced Glycation End Products/Raf Protein/Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase/Extracellular Signal-regulated Kinase (RAGE/Raf/MEK/ERK) Pathway.

    Science.gov (United States)

    Meng, Hong-Zheng; Zhang, Wei-Lin; Liu, Fei; Yang, Mao-Wei

    2015-11-20

    The interaction between advanced glycation end products (AGEs) and receptor of AGEs (RAGE) is associated with the development and progression of diabetes-associated osteoporosis, but the mechanisms involved are still poorly understood. In this study, we found that AGE-modified bovine serum albumin (AGE-BSA) induced a biphasic effect on the viability of hFOB1.19 cells; cell proliferation was stimulated after exposure to low dose AGE-BSA, but cell apoptosis was stimulated after exposure to high dose AGE-BSA. The low dose AGE-BSA facilitates proliferation of hFOB1.19 cells by concomitantly promoting autophagy, RAGE production, and the Raf/MEK/ERK signaling pathway activation. Furthermore, we investigated the effects of AGE-BSA on the function of hFOB1.19 cells. Interestingly, the results suggest that the short term effects of low dose AGE-BSA increase osteogenic function and decrease osteoclastogenic function, which are likely mediated by autophagy and the RAGE/Raf/MEK/ERK signal pathway. In contrast, with increased treatment time, the opposite effects were observed. Collectively, AGE-BSA had a biphasic effect on the viability of hFOB1.19 cells in vitro, which was determined by the concentration of AGE-BSA and treatment time. A low concentration of AGE-BSA activated the Raf/MEK/ERK signal pathway through the interaction with RAGE, induced autophagy, and regulated the proliferation and function of hFOB1.19 cells.

  6. Learned stressor resistance requires extracellular signal-regulated kinase in the prefrontal cortex

    Directory of Open Access Journals (Sweden)

    John Paul Christianson

    2014-10-01

    Full Text Available Behaviorally controllable stressors confer protection from the neurochemical and behavioral consequences of future uncontrollable stressors, a phenomenon termed behavioral immunization. Recent data implicate neuroplasticity within the ventromedial prefrontal cortex (mPFC as critical to behavioral immunization. Adult, male Sprague-Dawley rats were exposed to a series of controllable tailshocks and one week later to uncontrollable tailshocks, followed 24h later by social exploration and shuttlebox escape tests. To test the involvement of N-methyl-D-aspartate receptors (NMDAR and the extracellular signal-regulated kinase (ERK cascade in behavioral immunization, either D-AP5 or the MEK inhibitor U0126 was injected to the prelimbic (PL or infralimbic (IL mPFC prior to controllable stress exposure. Phosphorylated ERK and P70S6K, regulators of transcription and translation, were quantified by Western blot or immunohistochemistry after controllable or uncontrollable tailshocks. Prior controllable stress prevented the social exploration and shuttlebox performance deficits caused by the later uncontrollable stressor, and this effect was blocked by injections of D-AP5 into mPFC. A significant increase in phosphorylated ERK1 and ERK2, but not P70S6K, occurred within the PL and IL in rats exposed to controllable stress, but not to uncontrollable stress. However, U0126 only prevented behavioral immunization when injected to the PL. We provide evidence that NMDAR and ERK dependent plasticity within the PL region is required for behavioral immunization, a learned form of stressor resistance.

  7. Protein kinase C and extracellular signal-regulated kinase regulate movement, attachment, pairing and egg release in Schistosoma mansoni.

    Directory of Open Access Journals (Sweden)

    Margarida Ressurreição

    2014-06-01

    Full Text Available Protein kinases C (PKCs and extracellular signal-regulated kinases (ERKs are evolutionary conserved cell signalling enzymes that coordinate cell function. Here we have employed biochemical approaches using 'smart' antibodies and functional screening to unravel the importance of these enzymes to Schistosoma mansoni physiology. Various PKC and ERK isotypes were detected, and were differentially phosphorylated (activated throughout the various S. mansoni life stages, suggesting isotype-specific roles and differences in signalling complexity during parasite development. Functional kinase mapping in adult worms revealed that activated PKC and ERK were particularly associated with the adult male tegument, musculature and oesophagus and occasionally with the oesophageal gland; other structures possessing detectable activated PKC and/or ERK included the Mehlis' gland, ootype, lumen of the vitellaria, seminal receptacle and excretory ducts. Pharmacological modulation of PKC and ERK activity in adult worms using GF109203X, U0126, or PMA, resulted in significant physiological disturbance commensurate with these proteins occupying a central position in signalling pathways associated with schistosome muscular activity, neuromuscular coordination, reproductive function, attachment and pairing. Increased activation of ERK and PKC was also detected in worms following praziquantel treatment, with increased signalling associated with the tegument and excretory system and activated ERK localizing to previously unseen structures, including the cephalic ganglia. These findings support roles for PKC and ERK in S. mansoni homeostasis, and identify these kinase groups as potential targets for chemotherapeutic treatments against human schistosomiasis, a neglected tropical disease of enormous public health significance.

  8. Activation of extracellular signal-regulated kinase during silibinin-protected, isoproterenol-induced apoptosis in rat cardiac myocytes is tyrosine kinase pathway-mediated and protein kinase C-dependent

    Institute of Scientific and Technical Information of China (English)

    Bei ZHOU; Li-jun WU; Shin-ichi TASHIRO; Satoshi ONODERA; Fumiaki UCHIUMI; Takashi IKEJIMA

    2007-01-01

    Aim: To investigate the mechanism of silibinin-protected isoproterenol-induced apoptosis in rat cardiac myocytes.Methods: The viability of rat cardiac myocytes was measured by MTT method. The apoptotic ratio was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling. Protein kinase C (PKC) activity assay was carried out according to the instructions of the PepTag non-radioactive protein kinase C assay kit. Western blot analysis was used to evaluate the level of Ras, Raf-1 and mitogen-activated protein kinase (MAPK) expression.Results: The protective effects of silibinin were significantly sup-pressed by inhibitors, including genistein, manumycin A and GW5074 [inhibitors for protein tyrosine kinases (PTK), Ras and Raf- 1, respectively]. The exposure of rat cardiac myocytes to isoproterenol alone caused decreased PKC activity, which was prevented by pretreatment with silibinin dose-dependently. Simultaneously,the increased expression of Ras and Raf-1 activated by silibinin were blocked by the PKC inhibitor, stauroporine. In addition, the extracellularly responsive kinase (ERK) inhibitor, PD98059, suppressed silibinin-protected apoptosis, whereas the p38 MAPK inhibitor, SB203580, protected cardiac myocytes from isoproterenol-induced injury, and the c-Jun N-terminal kinase (JNK) inhibitor, SP600125 had no protective effects. Furthermore, Western blot analysis showed that the expres-sion of phosphorylated ERK was increased by silibinin, the expression of phos-phorylated p38 MAPK was decreased and total ERK, p38, JNK and phosphory-lated JNK MAPK did not change after treatment with both isoproterenol and silibinin. Furthermore, pretreatment of cardiac myocyte with PKC, Ras and Raf inhibitors significantly blocked ERK phosphorylation.Conclusion: Silibinin is suggested to protect isoproterenol-induced rat cardiac myocyte apoptosis by activating the tyrosine kinase pathway, PKC and MAPK pathways.

  9. Oncostatin M and leukaemia inhibitory factor trigger signal transducer and activator of transcription 3 and extracellular signal-regulated kinase 1/2 pathways but result in heterogeneous cellular responses in trophoblast cells.

    Science.gov (United States)

    Chaiwangyen, Wittaya; Ospina-Prieto, Stephanie; Morales-Prieto, Diana M; Pereira de Sousa, Francisco Lazaro; Pastuschek, Jana; Fitzgerald, Justine S; Schleussner, Ekkehard; Markert, Udo R

    2016-04-01

    Leukaemia inhibitory factor (LIF) and oncostatin M (OSM) are pleiotropic cytokines present at the implantation site that are important for the normal development of human pregnancy. These cytokines share the cell membrane receptor subunit gp130, resulting in similar functions. The aim of this study was to compare the response to LIF and OSM in several trophoblast models with particular regard to intracellular mechanisms and invasion. Four trophoblast cell lines with different characteristics were used: HTR-8/SVneo, JEG-3, ACH-3P and AC1-M59 cells. Cells were incubated with LIF, OSM (both at 10ngmL(-1)) and the signal transducer and activator of transcription (STAT) 3 inhibitor S3I-201 (200µM). Expression and phosphorylation of STAT3 (tyr(705)) and extracellular regulated kinase (ERK) 1/2 (thr(202/204)) and the STAT3 DNA-binding capacity were analysed by Western blotting and DNA-binding assays, respectively. Cell viability and invasiveness were assessed by the methylthiazole tetrazolium salt (MTS) and Matrigel assays. Enzymatic activity of matrix metalloproteinase (MMP)-2 and MMP-9 was investigated by zymography. OSM and LIF triggered phosphorylation of STAT3 and ERK1/2, followed by a significant increase in STAT3 DNA-binding activity in all tested cell lines. Stimulation with LIF but not OSM significantly enhanced invasion of ACH-3P and JEG-3 cells, but not HTR-8/SVneo or AC1-M59 cells. Similarly, STAT3 inhibition significantly decreased the invasiveness of only ACH-3P and JEG-3 cells concomitant with decreases in secreted MMP-2 and MMP-9. OSM shares with LIF the capacity to activate ERK1/2 and STAT3 pathways in all cell lines tested, but their resulting effects are dependent on cell type. This suggests that LIF and OSM may partially substitute for each other in case of deficiencies or therapeutic interventions. PMID:25247600

  10. Role of reactive oxygen species in extracellular signal-regulated protein kinase phosphorylation and 6-hydroxydopamine cytotoxicity

    Indian Academy of Sciences (India)

    Scott M Kulich; Charleen T Chu

    2003-02-01

    A number of reports indicate the potential for redox signalling via extracellular signal-regulated protein kinases (ERK) during neuronal injury. We have previously found that sustained ERK activation contributes to toxicity elicited by 6-hydroxydopamine (6-OHDA) in the B65 neuronal cell line. To determine whether reactive oxygen species (ROS) play a role in mediating ERK activation and 6-OHDA toxicity, we examined the effects of catalase, superoxide dismutase (SOD1), and metalloporphyrin antioxidants (‘SOD mimetics’) on 6-OHDA-treated cells. We found that catalase and metalloporphyrin antioxidants not only conferred protection against 6-OHDA but also inhibited development of sustained ERK phosphorylation in both differentiated and undifferentiated B65 cells. However, exogenously added SOD1 and heat-inactivated catalase had no effect on either toxicity or sustained ERK phosphorylation. This correlation between antioxidant protection and inhibition of 6-OHDA-induced sustained ERK phosphorylation suggests that redox regulation of ERK signalling cascades may contribute to neuronal toxicity.

  11. The Ras/Raf/MEK/Extracellular Signal-Regulated Kinase Pathway Induces Autocrine-Paracrine Growth Inhibition via the Leukemia Inhibitory Factor/JAK/STAT Pathway

    OpenAIRE

    Park, Jong-In; Strock, Christopher J.; Ball, Douglas W.; Nelkin, Barry D.

    2003-01-01

    Sustained activation of the Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) pathway can lead to cell cycle arrest in many cell types. We have found, with human medullary thyroid cancer (MTC) cells, that activated Ras or c-Raf-1 can induce growth arrest by producing and secreting an autocrine-paracrine factor. This protein was purified from cell culture medium conditioned by Raf-activated MTC cells and was identified by mass spectrometry as leukemia inhibitory factor (LIF). LIF express...

  12. Time-dependent effects of electroacupuncture at the Ren channel on extracellular signal-regulated kinases 1/2 expression in focal cerebral ischemia rats

    Institute of Scientific and Technical Information of China (English)

    Zhuoxin Yang; Lihong Diao; Haibo Yu; Wenshu Luo; Ling Wang; Min Pi; Xiaodan Rao; Junhua Peng

    2008-01-01

    BACKGROUND: The onset of focal cerebral ischemia activates extracellular signal-regulated kinases 1 and 2, regulates cell cycle, promotes cell proliferation and differentiation, and affects the normal stage and function of brain cells.OBJECTIVE: To observe the effects of electroacupuncture at the Ren channel on extracellular signal-regulated kinases 1/2 expression in the lateral cerebral ventricle wall of rats with focal cerebral ischemia. The effects were analyzed at different time points after intervention.DESIGN: Randomized controlled study.SETTING: Department of Anatomy, Sun Yat-Sen University. MATERIALS: A total of 60 healthy adult male Wistar rats weighing (250±10) g were provided by the Experimental Animal Center, Medical College of Sun Yat-Sen University. The animal experiment was conducted with confirmed consent by the local ethics committee. The GB6805-Ⅱ electric acupuncture apparatus was provided by Shanghai Medical Equipment High-techno Company. METHODS: The experiment was performed at the Laboratory of Anatomy, Sun Yat-Sen University, from February to July 2007. All experimental animals were randomly divided into the following groups: normal group (n = 6), sham operation group (n = 18), model group (n = 18), and electroacupuncture group (n = 18). Middle cerebral artery occlusion (MCAO) was performed in the model group and electroacupuncture group. Zea Longa's grading standard was used to assess neurological impairment after reperfusion; animals whose grades were between 1 and 4 were included in this study. The normal control group was not exposed to MCAO. In sham operation animals, the right common carotid artery (CCA) was isolated, and the external carotid artery (ECA) was damaged, but no embolism was induced. The electroacupuncture group was given acupuncture on the second day after surgery. The acupoint locations were chosen according to Experimental Acupuncture (People's Publishing House; 1997; First Edition). The Chengjiang, Qihai, and Guanyuan

  13. Effects of chronic sleep deprivation on the extracellular signal-regulated kinase pathway in the temporomandibular joint of rats.

    Directory of Open Access Journals (Sweden)

    Chuan Ma

    Full Text Available OBJECTIVES: To examine the possible involvement and regulatory mechanisms of extracellular signal-regulated kinase (ERK pathway in the temporomandibular joint (TMJ of rats subjected to chronic sleep deprivation (CSD. METHODS: Rats were subjected to CSD using the modified multiple platform method (MMPM. The serum levels of corticosterone (CORT and adrenocorticotropic hormone (ACTH were tested and histomorphology and ultrastructure of the TMJ were observed. The ERK and phospho-ERK (p-ERK expression levels were detected by Western blot analysis, and the MMP-1, MMP-3, and MMP-13 expression levels were detected by real-time quantitative polymerase chain reaction (PCR and Western blotting. RESULTS: The elevated serum CORT and ACTH levels confirmed that the rats were under CSD stress. Hematoxylin and eosin (HE staining and scanning electron microscopy (SEM showed pathological alterations in the TMJ following CSD; furthermore, the p-ERK was activated and the mRNA and protein expression levels of MMP-1, MMP-3, and MMP-13 were upregulated after CSD. In the rats administered with the selective ERK inhibitor U0126, decreased tissue destruction was observed. Phospho-ERK activation was visibly blocked and the MMP-1, MMP-3, and MMP-13 mRNA and protein levels were lower than the corresponding levels in the CSD without U0126 group. CONCLUSION: These findings indicate that CSD activates the ERK pathway and upregulates the MMP-1, MMP-3, and MMP-13 mRNA and protein levels in the TMJ of rats. Thus, CSD induces ERK pathway activation and causes pathological alterations in the TMJ. ERK may be associated with TMJ destruction by promoting the expression of MMPs.

  14. Both mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinases (ERK) 1/2 and phosphatidylinositide-3-OH kinase (PI3K)/Akt pathways regulate activation of E-twenty-six (ETS)-like transcription factor 1 (Elk-1) in U138 glioblastoma cells.

    Science.gov (United States)

    Mut, Melike; Lule, Sevda; Demir, Ozlem; Kurnaz, Isil Aksan; Vural, Imran

    2012-02-01

    Epidermal growth factor (EGF) and its receptor (EGFR) have been shown to play a significant role in the pathogenesis of glioblastoma. In our study, the EGFR was stimulated with EGF in human U138 glioblastoma cells. We show that the activated mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinases (ERK) 1/2 pathway phosphorylated the E twenty-six (ETS)-like transcription factor 1 (Elk-1) mainly at serine 383 residue. Mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, UO126 and ERK inhibitor II, FR180204 blocked the Elk-1 phosphorylation and activation. The phosphatidylinositide-3-OH kinase (PI3K)/Akt pathway was also involved in the Elk-1 activation. Activation of the Elk-1 led to an increased survival and a proliferative response with the EGF stimulation in the U138 glioblastoma cells. Knocking-down the Elk-1 using an RNA interference technique caused a decrease in survival of the unstimulated U138 glioblastoma cells and also decreased the proliferative response to the EGF stimulation. The Elk-1 transcription factor was important for the survival and proliferation of U138 glioblastoma cells upon the stimulation of EGFR with EGF. The MAPK/ERK1/2 and PI3K/Akt pathways regulated this response via activation of the Elk-1 transcription factor. The Elk-1 may be one of the convergence points for pathways located downstream of EGFR in glioblastoma cells. Utilization of the Elk-1 as a therapeutic target may lead to a novel strategy in treatment of glioblastoma.

  15. Expression of Extracellular Signal-regulated Kinase and Angiotensin-converting Enzyme in Human Atria during Atrial Fibrillation

    Institute of Scientific and Technical Information of China (English)

    戴友平; 王祥; 曹林生; 杨杪; 邬堂春

    2004-01-01

    Summary: In order to investigate the changes in the expression of extracellular signal-regulated kinase (ERK1/ERK2) and angiotensin-converting enzyme (ACE) in the patients with atrial fibrillation (AF), 52 patients with rheumatic heart diseases were examined. Nineteen patients had chronic persistent AF (AF≥6 months, CAF), 12 patients had paroxymal AF (PAF) and 21 patients had no history of AF. The ERK expression was detected at the mRNA level by reverse transcription polymerase chain reaction, at the protein level by Western blotting and at atrial tissue level by immunohistochemistry. ERK-activating kinases (MEK1/2) and ACE were determined by Western blotting techniques. The expression of ERK2-mRNA was increased in the patients with CAF (74±19 U vs sinus rhythm: 32±24 U, P<0.05). Activated ERK1/ERK2 and MEK1/2 were increased to more than 150 % in the patients with AF compared to those with sinus rhythm. No significant difference between CAF and PAF was found. The expression of ACE was three-fold increased in the patients with CAF compared to those with sinus rhythm. Patients with AF showed an increased expression of ERK1/ERK2 in atrial interstitial cells and marked atrial fibrosis. An ACE-dependent increase in the amounts of activated ERK1/ERK2 in atrial interstitial cells may be one of molecular mechanisms for the development of atrial fibrosis in the patients with AF. These findings may have important impact on the treatment of AF.

  16. Adenovirus-induced extracellular signal-regulated kinase phosphorylation during the late phase of infection enhances viral protein levels and virus progeny

    DEFF Research Database (Denmark)

    Schümann, Michael; Dobbelstein, Matthias

    2006-01-01

    . Hence, adenovirus induces the oncogenic Raf/MEK/ERK signaling pathway to enhance viral progeny by sustaining the levels of viral proteins. Concerning therapy, our results suggest that the use of Raf/MEK/ERK inhibitors will interfere with the propagation of oncolytic adenoviruses.......The Raf/mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK signaling cascade enhances tumor cell proliferation in many cases. Here, we show that adenovirus type 5, a small DNA tumor virus used in experimental cancer therapy, strongly induces ERK phosphorylation...

  17. Role of extracellular signal-regulated kinase in regulating expression of interleukin 13 in lymphocytes from an asthmatic rat model

    Institute of Scientific and Technical Information of China (English)

    LI Yuan-yuan; LIU Xian-sheng; LIU Chang; XU Yong-jian; XIONG Wei-xing

    2010-01-01

    Background The extracellular signal-regulated kinase (ERK) is widely expressed in mammal cells and involved in airway proliferation and remodeling in asthma. In this study, we intend to explore the role of ERK in the expression of the Th2 cytokine, interleukin 13 (IL-13) in lymphocytes in asthma.Methods Forty Sprague-Dawley rats were randomly divided into two groups: normal control and asthmatic groups. Peripheral blood lymphocytes were isolated and purified from the blood of each rat and divided into five groups: control, asthmatic lymphocytes, asthmatic cells stimulated with ERK activator epidermal growth factor (EGF), or with ERK inhibitor PD98059, or with EGF and PD98059 together. The expression of phosphorylated-ERK (p-ERK) was observed by immunocvtochemical staining, the expression of ERK mRNA was determined by reverse transcriptase-PCR, IL-13 protein in supernatants was measured by ELISA.Results (1) The ERK mRNA level and the percentage of cells with p-ERK in lymphocytes from asthmatic rats were significantly higher than those in normal controls, and were significantly increased by EGF administration. This effect of EGF was significantly inhibited by PD98059 pretreatment. (2) IL-13 protein in supematants of asthmatic lymphocytes was higher than that produced by normal control lymphocytes, and was significantly increased by EGF treatment. This EGF effect was partly blocked by PD98059 pretreatment. (3) There was a significant positive correlation between the percentage of cells with p-ERK in peripheral blood lymphocytes and IL-13 protein in supematants of lymphocytes from asthmatic rats.Conclusions In asthma the ERK expression and activation levels were increased, as was the protein level of IL-13. The ERK signaling pathway may be involved in the increased expression of the Th2 cytokine IL-13 in asthma.

  18. Cell division in the unicellular microalga Dunaliella viridis depends on phosphorylation of extracellular signal-regulated kinases (ERKs).

    Science.gov (United States)

    Jiménez, Carlos; Cossío, Belén R; Rivard, Christopher J; Berl, Tomás; Capasso, Juan M

    2007-01-01

    In mammalian cells, MAPKs are involved in both stress response (JNK and p38 pathways) and cell proliferation and differentiation [extracellular signal-regulated kinase (ERK)] through protein kinase cascades. Exposure of Dunaliella viridis cell cultures to PD98059, a very specific inhibitor of the ERK signalling pathway, resulted in a total arrest of cell proliferation and a complete dephosphorylation of ERK. As shown by flow cytometry analysis of propidium iodide-stained cells, PD98059 stopped mitosis at the G(2) phase after the S phase has been completed. Multiple physiological parameters such as cell motility and reducing power generation (NADPH) clearly indicate that the treated cells are wholly viable. Exposure of D. viridis to environmental stresses that impair cell division, such as hyperosmotic shock, nitrogen starvation, or sublethal UV irradiation, caused a marked decrease in the phospho-ERK levels as detected by western blot. Two 400 bp polynucleotides from D. viridis with high homologies to published sequences of ERK1 and ERK2 were cloned, sequenced, and submitted to GenBank. Northern blot analysis revealed two mRNA bands of approximately 1.9 kb, consistent with the expected size of ERK proteins ( approximately 40 kDa). Sequence analysis showed that they contained several mitogen-activated protein kinase (MAPK) conserved domains, including II, III, VIb, VII, and the double phosphorylation motif. Interestingly, in D. viridis, this motif was T*DY* instead of the canonic T*EY*. Based on this finding, ERK plant sequences can be divided into two groups, one termed the T*DY* branch and the other termed the T*EY* branch. The molecular and functional data presented here suggest that ERK is a very ancient signalling pathway and that it was already present in the last common ancestor of all eukaryotic cells. PMID:17220513

  19. 17-Beta-estradiol inhibits transforming growth factor-beta signaling and function in breast cancer cells via activation of extracellular signal-regulated kinase through the G protein-coupled receptor 30.

    Science.gov (United States)

    Kleuser, Burkhard; Malek, Daniela; Gust, Ronald; Pertz, Heinz H; Potteck, Henrik

    2008-12-01

    Breast cancer development and breast cancer progression involves the deregulation of growth factors leading to uncontrolled cellular proliferation, invasion and metastasis. Transforming growth factor (TGF)-beta plays a crucial role in breast cancer because it has the potential to act as either a tumor suppressor or a pro-oncogenic chemokine. A cross-communication between the TGF-beta signaling network and estrogens has been postulated, which is important for breast tumorigenesis. Here, we provide evidence that inhibition of TGF-beta signaling is associated with a rapid estrogen-dependent nongenomic action. Moreover, we were able to demonstrate that estrogens disrupt the TGF-beta signaling network as well as TGF-beta functions in breast cancer cells via the G protein-coupled receptor 30 (GPR30). Silencing of GPR30 in MCF-7 cells completely reduced the ability of 17-beta-estradiol (E2) to inhibit the TGF-beta pathway. Likewise, in GPR30-deficient MDA-MB-231 breast cancer cells, E2 achieved the ability to suppress TGF-beta signaling only after transfection with GPR30-encoding plasmids. It is most interesting that the antiestrogen fulvestrant (ICI 182,780), which possesses agonistic activity at the GPR30, also diminished TGF-beta signaling. Further experiments attempted to characterize the molecular mechanism by which activated GPR30 inhibits the TGF-beta pathway. Our results indicate that GPR30 induces the stimulation of the mitogen-activated protein kinases (MAPKs), which interferes with the activation of Smad proteins. Inhibition of MAPK activity prevented the ability of E2 from suppressing TGF-beta signaling. These findings are of great clinical relevance, because down-regulation of TGF-beta signaling is associated with the development of breast cancer resistance in response to antiestrogens.

  20. The forced swimming-induced behavioural immobility response involves histone H3 phospho-acetylation and c-Fos induction in dentate gyrus granule neurons via activation of the N-methyl-D-aspartate/extracellular signal-regulated kinase/mitogen- and stress-activated kinase signalling pathway.

    Science.gov (United States)

    Chandramohan, Yalini; Droste, Susanne K; Arthur, J Simon C; Reul, Johannes M H M

    2008-05-01

    The hippocampus is involved in learning and memory. Previously, we have shown that the acquisition of the behavioural immobility response after a forced swim experience is associated with chromatin modifications and transcriptional induction in dentate gyrus granule neurons. Given that both N-methyl-D-aspartate (NMDA) receptors and the extracellular signal-regulated kinases (ERK) 1/2 signalling pathway are involved in neuroplasticity processes underlying learning and memory, we investigated in rats and mice whether these signalling pathways regulate chromatin modifications and transcriptional events participating in the acquisition of the immobility response. We found that: (i) forced swimming evoked a transient increase in the number of phospho-acetylated histone H3-positive [P(Ser10)-Ac(Lys14)-H3(+)] neurons specifically in the middle and superficial aspects of the dentate gyrus granule cell layer; (ii) antagonism of NMDA receptors and inhibition of ERK1/2 signalling blocked forced swimming-induced histone H3 phospho-acetylation and the acquisition of the behavioural immobility response; (iii) double knockout (DKO) of the histone H3 kinase mitogen- and stress-activated kinases (MSK) 1/2 in mice completely abolished the forced swimming-induced increases in histone H3 phospho-acetylation and c-Fos induction in dentate granule neurons and the behavioural immobility response; (iv) blocking mineralocorticoid receptors, known not to be involved in behavioural immobility in the forced swim test, did not affect forced swimming-evoked histone H3 phospho-acetylation in dentate neurons; and (v) the pharmacological manipulations and gene deletions did not affect behaviour in the initial forced swim test. We conclude that the forced swimming-induced behavioural immobility response requires histone H3 phospho-acetylation and c-Fos induction in distinct dentate granule neurons through recruitment of the NMDA/ERK/MSK 1/2 pathway.

  1. Epidermal growth factor stimulates proliferation and migration of porcine trophectoderm cells through protooncogenic protein kinase 1 and extracellular-signal-regulated kinases 1/2 mitogen-activated protein kinase signal transduction cascades during early pregnancy.

    Science.gov (United States)

    Jeong, Wooyoung; Kim, Jinyoung; Bazer, Fuller W; Song, Gwonhwa

    2013-12-01

    For successful implantation and establishment of early epitheliochorial placentation, porcine conceptuses require histotroph, including nutrients and growth factors, secreted by or transported into the lumen of the uterus. Epidermal growth factor (EGF), an essential component of histotroph, is known to have potential growth-promoting activities on the conceptus and uterine endometrium. However, little is known about its effects to transactivate cell signaling cascades responsible for proliferation, growth and differentiation of conceptus trophectoderm. In the present study, therefore, we determined that EGFR mRNA and protein were abundant in endometrial luminal and glandular epithelia, stratum compactum stroma and conceptus trophectoderm on days 13-14 of pregnancy, but not in any other cells of the uterus or conceptus. In addition, primary porcine trophectoderm (pTr) cells treated with EGF exhibited increased abundance of phosphorylated (p)-AKT1, p-ERK1/2 MAPK and p-P90RSK over basal levels within 5min, and effect that was maintained to between 30 and 120min. Immunofluorescence microscopy revealed abundant amounts of p-ERK1/2 MAPK and p-AKT1 proteins in the nucleus and, to a lesser extent, in the cytoplasm of pTr cells treated with EGF as compared to control cells. Furthermore, the abundance of p-AKT1 and p-ERK1/2 MAPK proteins was inhibited in control and EGF-treated pTr cells transfected with EGFR siRNA. Compared to the control siRNA transfected pTr cells, pTr cells transfected with EGFR siRNA exhibited an increase in expression of IFND and TGFB1, but there was no effect of expression of IFNG. Further, EGF stimulated proliferation and migration of pTr cells through activation of the PI3K-AKT1 and ERK1/2 MAPK-P90RSK cell signaling pathways. Collectively, these results support the hypothesis that EGF coordinately activates multiple cell signaling pathways critical to proliferation, migration and survival of trophectoderm cells that are critical to development of

  2. Glucocorticoid modulation of extracellular signal-regulated protein kinase 1/2 and p38 in human ovarian cancer HO-8910 cells

    Institute of Scientific and Technical Information of China (English)

    夏冰; 卢建; 王钢

    2003-01-01

    Objective To investigate the signaling pathway through testing the effects of dexamethasone (Dex) on the activation of the extracellular signal-regulated protein kinase 1/2 (ERK1/2) and p38 kinase (p38) in HO-8910 cells.Methods Activation of the ERK1/2 and p38 was detected by Western blotting using the antibodies against the total ERK1/2 and p38 mitogen-activated protein kinases (MAPKs) protein and the phosphorylated forms of them. Results Dex could suppress the activation of ERK1/2, while enhance the activation of p38 rapidly and strongly in a dose- and time- dependent manner. Neither effect could be blocked by RU486, the antagonist of glucocorticoid receptor (GR).Conclusion Dex has rapid effects on the activation of ERK1/2 and p38, and these effects are not mediated by GR.

  3. The human Na(+)/H(+) exchanger 1 is a membrane scaffold protein for extracellular signal-regulated kinase 2

    DEFF Research Database (Denmark)

    Hendus-Altenburger, Ruth; Pedraz-Cuesta, Elena; Olesen, Christina W;

    2016-01-01

    BACKGROUND: Extracellular signal-regulated kinase 2 (ERK2) is an S/T kinase with more than 200 known substrates, and with critical roles in regulation of cell growth and differentiation and currently no membrane proteins have been linked to ERK2 scaffolding. METHODS AND RESULTS: Here, we identify...... the human Na(+)/H(+) exchanger 1 (hNHE1) as a membrane scaffold protein for ERK2 and show direct hNHE1-ERK1/2 interaction in cellular contexts. Using nuclear magnetic resonance (NMR) spectroscopy and immunofluorescence analysis we demonstrate that ERK2 scaffolding by hNHE1 occurs by one of three D...... and ERK2, and provides a molecular mechanism for the important ERK2 scaffolding function of the membrane protein hNHE1, which regulates the phosphorylation of both hNHE1 and ERK2....

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

    Institute of Scientific and Technical Information of China (English)

    Hongyan Wang; Yingquan Zhang; Mingqi Qiao

    2013-01-01

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

  5. Extracellular signal-regulated kinases 1/2 control claudin-2 expression in Madin-Darby canine kidney strain I and II cells.

    Science.gov (United States)

    Lipschutz, Joshua H; Li, Shixiong; Arisco, Amy; Balkovetz, Daniel F

    2005-02-01

    The tight junction of the epithelial cell determines the characteristics of paracellular permeability across epithelium. Recent work points toward the claudin family of tight junction proteins as leading candidates for the molecular components that regulate paracellular permeability properties in epithelial tissues. Madin-Darby canine kidney (MDCK) strain I and II cells are models for the study of tight junctions and based on transepithelial electrical resistance (TER) contain "tight" and "leaky" tight junctions, respectively. Overexpression studies suggest that tight junction leakiness in these two strains of MDCK cells is conferred by expression of the tight junction protein claudin-2. Extracellular signal-regulated kinase (ERK) 1/2 activation by hepatocyte growth factor treatment of MDCK strain II cells inhibited claudin-2 expression and transiently increased TER. This process was blocked by the ERK 1/2 inhibitor U0126. Transfection of constitutively active mitogen-activated protein kinase/extracellular signal-regulated kinase kinase into MDCK strain II cells also inhibited claudin-2 expression and increased TER. MDCK strain I cells have higher levels of active ERK 1/2 than do MDCK strain II cells. U0126 treatment of MDCK strain I cells decreased active ERK 1/2 levels, induced expression of claudin-2 protein, and decreased TER by approximately 20-fold. U0126 treatment also induced claudin-2 expression and decreased TER in a high resistance mouse cortical collecting duct cell line (94D). These data show for the first time that the ERK 1/2 signaling pathway negatively controls claudin-2 expression in mammalian renal epithelial cells and provide evidence for regulation of tight junction paracellular transport by alterations in claudin composition within tight junction complexes.

  6. Enhanced transcription of contractile 5-hydroxytryptamine 2A receptors via extracellular signal-regulated kinase 1/2 after organ culture of rat mesenteric artery

    DEFF Research Database (Denmark)

    Cao, Yong-Xiao; He, Lang-Chong; Xu, Cang-Bao;

    2005-01-01

    5-Hydroxytryptamine (5-HT) has been found to elicit enhanced contractile effects in some vascular disorders. The present study was designed to examine if vascular 5-HT2A receptors are up-regulated during organ culture and if the extracellular signal-regulated protein kinase 1/2 (ERK1/2) pathways ...

  7. Dopamine D1 Receptors Regulate Protein Synthesis-Dependent Long-Term Recognition Memory via Extracellular Signal-Regulated Kinase 1/2 in the Prefrontal Cortex

    Science.gov (United States)

    Nagai, Taku; Takuma, Kazuhiro; Kamei, Hiroyuki; Ito, Yukio; Nakamichi, Noritaka; Ibi, Daisuke; Nakanishi, Yutaka; Murai, Masaaki; Mizoguchi, Hiroyuki; Nabeshima, Toshitaka; Yamada, Kiyofumi

    2007-01-01

    Several lines of evidence suggest that extracellular signal-regulated kinase1/2 (ERK1/2) and dopaminergic system is involved in learning and memory. However, it remains to be determined if the dopaminergic system and ERK1/2 pathway contribute to cognitive function in the prefrontal cortex (PFC). The amount of phosphorylated ERK1/2 was increased in…

  8. Augmentation of RANTES-induced extracellular signal-regulated kinase mediated signaling and T cell adhesion by elastase-treated fibronectin.

    Science.gov (United States)

    Brill, A; Hershkoviz, R; Vaday, G G; Chowers, Y; Lider, O

    2001-06-15

    T cells migrating across extracellular matrix (ECM) barriers toward their target, the inflammatory site, should respond to chemoattractant cytokines and to the degradation of ECM by specific enzymes. In this study, we examined the effects of RANTES and ECM proteins treated with human leukocyte elastase on T cell activation and adhesion to the ECM. We found that human peripheral blood T cells briefly suspended with RANTES (0.1-100 ng/ml) had increased phosphorylation of their intracellular extracellular signal-regulated kinase (ERK), a mitogen-activated protein kinase involved in the activation of several intracellular downstream effector molecules implicated in cell adhesion and migration. Consequently, a small portion (12-20%) of the responding cells adhered to fibronectin (FN). However, when the T cells were exposed to RANTES in the presence of native immobilized FN, laminin, or collagen type I, ERK phosphorylation was partially inhibited, suggesting that this form of the ECM proteins can down-regulate RANTES-induced intracellular signaling. In contrast, when the T cells were exposed to RANTES in the presence of elastase-treated immobilized FN, but not to elastase-treated laminin, ERK phosphorylation was markedly increased. Furthermore, a large percentage (30%) of RANTES-activated T cells adhered to the enzymatically treated FN in a beta1 integrin-dependent fashion. Thus, while migrating along chemotactic gradients within the ECM, T cells can adapt their adhesive performance according to the level of cleavage induced by enzymes to the matrix. PMID:11390457

  9. Inhibition of Extracellular Signal-Regulated Kinases Ameliorates Hypertension-Induced Renal Vascular Remodeling in Rat Models

    Directory of Open Access Journals (Sweden)

    Li Jing

    2011-11-01

    Full Text Available The aim of this study is to investigate the effect of the extracellular signal-regulated kinases 1/2 (ERK1/2 inhibitor, PD98059, on high blood pressure and related vascular changes. Blood pressure was recorded, thicknesses of renal small artery walls were measured and ERK1/2 immunoreactivity and erk2 mRNA in renal vascular smooth muscle cells (VSMCs and endothelial cells were detected by immunohistochemistry and in situ hybridization in normotensive wistar kyoto (WKY rats, spontaneously hypertensive rats (SHR and PD98059-treated SHR. Compared with normo-tensive WKY rats, SHR developed hypertension at 8 weeks of age, thickened renal small artery wall and asymmetric arrangement of VSMCs at 16 and 24 weeks of age. Phospho-ERK1/2 immunoreactivity and erk2 mRNA expression levels were increased in VSMCs and endothelial cells of the renal small arteries in the SHR. Treating SHR with PD98059 reduced the spontaneous hypertension-induced vascular wall thickening. This effect was associated with suppressions of erk2 mRNA expression and ERK1/2 phosphorylation in VSMCs and endothelial cells of the renal small arteries. It is concluded that inhibition of ERK1/2 ameliorates hypertension induced vascular remodeling in renal small arteries.

  10. Proteomic and functional genomic landscape of receptor tyrosine kinase and ras to extracellular signal-regulated kinase signaling.

    Science.gov (United States)

    Friedman, Adam A; Tucker, George; Singh, Rohit; Yan, Dong; Vinayagam, Arunachalam; Hu, Yanhui; Binari, Richard; Hong, Pengyu; Sun, Xiaoyun; Porto, Maura; Pacifico, Svetlana; Murali, Thilakam; Finley, Russell L; Asara, John M; Berger, Bonnie; Perrimon, Norbert

    2011-10-25

    Characterizing the extent and logic of signaling networks is essential to understanding specificity in such physiological and pathophysiological contexts as cell fate decisions and mechanisms of oncogenesis and resistance to chemotherapy. Cell-based RNA interference (RNAi) screens enable the inference of large numbers of genes that regulate signaling pathways, but these screens cannot provide network structure directly. We describe an integrated network around the canonical receptor tyrosine kinase (RTK)-Ras-extracellular signal-regulated kinase (ERK) signaling pathway, generated by combining parallel genome-wide RNAi screens with protein-protein interaction (PPI) mapping by tandem affinity purification-mass spectrometry. We found that only a small fraction of the total number of PPI or RNAi screen hits was isolated under all conditions tested and that most of these represented the known canonical pathway components, suggesting that much of the core canonical ERK pathway is known. Because most of the newly identified regulators are likely cell type- and RTK-specific, our analysis provides a resource for understanding how output through this clinically relevant pathway is regulated in different contexts. We report in vivo roles for several of the previously unknown regulators, including CG10289 and PpV, the Drosophila orthologs of two components of the serine/threonine-protein phosphatase 6 complex; the Drosophila ortholog of TepIV, a glycophosphatidylinositol-linked protein mutated in human cancers; CG6453, a noncatalytic subunit of glucosidase II; and Rtf1, a histone methyltransferase.

  11. Epidermal Growth Factor Stimulates Extracellular-Signal Regulated Kinase Phosphorylation of a Novel Site on Cytoplasmic Dynein Intermediate Chain 2

    Directory of Open Access Journals (Sweden)

    Andrew D. Catling

    2013-02-01

    Full Text Available Extracellular-signal regulated kinase (ERK signaling is required for a multitude of physiological and patho-physiological processes. However, the identities of the proteins that ERK phosphorylates to elicit these responses are incompletely known. Using an affinity purification methodology of general utility, here we identify cytoplasmic dynein intermediate chain 2 (DYNC1I-2, IC-2 as a novel substrate for ERK following epidermal growth factor receptor stimulation of fibroblasts. IC-2 is a subunit of cytoplasmic dynein, a minus-end directed motor protein necessary for transport of diverse cargos along microtubules. Emerging data support the hypothesis that post-translational modification regulates dynein but the signaling mechanisms used are currently unknown. We find that ERK phosphorylates IC-2 on a novel, highly conserved Serine residue proximal to the binding site for the p150Glued subunit of the cargo adapter dynactin. Surprisingly, neither constitutive phosphorylation nor a phosphomimetic substitution of this Serine influences binding of p150Glued to IC-2. These data suggest that ERK phosphorylation of IC-2 regulates dynein function through mechanisms other than its interaction with dynactin.

  12. An experimental study of extracellular signal-regulated kinase and its inter ventional treatments in hepatic ifbrosis

    Institute of Scientific and Technical Information of China (English)

    Ming-De Jiang; Shu-Mei Zheng; Hui Xu; Wei-Zheng Zeng; Yong Zhang; Hao-Ping Sun; Yun-Xia Wang; Jian-Ping Qin; Xiao-Ling Wu

    2008-01-01

    BACKGROUND:The pathogenesis of hepatic ifbrosis and cirrhosis is still not fully understood. The extracellular signal-regulated kinase (ERK) pathway is involved in the regulation of cell proliferation and differentiation. The aim of this study was to investigate the effects of PD98059, a speciifc inhibitor of ERK, on the cell cycle, cell proliferation, secretion of typeⅠcollagen and expression of cyclin D1 mRNA, CDK4 mRNA and transforming growth factor-β1 (TGF-β1) mRNA in rat hepatic stellate cells (HSCs) stimulated by acetaldehyde. METHODS:Rat HSCs stimulated by acetaldehyde were incubated with PD98059 at different concentrations. The cell cycle was analysed by lfow cytometry. Cell proliferation was assessed by the methyl thiazolyl tetrazolium colorimetric assay. The mRNA expression of cyclin D1, CDK4 and TGF-β1 was examined using the reverse transcriptase-polymerase chain reaction. Type Ⅰcollagen in the culture medium was detected by enzyme-linked immunosorbent assay. RESULTS: 20, 50 and 100 μmol/L PD98059 signiifcantly inhibited the proliferation and provoked a G0/G1-phase arrest of acetaldehyde-induced HSCs in a dose-dependent manner. The secretion of typeⅠ collagen and the expression of cyclin D1, CDK4 and TGF-β1 mRNA in acetaldehyde-induced HSCs were markedly inhibited by 50 and 100μmol/L PD98059, respectively. CONCLUSIONS:The ERK pathway regulates the cell proliferation, secretion of type Ⅰ collagen and the expression of TGF-β1 mRNA in rat HSCs stimulated by acetaldehyde, which is likely related to its regulative effect on the cell cycle.

  13. Extracellular signal regulated kinases 1/2 signal pathway and responses of astrocytes after diffuse brain injury

    Institute of Scientific and Technical Information of China (English)

    Jinxing Li; Haimei Zhao; Yu Li; Chong Wang; Jiashan Zhao; Xianli Zhu

    2007-01-01

    BACKGROUND: The treatment of diffuse brain injury during an acute period is focused on relieving degrees of secondary brain injury. Generation and development of pathological changes of secondary brain injury depend on signal conduction, so down-regulating over response of astrocyte through interfering a key link of signal conduction pathway may bring a new thinking for the treatment of diffuse brain injury. OBJECTIVE: To observe the effect of over activity of extracellular signal regulated kinases 1/2 (ERK1/2) signal pathway on the response of astrocyte during an acute period of diffuse brain injury. DESIGN: Completely randomized grouping and controlled animal study.SETTINGS: Department of Neurosurgery, the Third Affiliated Hospital, Nanchang University; Department of Neurosurgery, Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: A total of 158 healthy male SD rats, of 11 weeks old, weighing 320 - 370 g, were provided by Experimental Animal Faulty, Tongji Medical College, Huazhong University of Science and Technology. Rabbit-anti-phosphorylated ERK1/2 (pERKl/2) polyclonal antibody was provided by R&D Company; rabbit-anti-glial fibrillary acidic protein (GFAP) polyclonal antibody, SP immunohistochemical kit and horseradish peroxidase (HRP)-labeled goat-anti-rabbit IgG by Santa Cruz Company; specific inhibitor U0126 of ERK1/2 signal pathway by Alexis Company. METHODS: The experiment was carried out in the Laboratory of Neurosurgery, Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology from September 2004 to March 2006. ①Detection of pERKl/2 expression: A total of 110 rats were randomly divided into sham operation group (n =5), model group (n =35), high-dosage U0126 group (n =35) and low-dosage U0126 group (n =35). Rats in the sham operation group were only treated with incision of epicranium and fixation of backup plate, but not hit. Rats in the model group

  14. Extracellular signal-regulated kinase 2 (ERK2) phosphorylation sites and docking domain on the nuclear pore complex protein Tpr cooperatively regulate ERK2-Tpr interaction.

    Science.gov (United States)

    Vomastek, Tomás; Iwanicki, Marcin P; Burack, W Richard; Tiwari, Divya; Kumar, Devanand; Parsons, J Thomas; Weber, Michael J; Nandicoori, Vinay Kumar

    2008-11-01

    Identifying direct substrates of mitogen-activated protein kinases (MAPKs) and understanding how those substrates are selected is central to understanding how these ubiquitously activated enzymes generate diverse biological responses. In previous work, we identified several new candidate substrates for the MAPK ERK2 (extracellular signal-regulated kinase 2), including the nuclear pore complex protein Tpr (translocated promoter region). In this report, we identify sites on Tpr for ERK2 phosphorylation and binding and demonstrate their functional interaction. ERK2 phosphorylation and dimerization are necessary for ERK2-Tpr binding, and this occurs through a DEF (docking site for ERK2, FXF) domain on Tpr. Surprisingly, the DEF domain and the phosphorylation sites displayed positive cooperativity to promote ERK2 binding to Tpr, in contrast to substrates where phosphorylation reduces binding. Ectopic expression or depletion of Tpr resulted in decreased movement of activated ERK2 from the cytoplasm to the nucleus, implying a role for Tpr in ERK2 translocation. Collectively, the data provide direct evidence that a component of the nuclear pore complex is a bona fide substrate of ERK2 in vivo and that activated ERK2 stably associates with this substrate after phosphorylation, where it could play a continuing role in nuclear pore function. We propose that Tpr is both a substrate and a scaffold for activated ERKs.

  15. N-glycosylation at Asn residues 554 and 566 of E-cadherin affects cell cycle progression through extracellular signal-regulated protein kinase signaling pathway

    Institute of Scientific and Technical Information of China (English)

    Hongbo Zhao; Xiliang Zha; Lidong Sun; Liying Wang; Zhibin Xu; Feng Zhou; Jianmin Su; Jiawei Jin; Yong Yang; Yali Hu

    2008-01-01

    E-cadherin, which has a widely acknowledged role in mediating calcium-dependent cell-cell adhesion between epithelial cells, also functions as a tumor suppressor. The ectodomain of human E-cadherin contains four potential N-glycosylation sites at Asn residues 554, 566, 618, and 633.We investigated the role of E-cadherin N-glycosylation in cell cycle progression by site-directed mutagenesis. We showed previously that all four potential N-glycosylation sites of E-cadherin were N-glycosylated in human breast carcinoma MDA-MB-435 cells. Removal of N-glycan at Asn633 dramatically affected E-cadherin stability. In this study we showed that E-cadherin mutant missing N-glycans at Asn554, Asn566 and Asn618 failed to induce cell cycle arrest in G1 phase and to suppress cell proliferation in comparison with wild-type E-cadherin. Moreover, N-glycans at Asn554 and Asn566, but not at Asn618, seemed to be indispensable for E-cadherin-mediated suppression of cell cycle progression.Removal of N-glycans at either Asn554 or Asn566 of E-cadherin was accompanied with the activation of the extracellular signal-regulated protein kinase signaling pathway. After treatment with PD98059, an inhibitor of the extraceilular signal-regulated protein kinase signaling pathway, wild-type E-cadherin transfected MDA-MB-435 and E-cadherin N-glycosylation-deficient mutant transfected MDA-MB-435 cells had equivalent numbers of cells in G1 phase. These findings implied that N-glycosylation might be crucial for E-cadherin-mediated suppression of cell cycle progression.

  16. Study on Effects of Extracts from Salvia Miltiorrhiza and Curcuma Longa in Inhibiting Phosphorylated Extracellular Signal Regulated Kinase Expression in Rat's Hepatic Stellate Cells

    Institute of Scientific and Technical Information of China (English)

    CHENG Yang; PING Jian; LIU Cheng; TAN Ying-zi; CHEN Gao-feng

    2006-01-01

    Objective: To study the effect of salvianolic acid B (SAB) and curcumin, the extracts of Salvia Miltiorrhiza and Curcuma Longa, on the proliferation and activation of hepatic stellate cell (HSC), and the extracellular signal regulated kinase (ERK) expression in it. Methods: Rat's HSC-T6 were cultured and treated by SAB or curcumin. The inhibitory effect on cell proliferation was determined by 3-(4,5-dimthyl-2-2thiazoly)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetry, and the expression levels of α smooth actin (α-SMA), collagen type Ⅰ , and ERK were determined by Western blot. Results: SAB and curcumin inhibited the proliferation and activation of rat's HSC-T6 in dose-dependent fashion and significantly reduced the expression level of α-SMA ( P<0.01 ). Curcumin significantly reduced the expression of collagen type Ⅰ( P<0.05). Both SAB and curcumin showed insignificant effect on the ERK expression level, but they could significantly reduce the level of phosphorylated-ERK expression, showing significant difference as compared with that in the control group ( P<0.01 and P<0.05 respectively). Conclusion: SAB and curcumin could significantly inhibit the proliferation, activation of HSC, and the production of type Ⅰ collagen in HSC, the mechanism may be associated with their inhibition on ERK phosphorylation.

  17. OVER-EXPRESSION OF EXTRACELLULAR SIGNAL-REGULATED KINASE IN VASCULAR SMOOTH MUSCLE CELL OF HYPERTENSIVE RATS

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Objective To investigate whether extracellular signal-regulated kinase (ERK1/2) was involved in changes of vascular smooth muscle cell (VSMC) under hypertension. Methods Two-kidney one clip Wistar hypertensive rats (WHR) were sacrificed and their right kidneys were harvested 4 weeks after surgery. The spontaneously hypertensive rats (SHR) were divided into 4, 8, and 16 weeks old groups (SHR4w, SHR8w, and SHR16w), respectively. The control group were sham operated age-matched Wistar rats. Immunohistochemical technique and Western blotting were applied to study ERK1/2 protein expression in VSMC of the renal vascular trees in WHR, SHR, and control rats. Results Blood pressure in two-kidney one clip WHR obviously increased at one week after surgery, and reached to 198.00 ± 33.00 mm Hg at the end of experiment, significantly higher than that in the control rats ( P < 0. 01 ). Blood pressure in SHR4w ( 108.00 ± 11.25 mm Hg) was similar to that in the controls. However, it rose to 122.25 ± 21.75 mm Hg in SHR8w, and even up to 201.75 ± 18.00 mm Hg in SHR16w, which were significantly higher than that of both the SHR4w and the controls ( P < 0. 01 ). The rate and degree of glomerular fibrosis in WHR were significantly higher than controls (P < 0. 05 ). Hyaline degeneration of the afferent arterioles was found in WHR. In contrast, either fibrosis of glomerulus or hyaline degeneration of the arterioles or protein casts was not observed in SHR4w, SHR8w,and SHR16w. Immunohistochemical staining results showed expression of ERK1 was similar to that of ERK2. The positive rates of ERK2 staining in VSMC of afferent arterioles, interlobular, interlobar, and arcuate arteries in two-kidney one clip WHR were significantly higher (7. 09% ± 1.75%, 14. 57% ± 4. 58%, 29.44% ± 7. 35%, and 13.63% ±3.85%, respectively) than that of the controls( P < 0. 01 ). The positive rates of ERK2 staining in VSMC at afferent arterioles, interlobular, interlobar, and arcuate arteries in SHR

  18. Cold-Inducible RNA-Binding Protein Bypasses Replicative Senescence in Primary Cells through Extracellular Signal-Regulated Kinase 1 and 2 Activation▿ †

    Science.gov (United States)

    Artero-Castro, Ana; Callejas, Francisco B.; Castellvi, Josep; Kondoh, Hiroshi; Carnero, Amancio; Fernández-Marcos, Pablo J.; Serrano, Manuel; Ramón y Cajal, Santiago; Lleonart, Matilde E.

    2009-01-01

    Embryonic stem cells are immortalized cells whose proliferation rate is comparable to that of carcinogenic cells. To study the expression of embryonic stem cell genes in primary cells, genetic screening was performed by infecting mouse embryonic fibroblasts (MEFs) with a cDNA library from embryonic stem cells. Cold-inducible RNA-binding protein (CIRP) was identified due to its ability to bypass replicative senescence in primary cells. CIRP enhanced extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation, and treatment with an MEK inhibitor decreased the proliferation caused by CIRP. In contrast to CIRP upregulation, CIRP downregulation decreased cell proliferation and resulted in inhibition of phosphorylated ERK1/2 inhibition. This is the first evidence that ERK1/2 activation, through the same mechanism as that described for a Val12 mutant K-ras to induce premature senescence, is able to bypass senescence in the absence of p16INK4a, p21WAF1, and p19ARF upregulation. Moreover, these results show that CIRP functions by stimulating general protein synthesis with the involvement of the S6 and 4E-BP1 proteins. The overall effect is an increase in kinase activity of the cyclin D1-CDK4 complex, which is in accordance with the proliferative capacity of CIRP MEFs. Interestingly, CIRP mRNA and protein were upregulated in a subgroup of cancer patients, a finding that may be of relevance for cancer research. PMID:19158277

  19. Matrine-induced apoptosis of human nasopharyngeal carcinoma cells via in vitro vascular endothelial growth factor-A/extracellular signal-regulated kinase1/2 pathway inactivation.

    Science.gov (United States)

    Xie, M; He, G; Wang, R; Shi, S; Chen, J; Ye, Y; Xie, L; Yi, X; Tang, A

    2014-07-01

    Matrine, a main active extract from Sophora flavescens Ait, has been demonstrated to exert anticancer effects on various cancer cell lines, such as malignant melanoma, breast cancer, and lung cancer. However, it is currently unclear whether matrine could also elicit an inhibitory effect on growth of nasopharyngeal carcinoma (NPC), let alone the possible molecular mechanisms. Therefore, in a previous study, we investigated matrine-induced proliferation inhibition and apoptosis in NPC cells. It was shown that proliferation of human NPC cells (CNE1 and CNE2) was significantly diminished by matrine in a dose- and time-dependent manner, and apoptosis was induced in both 2 NPC cells, particularly in CNE2 cells. Moreover, the increased apoptosis rate in matrine-treated CNE2 cells confirmed the proapoptotic activity of matrine. We further found that matrine treatment dose- and time-dependently reduced the levels of vascular endothelial growth factor-A (VEGF-A), and inactivated extracellular signal-regulated kinase1/2 (ERK1/2), followed by increased expression of downstream target caspase-3. Overall, we conclude that matrine could induce apoptosis of human NPC cells via VEGF-A/ERK1/2 pathway, which supports the potential use of matrine in clinically treating NPC.

  20. Differential expression of extracellular-signal-regulated kinase 5 (ERK5) in normal and degenerated human nucleus pulposus tissues and cells

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Weiguo, E-mail: liangweiguo@tom.com [Guangzhou Institute of Traumatic Surgery, The Fourth Affiliated Hospital of Medical College, Jinan University, Guangzhou 510220 (China); Fang, Dejian [Guangzhou Institute of Traumatic Surgery, The Fourth Affiliated Hospital of Medical College, Jinan University, Guangzhou 510220 (China); Ye, Dongping [Guangzhou Institute of Traumatic Surgery, The Fourth Affiliated Hospital of Medical College, Jinan University, Guangzhou 510220 (China); School of Pathology and Laboratory Medicine, University of Western Australia, Crawley, Western Australia 6009 (Australia); Zou, Longqiang; Shen, Yan; Dai, Libing [Guangzhou Institute of Traumatic Surgery, The Fourth Affiliated Hospital of Medical College, Jinan University, Guangzhou 510220 (China); Xu, Jiake, E-mail: jiake.xu@uwa.edu.au [Guangzhou Institute of Traumatic Surgery, The Fourth Affiliated Hospital of Medical College, Jinan University, Guangzhou 510220 (China); School of Pathology and Laboratory Medicine, University of Western Australia, Crawley, Western Australia 6009 (Australia)

    2014-07-11

    Highlights: • ERK5 involved in NP cells. • ERK5 involved in NP tissue. • It was important modulator. - Abstract: Extracellular-signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase (MAPK) family and regulates a wide variety of cellular processes such as proliferation, differentiation, necrosis, apoptosis and degeneration. However, the expression of ERK5 and its role in degenerated human nucleus pulposus (NP) is hitherto unknown. In this study, we observed the differential expression of ERK5 in normal and degenerated human nucleus pulposus tissues by using immunohistochemical staining and Western blot. Treatment of NP cells with Pro-inflammatory cytokine, TNF-α decreased ERK5 gene expression as well as NP marker gene expression; including the type II collagen and aggrecan. Suppression of ERK5 gene expression in NP cells by ERK5 siRNA resulted in decreased gene expression of type II collagen and aggrecan. Furthermore, inhibition of ERK5 activation by BIX02188 (5 μM) decreased the gene expression of type II collagen and aggrecan in NP cells. Our results document the expression of ERK5 in degenerated nucleus pulposus tissues, and suggest a potential involvement of ERK5 in human degenerated nucleus pulposus.

  1. Tissue kallikrein induces SH-SY5Y cell proliferation via epidermal growth factor receptor and extracellular signal-regulated kinase1/2 pathway

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Zhengyu [Department of Neurology, Huashan Hospital, State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai 200040 (China); Department of Neurology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200437 (China); Yang, Qi; Cui, Mei; Liu, Yanping [Department of Neurology, Huashan Hospital, State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai 200040 (China); Wang, Tao; Zhao, Hong [Department of Neurology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200437 (China); Dong, Qiang, E-mail: qiang_dong163@163.com [Department of Neurology, Huashan Hospital, State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai 200040 (China)

    2014-03-28

    Highlights: • TK promotes EGFR phosphorylation in SH-SY5Y cells. • TK activates ERK1/2 and p38 phosphorylation in SH-SY5Y cells. • TK mediates SH-SY5Y cell proliferation via EGFR and ERK1/2 pathway. - Abstract: Tissue kallikrein (TK) is well known to take most of its biological functions through bradykinin receptors. In the present study, we found a novel signaling pathway mediated by TK through epidermal growth factor receptor (EGFR) in human SH-SY5Y cells. We discovered that TK facilitated the activation of EGFR, extracellular signal-regulated kinase (ERK) 1/2 and p38 cascade. Interestingly, not p38 but ERK1/2 phosphorylation was severely compromised in cells depleted of EGFR. Nevertheless, impairment of signaling of ERK1/2 seemed not to be restricted to EGFR phosphorylation. We also observed that TK stimulation could induce SH-SY5Y cell proliferation, which was reduced by EGFR down-regulation or ERK1/2 inhibitor. Overall, our findings provided convincing evidence that TK could mediate cell proliferation via EGFR and ERK1/2 pathway in vitro.

  2. Spinal neurons that contain gastrin-releasing peptide seldom express Fos or phosphorylate extracellular signal-regulated kinases in response to intradermal chloroquine

    Science.gov (United States)

    Bell, Andrew M; Gutierrez-Mecinas, Maria; Polgár, Erika; Todd, Andrew J

    2016-01-01

    Background Gastrin-releasing peptide (GRP) is thought to play a role in the itch evoked by intradermal injection of chloroquine. Although some early studies suggested that GRP was expressed in pruriceptive primary afferents, it is now thought that GRP in the spinal cord is derived mainly from a population of excitatory interneurons in lamina II, and it has been suggested that these are involved in the itch pathway. To test this hypothesis, we used the transcription factor Fos and phosphorylation of extracellular signal-regulated kinases (ERK) to look for evidence that interneurons expressing GRP were activated following intradermal injection of chloroquine into the calf, in mice that express enhanced green fluorescent protein (EGFP) in these cells. Results Injection of chloroquine resulted in numerous Fos- or phospho-ERK (pERK) positive cells in the somatotopically appropriate part of the superficial dorsal horn. The proportion of all neurons in this region that showed Fos or pERK was 18% and 21%, respectively. However, among the GRP–EGFP, only 7% were Fos-positive and 3% were pERK-positive. As such, GRP–EGFP cells were significantly less likely than other neurons to express Fos or to phosphorylate ERK. Conclusions Both expression of Fos and phosphorylation of ERK can be used to identify dorsal horn neurons activated by chloroquine injection. However, these results do not support the hypothesis that interneurons expressing GRP are critical components in the itch pathway. PMID:27270268

  3. Tissue kallikrein induces SH-SY5Y cell proliferation via epidermal growth factor receptor and extracellular signal-regulated kinase1/2 pathway

    International Nuclear Information System (INIS)

    Highlights: • TK promotes EGFR phosphorylation in SH-SY5Y cells. • TK activates ERK1/2 and p38 phosphorylation in SH-SY5Y cells. • TK mediates SH-SY5Y cell proliferation via EGFR and ERK1/2 pathway. - Abstract: Tissue kallikrein (TK) is well known to take most of its biological functions through bradykinin receptors. In the present study, we found a novel signaling pathway mediated by TK through epidermal growth factor receptor (EGFR) in human SH-SY5Y cells. We discovered that TK facilitated the activation of EGFR, extracellular signal-regulated kinase (ERK) 1/2 and p38 cascade. Interestingly, not p38 but ERK1/2 phosphorylation was severely compromised in cells depleted of EGFR. Nevertheless, impairment of signaling of ERK1/2 seemed not to be restricted to EGFR phosphorylation. We also observed that TK stimulation could induce SH-SY5Y cell proliferation, which was reduced by EGFR down-regulation or ERK1/2 inhibitor. Overall, our findings provided convincing evidence that TK could mediate cell proliferation via EGFR and ERK1/2 pathway in vitro

  4. Vascular endothelial growth factor signaling regulates the segregation of artery and vein via ERK activity during vascular development

    International Nuclear Information System (INIS)

    Highlights: ► VEGF-A signaling regulates the segregation of axial vessels. ► VEGF-A signaling is mediated by PKC and ERK in this process. ► Ectopic activation of ERK is sufficient to rescue defects in vessel segregation. -- Abstract: Segregation of two axial vessels, the dorsal aorta and caudal vein, is one of the earliest patterning events occur during development of vasculature. Despite the importance of this process and recent advances in our understanding on vascular patterning during development, molecular mechanisms that coordinate the segregation of axial vessels remain largely elusive. In this report, we find that vascular endothelial growth factor-A (Vegf-A) signaling regulates the segregation of dorsal aorta and axial vein during development. Inhibition of Vegf-A pathway components including ligand Vegf-A and its cognate receptor Kdrl, caused failure in segregation of axial vessels in zebrafish embryos. Similarly, chemical inhibition of Mitogen-activated protein kinase kinase (Map2k1)/Extracellular-signal-regulated kinases (Erk) and phosphatidylinositol 3-kinases (PI3 K), which are downstream effectors of Vegf-A signaling pathway, led to the fusion of two axial vessels. Moreover, we find that restoring Erk activity by over-expression of constitutively active MEK in embryos with a reduced level of Vegf-A signaling can rescue the defects in axial vessel segregation. Taken together, our data show that segregation of axial vessels requires the function of Vegf-A signaling, and Erk may function as the major downstream effector in this process

  5. Vascular endothelial growth factor signaling regulates the segregation of artery and vein via ERK activity during vascular development

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Se-Hee [McAllister Heart Institute, Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 (United States); Schmitt, Christopher E.; Woolls, Melissa J. [McAllister Heart Institute, Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 (United States); Yale Cardiovascular Research Center and Section of Cardiovascular Medicine, Dept. of Internal Medicine, Yale University School of Medicine, New Haven, CT 06511 (United States); Holland, Melinda B. [McAllister Heart Institute, Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 (United States); Kim, Jun-Dae [Yale Cardiovascular Research Center and Section of Cardiovascular Medicine, Dept. of Internal Medicine, Yale University School of Medicine, New Haven, CT 06511 (United States); Jin, Suk-Won, E-mail: suk-won.jin@yale.edu [Yale Cardiovascular Research Center and Section of Cardiovascular Medicine, Dept. of Internal Medicine, Yale University School of Medicine, New Haven, CT 06511 (United States)

    2013-01-25

    Highlights: ► VEGF-A signaling regulates the segregation of axial vessels. ► VEGF-A signaling is mediated by PKC and ERK in this process. ► Ectopic activation of ERK is sufficient to rescue defects in vessel segregation. -- Abstract: Segregation of two axial vessels, the dorsal aorta and caudal vein, is one of the earliest patterning events occur during development of vasculature. Despite the importance of this process and recent advances in our understanding on vascular patterning during development, molecular mechanisms that coordinate the segregation of axial vessels remain largely elusive. In this report, we find that vascular endothelial growth factor-A (Vegf-A) signaling regulates the segregation of dorsal aorta and axial vein during development. Inhibition of Vegf-A pathway components including ligand Vegf-A and its cognate receptor Kdrl, caused failure in segregation of axial vessels in zebrafish embryos. Similarly, chemical inhibition of Mitogen-activated protein kinase kinase (Map2k1)/Extracellular-signal-regulated kinases (Erk) and phosphatidylinositol 3-kinases (PI3 K), which are downstream effectors of Vegf-A signaling pathway, led to the fusion of two axial vessels. Moreover, we find that restoring Erk activity by over-expression of constitutively active MEK in embryos with a reduced level of Vegf-A signaling can rescue the defects in axial vessel segregation. Taken together, our data show that segregation of axial vessels requires the function of Vegf-A signaling, and Erk may function as the major downstream effector in this process.

  6. The Ras/Raf/MEK/extracellular signal-regulated kinase pathway induces autocrine-paracrine growth inhibition via the leukemia inhibitory factor/JAK/STAT pathway.

    Science.gov (United States)

    Park, Jong-In; Strock, Christopher J; Ball, Douglas W; Nelkin, Barry D

    2003-01-01

    Sustained activation of the Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) pathway can lead to cell cycle arrest in many cell types. We have found, with human medullary thyroid cancer (MTC) cells, that activated Ras or c-Raf-1 can induce growth arrest by producing and secreting an autocrine-paracrine factor. This protein was purified from cell culture medium conditioned by Raf-activated MTC cells and was identified by mass spectrometry as leukemia inhibitory factor (LIF). LIF expression upon Raf activation and subsequent activation of JAK-STAT3 was also observed in small cell lung carcinoma cells, suggesting that this autocrine-paracrine signaling may be a common response to Ras/Raf activation. LIF was sufficient to induce growth arrest and differentiation of MTC cells. This effect was mediated through the gp130/JAK/STAT3 pathway, since anti-gp130 blocking antibody or dominant-negative STAT3 blocked the effects of LIF. Thus, LIF expression provides a novel mechanism allowing Ras/Raf signaling to activate the JAK-STAT3 pathway. In addition to this cell-extrinsic growth inhibitory pathway, we find that the Ras/Raf/MEK/ERK pathway induces an intracellular growth inhibitory signal, independent of the LIF/JAK/STAT3 pathway. Therefore, activation of the Ras/Raf/MEK/ERK pathway can lead to growth arrest and differentiation via at least two different signaling pathways. This use of multiple pathways may be important for "fail-safe" induction and maintenance of cell cycle arrest.

  7. Mesenchymal stem cells cultured under hypoxia escape from senescence via down-regulation of p16 and extracellular signal regulated kinase

    International Nuclear Information System (INIS)

    Hypoxia has been considered to affect the properties of tissue stem cells including mesenchymal stem cells (MSCs). Effects of long periods of exposure to hypoxia on human MSCs, however, have not been clearly demonstrated. MSCs cultured under normoxic conditions (20% pO2) ceased to proliferate after 15-25 population doublings, while MSCs cultured under hypoxic conditions (1% pO2) retained the ability to proliferate with an additional 8-20 population doublings. Most of the MSCs cultured under normoxic conditions were in a senescent state after 100 days, while few senescent cells were found in the hypoxic culture, which was associated with a down-regulation of p16 gene expression. MSCs cultured for 100 days under hypoxic conditions were superior to those cultured under normoxic conditions in the ability to differentiate into the chondro- and adipogenic, but not osteogenic, lineage. Among the molecules related to mitogen-activated protein kinase (MAPK) signaling pathways, extracellular signal regulated kinase (ERK) was significantly down-regulated by hypoxia, which helped to inhibit the up-regulation of p16 gene expression. Therefore, the hypoxic culture retained MSCs in an undifferentiated and senescence-free state through the down-regulation of p16 and ERK.

  8. Growth arrest- and DNA-damage-inducible 45beta gene inhibits c-Jun N-terminal kinase and extracellular signal-regulated kinase and decreases IL-1beta-induced apoptosis in insulin-producing INS-1E cells

    DEFF Research Database (Denmark)

    Larsen, Claus Morten; Døssing, M G; Papa, S;

    2006-01-01

    IL-1beta is a candidate mediator of apoptotic beta cell destruction, a process that leads to type 1 diabetes and progression of type 2 diabetes. IL-1beta activates beta cell c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38, all of which are members of the mitogen......-activated protein kinase (MAPK) family. Inhibition of JNK prevents IL-1beta-mediated beta cell destruction. In mouse embryo fibroblasts and 3DO T cells, overexpression of the gene encoding growth arrest and DNA-damage-inducible 45beta (Gadd45b) downregulates pro-apoptotic JNK signalling. The aim of this study...

  9. Homogeneous Time-Resolved Fluorescence-Based Assay to Monitor Extracellular Signal-Regulated Kinase Signalling in a High-Throughput Format

    Directory of Open Access Journals (Sweden)

    Mohammed Akli eAyoub

    2014-06-01

    Full Text Available The extracellular signal-regulated kinases (ERKs are key components of multiple important cell signalling pathways regulating diverse biological responses. This signalling is characterized by phosphorylation cascades leading to ERK1/2 activation and promoted by various cell surface receptors including G protein-coupled receptors (GPCRs and receptor tyrosine kinases (RTKs. We report the development of a new cell-based phospho-ERK1/2 assay (designated Phospho-ERK, which is a sandwich proximity-based assay using the homogeneous time-resolved fluorescence technology. We have validated the assay on endogenously expressed ERK1/2 activated by the epidermal growth factor (EGFR as a prototypical RTK, as well as various GPCRs belonging to different classes and coupling to different heterotrimeric G proteins. The assay was successfully miniaturized in 384-well plates using various cell lines endogenously, transiently or stably expressing the different receptors. The validation was performed for agonists, antagonists and inhibitors in dose-response as well as kinetic analysis, and the signalling and pharmacological properties of the different receptors were reproduced. Furthermore, the determination of a Z’-factor value of 0.7 indicates the potential of the Phospho-ERK assay for high-throughput screening of compounds that may modulate ERK1/2 signalling. Finally, our study is of great interest in the current context of investigating ERK1/2 signalling with respect to the emerging concepts of biased ligands, G protein-dependent/independent ERK1/2 activation, and functional transactivation between GPCRs and RTKs, illustrating the importance of considering the ERK1/2 pathway in cell signalling.

  10. Nitric oxide production by Biomphalaria glabrata haemocytes: effects of Schistosoma mansoni ESPs and regulation through the extracellular signal-regulated kinase pathway

    Directory of Open Access Journals (Sweden)

    Kirk Ruth S

    2009-04-01

    Full Text Available Abstract Background Schistosoma mansoni uses Biomphalaria glabrata as an intermediate host during its complex life cycle. In the snail, the parasite initially transforms from a miracidium into a mother sporocyst and during this process excretory-secretory products (ESPs are released. Nitric oxide (NO and its reactive intermediates play an important role in host defence responses against pathogens. This study therefore aimed to determine the effects of S. mansoni ESPs on NO production in defence cells (haemocytes from schistosome-susceptible and schistosome-resistant B. glabrata strains. As S. mansoni ESPs have previously been shown to inhibit extracellular signal-regulated kinase (ERK phosphorylation (activation in haemocytes from susceptible, but not resistant, B. glabrata the regulation of NO output by ERK in these cells was also investigated. Results Haemocytes from resistant snails challenged with S. mansoni ESPs (20 μg/ml over 5 h displayed an increase in NO production that was 3.3 times greater than that observed for unchallenged haemocytes; lower concentrations of ESPs (0.1–10 μg/ml did not significantly increase NO output. In contrast, haemocytes from susceptible snails showed no significant change in NO output following challenge with ESPs at any concentration used (0.1–20 μg/ml. Western blotting revealed that U0126 (1 μM or 10 μM blocked the phosphorylation (activation status of ERK in haemocytes from both snail strains. Inhibition of ERK signalling by U0126 attenuated considerably intracellular NO production in haemocytes from both susceptible and resistant B. glabrata strains, identifying ERK as a key regulator of NO output in these cells. Conclusion S. mansoni ESPs differentially influence intracellular NO levels in susceptible and resistant B. glabrata haemocytes, possibly through modulation of the ERK signalling pathway. Such effects might facilitate survival of S. mansoni in its intermediate host.

  11. Tissue Strain Reorganizes Collagen With a Switchlike Response That Regulates Neuronal Extracellular Signal-Regulated Kinase Phosphorylation In Vitro: Implications for Ligamentous Injury and Mechanotransduction.

    Science.gov (United States)

    Zhang, Sijia; Cao, Xuan; Stablow, Alec M; Shenoy, Vivek B; Winkelstein, Beth A

    2016-02-01

    Excessive loading of ligaments can activate the neural afferents that innervate the collagenous tissue, leading to a host of pathologies including pain. An integrated experimental and modeling approach was used to define the responses of neurons and the surrounding collagen fibers to the ligamentous matrix loading and to begin to understand how macroscopic deformation is translated to neuronal loading and signaling. A neuron-collagen construct (NCC) developed to mimic innervation of collagenous tissue underwent tension to strains simulating nonpainful (8%) or painful ligament loading (16%). Both neuronal phosphorylation of extracellular signal-regulated kinase (ERK), which is related to neuroplasticity (R2 ≥ 0.041; p ≤ 0.0171) and neuronal aspect ratio (AR) (R2 ≥ 0.250; p element based discrete fiber network (DFN) model predicted that at bulk strains above the transition point, heterogeneous fiber strains were both tensile and compressive and increased, with strains in some fibers along the loading direction exceeding the applied bulk strain. The transition point identified for changes in collagen fiber realignment was consistent with the measured strain threshold (11.7% with a 95% confidence interval of 10.2-13.4%) for elevating ERK phosphorylation after loading. As with collagen fiber realignment, the greatest degree of neuronal reorientation toward the loading direction was observed at the NCC distraction corresponding to painful loading. Because activation of neuronal ERK occurred only at strains that produced evident collagen fiber realignment, findings suggest that tissue strain-induced changes in the micromechanical environment, especially altered local collagen fiber kinematics, may be associated with mechanotransduction signaling in neurons. PMID:26549105

  12. Dexamethasone suppresses DU145 cell proliferation and cell cycle through inhibition of the extracellular signal-regulated kinase 1/2 pathway and cyclin D1 expression

    Institute of Scientific and Technical Information of China (English)

    Qing-Zhen Gao; Jia-Ju Lu; Zi-Dong Liu; Hui Zhang; Shao-Mei Wang; He Xu

    2008-01-01

    Aim: To determine the mechanisms of glucocorticoids in inhibiting advanced prostate cancer growth. Methods: The cell proliferation and cell cycle of prostate cancer DU145 cells following dexamethasone treatment were determined by proliferation assay and fluorescence-activated cell sorter. Western blot analysis was carried out to evaluate the effects of dexamethasone on phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and expression of cyclin D1 in DU145 cells with or without glucocorticoid receptor (GR) antagonist RU486. Reverse transcription- polymerase chain reaction verified the expression of GR mRNA in DU145 cells. Results: Dexamethasone signifi- cantly inhibited DU145 cell proliferation at the G0/G1 phase. Western blot analysis showed a dramatic reduction of ERK1/2 activity and cyclin D1 expression in dexamethasone-treated cells. The decreased phosphorylation of ERK1/2 in dexamethasone-treated cells was attenuated by GR blockade. Additionally, the effects of dexamethasone in inhibiting cyclin D1 expression were altered by GR blockade. Conclusion: Dexamethasone suppresses DU 145 cell prolifera- tion and cell cycle, and the underlying mechanisms are through the inhibition of phosphorylation of ERK1/2 and cyclin D1 expression. The inhibition of ERK1/2 phosphorylation and cyclin D1 expression is attenuated by GR blockade, suggesting that GR regulates ERK1/2 and cyclin D 1 pathways. These observations suggest that dexamethasone has a potential clinical application in prostate cancer therapy. (Asian JAndrol 2008 Jul; 10: 635-641)

  13. Extracellular signal-regulated kinase mediates gonadotropin subunit gene expression and LH release responses to endogenous gonadotropin-releasing hormones in goldfish.

    Science.gov (United States)

    Klausen, Christian; Booth, Morgan; Habibi, Hamid R; Chang, John P

    2008-08-01

    The possible involvement of extracellular signal-regulated kinase (ERK) in mediating the stimulatory actions of two endogenous goldfish gonadotropin-releasing hormones (salmon (s)GnRH and chicken (c)GnRH-II) on gonadotropin synthesis and secretion was examined. Western blot analysis revealed the presence of ERK and phosphorylated (p)ERK in goldfish brain, pituitary, liver, ovary, testis and muscle tissue extracts, as well as extracts of dispersed goldfish pituitary cells and HeLa cells. Interestingly, a third ERK-like immunoreactive band of higher molecular mass was detected in goldfish tissue and pituitary cell extracts in addition to the ERK1-p44- and ERK2-p42-like immunoreactive bands. Incubation of primary cultures of goldfish pituitary cells with either a PKC-activating 4beta-phorbol ester (TPA) or a synthetic diacylglycerol, but not a 4alpha-phorbol ester, elevated the ratio of pERK/total (t)ERK for all three ERK isoforms. The stimulatory effects of TPA were attenuated by the PKC inhibitor GF109203X and the MEK inhibitor PD98059. sGnRH and cGnRH-II also elevated the ratio of pERK/tERK for all three ERK isoforms, in a time-, dose- and PD98059-dependent manner. In addition, treatment with PD98059 reduced the sGnRH-, cGnRH-II- and TPA-induced increases in gonadotropin subunit mRNA levels in Northern blot studies and sGnRH- and cGnRH-II-elicited LH release in cell column perifusion studies with goldfish pituitary cells. These results indicate that GnRH and PKC can activate ERK through MEK in goldfish pituitary cells. More importantly, the present study suggests that GnRH-induced gonadotropin subunit gene expression and LH release involve MEK/ERK signaling in goldfish. PMID:18558406

  14. Osthole-mediated cell differentiation through bone morphogenetic protein-2/p38 and extracellular signal-regulated kinase 1/2 pathway in human osteoblast cells.

    Science.gov (United States)

    Kuo, Po-Lin; Hsu, Ya-Ling; Chang, Cheng-Hsiung; Chang, Jiunn-Kae

    2005-09-01

    The survival of osteoblast cells is one of the determinants of the development of osteoporosis in patients. Osthole (7-methoxy-8-isopentenoxycoumarin) is a coumarin derivative present in many medicinal plants. By means of alkaline phosphatase (ALP) activity, osteocalcin, osteopontin, and type I collagen, enzyme-linked immunosorbent assay, we have shown that osthole exhibits a significant induction of differentiation in two human osteoblast-like cell lines, MG-63 and hFOB. Induction of differentiation by osthole was associated with increased bone morphogenetic protein (BMP)-2 production and the activations of SMAD1/5/8 and p38 and extracellular signal-regulated kinase (ERK) 1/2 kinases. Addition of purified BMP-2 protein did not increase the up-regulation of ALP activity and osteocalcin by osthole, whereas the BMP-2 antagonist noggin blocked both osthole and BMP-2-mediated ALP activity enhancement, indicating that BMP-2 production is required in osthole-mediated osteoblast maturation. Pretreatment of osteoblast cells with noggin abrogated p38 activation but only partially decreased ERK1/2 activation, suggesting that BMP-2 signaling is required in p38 activation and is partially involved in ERK1/2 activation in osthole-treated osteoblast cells. Cotreatment of p38 inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole] or p38 small interfering RNA (siRNA) expression inhibited osthole-mediated activation of ALP but only slightly affected osteocalcin production. In contrast, the production of osteocalcin induced by osthole was inhibited by the mitogen-activated protein kinase kinase inhibitor PD98059 (2'-amino-3'-methoxyflavone) or by expression of an ERK2 siRNA. These data suggest that BMP-2/p38 pathway links to the early phase, whereas ERK1/2 pathway is associated with the later phase in osthole-mediated differentiation of osteoblast cells. In this study, we demonstrate that osthole is a promising agent for treating osteoporosis

  15. Effect of Sodium Tanshinone Ⅱ A Sulfonate on Phosphorylation of Extracellular Signal-regulated Kinase1/2 in Angiotensin Ⅱ-induced Hypertrophy of Myocardial Cells

    Institute of Scientific and Technical Information of China (English)

    LI Shu-sheng; FENG Jun; ZHENG Zhi; LIANG Qian-sheng

    2008-01-01

    Objective:To observe the effects of sodium tanshinone Ⅱ A sulfonate(STS)on angiotensin Ⅱ(Ang Ⅱ)-induced hypertrophy of myocardial cells through the expression of phosphorylated extracellular signal-regulated kinase(P-ERK1/2).Methods:In the primary culture of neonatal rat myocardial cells.the total protein content in myocardial cells was determined by coomassie brilliant blue and the protein synthesis rate was measured by[3H]-Leucine incorporation as indexes for hypertrophy of myocardial cells.The expression of p-ERK1/2 was determined using Western blot and immunofluorescence Iabeling.Results:(1)The totaI protein and protein synthesis rate increased significantly in contrast to the control group after the myocardial cells were stimulated by Ang Ⅱ (1 μmol/L)for 24 h;STS markedly inhibited the increment of the total protein level induced by Ang Ⅱ and the syntheses of protein.(2)After pretreatment of myocardial cells with Ang Ⅱ(1 μ mol/L)for 5 min,the p-ERK1/2 protein expression was increased,with the most obvious effect shown at about 10 min;pretreatment of myocardial cells with STS at different doses(2,10,50 μ mol/L)for 30 min resulted in obvious inhibition of the expression of p-ERK1/2 stimulated by Ang Ⅱ in a dose-dependent manner.(3)After the myocardial cells were stimulated by Ang Ⅱ(1 μ mol/L),the immunofluorescence of ERK1/2 rapidly appeared in the nucleus.The activation and translocation process of ERK1/2 induced by Ang Ⅱ was blocked distinctly by STS.Conclusion:STS inhibited the myocardial cell hypertrophy induced by Ang Ⅱ,and the mechanism may be associated with the inhibition of p-ERK1/2 expression.

  16. Angiotensin Ⅱ induced upregulation of Gαq/11, phospholipase Cβ3 and extracellular signal-regulated kinase 1/2 via angiotensin Ⅱtype 1 receptor

    Institute of Scientific and Technical Information of China (English)

    白桦; 吴立玲; 邢东琦; 刘洁; 赵亚莉

    2004-01-01

    Background The role of the Gαq/11-mediated signal transduction pathway in angiotensin Ⅱ (Ang Ⅱ) induced cardiac hypertrophy remains unclear. This study was to investigate the role of the Gαq/11 signal transduction pathway in the development of cardiac hypertrophy in 2K1C hypertensive rats and in cultured neonatal rat ventricular myocytes (NRVMs) and to elucidate the effects of the pathway on Ang Ⅱ induced cardiac hypertrophy.Methods Renal hypertension was induced in 2K1C hypertensive rats by placing a silver clip around the left renal artery. At 8 weeks after operation, the systolic blood pressure, the ratio of left ventricular weight to body weight (LV/BW), and the concentration of AngⅡ in the heart were measured. The protein levels of Gαq/11 and extracellular signal-regulated kinase 1/2 (ERK1/2) were assayed by Western blot analysis, and the activity of phospholipase C (PLC) in the myocardium was detected using [3H]-PIP2 as a substrate. Changes in [3H]-leucine incorporation and in the protein levels of the signal molecules Gαq/11, PLCβ3, and ERK1/2 were measured after NRVMs were stimulated with 10-7mol/L AngⅡ. Results The protein levels of Gαq/11 and ERK1/2 in the hearts of 2K1C rats increased by 35.8% and 31.9%, respectively, compared with the sham group. The PLC activity in the 2K1C group was also significantly increased (P<0.05). The levels of Gαq/11, PLCβ3, and ERK1/2 increased significantly after NRVMs were stimulated by AngⅡ. The upregulation of Gαq/11, PLCβ3 and ERK1/2 in NRVMs occurred prior to [3H]-leucine incorporation increases, and could be inhibited with losartan. Conclusion AngⅡ can initiate cardiac hypertrophy and upregulate signal molecules in the Gαq/11-mediated signal transduction pathway, such as Gαq/11, PLCβ3 and ERK1/2, at both tissue and cellular levels.

  17. Constitutive hypophosphorylation of extracellular signal-regulated kinases-1/2 and down-regulation of c-Jun in human gastric adenocarcinoma

    International Nuclear Information System (INIS)

    Hyperphosphorylation of extracellular signal-regulated protein kinases-1/2 (ERK1/2) is known to promote cancer cell proliferation. We therefore investigated the constitutive phosphorylation levels of ERK1/2 and the expression of its downstream targets c-Fos, c-Jun, and cyclooxygenase-2 (COX-2) in biopsied human gastric cancer tissues. Results showed that ERK1/2 phosphorylation and c-Jun expression were significantly lowered in gastric cancer compared with the non-cancer adjacent tissues. The expression of c-Fos, however, was not altered while COX-2 was significantly up-regulated. To conclude, we demonstrate that hypophosphorylation of ERK1/2 may occur in gastric cancer. Such discovery may have implication in the application of pathway-directed therapy for this malignant disease

  18. Metabotropic glutamate receptor 5 contributes to inflammatory tongue pain via extracellular signal-regulated kinase signaling in the trigeminal spinal subnucleus caudalis and upper cervical spinal cord

    Directory of Open Access Journals (Sweden)

    Liu Ming-Gang

    2012-11-01

    Full Text Available Abstract Background In the orofacial region, limited information is available concerning pathological tongue pain, such as inflammatory pain or neuropathic pain occurring in the tongue. Here, we tried for the first time to establish a novel animal model of inflammatory tongue pain in rats and to investigate the roles of metabotropic glutamate receptor 5 (mGluR5-extracellular signal-regulated kinase (ERK signaling in this process. Methods Complete Freund’s adjuvant (CFA was submucosally injected into the tongue to induce the inflammatory pain phenotype that was confirmed by behavioral testing. Expression of phosphorylated ERK (pERK and mGluR5 in the trigeminal subnucleus caudalis (Vc and upper cervical spinal cord (C1-C2 were detected with immunohistochemical staining and Western blotting. pERK inhibitor, a selective mGluR5 antagonist or agonist was continuously administered for 7 days via an intrathecal (i.t. route. Local inflammatory responses were verified by tongue histology. Results Submucosal injection of CFA into the tongue produced a long-lasting mechanical allodynia and heat hyperalgesia at the inflamed site, concomitant with an increase in the pERK immunoreactivity in the Vc and C1-C2. The distribution of pERK-IR cells was laminar specific, ipsilaterally dominant, somatotopically relevant, and rostrocaudally restricted. Western blot analysis also showed an enhanced activation of ERK in the Vc and C1-C2 following CFA injection. Continuous i.t. administration of the pERK inhibitor and a selective mGluR5 antagonist significantly depressed the mechanical allodynia and heat hyperalgesia in the CFA-injected tongue. In addition, the number of pERK-IR cells in ipsilateral Vc and C1-C2 was also decreased by both drugs. Moreover, continuous i.t. administration of a selective mGluR5 agonist induced mechanical allodynia in naive rats. Conclusions The present study constructed a new animal model of inflammatory tongue pain in rodents, and

  19. Sexual differences of the effects of prenatal stress on the expression of extracellular signal-regulated kinaseas in the hippocampus of offspring rats

    Institute of Scientific and Technical Information of China (English)

    Qing Cai; Zhongliang Zhu; Xiaoli Fan; Ning Jia; Qinghong Li; Liang Song; Hui Li; Jiankang Liu

    2006-01-01

    were separated and housed four in each cage respectively until test at 30 days of age. At the end of postnatal day 30, one male and female offspring rats from the same dam were selected with a random choice and a total of 24 animals from 12 different dams were used. The experimental rats were sacrificed by decapitation under anesthesia. Bilateral hippocampal tissues were isolated and homogenized in cold condition. Alkaline carbonate buffer (BCA)method was used to detect the concentration of extracellular signal-regulated kinases (ERK), then mixed with loading buffer, the constant voltage was 100 V. Finally, BCIP/NBT staining and electrophoresis were performed, the absorbance (A) value for the bands was detected with the Bandscan analytical software,and the expression of ERK in hippocampus of offspring rats of different genders in each group was quantitatively analyzed.MAIN OUTCOME MEASURES: The level of ERK expression in hippocampus of offspring rats of different genders in each group was observed.RESULTS: All the 24 offspring rats were involved in the analysis of results. ① The staining results of ERP activity in the extract of brain tissue detected with Western blotting technique and specific antibody analysis showed that the ERP in hippocampus of offspring rats had two subtypes of ERK-1 and ERK-2, and the latter was the main type. ② Standardized by the average A value in the control group, the quantitative data of the general A value of total ERK showed that the expression of ERK-2 in hippocampus of female offspring rats was obviously higher in the PNS group than in the control group (A value: 126±6.76,100±4.89,P < 0.01). ③The expression of ERK-2 had no obvious difference between the female and male offspring rats in the control group. ④ The expression of ERK-2 in hippocampus of male offspring rats was a little higher in the PNS group than in the control group (A value: 104±6.27,102±5.48,P > 0.05).CONCLUSION: PNS significantly affects the increase of

  20. Acidic pH stimulates the production of the angiogenic CXC chemokine, CXCL8 (interleukin-8), in human adult mesenchymal stem cells via the extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and NF-kappaB pathways.

    Science.gov (United States)

    Bischoff, David S; Zhu, Jian-Hua; Makhijani, Nalini S; Yamaguchi, Dean T

    2008-07-01

    Blood vessel injury results in limited oxygen tension and diffusion leading to hypoxia, increased anaerobic metabolism, and elevated production of acidic metabolites that cannot be easily removed due to the reduced blood flow. Therefore, an acidic extracellular pH occurs in the local microenvironment of disrupted bone. The potential role of acidic pH and glu-leu-arg (ELR(+)) CXC chemokines in early events in bone repair was studied in human mesenchymal stem cells (hMSCs) treated with medium of decreasing pH (7.4, 7.0, 6.7, and 6.4). The cells showed a reciprocal increase in CXCL8 (interleukin-8, IL-8) mRNA levels as extracellular pH decreased. At pH 6.4, CXCL8 mRNA was induced >60x in comparison to levels at pH 7.4. hMSCs treated with osteogenic medium (OGM) also showed an increase in CXCL8 mRNA with decreasing pH; although, at a lower level than that seen in cells grown in non-OGM. CXCL8 protein was secreted into the medium at all pHs with maximal induction at pH 6.7. Inhibition of the G-protein-coupled receptor alpha, G(alphai), suppressed CXCL8 levels in response to acidic pH; whereas phospholipase C inhibition had no effect on CXCL8. The use of specific mitogen-activated protein kinase (MAPK) signal transduction inhibitors indicated that the pH-dependent increase in CXCL8 mRNA is due to activation of ERK and p38 pathways. The JNK pathway was not involved. NF-kappaB inhibition resulted in a decrease in CXCL8 levels in hMSCs grown in non-OGM. However, OGM-differentiated hMSCs showed an increase in CXCL8 levels when treated with the NF-kappaB inhibitor PDTC, a pyrrolidine derivative of dithiocarbamate.

  1. Light induces Fos expression via extracellular signal-regulated kinases 1/2 in melanopsin-expressing PC12 cells

    DEFF Research Database (Denmark)

    Moldrup, Marie-Louise Bülow; Georg, Birgitte; Falktoft, Birgitte;

    2010-01-01

    -regulated protein kinase 1/2 (ERK1/2) was found as pharmacological blockage of this kinase suppressed the light-induced Fos expression. Illumination increased the inositol phosphate turnover and induced phosphorylation of ERK1/2 and p38 but not the c-Jun N-terminal kinase. The Galpha(q/11) protein inhibitor YM......254890 attenuated these intracellular light responses. Our data strongly indicate that Galpha(q/11)-mediated ERK1/2 activation is essential for expression of Fos upon illumination of melanopsin-expressing PC12 cells....

  2. PtdIns(3,4,5)P3-dependent Rac Exchanger 1 (PREX1) Rac-Guanine Nucleotide Exchange Factor (GEF) Activity Promotes Breast Cancer Cell Proliferation and Tumor Growth via Activation of Extracellular Signal-regulated Kinase 1/2 (ERK1/2) Signaling.

    Science.gov (United States)

    Liu, Heng-Jia; Ooms, Lisa M; Srijakotre, Nuthasuda; Man, Joey; Vieusseux, Jessica; Waters, JoAnne E; Feng, Yue; Bailey, Charles G; Rasko, John E J; Price, John T; Mitchell, Christina A

    2016-08-12

    PtdIns(3,4,5)P3-dependent Rac exchanger 1 (PREX1) is a Rac-guanine nucleotide exchange factor (GEF) overexpressed in a significant proportion of human breast cancers that integrates signals from upstream ErbB2/3 and CXCR4 membrane surface receptors. However, the PREX1 domains that facilitate its oncogenic activity and downstream signaling are not completely understood. We identify that ERK1/2 MAPK acts downstream of PREX1 and contributes to PREX1-mediated anchorage-independent cell growth. PREX1 overexpression increased but its shRNA knockdown decreased ERK1/2 phosphorylation in response to EGF/IGF-1 stimulation, resulting in induction of the cell cycle regulators cyclin D1 and p21(WAF1/CIP1) PREX1-mediated ERK1/2 phosphorylation, anchorage-independent cell growth, and cell migration were suppressed by inhibition of MEK1/2/ERK1/2 signaling. PREX1 overexpression reduced staurosporine-induced apoptosis whereas its shRNA knockdown promoted apoptosis in response to staurosporine or the anti-estrogen drug tamoxifen. Expression of wild-type but not GEF-inactive PREX1 increased anchorage-independent cell growth. In addition, mouse xenograft studies revealed that expression of wild-type but not GEF-dead PREX1 resulted in the formation of larger tumors that displayed increased phosphorylation of ERK1/2 but not AKT. The impaired anchorage-independent cell growth, apoptosis, and ERK1/2 signaling observed in stable PREX1 knockdown cells was restored by expression of wild-type but not GEF-dead-PREX1. Therefore, PREX1-Rac-GEF activity is critical for PREX1-dependent anchorage-independent cell growth and xenograft tumor growth and may represent a possible therapeutic target for breast cancers that exhibit PREX1 overexpression. PMID:27358402

  3. Roles of extracellular signal-regulated kinase 1/2 on the suppression of myostatin gene expression induced by basic fibroblast growth factor

    Institute of Scientific and Technical Information of China (English)

    Huazhoag Liu; Xiaorong An; Yongfu Chen; Jieping Zhong

    2008-01-01

    Basic fibmblast growth factor (bFGF, FG F-2 ) has an inhibitory effect on the expression of the myostatin gene in murine C2C12 myoblasts, as shown in our recent investigation. To further verify the regulatory effects of bFGF on the myostalin gene and to better understand its mechanism in skeletal muscle, and to promote clinical applications of bFGF to treat skeletal muscle diseases correlated to muscular dystrophy or AIDS and so on, recombinant human bFGF (rh-bFGF) was added into media and stimulated murine C2C12 myoblasts to investigate the dose-dependent effect ofbFGF on suppression of myostatin gene expression and the role of extracellular signal-regulated kinase 1/2 (ERK1/2) in the regulatory mechanism. Simultaneously, complete coding sequence of ovine 18 kDa-bFGF gene was inserted into eukaryotic vector pCMV-neo (originated from pEGFP-N1 vector, from which the EGFP gene has been removed), the recombinant plasmid pCMV-neo-bFGF was harvested and injected into the mouse skeletal muscle of posterior limb. Expression levels of bFGF,myostatin, and ERKI/2 genes in murine C2C12 myoblasts and the skeletal muscle were analyzed by real-time reverse transcription-polymerase chain reaction and Western blotting analysis, respectively. The results showed that bFGFimpaired the expression ofmyostatin gene in a dose-dependent manner in C2C12 cells, with increasing concentration of rh-bFGF,myostatin mRNA declined gradually. In addition, results in skeletal muscle indicated that bFGF also suppressed the expression of the myostatin gene in vivo. Furthermore, we found ERKI/2 participated in the regulatory mechanism of bFGF on the expression of the myostatin gene.

  4. Role of extracellular signal-regulated kinase 1/2 in cigarette smoke-induced mucus hypersecretion in a rat model

    Institute of Scientific and Technical Information of China (English)

    XIAO Jun; WANG Ke; FENG Yu-lin; CHEN Xue-rong; XU Dan; ZHANG Ming-ke

    2011-01-01

    Background Airway mucus hypersecretion is an important pathophysiological feature of chronic obstructive pulmonary disease,which is closely associated with cigarette smoking.However,the signal transduction pathway from the cell surface to the nucleus through which cigarette smoke causes upregulation of mucin gene expression is not well known.This study was designed to investigate the role of extracellular signal-regulated Kinase 1/2 (ERK 1/2) in airway mucus hypersecretion induced by cigarette smoke in rats.Methods A rat model of airway mucus hypersecretion was induced by exposure to cigarette smoke for 4 weeks.Rats exposed to inhalation of cigarette smoke or normal saline were given an intraperitoneal injection of U0126,a specific MEK1 kinase inhibitor,at doses of 0.25 mg/kg,0.5 mg/kg and 1 mg/kg for 14 days.Expression of MUC5AC mRNA and protein,ERK 1/2 and phosphorylated-ERK 1/2 (p-ERK 1/2) were detected by RT-PCR,immunohistochemistry and Western blotting.Results Cigarette smoke significantly increased airway goblet cells metaplasia,induced the overexpression of MUG5AC mRNA and protein in bronchial epithelia,and increased the ratio of p-ERK 1/2 and ERK 1/2.U0126 significantly attentuated the expression of MUC5AC mRNA and protein induced by cigarette smoke (P <0.05).Moreover,there was a significant positive correlation between the ratio of p-ERK1/2 to ERK1/2 and the expression of MUC5AC mRNA and protein (P<0.05).Conclusions Inhibition of ERK 1/2 by U0126 decreased the ratio of p-ERK 1/2 to ERK 1/2 and expression of MUC5AC mRNA and protein.ERK 1/2 may play an essential role in cigarette smoke-induced mucus hypersecretion in vivo.

  5. Glucose- and interleukin-1beta-induced beta-cell apoptosis requires Ca2+ influx and extracellular signal-regulated kinase (ERK) 1/2 activation and is prevented by a sulfonylurea receptor 1/inwardly rectifying K+ channel 6.2 (SUR/Kir6.2) selective potassium channel opener in human islets

    DEFF Research Database (Denmark)

    Maedler, Kathrin; Størling, Joachim; Sturis, Jeppe;

    2004-01-01

    -regulated kinase (ERK) 1/2, an effect that was abrogated by 3 micromol/l NN414. Similarly, 1 micromol/l of the mitogen-activated protein kinase/ERK kinase 1/2 inhibitor PD098059 or 1 micromol/l of the l-type Ca(2+) channel blocker nimodipine prevented glucose- and IL-1beta-induced ERK activation, beta......-cell apoptosis, and impaired function. Finally, islet release of IL-1beta in response to high glucose could be abrogated by nimodipine, NN414, or PD098059. Thus, in human islets, glucose- and IL-1beta-induced beta-cell secretory dysfunction and apoptosis are Ca(2+) influx and ERK dependent and can be prevented...

  6. Mechanism of the activation of extracellular signal-regulated kinase (ERK) in prostate cancer cell lines with different metastatic potential%细胞外信号调节激酶在不同转移性人前列腺癌细胞中被激活的 调节机制

    Institute of Scientific and Technical Information of China (English)

    李红梅; 何春生; 郑杰; 由江峰; 吴秉铨; 方伟岗

    2001-01-01

    目的 探讨不同转移潜能的人前列腺癌细胞中与增殖分化密切相关的细胞外信号调节激酶(ERK)信号传导途径被激活的机制。方法 用细胞计数及MTT法检测外源性P2嘌呤受体激动剂ATP 对人前列腺癌细胞PC-3M亚系1E8(高转移)和2B4(低转移)体外生长的影响。用特异性识别双磷酸化ERK1/2(p44/p42)的抗体及蛋白质印迹方法,检测细胞经ATP作用后ERK1/2的活化情况并研究其调节机制。结果 ATP可明显抑制1E8和2B4细胞的体外生长,第6和第8天的抑制率分别为:1E8:54% 和59%;2B4:67% 和39%。ATP可激活1E8和2B4细胞内的ERK1/2 激酶。ATP诱导的ERK1/2活化可被P2嘌呤受体拮抗剂苏拉明抑制,抑制率:1E8:82%±9%;2B4:81%±6%。ERK1/2上游激酶MEK 抑制剂 PD98059可有效抑制ATP对ERK1/2的激活,抑制率:1E8:94%±4%;2B4:91%±4%。ATP对ERK的激活受到G蛋白活性调节剂PTX的抑制,抑制率:1E8:50%±3%,2B4:51%±4%。ATP对1E8细胞ERK1/2的激活水平高于2B4细胞。两种细胞对蛋白激酶C活性调节剂TPA作用的反应性不同。结论 不同转移性的人前列腺癌细胞亚系对外源性ATP 激活ERK1/2信号传导通路的反应性间存在差异,提示肿瘤转移受到不同信号传导机制调节。%Objective To investigate the mechanism of the activation of signal transduction of ERK induced by purinergic receptor agonist ATP in prostate cancer cells with different metastatic potential. Methods Cell counts and MTT method were used to detect the influence of ATP on the growth of 1E8 (metastatic) and 2B4 (non-metastatic) cells derived from human prostate cancer cell line PC3M. The activity of ERK1/2 was analyzed with phosphospecific antibodies directed against the dually phosphorylated, active forms of ERK1/2 (p44/p42) by Western Blot. Results ATP can significantly inhibit the growth of 1E8 and 2B4 cells in vitro

  7. Involvement of extracellular signal-regulated kinase (ERK1/2)-p53-p21 axis in mediating neural stem/progenitor cell cycle arrest in co-morbid HIV-drug abuse exposure.

    Science.gov (United States)

    Malik, Shaily; Saha, Rinki; Seth, Pankaj

    2014-06-01

    Neurological complications in opioid abusing Human Immunodeficiency Virus-1 (HIV-1) patients suggest enhanced neurodegeneration as compared to non-drug abusing HIV-1 infected population. Neural precursor cells (NPCs), the multipotent cells of the mammalian brain, are susceptible to HIV-1 infection and as opiates also perturb their growth kinetics, detailed mechanistic studies for their co-morbid exposure are highly warranted. Using a well characterized in vitro model of human fetal brain-derived neural precursor cells, we investigated alterations in NPC properties at both acute and chronic durations. Chronic morphine and Tat treatment attenuated proliferation in NPCs, with cells stalled at G1-phase of the cell cycle. Furthermore HIV-Tat and morphine exposure increased activation of extracellular signal-regulated kinase-1/2 (ERK1/2), enhanced levels of p53 and p21, and decreased cyclin D1 and Akt levels in NPCs. Regulated by ERK1/2 and p53, p21 was found to be indispensible for Tat and morphine mediated cell cycle arrest. Our study elaborates on the cellular and molecular machinery in NPCs and provides significant mechanistic details into HIV-drug abuse co-morbidity that may have far reaching clinical consequences both in pediatric as well as adult neuroAIDS.

  8. Induction of apoptosis in renal cell carcinoma by reactive oxygen species: involvement of extracellular signal-regulated kinase 1/2, p38delta/gamma, cyclooxygenase-2 down-regulation, and translocation of apoptosis-inducing factor.

    LENUS (Irish Health Repository)

    Ambrose, Monica

    2012-02-03

    Renal cell carcinoma (RCC) is the most common malignancy of the kidney. Unfortunately, RCCs are highly refractory to conventional chemotherapy, radiation therapy, and even immunotherapy. Thus, novel therapeutic targets need to be sought for the successful treatment of RCCs. We now report that 6-anilino-5,8-quinolinequinone (LY83583), an inhibitor of cyclic GMP production, induced growth arrest and apoptosis of the RCC cell line 786-0. It did not prove deleterious to normal renal epithelial cells, an important aspect of chemotherapy. To address the cellular mechanism(s), we used both genetic and pharmacological approaches. LY83583 induced a time- and dose-dependent increase in RCC apoptosis through dephosphorylation of mitogen-activated protein kinase kinase 1\\/2 and its downstream extracellular signal-regulated kinases (ERK) 1 and -2. In addition, we observed a decrease in Elk-1 phosphorylation and cyclooxygenase-2 (COX-2) down-regulation. We were surprised that we failed to observe an increase in either c-Jun NH(2)-terminal kinase or p38alpha and -beta mitogen-activated protein kinase activation. In contradiction, reintroduction of p38delta by stable transfection or overexpression of p38gamma dominant negative abrogated the apoptotic effect. Cell death was associated with a decrease and increase in Bcl-x(L) and Bax expression, respectively, as well as release of cytochrome c and translocation of apoptosis-inducing factor. These events were associated with an increase in reactive oxygen species formation. The antioxidant N-acetyl l-cysteine, however, opposed LY83583-mediated mitochondrial dysfunction, ERK1\\/2 inactivation, COX-2 down-regulation, and apoptosis. In conclusion, our results suggest that LY83583 may represent a novel therapeutic agent for the treatment of RCC, which remains highly refractory to antineoplastic agents. Our data provide a molecular basis for the anticancer activity of LY83583.

  9. Diallyl trisulfide-induced apoptosis in human prostate cancer cells involves c-Jun N-terminal kinase and extracellular-signal regulated kinase-mediated phosphorylation of Bcl-2.

    Science.gov (United States)

    Xiao, Dong; Choi, Sunga; Johnson, Daniel E; Vogel, Victor G; Johnson, Candace S; Trump, Donald L; Lee, Yong J; Singh, Shivendra V

    2004-07-22

    Garlic-derived organosulfides (OSCs) including diallyl trisulfide (DATS) are highly effective in affording protection against chemically induced cancer in animals. Evidence is also mounting to indicate that some naturally occurring OSCs can suppress proliferation of cancer cells by causing apoptosis, but the sequence of events leading to proapoptotic effect of OSCs is poorly defined. Using PC-3 and DU145 human prostate cancer cells as a model, we now demonstrate that DATS is a significantly more potent apoptosis inducer than diallyl sulfide (DAS) or diallyl disulfide (DADS). DATS-induced apoptosis in PC-3 cells was associated with phosphorylation of Bcl-2, reduced Bcl-2 : Bax interaction, and cleavage of procaspase-9 and -3. Bcl-2 overexpressing PC-3 cells were significantly more resistant to apoptosis induction by DATS compared with vector-transfected control cells. DATS treatment resulted in activation of extracellular-signal regulated kinase 1/2 (ERK1/2) and c-jun N-terminal kinase 1 (JNK1) and/or JNK2, but not p38 mitogen-activated protein kinase. Phosphorylation of Bcl-2 in DATS-treated PC-3 cells was fully blocked in the presence of JNK-specific inhibitor SP600125. Moreover, JNK inhibitor afforded significant protection against DATS-induced apoptosis in both cells. DATS-induced Bcl-2 phosphorylation and apoptosis were partially attenuated by pharmacological inhibition of ERK1/2 using PD98059 or U0126. Overexpression of catalase inhibited DATS-mediated activation of JNK1/2, but not ERK1/2, and apoptosis induction in DU145 cells suggesting involvement of hydrogen peroxide as a second messenger in DATS-induced apoptosis. In conclusion, our data point towards important roles for Bcl-2, JNK and ERK in DATS-induced apoptosis in human prostate cancer cells.

  10. The Role of Matrine and Mitogen-Ativated Protein Kinase/Extracellular Signal-Regulated Kinase Signal Transduction in the Inhibition of the Proliferation and Migration of Human Umbilical Veins Endothelial Cells Induced by Lung Cancer cells

    Directory of Open Access Journals (Sweden)

    Ming BAI

    2009-07-01

    Full Text Available Background and objective Matrine, one of the major alkaloid components of the traditional Chinese medicine Sophora roots, has a wide range of pharmacological effects including anti-inflammatory activities, growth inhibition and induction of cell differentiation and apoptosis. Motigen-activated protein kinase (MAPK/extracellular signal-regulated kinase (ERK has found to be a crucial signaling pathway in endothelial cells. The aim of this study is to investigate the role of Matrine and MAPK/ERK signal transduction in the inhibition of the proliferation and migration of human umbilical veins endothelial cells (HUVECs induced by lung cancer cells. Methods HUVECs were cultured with A549CM. Mat or PD98059 (i.e PD, specific inhibitor of MAPK/ERK, was added into the A549CM. The proliferation of the HUVECs was measured by cell counting. The migration of the HUVECs was observed by wound healing assay. The expression levels of ERK and p-ERK protein were detected by Western Blot analysis. Results On 24 hours after intervention, the A549CM significantly stimulated the proliferation, migration and expression of p-ERK of HUVECs. Compared with the A549CM group, Mat significantly inhibited the proliferation, migration and p-ERK expression of HUVECs induced by A549CM. While PD only decreased the proliferation and p-ERK expression of HUVECs induced by A549CM. PD had no effect in the migration of HUVECs. Conclusion The results demonstrated that Mat and PD98059 can effectively decrease proliferation and expression of p-ERK of HUVECs induced by A549CM. Furthermore Mat can also inhibit migration of HUVECs induced by A549CM that did not changed by PD98059. These data implied that suppressing MAPK/ERK signal transduction may play the crucial role in resisting lung cacinoma angiogenesis with Mat.

  11. Depletion of Serotonin and Selective Inhibition of 2B Receptor Suppressed Tumor Angiogenesis by Inhibiting Endothelial Nitric Oxide Synthase and Extracellular Signal-Regulated Kinase 1/2 Phosphorylation

    Directory of Open Access Journals (Sweden)

    Masanori Asada

    2009-04-01

    Full Text Available The effects of serotonin (5-HT on tumor growth are inconsistent. We investigated whether a decreased level of 5-HT affected tumor growth using 5-HT transporter knockout (5-HTT-/- mice, which showed 5-HT depletion. When cancer cells were injected subcutaneously into both 5-HTT-/- and 5-HTT+/+ mice, the tumor growth was markedly attenuated in 5-HTT-/- mice. Serotonin levels in the blood, forebrain, and tumors of 5-HTT-/- mice bearing tumors were significantly smaller than those of their 5-HTT+/+ littermates. However, 5-HT did not increase cancer cells' proliferation in vitro. When we applied 5-HTT inhibitors to the wild mice bearing tumors, they did not inhibit tumor growth. The endothelial nitric oxide synthase (eNOS expressions in tumors were reduced in 5-HTT-/- mice compared with 5-HTT+/+ mice. Stimulations with 5-HT (1–50 µM induced eNOS expressions in human umbilical vein endothelial cell (HUVEC in a concentration-dependent manner. When we measured activations of multiple signaling pathways by using a high-throughput phosphospecific antibodies platform, 5-HT stimulated the extracellular signal-regulated kinase 1/2 (ERK1/2 in HUVEC. Moreover, we found that the physiological level of 5-HT induced phosphorylation of both ERK1/2 and eNOS in HUVEC. Human umbilical vein endothelial cell expressed both 5-HT2B and 5-HT2C receptors. SB204741, a specific 5-HT2B receptor inhibitor, blocked 5-HT-induced ERK1/2 and eNOS phosphorylations, whereas RS102221, a specific 5-HT2C receptor inhibitor, did not in HUVEC. SB204741 reduced microvessel density in tumors and inhibited the proliferation of HUVEC in vitro. These results suggest that regulation of 5-HT and 5-HT receptors, especially the 5-HT2B receptor, may serve as a therapeutic strategy in cancer therapy.

  12. c-Src regulates cell cycle proteins expression through protein kinase B/glycogen synthase kinase 3 beta and extracellular signal-regulated kinases 1/2 pathways in MCF-7 cells.

    Science.gov (United States)

    Liu, Xiang; Du, Liying; Feng, Renqing

    2013-07-01

    We have demonstrated that c-Src suppression inhibited the epithelial to mesenchymal transition in human breast cancer cells. Here, we investigated the role of c-Src on the cell cycle progression using siRNAs and small molecule inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Western blot analysis demonstrated the down-regulation of cyclin D1 and cyclin E and up-regulation of p27 Kip1 after c-Src suppression by PP2. Incubation of cells in the presence of PP2 significantly blocked the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), protein kinase B (AKT), and glycogen synthase kinase 3 beta (GSK3β). Specific pharmacological inhibitors of MEK1/2/ERK1/2 and phosphatidylinositide 3-kinase/AKT pathways were used to demonstrate the relationship between the signal cascade and cell cycle proteins expression. The expression of cyclin D1 and cyclin E were decreased after inhibition of ERK1/2 or AKT activity, whereas the p27 Kip1 expression was increased. In addition, knockdown of c-Src by siRNAs reduced cell proliferation and phosphorylation of ERK1/2, AKT, and GSK3β. After c-Src depletion by siRNAs, we observed significant down-regulation of cyclin D1 and cyclin E, and up-regulation of p27 Kip1. These results suggest that c-Src suppression by PP2 or siRNAs may regulate the progression of cell cycle through AKT/GSK3β and ERK1/2 pathways.

  13. c-Src regulates cell cycle proteins expression through protein kinase B/glycogen synthase kinase 3 beta and extracellular signal-regulated kinases 1/2 pathways in MCF-7 cells

    Institute of Scientific and Technical Information of China (English)

    Xiang Liu; Liying Du; Renqing Feng

    2013-01-01

    We have demonstrated that c-Src suppression inhibited the epithelial to mesenchymal transition in human breast cancer cells.Here,we investigated the role of c-Src on the cell cycle progression using siRNAs and small molecule inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4-d]pyrimidine (PP2).Western blot analysis demonstrated the downregulation of cyclin D1 and cyclin E and up-regulation of p27 Kip1 after c-Src suppression by PP2.Incubation of cells in the presence of PP2 significantly blocked the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2),protein kinase B (AKT),and glycogen synthase kinase 3 beta (GSK3β).Specific pharmacological inhibitors of MEK1/2/ERK1/2 and phosphatidylinositide 3-kinase/AKTpathways were used to demonstrate the relationship between the signal cascade and cell cycle proteins expression.The expression of cyclin D1 and cyclin E were decreased after inhibition of ERK1/2 or AKT activity,whereas the p27 Kip1 expression was increased.In addition,knockdown of c-Src by siRNAs reduced cell proliferation and phosphorylation of ERK1/2,AKT,and GSK3β.After c-Src depletion by siRNAs,we observed significant down-regulation of cyclin D1 and cyclin E,and up-regulation of p27 Kip1.These results suggest that c-Src suppression by PP2 or siRNAs may regulate the progression of cell cycle through AKT/GSK3β and ERK1/2 pathways.

  14. Overexpression of KAI1 induces autophagy and increases MiaPaCa-2 cell survival through the phosphorylation of extracellular signal-regulated kinases

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Chun-Yan [State Key Laboratory of Cancer Biology and Institute of Digestive Diseases, Xijing Hospital of Digestive Disease, Fourth Military Medical University, Xi' an 710032 (China); Department of Gastroenterology, Shenyang General Hospital of PLA, 83 Wenhua Road, Shenyang 110016 (China); Yan, Jun; Yang, Yue-Feng; Xiao, Feng-Jun; Li, Qing-Fang; Zhang, Qun-Wei; Wang, Li-Sheng [Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850 (China); Guo, Xiao-Zhong, E-mail: guoxiaozhong1962@163.com [Department of Gastroenterology, Shenyang General Hospital of PLA, 83 Wenhua Road, Shenyang 110016 (China); Wang, Hua, E-mail: wanghua@bmi.ac.cn [Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850 (China)

    2011-01-21

    Research highlights: {yields} We first investigate the effects of KAI1 on autophagy in MiaPaCa-2 cells. {yields} Our findings demonstrate that KAI1 induces autophagy, which in turn inhibits KAI1-induced apoptosis. {yields} This study also supplies a possible novel therapeutic method for the treatment of pancreatic cancer using autophagy inhibitors. -- Abstract: KAI1, a metastasis-suppressor gene belonging to the tetraspanin family, is known to inhibit cancer metastasis without affecting the primary tumorigenicity by inhibiting the epidermal growth factor (EGF) signaling pathway. Recent studies have shown that hypoxic conditions of solid tumors induce high-level autophagy and KAI1 expression. However, the relationship between autophagy and KAI1 remains unclear. By using transmission electron microscopy, confocal microscopy, and Western blotting, we found that KAI1 can induce autophagy in a dose- and time-dependent manner in the human pancreatic cell line MiaPaCa-2. KAI1-induced autophagy was confirmed by the expression of autophagy-related proteins LC3 and Beclin 1. KAI1 induces autophagy through phosphorylation of extracellular signal-related kinases rather than that of AKT. KAI1-induced autophagy protects MiaPaCa-2 cells from apoptosis and proliferation inhibition partially through the downregulation of poly [adenosine diphosphate (ADP)-ribose] polymerase (PARP) cleavage and caspase-3 activation.

  15. Role of the extracellular signal-regulated kinase 1/2 signaling pathway in regulating the secretion of bronchial smooth muscle cells in a rat model of chronic asthma

    Institute of Scientific and Technical Information of China (English)

    XIE Min; LIU Xian-sheng; XU Yong-jian; ZHANG Zhen-xiang; BAI Jing; NI Wang; CHEN Shi-xin

    2008-01-01

    Background Although it is recognized that bronchial smooth muscle cells (BSMCs) play a key role. In airway remodeling during chronic asthma, it is not well understood how BSMCs exert their inflammatory functions. The extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway is an important signaling pathway in chronic asthma, but its influence on secretion by BSMCs has not been well-studied. We investigated the impact of ERK1/2 signaling pathway on secretion by BSMCs in a rat model of chronic asthma in this study.Methods To create a rat model of chronic asthma, Wistar rats underwent ovalbumim (OVA) injection and eight weeks of inhalation. BSMCs were isolated and cultured in vitro. Epidermal growth factor, PD98059 and ERK1/2 antisense oligonucleotide were used to explore the role of ERK1/2 signaling pathway. The expression of P-ERK1/2 (phospho-ERK1/2) in BSMCs was analyzed by Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR). Secretion of BSMCs was detected by enzyme-linked immunosorbent assay (ELISA).Results Phospho-ERK1/2 expression was increased in BSMCs of chronic asthmatic rats compared with the controls. PD98059 inhibited expression of phospho-ERK1/2 protein, while treatment with an antisense oligonucleotide inhibited the expression of P-ERK1/2 mRNA and protein. BSMCs obtained from the chronic asthma group secreted significantly greater quantities of growth factors (transforming growth factor (TGF)-β1, vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF)), cytokines (regulated upon activation, normal T cell-expressed and secreted (RANTES) and eotaxin), and extracellular matrix (fibronectin and collagen l) compared with normal controls. Epidermal growth factor stimulated secretion in both groups, but the response of the chronic asthma group was more intense. Both PD98059 and antisense oligonucleotide suppressed secretion by BSMCs in chronic ashmatic rats. Antisense oligonucleotide reduced the

  16. Cyclic dermal BMP signalling regulates stem cell activation during hair regeneration

    OpenAIRE

    Plikus, M. V.; Mayer, J. A.; de La Cruz, D.; Baker, Ruth E.; Maini, P.K.; Maxson, R.; Chuong, C M

    2008-01-01

    In the age of stem cell engineering it is critical to understand how stem cell activity is regulated during regeneration. Hairs are mini-organs that undergo cyclic regeneration throughout adult life1, and are an important model for organ regeneration. Hair stem cells located in the follicle bulge2 are regulated by the surrounding microenvironment, or niche3. The activation of such stem cells is cyclic, involving periodic -catenin activity4, 5, 6, 7. In the adult mouse, regeneration occurs in ...

  17. Cyclic dermal BMP signaling regulates stem cell activation during hair regeneration

    OpenAIRE

    Plikus, Maksim V; Mayer, Julie; de la Cruz, Damon; Baker, Ruth E.; Maini, Philip K.; Maxson, Robert; Chuong, Cheng-ming

    2008-01-01

    In the age of stem cell engineering, it is critical to understand how stem cell activity is regulated during regeneration. Hairs are mini-organs that undergo cyclic regeneration throughout adult life,1 and are an important model for organ regeneration. Hair stem cells located in the follicle bulge2 are regulated by the surrounding micro-environment, or niche3. The activation of such stem cells is cyclic, involving periodic β-catenin activity4–7. In adult mouse, regeneration occurs in waves in...

  18. Cyclic dermal BMP signalling regulates stem cell activation during hair regeneration.

    Science.gov (United States)

    Plikus, Maksim V; Mayer, Julie Ann; de la Cruz, Damon; Baker, Ruth E; Maini, Philip K; Maxson, Robert; Chuong, Cheng-Ming

    2008-01-17

    In the age of stem cell engineering it is critical to understand how stem cell activity is regulated during regeneration. Hairs are mini-organs that undergo cyclic regeneration throughout adult life, and are an important model for organ regeneration. Hair stem cells located in the follicle bulge are regulated by the surrounding microenvironment, or niche. The activation of such stem cells is cyclic, involving periodic beta-catenin activity. In the adult mouse, regeneration occurs in waves in a follicle population, implying coordination among adjacent follicles and the extrafollicular environment. Here we show that unexpected periodic expression of bone morphogenetic protein 2 (Bmp2) and Bmp4 in the dermis regulates this process. This BMP cycle is out of phase with the WNT/beta-catenin cycle, thus dividing the conventional telogen into new functional phases: one refractory and the other competent for hair regeneration, characterized by high and low BMP signalling, respectively. Overexpression of noggin, a BMP antagonist, in mouse skin resulted in a markedly shortened refractory phase and faster propagation of the regenerative wave. Transplantation of skin from this mutant onto a wild-type host showed that follicles in donor and host can affect their cycling behaviours mutually, with the outcome depending on the equilibrium of BMP activity in the dermis. Administration of BMP4 protein caused the competent region to become refractory. These results show that BMPs may be the long-sought 'chalone' inhibitors of hair growth postulated by classical experiments. Taken together, results presented in this study provide an example of hierarchical regulation of local organ stem cell homeostasis by the inter-organ macroenvironment. The expression of Bmp2 in subcutaneous adipocytes indicates physiological integration between these two thermo-regulatory organs. Our findings have practical importance for studies using mouse skin as a model for carcinogenesis, intra-cutaneous drug

  19. Suppression of hedgehog signaling regulates hepatic stellate cell activation and collagen secretion

    OpenAIRE

    Li, Tao; Leng, Xi-Sheng; Zhu, Ji-Ye; Wang, Gang

    2015-01-01

    Hepatic stellate cells (HSCs) play an important role in liver fibrosis. This study investigates the expression of hedgehog in HSC and the role of hedgehog signaling on activation and collagen secretion of HSC. Liver ex vivo perfusion with collagenase IV and density gradient centrifugation were used to isolate HSC. Expression of hedgehog signaling components Ihh, Smo, Ptc, Gli2 and Gli3 in HSC were detected by RT-PCR. Hedgehog siRNA vectors targeting Ihh, Smo and Gli2 were constructed and tran...

  20. Translocated signals regulating root meristem activity in lupins (Lupinus albus and L. angustifolius)

    International Nuclear Information System (INIS)

    Pluripotent stem cells in flowering plants occur at the root and shoot apices, at the cambium of shoot organs and the root pericycle. These meristematic cells provide sites for cell division and postembryonic organ differentiation. Their activity responds to environmental and endogenous cues that determine rate and direction of growth, developmental pattern and change in organ function. Recent analysis of gene expression in the shoot apical meristem (SAM) of Arabidopsis has revealed close cell/cell interactions and an exchange of signals between differentiating cell types. However, it is clear that the long distance translocation channels of vascular plants, phloem and xylem, also provide regulatory signals that influence the course of events in the SAM, such as the transition from vegetative to floral development. These channels serve as pathways for translocation of assimilates providing the vascular link between 'sources' and 'sinks' on the plant. Similarly, the below ground meristems responsible for root growth, lateral root initiation and branching as well as the initiation of nodules on legumes receive translocated shoot-derived 'signals' as well as assimilates in phloem. Physiological studies have established that such signals are integral components of meristem activity but their nature has not been clearly established. (author)

  1. Opposing Activities of Notch and Wnt Signaling Regulate Intestinal Stem Cells and Gut Homeostasis

    Directory of Open Access Journals (Sweden)

    Hua Tian

    2015-04-01

    Full Text Available Proper organ homeostasis requires tight control of adult stem cells and differentiation through the integration of multiple inputs. In the mouse small intestine, Notch and Wnt signaling are required both for stem cell maintenance and for a proper balance of differentiation between secretory and absorptive cell lineages. In the absence of Notch signaling, stem cells preferentially generate secretory cells at the expense of absorptive cells. Here, we use function-blocking antibodies against Notch receptors to demonstrate that Notch blockade perturbs intestinal stem cell function by causing a derepression of the Wnt signaling pathway, leading to misexpression of prosecretory genes. Importantly, attenuation of the Wnt pathway rescued the phenotype associated with Notch blockade. These studies bring to light a negative regulatory mechanism that maintains stem cell activity and balanced differentiation, and we propose that the interaction between Wnt and Notch signaling described here represents a common theme in adult stem cell biology.

  2. Chloroplasts activity and PAP-signaling regulate programmed cell death in Arabidopsis

    KAUST Repository

    Bruggeman, Quentin

    2016-01-09

    Programmed cell death (PCD) is a crucial process both for plant development and responses to biotic and abiotic stress. There is accumulating evidence that chloroplasts may play a central role during plant PCD as for mitochondria in animal cells, but it is still unclear whether they participate in PCD onset, execution, or both. To tackle this question, we have analyzed the contribution of chloroplast function to the cell death phenotype of the myoinositol phosphate synthase1 (mips1) mutant that forms spontaneous lesions in a light-dependent manner. We show that photosynthetically active chloroplasts are required for PCD to occur in mips1, but this process is independent of the redox state of the chloroplast. Systematic genetic analyses with retrograde signaling mutants reveal that 3’-phosphoadenosine 5’-phosphate, a chloroplast retrograde signal that modulates nuclear gene expression in response to stress, can inhibit cell death and compromises plant innate immunity via inhibition of the RNA-processing 5’-3’ exoribonucleases. Our results provide evidence for the role of chloroplast-derived signal and RNA metabolism in the control of cell death and biotic stress response. © 2016 American Society of Plant Biologists. All Rights Reserved.

  3. 5-Hydroxytryptamine-induced proliferation of pulmonary artery smooth muscle cells are extracellular signal-regulated kinase pathway depen-dent

    Institute of Scientific and Technical Information of China (English)

    Dan SONG; Huai-liang WANG; Shuang WANG; Xin-hua ZHANG

    2005-01-01

    Aim:To investigate the effect of 5-hydroxytryptamine transporter (5-HTT) inhibitor fluoxetine and antisense oligodeoxynucleotide (ODN) to extracelluar signal regulated kinases (ERKs) on pulmonary arterial smooth muscle cells (PASMCs) proliferation induced by 5-HT. Methods: Liposomal transfection was used to introduce ODNs to ERK1/2 into cultured rat PASMCs and the transfection effi ciency was measured by observing the uptake of the fluorecein isothiocynate (FITC)-labeled antisense ODN in PASMCs. The effects of 5-HTT selective inhibi tor fluoxetine and ODNs on the proliferation of PASMCs were evaluated by cell number counting and cell cycle analysis, and measured by microculture tetrazo lium (MTT) assay and flow cytometry (FCM), respectively. Results: Liposomes mediated the transfection of ODNs into PASMCs with high efficiency. MTT assay showed fluoxetine (10 μmol/L, 1 μmol/L, and 100 nmol/L) concentration dependently inhibited the proliferation of PASMCs induced by 5-HT (1 μmol/L) in vitro. The proliferation rate of PASMCs by 5-HT was significantly inhibited by pretreatment with ERK1/2 antisense ODN (0.2 μmol/L) from 251%± 18% to 86%±5% (P<0.01). Flow cytometric analysis of cell cycle distribution showed that the increase of 5-HT induced S phase fraction (SPF) and proliferation index (PI) were significantly inhibited by fluoxetine (1 μmol/L) or antisense ODN with SPF from 36%±4% to 26%±3% and 24%±4%, and PI from 34%±2% to 29%±2% and 24%±2%,respectively. Conclusion: 5-HTT mediates the mitogenic effect of 5-HT on PASMCs and the proliferation of PASMCs induced by 5-HT is dependent on ERKs signal pathway.

  4. The extracellular signal-regulated kinase was promoted by Pyrroloquinoline Quinine in cultured Schwann cells%MEK/ERK信号通路在吡咯喹啉醌促雪旺细胞增殖中的作用

    Institute of Scientific and Technical Information of China (English)

    贺斌; 刘世清; 李皓桓

    2010-01-01

    目的 探讨MEK/ERK信号通路在吡咯喹啉醌促雪旺细胞增殖过程中的作用. 方法 体外培养雪旺细胞,S-100免疫荧光鉴定;Western blot检测MEK下游因子ERK1/2磷酸化激活形式(p-ERK1/2)的表达;MEK抑制剂(PD98059)阻断该通路后检测p-ERK1/2的表达;MTT法检测经PD98059阻断MEK通路后雪旺细胞的增殖情况. 结果 吡咯喹啉醌可激活雪旺细胞内MEK/ERK信号通路,在加入吡咯喹啉醌1 h后p-ERK1/2表达最高;吡咯喹啉醌在1~500 nmol/L范围内可使p-ERK1/2表达增加,1 000 nmol/L时与对照组比较差异无统计学意义,10 000 nmol/L时则表现为抑制作用(P<0.05);经PD98059阻断MEK通路后p-ERK1/2的上调效应消失(P<0.05).而且加入PD98059阻断MEK通路后吡咯喹啉醌对雪旺细胞的促增殖效果减弱. 结论 吡咯喹啉醌可激活雪旺细胞MEK/ERK信号通路,且该通路在吡咯喹啉醌促雪旺细胞增殖过程中发挥作用.%Objective To investigate the effect of mitogen-activated protein kinase(MEK)kinase cascade,extracellular signal-regulated kinase(ERK1/2)signal pathway on Schwann cells proliferation promoted by Pyrroloquinoline Quinine(PQQ)and its molecular mechanisms. Methods Schwann cells were cultured and purified in vitro.The purity was identified by S-100.Different time and concentration of PQQ was added into culture medium.The expression of ERK1/2 and phosphorylated-ERK1/2 was detected by western blot.The expression of p-ERK1/2 after blocking of MEK signal pathway by specific inhibitor PD98059 was detected by western blot. Results Morphological change was observed in PQQ treated Schwann cells.1 ~ 500 nmol/L PQQ could up-regulate the expression of p-ERK1/2,and 1 000 nmol/Lhad no effects,while 10 000 nmol/L exhibited inhibitory effect(P<0.05).p-ERKI/2 increased to peak 1 h after PQQ added,and this up-regulation of p-ERKI/2 was inhibited by PD98059(P<0.05). Conclusions PQQ could affect morphology of Schwann cells and activation of ERKI/2

  5. Rosiglitazone attenuates the metalloprotease/anti-metalloprotease imbalance in emphysema induced by cigarette smoke: involvement of extracellular signal-regulated kinase and NFκB signaling

    Directory of Open Access Journals (Sweden)

    Hou G

    2015-04-01

    Full Text Available Gang Hou, Yan Yin, Dan Han, Qiu-yue Wang, Jian Kang Department of Respiratory Medicine, the First Hospital of China Medical University, Shenyang, People’s Republic of China Objective: We investigated how rosiglitazone attenuated cigarette smoke (CS-induced emphysema in a rat model. In particular, we focused on its possible effects on the imbalance between metalloprotease (MMP and anti-MMP activity, mitogen-activated protein kinase (MAPK phosphorylation, and nuclear factor kappa-light-chain-enhancer of activated B cell (NFκB signaling pathway over-activation.Methods: A total of 36 Wistar rats were divided into three groups (n=12 each: animals were exposed to CS for 12 weeks in the absence (the CS group or presence of 30 mg/kg rosiglitazone (the rosiglitazone-CS [RCS] group; a control group was treated with the rosiglitazone vehicle only, without any CS exposure. Histopathology of lung tissue in all groups was evaluated to grade severity of the disease. Expression levels of peroxisome proliferator-activated receptor γ (PPARγ, MMP2, and MMP9 in lung tissue were determined and compared using Western blotting and immunohistochemistry. Activation of MAPKs, NFκB, and the nuclear factor of kappa light polypeptide gene enhancer in B-cell inhibitor, alpha (IκBα phosphorylation in lung tissue was examined by Western blotting.Results: Emphysema-related pathology, based on inter-alveolar wall distance and alveolar density, was less severe in the RCS group than in the CS group. Compared with the CS group, levels of PPARγ were higher in the RCS group, and levels of MMP2 and MMP9 proteins were lower in the RCS rats. Levels of activated MAPKs and NFκB were also lower, while the IκBαphosphorylation was increased in the lung tissue of RCS rats.Conclusion: Our findings suggest that oral administration of rosiglitazone attenuates the metalloprotease activity induced by CS, and the underlying mechanism might involve the activation of signaling pathways

  6. Extracellular signal regulated kinase and SMAD signaling both mediate the angiotensin II driven progression towards overt heart failure in homozygous TGR(mRen2)27

    NARCIS (Netherlands)

    de Boer, RA; Pokharel, S; Flesch, M; van Kampen, DA; Suurmeijer, AJH; Boomsma, F; van Gilst, WH; van Veldhuisen, DJ; Pinto, YM

    2004-01-01

    Angiotensin (Ang) II is a key player in left ventricular (LV) remodeling and cardiac fibrosis. Its effects are thought to be transferred at least in part by mitogen-activated protein kinases (MAPK), transforming growth factor (TGF) beta(1), and the Smad pathway. In this study we sought to elucidate

  7. Extracellular signal-regulated kinase 2 (ERK-2) mediated phosphorylation regulates nucleo-cytoplasmic shuttling and cell growth control of Ras-associated tumor suppressor protein, RASSF2

    Energy Technology Data Exchange (ETDEWEB)

    Kumari, Gita [Laboratory of Molecular Virology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad 500076 (India); Mahalingam, S., E-mail: mahalingam@iitm.ac.in [Laboratory of Molecular Virology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad 500076 (India); Department of Biotechnology, Laboratory of Molecular Virology and Cell Biology, Indian Institute of Technology-Madras, Chennai 600 036 (India)

    2009-10-01

    nucleo-cytoplasmic transport and cell growth arrest activity of RASSF2. Taken together, the present study suggests that active transport between nucleus and cytoplasm may constitute an important regulatory mechanism for RASSF2 function.

  8. Lactoferrin inhibits dexamethasone-induced chondrocyte impairment from osteoarthritic cartilage through up-regulation of extracellular signal-regulated kinase 1/2 and suppression of FASL, FAS, and Caspase 3

    Energy Technology Data Exchange (ETDEWEB)

    Tu, Yihui [Department of Orthopaedics, Yangpu District Central Hospital Affiliated to Tongji University School of Medicine, 450 Tengyue Road, Shanghai (China); Xue, Huaming [Department of Orthopaedics, Yangpu District Central Hospital Affiliated to Tongji University School of Medicine, 450 Tengyue Road, Shanghai (China); Institute of Life Science, College of Medicine, Swansea University, Singleton Park (United Kingdom); Francis, Wendy [Institute of Life Science, College of Medicine, Swansea University, Singleton Park (United Kingdom); Davies, Andrew P. [Department of Orthopaedics and Trauma, Moriston Hospital, Swansea (United Kingdom); Pallister, Ian; Kanamarlapudi, Venkateswarlu [Institute of Life Science, College of Medicine, Swansea University, Singleton Park (United Kingdom); Xia, Zhidao, E-mail: zhidao.xia@gmail.com [Institute of Life Science, College of Medicine, Swansea University, Singleton Park (United Kingdom)

    2013-11-08

    Highlights: •Dex exerts dose-dependant inhibition of HACs viability and induction of apoptosis. •Dex-induced impairment of chondrocytes was attenuated by rhLF. •ERK and FASL/FAS signaling are involved in the effects of rhLF. •OA patients with glucocorticoid-induced cartilage damage may benefit from treatment with rhLF. -- Abstract: Dexamethasone (Dex) is commonly used for osteoarthritis (OA) with excellent anti-inflammatory and analgesic effect. However, Dex also has many side effects following repeated use over prolonged periods mainly through increasing apoptosis and inhibiting proliferation. Lactoferrin (LF) exerts significantly anabolic effect on many cells and little is known about its effect on OA chondrocytes. Therefore, the aim of this study is to investigate whether LF can inhibit Dex-induced OA chondrocytes apoptosis and explore its possible molecular mechanism involved in. MTT assay was used to determine the optimal concentration of Dex and recombinant human LF (rhLF) on chondrocytes at different time and dose points. Chondrocytes were then stimulated with Dex in the absence or presence of optimal concentration of rhLF. Cell proliferation and viability were evaluated using MTT and LIVE/DEAD assay, respectively. Cell apoptosis was evaluated by multi-parameter apoptosis assay kit using both confocal microscopy and flow cytometry, respectively. The expression of extracellular signal-regulated kinase (ERK), FAS, FASL, and Caspase-3 (CASP3) at the mRNA and protein levels were examined by real-time polymerase chain reaction (PCR) and immunocytochemistry, respectively. The optimal concentration of Dex (25 μg/ml) and rhLF (200 μg/ml) were chosen for the following experiments. rhLF significantly reversed the detrimental effect of Dex on chondrocytes proliferation, viability, and apoptosis. In addition, rhLF significantly prevented Dex-induced down-regulation of ERK and up-regulation of FAS, FASL, and CASP3. These findings demonstrated that rhLF acts as

  9. Extracellular polymers of ozonized waste activated sludge.

    Science.gov (United States)

    Liu, J C; Lee, C H; Lai, J Y; Wang, K C; Hsu, Y C; Chang, B V

    2001-01-01

    Effect of ozonation on characteristics of waste activated sludge was investigated in the current study. Concentrations of cell-bound extracellular polymers (washed ECPs) did not change much upon ozonation, whereas the sum of cell-bound and soluble extracellular polymers (unwashed ECPs) increased with increasing ozone dose. Washed ECPs in original sludge as divided by molecular weight distribution was 39% 10,000 Da (high MW). It was observed that the low-MW fraction decreased, and the high-MW fraction increased in ozonized sludge. The unwashed ECPs were characterized as 44% in low MW, 30% in medium MW, and 26% in high MW. Both low-MW and medium-MW fractions of unwashed ECPs decreased while high-MW fraction increased in ozonized sludge. The dewaterability of ozonized sludge, assessed by capillary suction time (CST) and specific resistance to filtration (SRF), deteriorated with ozone dose. The optimal dose of cationic polyelectrolyte increased with increasing ozone dose. The production rate and the accumulated amount of methane gas of ozonized sludge were also higher.

  10. Pneumolysin activates neutrophil extracellular trap formation.

    Science.gov (United States)

    G Nel, J; Theron, A J; Durandt, C; Tintinger, G R; Pool, R; Mitchell, T J; Feldman, C; Anderson, R

    2016-06-01

    The primary objective of the current study was to investigate the potential of the pneumococcal toxin, pneumolysin (Ply), to activate neutrophil extracellular trap (NET) formation in vitro. Isolated human blood neutrophils were exposed to recombinant Ply (5-20 ng ml(-1) ) for 30-90 min at 37°C and NET formation measured using the following procedures to detect extracellular DNA: (i) flow cytometry using Vybrant® DyeCycle™ Ruby; (ii) spectrofluorimetry using the fluorophore, Sytox(®) Orange (5 μM); and (iii) NanoDrop(®) technology. These procedures were complemented by fluorescence microscopy using 4', 6-diamino-2-phenylindole (DAPI) (nuclear stain) in combination with anti-citrullinated histone monoclonal antibodies to visualize nets. Exposure of neutrophils to Ply resulted in relatively rapid (detected within 30-60 min), statistically significant (P DNA impregnated with both citrullinated histone and myeloperoxidase. Microscopy revealed that NETosis appeared to be restricted to a subpopulation of neutrophils, the numbers of NET-forming cells in the control and Ply-treated systems (10 and 20 ng ml(-1) ) were 4·3 (4·2), 14.3 (9·9) and 16·5 (7·5), respectively (n = 4, P < 0·0001 for comparison of the control with both Ply-treated systems). Ply-induced NETosis occurred in the setting of retention of cell viability, and apparent lack of involvement of reactive oxygen species and Toll-like receptor 4. In conclusion, Ply induces vital NETosis in human neutrophils, a process which may either contribute to host defence or worsen disease severity, depending on the intensity of the inflammatory response during pneumococcal infection. PMID:26749379

  11. 细胞外信号调节激酶1/2在谷氨酸引起的星形胶质细胞炎症反应中的作用%Effect of extracellular signal-regulated kinase 1/2 in glutamate-induced inflammatory reaction of culture astroglia in vitro

    Institute of Scientific and Technical Information of China (English)

    魏红燕

    2009-01-01

    Objective It is to observe the expression of glutamate induce astroglia activated inflammatory and extracellular signal-regulated kinase 1/2 (ERK 1/2), and explore the effect of extracellular signal-regulated kinase1/2 in astroglia activated inflammatory. Methods The passage astroglia of rats cultured in vitro was treated by glutamate for 30 minutes with the density of 20 μmol/L and 50 μmol/L. The signal transduction pathway of MEK was inhibited by upstream kinase of ERK PD98059. The expressions changes of extracellular regulated protein kinases1/2 (ERK1/2), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) were observed before and after inhibited.Results Glutamate could increase the level of P-ERK 1/2 obviously in astroglia cultured in vitro, and also increase the level of iNos, COX-2, IL-1β obviously. PD98059 could completely inhibit the increase of ERK 1/2 induced by glutamate, and also inhibit the increase of iNos, COX-2, IL-1β induced by glutamate. Conclusion ERK 1/2 signal transduction pathway is involved in glutamate induce astroglia activated inflammatory reaction.%目的 观察谷氨酸诱导星形胶质细胞激活炎症细胞因子表达及细胞外信号调节激酶1/2(ERK1/2)的表达情况,探讨ERK1/2在星形胶质细胞激活炎症细胞因子中的作用.方法 传代体外培养的大鼠星形胶质细胞,分别用终浓度为20μmol/L 和50μmol/L的谷氨酸作用30min.应用ERK上游激酶MEK特异性阻断剂PD98059(10 μmol/L)阻断ERK信号转导通路,Western blot观察阻断前后星形胶质细胞磷酸化ERK1/2、白细胞介素-1β(IL-1β)、诱导型一氧化氮合酶(iNOS)和环氧合酶-2(COX-2)蛋白表达水平的改变.结果 谷氨酸使体外培养星形胶质细胞磷酸化ERK1/2蛋白表达明显增加,同时,使iNOS、COX-2、IL-1β表达明显增加;PD98059可完全阻断谷氨酸引起的ERK1/2表达增加,也可抑制谷氨酸引起的iNOS、COX-2、IL-1β蛋白表达增

  12. Macrophage activation and differentiation signals regulate schlafen-4 gene expression: evidence for Schlafen-4 as a modulator of myelopoiesis.

    Directory of Open Access Journals (Sweden)

    Wendy J van Zuylen

    Full Text Available BACKGROUND: The ten mouse and six human members of the Schlafen (Slfn gene family all contain an AAA domain. Little is known of their function, but previous studies suggest roles in immune cell development. In this report, we assessed Slfn regulation and function in macrophages, which are key cellular regulators of innate immunity. METHODOLOGY/PRINCIPAL FINDINGS: Multiple members of the Slfn family were up-regulated in mouse bone marrow-derived macrophages (BMM by the Toll-like Receptor (TLR4 agonist lipopolysaccharide (LPS, the TLR3 agonist Poly(I∶C, and in disease-affected joints in the collagen-induced model of rheumatoid arthritis. Of these, the most inducible was Slfn4. TLR agonists that signal exclusively through the MyD88 adaptor protein had more modest effects on Slfn4 mRNA levels, thus implicating MyD88-independent signalling and autocrine interferon (IFN-β in inducible expression. This was supported by the substantial reduction in basal and LPS-induced Slfn4 mRNA expression in IFNAR-1⁻/⁻ BMM. LPS causes growth arrest in macrophages, and other Slfn family genes have been implicated in growth control. Slfn4 mRNA levels were repressed during macrophage colony-stimulating factor (CSF-1-mediated differentiation of bone marrow progenitors into BMM. To determine the role of Slfn4 in vivo, we over-expressed the gene specifically in macrophages in mice using a csf1r promoter-driven binary expression system. Transgenic over-expression of Slfn4 in myeloid cells did not alter macrophage colony formation or proliferation in vitro. Monocyte numbers, as well as inflammatory macrophages recruited to the peritoneal cavity, were reduced in transgenic mice that specifically over-expressed Slfn4, while macrophage numbers and hematopoietic activity were increased in the livers and spleens. CONCLUSIONS: Slfn4 mRNA levels were up-regulated during macrophage activation but down-regulated during differentiation. Constitutive Slfn4 expression in the

  13. Identification of two novel activities of the Wnt signaling regulator Dickkopf 3 and characterization of its expression in the mouse retina

    Directory of Open Access Journals (Sweden)

    Yi Hyun

    2007-12-01

    Full Text Available Abstract Background The Wnt signaling pathway is a cellular communication pathway that plays critical roles in development and disease. A major class of Wnt signaling regulators is the Dickkopf (Dkk family of secreted glycoproteins. Although the biological properties of Dickkopf 1 (Dkk1 and Dickkopf 2 (Dkk2 are well characterized, little is known about the function of the related Dickkopf 3 (Dkk3 protein in vivo or in cell lines. We recently demonstrated that Dkk3 transcripts are upregulated during photoreceptor death in a mouse model of retinal degeneration. In this study, we characterized the activity of Dkk3 in Wnt signaling and cell death. Results Dkk3 was localized to Müller glia and retinal ganglion cells in developing and adult mouse retina. Western blotting confirmed that Dkk3 is secreted from Müller glia cells in culture. We demonstrated that Dkk3 potentiated Wnt signaling in Müller glia and HEK293 cells but not in COS7 cells, indicating that it is a cell-type specific regulator of Wnt signaling. This unique Dkk3 activity was blocked by co-expression of Dkk1. Additionally, Dkk3 displayed pro-survival properties by decreasing caspase activation and increasing viability in HEK293 cells exposed to staurosporine and H2O2. In contrast, Dkk3 did not protect COS7 cells from apoptosis. Conclusion These data demonstrate that Dkk3 is a positive regulator of Wnt signaling, in contrast to its family member Dkk1. Furthermore, Dkk3 protects against apoptosis by reducing caspase activity, suggesting that Dkk3 may play a cytoprotective role in the retina.

  14. Blockade of Apoptosis Signal-Regulating Kinase 1 Attenuates Matrix Metalloproteinase 9 Activity in Brain Endothelial Cells and the Subsequent Apoptosis in Neurons after Ischemic Injury.

    Science.gov (United States)

    Cheon, So Y; Cho, Kyoung J; Kim, So Y; Kam, Eun H; Lee, Jong E; Koo, Bon-Nyeo

    2016-01-01

    Conditions of increased oxidative stress including cerebral ischemia can lead to blood-brain barrier dysfunction via matrix metalloproteinase (MMP). It is known that MMP-9 in particular is released from brain endothelial cells is involved in the neuronal cell death that occurs after cerebral ischemia. In the intracellular signaling network, apoptosis signal-regulating kinase 1 (ASK1) is the main activator of the oxidative stress that is part of the pathogenesis of cerebral ischemia. ASK1 also promotes apoptotic cell death and brain infarction after ischemia and is associated with vascular permeability and the formation of brain edema. However, the relationship between ASK1 and MMP-9 after cerebral ischemia remains unknown. Therefore, the aim of the present study was to determine whether blocking ASK1 would affect MMP-9 activity in the ischemic brain and cultured brain endothelial cells. Our results showed that ASK1 inhibition efficiently reduced MMP-9 activity in vivo and in vitro. In endothelial cell cultures, ASK1 inhibition upregulated phosphatidylinositol 3-kinase/Akt/nuclear factor erythroid 2 [NF-E2]-related factor 2/heme oxygenase-1 signals and downregulated cyclooxygenase-2 signals after hypoxia/reperfusion. Additionally, in neuronal cell cultures, cell death occurred when neurons were incubated with endothelial cell-conditioned medium (EC-CM) obtained from the hypoxia/reperfusion group. However, after incubation with EC-CM and following treatment with the ASK1 inhibitor NQDI-1, neuronal cell death was efficiently decreased. We conclude that suppressing ASK1 decreases MMP-9 activity in brain endothelial cells, and leads to decreased neuronal cell death after ischemic injury. PMID:27642277

  15. Activation of epidermal growth factor receptor by metal-ligand complexes decreases levels of extracellular amyloid beta peptide.

    Science.gov (United States)

    Price, Katherine A; Filiz, Gulay; Caragounis, Aphrodite; Du, Tai; Laughton, Katrina M; Masters, Colin L; Sharples, Robyn A; Hill, Andrew F; Li, Qiao-Xin; Donnelly, Paul S; Barnham, Kevin J; Crouch, Peter J; White, Anthony R

    2008-01-01

    The epidermal growth factor receptor is a receptor tyrosine kinase expressed in a range of tissues and cell-types. Activation of the epidermal growth factor receptor by a number of ligands induces downstream signalling that modulates critical cell functions including growth, survival and differentiation. Abnormal epidermal growth factor receptor expression and activation is also involved in a number of cancers. In addition to its cognate ligands, the epidermal growth factor receptor can be activated by metals such as zinc (Zn) and copper (Cu). Due to the important role of these metals in a number of diseases including neurodegenerative disorders, therapeutic approaches are being developed based on the use of lipid permeable metal-complexing molecules. While these agents are showing promising results in animal models and clinical trials, little is known about the effects of metal-ligand complexes on cell signalling pathways. In this study, we investigated the effects of clioquinol (CQ)-metal complexes on activation of epidermal growth factor receptor. We show here that CQ-Cu complexes induced potent epidermal growth factor receptor phosphorylation resulting in downstream activation of extracellular signal-regulated kinase. Similar levels of epidermal growth factor receptor activation were observed with alternative lipid permeable metal-ligands including neocuproine and pyrrolidine dithiocarbamate. We found that CQ-Cu complexes induced a significant reduction in the level of extracellular Abeta1-40 in cell culture. Inhibition of epidermal growth factor receptor activation by PD153035 blocked extracellular signal-regulated kinase phosphorylation and restored Abeta1-40 levels. Activation of the epidermal growth factor receptor by CQ-Cu was mediated through up-regulation of src kinase activity by a cognate ligand-independent process involving membrane integrins. These findings provide the first evidence that metal-ligand complexes can activate the epidermal growth

  16. Extracellular enzymatic activities of Bipolaris sorokiniana isolates.

    Science.gov (United States)

    Geimba, Mercedes P; Brandelli, Adriano

    2002-01-01

    Several enzymatic activities were investigated in six isolates of the fungus Bipolaris sorokiniana, originating from different areas of Brazil. Among the glycosidases studied, beta-glucosidase, beta-N-acetylglucosaminidase, beta-xylosidase, cellobiohydrolase, and chitobiohydrolase were the major activities. In some isolates, beta-glucuronidase, beta-galactosidase, and alpha-mannosidase activities were also present. Polysaccharide-hydrolyzing enzymes, such as pectin lyase and carboxymethyl cellulase were detected in significant amounts, and their activities were variable among the different isolates. Other enzymes, namely phosphatases, proteinases and phenol oxidase, were also examined, showing variable amounts depending on the isolate. The pH dependence of all enzymes tested was investigated. Endoproteinase, carboxymethyl cellulase, and phenoloxidase had maximum activity in the pH range of 6-8, whilst all other enzymes showed maximum activity at pH 4-6.

  17. Rearrangement of the Extracellular Domain/Extracellular Loop 1 Interface Is Critical for Thyrotropin Receptor Activation.

    Science.gov (United States)

    Schaarschmidt, Joerg; Nagel, Marcus B M; Huth, Sandra; Jaeschke, Holger; Moretti, Rocco; Hintze, Vera; von Bergen, Martin; Kalkhof, Stefan; Meiler, Jens; Paschke, Ralf

    2016-07-01

    The thyroid stimulating hormone receptor (TSHR) is a G protein-coupled receptor (GPCR) with a characteristic large extracellular domain (ECD). TSHR activation is initiated by binding of the hormone ligand TSH to the ECD. How the extracellular binding event triggers the conformational changes in the transmembrane domain (TMD) necessary for intracellular G protein activation is poorly understood. To gain insight in this process, the knowledge on the relative positioning of ECD and TMD and the conformation of the linker region at the interface of ECD and TMD are of particular importance. To generate a structural model for the TSHR we applied an integrated structural biology approach combining computational techniques with experimental data. Chemical cross-linking followed by mass spectrometry yielded 17 unique distance restraints within the ECD of the TSHR, its ligand TSH, and the hormone-receptor complex. These structural restraints generally confirm the expected binding mode of TSH to the ECD as well as the general fold of the domains and were used to guide homology modeling of the ECD. Functional characterization of TSHR mutants confirms the previously suggested close proximity of Ser-281 and Ile-486 within the TSHR. Rigidifying this contact permanently with a disulfide bridge disrupts ligand-induced receptor activation and indicates that rearrangement of the ECD/extracellular loop 1 (ECL1) interface is a critical step in receptor activation. The experimentally verified contact of Ser-281 (ECD) and Ile-486 (TMD) was subsequently utilized in docking homology models of the ECD and the TMD to create a full-length model of a glycoprotein hormone receptor. PMID:27129207

  18. Sulfur activation-related extracellular proteins of Acidithiobacillus ferrooxidans

    Institute of Scientific and Technical Information of China (English)

    ZHANG Cheng-gui; ZHANG Rui-yong; XIA Jin-lan; ZHANG Qian; NIE Zhen-yuan

    2008-01-01

    The fractions of the extracellular proteins of Acidithiobacillus ferrooxidans grown on two different energy substrates,elemental sulfur and ferrous sulfate,were selectively prepared with hot water treatment and distinctly shown by two-dimensional gel electrophoresis.Some protein spots with apparently higher abundance in sulfur energy substrate than in ferrous sulfate energy substrate were identified by using MALDI-TOF/TOF.Based on peptide mass fingerprints and bioinformatical analysis,the extracellular proteins were classified according to their functions as conjugal transfer protein,pilin,vacJ lipoprotein,polysaccharide deacetylase family protein,Ser/Thr protein phosphatase family protein and hypothetical proteins.Several extracellular proteins were found abundant in thiol groups and with CXXC functional motif,these proteins may be directly involved in the sulfur activation by use of their thiol group (Pr-SH) to bond the elemental sulfur.

  19. The Phosphorylation of Extracellular Signal-regulated Kinase in the Microvascular in Rats with Diabetes%糖尿病大鼠脑内微血管细胞外信号调节激酶磷酸化的研究

    Institute of Scientific and Technical Information of China (English)

    黄艳; 吴兴临; 张建中; 王岩

    2012-01-01

    Objective To investigate the phosphorylation of extracellular signal -regulated kinase (ERK1/2) and ets like gene - 1 ( ELK - 1 ) in meningeal and brain microvascular endothelial cells and smooth muscle cells with diabetic rats and to explore the mechanism of diabetic brain microangiopathy. Methods After modelling 0. 5 weeks, two weeks, four weeks, eight weeks, the spleen central artery angiopathy of Type I diabetic rats , which was induced using intraperitoneal injection of STZ , was respectively observated . The phosphorylation of extracellular signal - regulated kinase( ERK1/2) and ets like gene - 1 ( ELK - 1) in the brain micro-vascular were detected by means of HE staining,and immunohistochemistry techniques. Results p - ERK, p - ELK expressed with different levels in meningeal microvascular endothelial cells, smooth muscle cells, brain microvascular endothelial cells of diabetic groups and normal control group. The up - regulation of phosphorylation ERK1/2 was found in meningeal microvascular endothelial cells, smooth muscle cells, brain microvascular endothelial cells of 8 week,4 week diabetic group. The up - regulation of phosphorylation Elk - 1 expression was found in meningeal and brain microvascular endothelial cells of 8 week, 4 week diabetic group. But there were no significant difference in meningeal microvascular smooth muscle cells between diabetes groups and the normal control groups (P>0.05). Conclusion In 8 week, 4 week diabetic meningeal and brain microvascular exist ERK1 / 2 signal transduction pathway activation.%目的 研究链脲佐菌素诱导的Ⅰ型糖尿病大鼠脑膜、脑实质微血管的病理改变及其中细胞外信号调节激酶ERK1/2及其下游作用底物转录因子Elk-1的磷酸化状态,探讨糖尿病血管的发病机制.方法 应用链脲佐菌素制备Ⅰ型糖尿病大鼠模型,HE染色观察制模后3d和2、4、8周脑膜、脑实质微血管病变,免疫组织化学观察高血糖状

  20. 细胞外信号激酶蛋白5在卵泡刺激素介导的卵泡颗粒细胞甾体激素生成中的作用%Role of Extracellular Signal-regulated Protein Kinase 5 in the Biosynthesis of Follicle-stimulating Hormone-stimulated Progesterone in Primary Granulosa Cells

    Institute of Scientific and Technical Information of China (English)

    高小博; 姚楠; 马旭; 陆彩玲; 杨伯清; 彭小忠

    2011-01-01

    Objective To study the role of extracellular signal-regulated protein kinase 5 ( ERK5 ) during the biosynthesis of follicle-stimulating hormone ( FSH ) -mediated progesterone in primary granulosa cells. Methods The expressions of phophorylated and general forms of ERK5 in primary granulosa cells afterthe treatment of FSH were detected by Western blot analysis. The subcellular localization of ERK5 was observed by confocal microscopy. The effect of ERK5 on FSH-mediated progesterone biosynthesis in primary granulosa cells was analyzed using recombinant adenovirus vectors. Results ERK5 activation was induced by FSH in a time-dependent manner in primary cultured granulosa cells, although the general ERK5 protein level decreased also in a time-dependent manner. The treatment of FSH showed no remarkable effect on the subcellular distribution of endogenous ERK5, which was mainly in the cytoplasm of granulosa cells. The co-infection of Ad-caM-EK5 and Ad-wtERK5 increased the progesterone production and StAR expression in primary cultured granulosa cells, whereas inhibition of ERK5 activation suppressed the FSH-stimulated progesterone production. Conclusion ERK5 may stimulate FSH-mediated progesterone production in primary cultured granulosa cells.%目的 研究细胞外信号激酶蛋白5(ERK5)在卵巢颗粒细胞甾体激素生成中的作用.方法 采用Western blot检测在卵泡刺激素(FSH)刺激原代颗粒细胞甾体激素生成过程中ERK5的表达变化和激活情况,荧光共聚焦技术分析ERK5的亚细胞定位,腺病毒技术观察活化型ERK5和显性负性ERK5对颗粒细胞甾体激素生成的影响.结果 Western blot检测结果表明,FSH处理颗粒细胞后ERK5的表达随处理时间延长而下调,而ERK5活性形式的表达随处理时间延长而逐渐上升.荧光共聚焦技术分析结果显示,FSH的处理并没有明显影响ERK5在颗粒细胞中的定位.腺病毒技术观察结果发现,在细胞中联合感染活化型MEK5

  1. Serine/threonine kinase-protein kinase B and extracellular signal-regulated kinase regulate ventilator-induced pulmonary fibrosis after bleomycin-induced acute lung injury: a prospective, controlled animal experiment

    OpenAIRE

    Li, Li-Fu; Liao, Shuen-Kuei; Huang, Chung-Chi; Hung, Ming-Jui; Quinn, Deborah A

    2008-01-01

    Introduction Lung fibrosis, reduced lung compliance, and severe hypoxemia found in patients with acute lung injury often result in a need for the support of mechanical ventilation. High-tidal-volume mechanical ventilation can increase lung damage and fibrogeneic activity but the mechanisms regulating the interaction between high tidal volume and lung fibrosis are unclear. We hypothesized that high-tidal-volume ventilation increased pulmonary fibrosis in acute lung injury via the serine/threon...

  2. Extracellular electrical activity from the photoreceptors of midge

    Indian Academy of Sciences (India)

    A A Babrekar; G R Kulkarni; B B Nath; P B Vidyasagar

    2004-09-01

    The ontogeny of photosensitivity has been studied in a holometabolous insect, the midge Chironomus ramosus. The life cycle of midges shifts from an aquatic environment to a non-aquatic environment. Extracellular electrical activity of photoreceptor organs was recorded at larval and adult stages. We found an increase in photosensitivity as the larva metamorphosed to the adult stage. This is the first report of changes in photosensitivity during the development of any insect described in an ecological context.

  3. Evidence for cross-talk between M2 and M3 muscarinic acetylcholine receptors in the regulation of second messenger and extracellular signal-regulated kinase signalling pathways in Chinese hamster ovary cells

    OpenAIRE

    Hornigold, David C; Mistry, Rajendra; Raymond, Pamela D; Blank, Jonathan L; John Challiss, R A

    2003-01-01

    We have examined possible mechanisms of cross-talk between the Gq/11-linked M3 muscarinic acetylcholine (mACh) receptor and the Gi/o-linked M2 mACh receptor by stable receptor coexpression in Chinese hamster ovary (CHO) cells. A number of second messenger (cyclic AMP, Ins(1,4,5)P3) and mitogen-activated protein kinase (ERK and JNK) responses stimulated by the mACh receptor agonist methacholine were examined in CHO-m2m3 cells and compared to those stimulated in CHO-m2 and CHO-m3 cell-lines, ex...

  4. Classification of the extracellular fields produced by activated neural structures

    Directory of Open Access Journals (Sweden)

    Perry Danielle

    2005-09-01

    Full Text Available Abstract Background Classifying the types of extracellular potentials recorded when neural structures are activated is an important component in understanding nerve pathophysiology. Varying definitions and approaches to understanding the factors that influence the potentials recorded during neural activity have made this issue complex. Methods In this article, many of the factors which influence the distribution of electric potential produced by a traveling action potential are discussed from a theoretical standpoint with illustrative simulations. Results For an axon of arbitrary shape, it is shown that a quadrupolar potential is generated by action potentials traveling along a straight axon. However, a dipole moment is generated at any point where an axon bends or its diameter changes. Next, it is shown how asymmetric disturbances in the conductivity of the medium surrounding an axon produce dipolar potentials, even during propagation along a straight axon. Next, by studying the electric fields generated by a dipole source in an insulating cylinder, it is shown that in finite volume conductors, the extracellular potentials can be very different from those in infinite volume conductors. Finally, the effects of impulses propagating along axons with inhomogeneous cable properties are analyzed. Conclusion Because of the well-defined factors affecting extracellular potentials, the vague terms far-field and near-field potentials should be abandoned in favor of more accurate descriptions of the potentials.

  5. Extracellular Signal-regulated Kinase (ERK)-dependent Phosphorylation of Y-Box-binding Protein 1 (YB-1) Enhances Gene Expression in Granulosa Cells in Response to Follicle-stimulating Hormone (FSH).

    Science.gov (United States)

    Donaubauer, Elyse M; Hunzicker-Dunn, Mary E

    2016-06-01

    Within the ovarian follicle, immature oocytes are surrounded and supported by granulosa cells (GCs). Stimulation of GCs by FSH leads to their proliferation and differentiation, events that are necessary for fertility. FSH activates multiple signaling pathways to regulate genes necessary for follicular maturation. Herein, we investigated the role of Y-box-binding protein-1 (YB-1) within GCs. YB-1 is a nucleic acid binding protein that regulates transcription and translation. Our results show that FSH promotes an increase in the phosphorylation of YB-1 on Ser(102) within 15 min that is maintained at significantly increased levels until ∼8 h post treatment. FSH-stimulated phosphorylation of YB-1(Ser(102)) is prevented by pretreatment of GCs with the PKA-selective inhibitor PKA inhibitor (PKI), the MEK inhibitor PD98059, or the ribosomal S6 kinase-2 (RSK-2) inhibitor BI-D1870. Thus, phosphorylation of YB-1 on Ser(102) is PKA-, ERK-, and RSK-2-dependent. However, pretreatment of GCs with the protein phosphatase 1 (PP1) inhibitor tautomycin increased phosphorylation of YB-1(Ser(102)) in the absence of FSH; FSH did not further increase YB-1(Ser(102)) phosphorylation. This result suggests that the major effect of RSK-2 is to inhibit PP1 rather than to directly phosphorylate YB-1 on Ser(102) YB-1 coimmunoprecipitated with PP1β catalytic subunit and RSK-2. Transduction of GCs with the dephospho-adenoviral-YB-1(S102A) mutant prevented the induction by FSH of Egfr, Cyp19a1, Inha, Lhcgr, Cyp11a1, Hsd17b1, and Pappa mRNAs and estradiol-17β production. Collectively, our results reveal that phosphorylation of YB-1 on Ser(102) via the ERK/RSK-2 signaling pathway is necessary for FSH-mediated expression of target genes required for maturation of follicles to a preovulatory phenotype. PMID:27080258

  6. 细胞外信号调节激酶信号转导通路在牙发生和再生中的作用%Extracellular signal-regulated kinase signal transduction pathway in dentification and tooth regeneration

    Institute of Scientific and Technical Information of China (English)

    班华杰; 王代友

    2015-01-01

    Extracellular signal-regulated kinase(ERK) is an important signaling transduction pathway involved in tooth development. The pathway widely regulates dentification and tooth morphogenesis in each developmental stage. Fibroblast growth factor-10 promotes the initiation and development of tooth germ by activating the ERK signaling transduction pathway in dental epithelium. The pathway also regulates the differentiation of mesenchymal stem cells into odontoblasts and the initiation and differentiation of periodontium. Mineral trioxide aggregate can induce odontoblast differentiation by phosphorylating and activating ERK. The pathway is critical in inducing the differentiation of dental pulp stem cell(DPSC) into odontoblasts; hence, activating the ERK signaling pathway in DPSC may be an effective way to regenerate odontoblasts. Hydrostatic pressures during dentification promote mesenchymal osteodifferentiation. By contrast, mechanical stretch inhibits osteogenic differentiation by increasing pERK-mediated cell proliferation. The ERK signaling pathway facilitates the migration of DPSC and the regeneration of dental pulp tissue. This review enumerates the functions of the ERK pathway in dentification and discusses how ERK influences tooth regeneration.%细胞外信号调节激酶(ERK)信号转导通路广泛调控牙发生及其形态发育,且其作用伴随在牙发生的各个时期。成纤维细胞生长因子-10通过激活牙上皮细胞中的ERK信号转导通路促进牙胚发生发育。在间质细胞的牙向分化以及牙周组织的发生和分化过程中,ERK信号转导通路皆起着重要的作用。三氧化聚合物可通过磷

  7. The human angiotensin AT(1) receptor supports G protein-independent extracellular signal-regulated kinase 1/2 activation and cellular proliferation

    DEFF Research Database (Denmark)

    Hansen, Jakob Lerche; Aplin, Mark; Hansen, Jonas Tind;

    2008-01-01

    (1) receptor actions. However, it is currently unknown whether the human angiotensin AT(1) receptor can signal through G protein-independent mechanisms - and if so, what the physiological impact of such signalling is. We have performed a detailed pharmacological analysis of the human angiotensin AT(1......) receptor using a battery of angiotensin analogues and registered drugs targeting this receptor. We show that the human angiotensin AT(1) receptor signals directly through G protein-independent pathways and supports NIH3T3 cellular proliferation. The realization of G protein-independent signalling...

  8. Role of extracellular signal-regulated kinases in aldosterone-induced rat mesangial cells proliferation%细胞外信号调节蛋白激酶介导醛固酮诱导的肾小球系膜细胞增殖

    Institute of Scientific and Technical Information of China (English)

    姚丽; 孙立; 魏敏; 葛丹梅; 王力宁

    2011-01-01

    目的 探讨细胞外信号调节蛋白激酶(ERK1/2)在醛固酮诱导肾小球系膜细胞(RMC)增殖中的作用.方法 获取6~8周健康雄性SD大鼠RMC并鉴定,取第5~10代的细胞用于实验.细胞分为6组:对照组;PD98059(10 μmol/L)组;依普利酮(1 μmol/L)组;醛固酮(100 nmol/L)组;醛固酮(100 nmol/L)+PD98059(10 μmol/L)组;醛固酮(100 nmol/L)+依普利酮(1 μmol/L)组.采用Western印迹技术检测SD大鼠RMC盐皮质激素受体(MR)表达状况,以及醛固酮刺激后RMC ERK1/2活性状态.采用3H-胸腺嘧啶核苷(3H-TdR)掺入法检测RMC增殖状况.结果 体外培养的SD大鼠RMC有MR蛋白表达.醛固酮(100 nmol/L)刺激RMC 10 min使ERK1/2活性显著增高(P<0.05);刺激30 h时使RMC的3H-TdR掺入量显著增加[(1.35±0.08)倍,P<0.05].选择性醛固酮受体拮抗剂依普利酮(1 μmol/L)及MAPKERK激酶(MEK)特异性抑制剂PD98059(10 μmol/L)可阻止醛固酮诱导的RMC ERK1/2激活以及3H-TdR掺入量增加.结论 醛固酮通过激活ERK1/2信号转导通路诱导RMC增殖.%Objective To determine the role of extracellular signal-regulated kinases (ERK1/2) in aldosterone-induced rat mesangial cells (RMCs) proliferation. Methods RMCs were obtained from intact glomeruli of 4- to 6-week-old Sprague-Dawley rats and characterized according to published methods. RMCs between passages 5 and passages 10 were used. Protein levels of mineralocorticoid receptor (MR) in RMCs were analyzed by Western blotting. The cells were divided into the following groups: control group, PD98059 (10 (μmol/L) group, eplerenone (1 μmol/L) group, aldosterone (100 nmol/L) group, aldosterone (100 nmol/L) +PD98059 (10 μmol/L) group, aldosterone (100 nmol/L)+eplerenone (1 μmol/L) group. ERK1/2 activity was measured by Western blotting. Cell proliferation of RMCs was evaluated by [3H]-thymidine uptake measurements.Results MR protein expression in RMCs was confirmed by Western blotting. Aldosterone activated ERK1/2, and the maximal

  9. Extracellular melanogenesis inhibitory activity and the structure-activity relationships of ugonins from Helminthostachys zeylanica roots.

    Science.gov (United States)

    Yamauchi, Kosei; Mitsunaga, Tohru; Itakura, Yuki; Batubara, Irmanida

    2015-07-01

    Ugonin J, K, and L, which are luteolin derivatives, were isolated from Helminthostachys zeylanica roots by a series of chromatographic separations of a 50% ethanol/water extract. They were identified using nuclear magnetic resonance (NMR), ultraviolet (UV) spectra, and ultra-performance liquid chromatography coupled to time-of-flight mass spectrometry (UPLC-TOF-MS). In this study, the intra and extracellular melanogenic activity of the ugonins were determined using B16 melanoma cells. The results showed that ugonin J at 12.5, 25, and 50μM reduced extracellular melanin contents to 75, 16, and 14%, respectively, compared to the control. This indicates that ugonin J showed a stronger activity than arbutin, used as the positive control. Moreover, ugonin K showed a more potent inhibition with 19, 8, and 9% extracellular melanin reduction at the same concentrations, than that shown by ugonin J. In contrast, ugonin L did not inhibit intra- or extracellular melanogenic activity. Furthermore, in order to investigate the structure-activity relationships of the ugonins, the intra- and extracellular melanogenic activity of luteolin, methylluteolin, quercetin, eriodictyol, apigenin, and chrysin were determined. Consequently, it was suggested that the catechol and flavone skeleton of ugonin K is essential for the extracellular melanogenic inhibitory activity, and the low polarity substituent groups on the A ring of ugonin K may increase the activity. PMID:25979512

  10. Extracellular Matrix Stiffness Regulates Osteogenic Differentiation through MAPK Activation.

    Directory of Open Access Journals (Sweden)

    Jun-Ha Hwang

    Full Text Available Mesenchymal stem cell (MSC differentiation is regulated by the extracellular matrix (ECM through activation of intracellular signaling mediators. The stiffness of the ECM was shown to be an important regulatory factor for MSC differentiation, and transcriptional coactivator with PDZ-binding motif (TAZ was identified as an effector protein for MSC differentiation. However, the detailed underlying mechanism regarding the role of ECM stiffness and TAZ in MSC differentiation is not yet fully understood. In this report, we showed that ECM stiffness regulates MSC fate through ERK or JNK activation. Specifically, a stiff hydrogel matrix stimulates osteogenic differentiation concomitant with increased nuclear localization of TAZ, but inhibits adipogenic differentiation. ERK and JNK activity was significantly increased in cells cultured on a stiff hydrogel. TAZ activation was induced by ERK or JNK activation on a stiff hydrogel because exposure to an ERK or JNK inhibitor significantly decreased the nuclear localization of TAZ, indicating that ECM stiffness-induced ERK or JNK activation is important for TAZ-driven osteogenic differentiation. Taken together, these results suggest that ECM stiffness regulates MSC differentiation through ERK or JNK activation.

  11. Extracellular Matrix Stiffness Regulates Osteogenic Differentiation through MAPK Activation.

    Science.gov (United States)

    Hwang, Jun-Ha; Byun, Mi Ran; Kim, A Rum; Kim, Kyung Min; Cho, Hang Jun; Lee, Yo Han; Kim, Juwon; Jeong, Mi Gyeong; Hwang, Eun Sook; Hong, Jeong-Ho

    2015-01-01

    Mesenchymal stem cell (MSC) differentiation is regulated by the extracellular matrix (ECM) through activation of intracellular signaling mediators. The stiffness of the ECM was shown to be an important regulatory factor for MSC differentiation, and transcriptional coactivator with PDZ-binding motif (TAZ) was identified as an effector protein for MSC differentiation. However, the detailed underlying mechanism regarding the role of ECM stiffness and TAZ in MSC differentiation is not yet fully understood. In this report, we showed that ECM stiffness regulates MSC fate through ERK or JNK activation. Specifically, a stiff hydrogel matrix stimulates osteogenic differentiation concomitant with increased nuclear localization of TAZ, but inhibits adipogenic differentiation. ERK and JNK activity was significantly increased in cells cultured on a stiff hydrogel. TAZ activation was induced by ERK or JNK activation on a stiff hydrogel because exposure to an ERK or JNK inhibitor significantly decreased the nuclear localization of TAZ, indicating that ECM stiffness-induced ERK or JNK activation is important for TAZ-driven osteogenic differentiation. Taken together, these results suggest that ECM stiffness regulates MSC differentiation through ERK or JNK activation.

  12. 细粒棘球蚴细胞外信号调节激酶基因克隆、序列分析及功能的初步鉴定%Molecular cloning, sequencing and function of extracellular signal regulated kinase of Echinococcus granulosus

    Institute of Scientific and Technical Information of China (English)

    吕国栋; 纪静; 王俊华; 李亮; 王红丽; 卢晓梅; 王星; 温浩; 林仁勇

    2010-01-01

    Objective To perform molecular cloning and sequencing, bioinformatics analysis,protein expression and function of extracellular signal regulated kinase (EgERK1) of Echinococcus granulosus in Xinjiang. Methods The specific primers of EgERK1 were designed and total RNA was extracted from Echinococcus granulosus in Xinjiang. EgERK1 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and prokaryotic expression plasmid pET28a-EgERK1 was constructed and sequenced. The sequences were analyzed by DNA sequencing and bioinformatics technology. The recombinant EgERK1 protein was induced and expressed. The biological function was detected using sodium dodecyl sulfate polyacrylamide gel electropheresis and Western blot. Results The sequence of RT-PCR product was 1125 bp, encoding 374 amino acids with isoelectric point of 6.34.This gene was a new ERK-homologues gene indicated by BLAST, named EgERK1(EU701008).Homology comparisons indicated that the homology of EgERK1 and EmMPK1from Echinococcus multilocularis was 95.45%, and was 43.04%-61.88% to ERK from Caenorhabditis elegans, S. cerevisiae, D. melanogaster and human. Phylogenetic analysis showed that EgERK1 clustered with EmMPK1. Bioinformatics analysis predicted that EgERK1 contained a highly conserved T-X-Y motif and activation loop segment of ERK-like kinase.Western blot results showed the EgERK1 recombinant protein could reacted specifically with anti-human ERK monoclonal antibody. Conclusion A new EgERK1 gene of Echinococcus granulosus is successfully cloned and its recombinant protein could reacted specifically with ERK1/2 antibody, which provides the basis for further study of EgERK1 function in the host-parasite interaction.%目的 从新疆株细粒棘球蚴中克隆细胞外信号调节激酶(EgERK1)基因,进行序列测定、生物信息学分析,蛋白表达及功能初步鉴定.方法 设计特异性引物,从新疆株细粒棘球蚴中提取总RNA,RT-PCR法扩增EgERK1基因,构建pET28

  13. Endocannabinoid Signaling Regulates Sleep Stability.

    Directory of Open Access Journals (Sweden)

    Matthew J Pava

    Full Text Available The hypnogenic properties of cannabis have been recognized for centuries, but endogenous cannabinoid (endocannabinoid regulation of vigilance states is poorly characterized. We report findings from a series of experiments in mice measuring sleep with polysomnography after various systemic pharmacological manipulations of the endocannabinoid system. Rapid, unbiased scoring of vigilance states was achieved using an automated algorithm that we devised and validated. Increasing endocannabinoid tone with a selective inhibitor of monoacyglycerol lipase (JZL184 or fatty acid amide hydrolase (AM3506 produced a transient increase in non-rapid eye movement (NREM sleep due to an augmentation of the length of NREM bouts (NREM stability. Similarly, direct activation of type 1 cannabinoid (CB1 receptors with CP47,497 increased NREM stability, but both CP47,497 and JZL184 had a secondary effect that reduced NREM sleep time and stability. This secondary response to these drugs was similar to the early effect of CB1 blockade with the antagonist/inverse agonist AM281, which fragmented NREM sleep. The magnitude of the effects produced by JZL184 and AM281 were dependent on the time of day this drug was administered. While activation of CB1 resulted in only a slight reduction in gamma power, CB1 blockade had dramatic effects on broadband power in the EEG, particularly at low frequencies. However, CB1 blockade did not significantly reduce the rebound in NREM sleep following total sleep deprivation. These results support the hypothesis that endocannabinoid signaling through CB1 is necessary for NREM stability but it is not necessary for sleep homeostasis.

  14. Afferent signals regulating food intake.

    Science.gov (United States)

    Bray, G A

    2000-08-01

    Food intake is a regulated system. Afferent signals provide information to the central nervous system, which is the centre for the control of satiety or food seeking. Such signals can begin even before food is ingested through visual, auditory and olfactory stimuli. One of the recent interesting findings is the demonstration that there are selective fatty acid taste receptors on the tongue of rodents. The suppression of food intake by essential fatty acids infused into the stomach and the suppression of electrical signals in taste buds reflect activation of a K rectifier channel (K 1.5). In animals that become fat eating a high-fat diet the suppression of this current by linoleic acid is less than that in animals that are resistant to obesity induced by dietary fat. Inhibition of fatty acid oxidation with either mercaptoacetate (which blocks acetyl-CoA dehydrogenase) or methylpalmoxirate will increase food intake. When animals have a choice of food, mercaptoacetate stimulates the intake of protein and carbohydrate, but not fat. Afferent gut signals also signal satiety. The first of these gut signals to be identified was cholecystokinin (CCK). When CCK acts on CCK-A receptors in the gastrointestinal tract, food intake is suppressed. These signals are transmitted by the vagus nerve to the nucleus tractus solitarius and thence to higher centres including the lateral parabrachial nucleus, amygdala, and other sites. Rats that lack the CCK-A receptor become obese, but transgenic mice lacking CCK-A receptors do not become obese. CCK inhibits food intake in human subjects. Enterostatin, the pentapeptide produced when pancreatic colipase is cleaved in the gut, has been shown to reduce food intake. This peptide differs in its action from CCK by selectively reducing fat intake. Enterostatin reduces hunger ratings in human subjects. Bombesin and its human analogue, gastrin inhibitory peptide (also gastrin-insulin peptide), reduce food intake in obese and lean subjects. Animals

  15. Sphingosine 1-phosphate (S1P)/S1P receptor 1 signaling regulates receptor activator of NF-{kappa}B ligand (RANKL) expression in rheumatoid arthritis

    Energy Technology Data Exchange (ETDEWEB)

    Takeshita, Harunori [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan); Kitano, Masayasu, E-mail: mkitano6@hyo-med.ac.jp [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan); Iwasaki, Tsuyoshi [Department of Pharmacy, Hyogo University of Health Sciences, 1-3-6 Minatojima Kobe, Hyogo 650-8530 (Japan); Kitano, Sachie; Tsunemi, Sachi; Sato, Chieri; Sekiguchi, Masahiro; Azuma, Naoto [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan); Miyazawa, Keiji [Discovery Research III, Research and Development, Kissei Pharmaceutical Company, 4365-1 Hodakakashiwara, Azumino, Nagano 399-8304 (Japan); Hla, Timothy [Center for Vascular Biology, Department of Pathology and Laboratory Medicine, Weill Medical College of Cornell University, 1300 York Avenue, Box 69, NY 10065 (United States); Sano, Hajime [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan)

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer MH7A cells and CD4{sup +} T cells expressed S1P1 and RANKL. Black-Right-Pointing-Pointer S1P increased RANKL expression in MH7A cells and CD4{sup +} T cells. Black-Right-Pointing-Pointer The effect of S1P in MH7A cells was inhibited by specific Gi/Go inhibitors. -- Abstract: Sphingosine 1-phosphate (S1P)/S1P receptor 1 (S1P1) signaling plays an important role in synovial cell proliferation and inflammatory gene expression by rheumatoid arthritis (RA) synoviocytes. The purpose of this study is to clarify the role of S1P/S1P1 signaling in the expression of receptor activator of NF-{kappa}B ligand (RANKL) in RA synoviocytes and CD4{sup +} T cells. We demonstrated MH7A cells, a human RA synovial cell line, and CD4{sup +} T cells expressed S1P1 and RANKL. Surprisingly, S1P increased RANKL expression in MH7A cells and CD4{sup +} T cells in a dose-dependent manner. Moreover, S1P enhanced RANKL expression induced by stimulation with TNF-{alpha} in MH7A cells and CD4{sup +} T cells. These effects of S1P in MH7A cells were inhibited by pretreatment with PTX, a specific Gi/Go inhibitor. These findings suggest that S1P/S1P1 signaling may play an important role in RANKL expression by MH7A cells and CD4{sup +} T cells. S1P/S1P1 signaling of RA synoviocytes is closely connected with synovial hyperplasia, inflammation, and RANKL-induced osteoclastogenesis in RA. Thus, regulation of S1P/S1P1 signaling may become a novel therapeutic target for RA.

  16. Neovibsanin B increases extracellular matrix proteins in optic nerve head cells via activation of Smad signalling pathway.

    Science.gov (United States)

    Wang, Zhen; Xu, Wei; Rong, Ao; Lin, Yan; Qiu, Xu-Ling; Qu, Shen; Lan, Xian-Hai

    2015-01-01

    The present study demonstrates the effect of neovibsanin B on the synthesis and deposition of ECM proteins and the signalling pathways used in optic nerve head (ONH) astrocytes and lamina cribrosa (LC) cells. For investigation of the signalling pathway used by neovibsanin B, ONH cells were treated with neovibsanin B. Western blot and immunostaining analyses were used to examine the phosphorylation of proteins involved in Smad and non-Smad signalling pathway. The results revealed that ONH cells on treatment with neovibsanin B showed enhanced synthesis of extracellular matrix (ECM) proteins. Neovibsanin B induced phosphorylation of canonical signalling proteins, Smad2/3. However phosphorylation of non-canonical signalling proteins, extracellular signal-regulated kinases, p38, and c-Jun N-terminal kinases (JNK) 1/2 remained unaffected. There was also increase in co-localization of pSmad2/3 with Co-Smad4 in the nucleus of ONH astrocytes and LC cells indicating activation of the canonical Smad signalling pathway. Treatment of ONH cells with SIS3, inhibitor of Smad3 phosphorylation reversed the neovibsanin B stimulated ECM expression as well as activation of canonical pathway signalling molecules. In addition, inhibition of Smad2 or Smad3 using small interfering RNA (siRNA) also suppressed neovibsanin B stimulated ECM protein synthesis in ONH astrocytes and LC cells. Thus neovibsanin B utilizes the canonical Smad signalling pathway to stimulate ECM synthesis in human ONH cells. The neovibsanin B induced ECM synthesis and activation of the canonical Smad signalling pathway may be due to its effect on transforming growth factor-β2 (TGF-β2). However, further studies are under process to understand the mechanism.

  17. Extracellular Streptomyces lividans vesicles: composition, biogenesis and antimicrobial activity

    Science.gov (United States)

    Schrempf, Hildgund; Merling, Philipp

    2015-01-01

    We selected Streptomyces lividans to elucidate firstly the biogenesis and antimicrobial activities of extracellular vesicles that a filamentous and highly differentiated Gram-positive bacterium produces. Vesicle types range in diameter from 110 to 230 nm and 20 to 60 nm, respectively; they assemble to clusters, and contain lipids and phospholipids allowing their in situ imaging by specific fluorescent dyes. The presence of the identified secondary metabolite undecylprodigiosin provokes red fluorescence of a portion of the heterogeneous vesicle populations facilitating in vivo monitoring. Protuberances containing vesicles generate at tips, and alongside of substrate hyphae, and enumerate during late vegetative growth to droplet-like exudates. Owing to in situ imaging in the presence and absence of a green fluorescent vancomycin derivative, we conclude that protuberances comprising vesicles arise at sites with enhanced levels of peptidoglycan subunits [pentapeptide of lipid II (C55)-linked disaccharides], and reduced levels of polymerized and cross-linked peptidoglycan within hyphae. These sites correlate with enhanced levels of anionic phospholipids and lipids. Vesicles provoke pronounced damages of Aspergillus proliferans, Verticillium dahliae and induced clumping and distortion of Escherichia coli. These harmful effects are likely attributable to the action of the identified vesicular compounds including different enzyme types, components of signal transduction cascades and undecylprodigiosin. Based on our pioneering findings, we highlight novel clues with environmental implications and application potential. PMID:25851532

  18. Differentiating Intracellular from Extracellular Alkaline Phosphatase Activity in Soil by Sonication

    NARCIS (Netherlands)

    Qin, S.P.; Hu, C.S.; Oenema, O.

    2013-01-01

    Differentiating intracellular from extracellular enzyme activity is important in soil enzymology, but not easy. Here, we report on an adjusted sonication method for the separation of intracellular from extracellular phosphatase activity in soil. Under optimal sonication conditions [soil:water ratio

  19. Extracellular Enzyme Activity assay as indicator of soil microbial functional diversity and activity

    DEFF Research Database (Denmark)

    Hendriksen, Niels Bohse; Winding, Anne

    2012-01-01

    Extracellular Enzyme Activity assay as indicator of soil microbial functional diversity and activity Niels Bohse Hendriksen, Anne Winding. Department of Environmental Science, Aarhus University, 4000 Roskilde, Denmark Soil enzymes originate from a variety of organisms, notably fungi and bacteria...... and especially hydrolytic extracellular enzymes are of pivotal importance for decomposition of organic substrates and biogeochemical cycling. Their activity reflects the functional diversity and activity of the microorganisms involved in decomposition processes which are essential processes for soil functioning...... and soil ecosystem services. The soil enzyme activity has been measured by the use of fluorogenic model substrates e.g. methylumbelliferyl (MUF) substrates for a number of enzymes involved in the degradation of polysaccharides as cellulose, hemicellulose and chitin, while degradation of proteins has been...

  20. Extracellular enzyme activity assay as indicator of soil microbial functional diversity and activity

    DEFF Research Database (Denmark)

    Hendriksen, Niels Bohse; Winding, Anne

    2012-01-01

    Extracellular enzyme activity assay as indicator of soil microbial functional diversity and activity Niels Bohse Hendriksen, Anne Winding. Department of Environmental Science, Aarhus University, 4000 Roskilde, Denmark Soils provide numerous essential ecosystem services such as carbon cycling...... of soil microbial functions is still needed. In soil, enzymes originate from a variety of organisms, notably fungi and bacteria and especially hydrolytic extracellular enzymes are of pivotal importance for decomposition of organic substrates and biogeochemical cycling. Their activity will reflect...... the functional diversity and activity of the microorganisms involved in decomposition processes. Their activity has been measured by the use of fluorogenic model substrates e.g. methylumbelliferyl (MUF) substrates for a number of enzymes involved in the degradation of polysacharides as cellulose, hemicellulose...

  1. Phosphatase of regenerating liver-3 promotes Lovo cells invasion by inducing serine protease inhibitor E3 through extracellular signal-regulated kinase%肝再生磷酸酶-3通过细胞外调节蛋白激酶上调丝氨酸蛋白酶抑制剂E3促进Lovo细胞侵袭

    Institute of Scientific and Technical Information of China (English)

    许鹤洋; 林显敢; 罗兴喜; 张旸; 蓝球生; 褚忠华

    2014-01-01

    Objective To investigate the influence and mechanisms of phosphatase of regenerating liver-3 (PRL-3) on serine protease inhibitor E3 (serpinE3),and their effects on Lovo cells invasion.Methods Western blotting was used to detect the serpinE3 of Lovo-P and Lovo-C cells.Transwell chamber was used to detect the invasion of Lovo-P and Lovo-C cells.The Lovo-P cells were treated with the extracellular signal-regulated kinase (ERK) inhibitor U0126 (10 μmol/L) for 6 h,and the expression of serpinE3 and invasion of Lovo-P cells were examined.Results The expression of serpinE3 was increased in the Lovo-P cells transfected with human PRL-3.Lovo-P cells exhibited stronger invasion ability than Lovo-Ccells (378 ± 13 vs.269 ± 15,P < 0.05).SerpinE3 was abrogated when Lovo-P treated with U0126 and the invasion ability of the cells was decreased either (211-±9 vs.358 ± 19,P <0.05).Conclusion PRL-3 could induce serpinE3 expression by ERK,and then promotes Lovo cells invasion.%目的 探讨肝再生磷酸酶-3(PRL-3)对丝氨酸蛋白酶抑制剂E3(serpinE3)的影响及机制,以及PRL-3和serpinF3对结肠癌Lovo细胞侵袭性的影响.方法 通过Western blot方法,分别检测已经稳转入PRL-3载体的Lovo-P细胞和对照组Lovo-C细胞中serpinE3的表达水平,Transwell小室检测Lovo细胞侵袭性.再给予细胞外调节蛋白激酶(ERK)特异性抑制剂U0126(10 μmol/L)预处理Lovo-P细胞6h,观察serpinE3表达的变化,检测Lovo-P细胞侵袭性的改变.结果 Western blot检测结果显示在转染人PRL-3的Lovo-P细胞中,serpinE3表达明显上调,Lovo-P细胞侵袭性增强[(378±13)个比(269±15)个,P<0.05];而当特异性阻断ERK后,Lovo-P细胞中的serpinE3表达下调,并且细胞侵袭性也降低[(21l±9)个比(358±19)个,P<0.05].结论 PRL-3能够通过ERK诱导serpinE3表达上调的方式,增加Lovo细胞的侵袭性.

  2. CD133在甲状腺乳头状癌中的表达及其与细胞外信号调节激酶1表达的关系%Expressions of CD133 and extracellular signal-regulated protein kinase 1 in papillary thyroid carcinoma and their correlations

    Institute of Scientific and Technical Information of China (English)

    张振寰; 王丹娜; 武世伍

    2014-01-01

    目的:检测肿瘤干细胞标志物CD133在甲状腺乳头状癌(PTC)组织中表达及其与细胞外信号调节激酶1(ERK1)的关系及其临床病理意义。方法:采用免疫组织化学EliVisionTM plus法检测68例PTC组织和40例结节性甲状腺肿组织中CD133和ERK1的表达情况。结果:在结节性甲状腺肿组织和PTC组织中,CD133和ERK1的阳性率分别为10.0%与50.0%和12.5%与63.2%,2组差异均有统计学意义(P<0.01)。 CD133的表达水平在PTC组织的不同分化程度、淋巴结转移和临床分期间差异均有统计学意义(P<0.05~P<0.01);PTC组织的分化越差,淋巴结发生转移及临床分期越高,ERK1的阳性率亦越高(P<0.01)。 CD133与ERK1的表达呈正相关关系(P<0.05)。结论:CD133和ERK1的异常表达参与了PTC的发生、发展、浸润及转移;早期联合检查CD133和ERK1对PTC的进展及预后判断均有临床意义。%Objective:To detect the expressions of CD133, cancer stem cells marker, and extracellular signal-regulated protein kinase 1(ERK1) in papillary thyroid carcinoma ( PTC), and their correlations and clinicopathological significance. Methods:The expressions of CD133 and ERK1 in 68 samples of PTC tissue and 40 samples of nodular goiter tissue were detected by immunohistochemistry. Results:The positive rates of CD133 and ERK1 in PTC and nodular goiter tissues were 10. 0% &50. 0% and 12.5% & 63. 2%,respectively,the differences of which were statistically significant(P <0. 01). The differences of the expression levels of CD133 in tumor grade,lymph node metastasis and TNM stage of PTC were statistically significant(P<0. 05 to P<0. 01). With the PTC worsening differentiation,lymph node transferring and clinical stage increasing,the positive rates of ERK1 increased( P<0.01). There was a positive correlation between the expression of CD133 and ERK1(P < 0. 05). Conclusions:The abnormal expressions of CD133 and ERK1 may be involved in the occurrence, development

  3. Variation in activity of root extracellular phytase between genotypes of barley

    DEFF Research Database (Denmark)

    Asmar, Mohammad Farouq

    1997-01-01

    Barley genotypes grown in nutrient solution under P nutrient stress and sterile conditions were compared in activity of root-associated and root-released extracellular phytase. The activity of root-associated phytase of all genotypes was about 10 times higher than that of root-released phytase...... and the genotypes performed differently with regard to the activity of the enzymes. The winter barley genotype, Marinka had the highest activity of root-associated extracellular phytase which differed significantly from Alexis and Senate, but not from Regatta. Alexis showed the lowest activity of root......-released extracellular phytase which differed significantly from those of Marinka and Regatta, but not from Senate. Generally, there was a significant correlation between the activity of root-associated and released extracellular phytase....

  4. Arginine vasopressin stimulates proliferation of adult rat cardiac fibroblasts via protein kinase C-extracellular signal-regulated kinase 1/2 pathway%蛋白激酶C-细胞外信号调节激酶1/2通路介导精氨酸升压素对成年大鼠心肌成纤维细胞的促增殖作用

    Institute of Scientific and Technical Information of China (English)

    何燕萍; 赵连友; 郑强荪; 刘少伟; 赵晓燕; 陆晓龙; 牛晓琳

    2008-01-01

    demonstrated that AVP is a mitogen for neonatal rat cardiac fibroblasts (CFs). In the present study, we extended our investigations to adult rat CFs to explore whether AVP could induce adult rat CF proliferation and, if so, to identify the mechanism involved. Adult rat CFs were isolated, cultured and subjected to AVP treatment. DNA synthesis and cell cycle distribution were analyzed by [3H]-thymidine incorporation and flow cytometry. Cellular extracellular signal-regulated kinase 1/2 (ERK 1/2) activity was measured by in vitro kinase assay using myelin basic protein (MBP) as a substrate. Protein expressions of total-and phospho-ERK1/2, p27Kip1, cyclins D1, A, E were assessed by Western blot. The results showed that AVP stimulated DNA synthesis in adult rat CFs, and the effect was abolished by a V1 receptor antagonist, d(CH2)5[Tyr2(Me),Arg8-vasopressin (0.1μmol/L), but not by a V2 receptor antagonist, desglycinamide-[d(CH2)5, D-Ile2, Ile4, Arg8-vasopressin (0.1μmol/L). AVP induced an activation of ERK1/2, which could be mimicked by the protein kinase C (PKC) activator, phorbol 12-myfistate 13-acetate (PMA, 30nmol/L, 5min), but abolished by depiction of PKC via chronic PMA incubation (2.5μmol/L,24h). In addition, AVP down-regulated protein expression of p27Kip1, increased protein expressions of cyclins D1, A and E, and induced cell cycle progression from G0/G1 into S stage. Inhibition of ERK1/2 activation by PD98059 (30μmol/L) abolished the effect of AVP on DNA synthesis, protein expressions of p27Klp1, cyclins D1, A and E as well as cell cycle progression. These results suggest that AVP is also a growth factor for adult rat CFs. The mitogenic effect of AVP is mediated via V1 receptors and PKC-ERK 1/2 pathway. Moreover, AVP modulates the expressions of cell cycle regulatory proteins p27Kip1 and cyclins D1, A and E, which lie downstream of ERK 1/2 activation, and induces cell cycle progression in adult rat CFs.

  5. Extracellular enzyme activity in anaerobic bacterial cultures: evidence of pullulanase activity among mesophilic marine bacteria.

    OpenAIRE

    C. Arnosti; Repeta, D. J.

    1994-01-01

    The extracellular enzymatic activity of a mixed culture of anaerobic marine bacteria enriched on pullulan [alpha(1,6)-linked maltotriose units] was directly assessed with a combination of gel permeation chromatography (GPC) and nuclear magnetic resonance spectroscopy (NMR). Hydrolysis products of pullulan were separated by GPC into three fractions with molecular weights of > or = 10,000, approximately 5,000, and < or = 1,200. NMR spectra of these fractions demonstrated that pullulan was rapid...

  6. Modulation of Network Activity in Dissociated Hippocampal Cultures by Enzymatic Digestion of Extracellular Matrix

    OpenAIRE

    Mukhina I.V.; Vedunova М.V.; Sakharnova Т.А.; Dityatev А.E.

    2012-01-01

    To investigate the role of extracellular matrix in spontaneous neuronal network activity, we used microelectrode array technology and enzymatic treatment of hippocampal culture with hyaluronidase, which digests the major component of extracellular matrix, hyaluronic acid. Studies were performed using hippocampal cells that were dissociated from embryonic С57ВL6 mice (E18) and plated on microelectrode arrays (MEAs). Our findings revealed that hyaluronidase promoted seizure-like activity during...

  7. 缺血后处理对肝缺血再灌注损伤后磷脂酰肌醇-3激酶和细胞外信号调节激酶的影响和意义%Effect of ischemic postconditioning on phosphatidylinositol-3-OH kinase and extracellular signal-regulated protein kinase in rats with hepatic ischemia-reperfusion injury

    Institute of Scientific and Technical Information of China (English)

    朱宇麟; 李静; 吕毅; 景桂霞

    2012-01-01

    目的 观察缺血后处理对肝缺血再灌注后磷脂酰肌醇-3激酶(PI3K)和细胞外信号调节激酶1/2(ERK1/2)表达的影响,探讨肝缺血后处理的作用机制 方法 采用大鼠70%肝缺血再灌注损伤模型,进行3次循环的再灌注1 min-阳断1 min的缺血后处理,观察假手术(S)组、LY294002+假手术(LY+S)组、PD98059+假手术(PD+S)组,缺血再灌注(IR)组、缺血后处理(IPO)组、LY294002+缺血后处理(LY +IPO )组和PD98059+IPO( PD+IPO)组的肝功能、细胞凋亡、Akt和ERK1/2磷酸化程度的变化.结果 缺血后处理能明显减轻缺血再灌注造成的肝功能损害,增加Akt和ERK1/2的磷酸化程度,应用LY294002或PD98059后都可以取消IPO的作用 结论 缺血后处理可能通过澈活PI3K和ERK1/2减轻肝缺血再灌注损伤.%Objective To observe the effect of ischemic postconditioning on phosphatidylinositol-3-OH kinase (PI3K) and extracellular signal-regulated protein kinase l/2(ERKl/2) in rats after hepatic ischemia reperfusion in rats and investigate the mechanism of ischemic postcoditioning of the liver.Methods Three cycles of 1 min-off-1 min-on ischemic postconditioning regime were used in a rat model of 70% hepatic ischemia-reperfusion injury.The changes in the liver function,he-patocyte apoptosis,phosphorylation of Akt and ERK1/2 were assessed in rats treated with sham operation,LY294002+sham operation (LY + S),PD98059 + sham operation (PD + S),ischemia reperfusion (IR),ischemic postconditioning (IPO),LY294002+ ischemic postconditioning (LY+IPO),or PD98059+ischemic postconditioning (PD+IPO).Results Ischemic postconditioning significantly alleviated hepatic ischemia-reperfusion-induced liver function injury and hepatocyte apoptosis and increased phophorylation of Akt and ERK1/2.LY294002 and PD98059 antagonized the effects of ischemic postconditioning in the liver.Conclusion Activation of PI3K and ERK1/2 may mediate the protective effect of ischemic postconditioning against hepatic

  8. 1,2,3,4,6-penta-O-galloyl-β-D-glucose up-regulates heme oxygenase-1 expression by stimulating Nrf2 nuclear translocation in an extracellular signal-regulated kinase-dependent manner in HepG2 cells

    Institute of Scientific and Technical Information of China (English)

    Hyun-Ock Pae; Gi-Su Oh; Sun-Oh Jeong; Gil-Saeng Jeong; Bok-Soo Lee; Byung-Min Choi; Ho-Sub Lee; Hun-Taeg Chung

    2006-01-01

    AIM: To examine the potency of 1,2,3,4,6-penta-O-galloyl-β-D-glucose (PGG) as a hepatic heme oxygenase-1 (HO-1) inducer and its regulation in HepG2 cells.METHODS: Expression of HO-1 and NF-E2-related factor 2 (Nrf2) and activation of mitogen-activated protein (MAP) kinases were analyzed by Western blot, immunofluorescence assay, and flow cytometry. Transfections of HO-1 gene, small interfering RNAs for HO-1 and Nrf2,and dominant-negative gene for MAP/extracellular signalregulated kinase (ERK) were carried out to dissect the signaling pathways leading to HO-1 expression in HepG 2 cells.RESULTS: PGG up-regulated HO-1 expression and this expression conferred cytoprotection against oxidative injury induced by t-butyl hydroperoxide. Moreover, PGG induced Nrf2 nuclear translocation, which was found to be an upstream step of PGG-induced HO-1 expression, and ERK activation, of which pathway was involved in PGG-induced Nrf2 nuclear translocation, HO-1 expression and cytoprotection.CONCLUSION: PGG up-regulates HO-1 expression by stimulating Nrf2 nuclear translocation in an ERK-dependent manner, and HO-1 expression by PGG may serve as one of the important mechanisms for its hepatoprotective effects.

  9. Phosphorylation of extracellular signal-regulated kinase in the medial prefrontal cortex is increased in the single-prolonged stress rats%单一连续应激大鼠内侧前额皮质细胞外信号调节激酶磷酸化增高

    Institute of Scientific and Technical Information of China (English)

    田艳霞; 徐爱军; 李冉; 吕翠平; 王海涛

    2011-01-01

    Objective To investigate the change of phosphorylated p44/42 extracellular signal-regulated kinase ( pERK1/2 ) and c-fos expression induced by single-prolonged stress (SPS) in medial prefrontal cortex (mPFC). Methods Forty-five male Wistar rats were divided into control group, SPS group and PD98059-SPS group. The rats of SPS group and PD98059-SPS group were exposed to single-prolonged stress ( SPS ), and that of PD98059-SPS group were bilaterally infused the PD98059, inhibitor of ERK, into the mPFC. The behavior was examined using open-field test, elevated plusmaze and Morris water maze. The expression of pERK1/2 in mPFC was detected with immunohistochemical staining and Western blotting. And reverse transcription-polymerase chain reaction ( RT-PCR ) was employed to detect the c-fos mRNA.Results SPS exposure resulted in pronounced anxiety-like behavior and learning and spatial memory impairments.PD98059 significantly ameliorated the behavior alteration. Absorbance of pERK1/2 positive cell, expression of pERK1/2 and expression of c-fos mRNA of SPS rats were 51.54 ± 5.41, 89.61 ± 3.25 and 0.91 ± 0. 13 respectively, while the control group rats were 12. 18 ± 1.61, 34. 22±5.83 and 0. 13 ±0. 03, and PD98059-SPS group rats were 26. 26 ± 1.42,60. 59 ±5.88 and O. 35 ± 0. 11. These data suggest that expressions of pERK1/2 and c-fos mRNA in mPFC increased significantly after rats were exposed to SPS ( P < 0. 01 ), and the increase was siguifieantly abolished by PD98059 ( P < 0. 01 ). Conlusion The results suggest that pERK1/2 may be related to signal transduction pathway in single-prolonged stress.%目的 探讨单一连续应激(SPS)大鼠内侧前额皮质(mPFC)磷酸化细胞外信号调节激酶(pERK1/2)和c-fos表达的变化.方法将45只雄性Wistar大鼠随机分为对照组、应激组和干预组.应激组和干预组大鼠接受SPS,干预组大鼠接受SPS前30min前额皮质局部注射ERK抑制剂2′-氨基-3′-甲氧黄酮(PD98059).利用开场

  10. 转染Cox7a2重组质粒对大鼠支持细胞分泌功能和ERK磷酸化水平的影响%Analysis of cell secretion function and the phosphorylation level of extracellular signal-regulated kinase in rat sertoli cell transfected with Cox7a2 recombinant plasmid

    Institute of Scientific and Technical Information of China (English)

    张铁; 刘保兴; 张秀平; 徐亚平

    2014-01-01

    Objective To investigate cell secretion function and the phosphorylation level of extracellular signal-regulated kinase in rat sertoli cell transfected with Cox7a2 recombinant plasmid. Methods Rat SCs were cultured and divided into three groups such as the control group, the mock group and the recombinant plasmid group. After 24h culture, the culture supernatants of the transfected cells were collected and the levels of transferrin (TF) , interleukin(IL)-1β and IL-6 in culture medium were measured by ELISA. The level of ERK phosphorylation in the transfected cells was detected by Western Blot. Results The level of TF was significantly decreased in the recombinant plasmid group as compared with that of the control group and the mock group (P<0.05) .The levels of IL-1βand IL-6 (157.14±12.69 pg/mL and 0.79±0.04 pg/mL) in the recombinant plasmid group were significantly higher than those of the control group and the mock group(33.05±1.92 pg/mL, 0.25±0.01pg/mL;40.46±6.69pg/mL, 0.37±0.03pg/mL, P<0.01), and the level of ERK phosphorylation was also increased significantly(P<0.01). Conclusion The cell secretion function was impaired in rat SCs transfected with Cox7a2 recombinant plasmid but the phosphorylation level of ERK was increased.%目的:观察大鼠睾丸支持细胞转染细胞色素C氧化酶7a2(Cox7a2)重组质粒后对支持细胞分泌功能及细胞外信号调节激酶(ERK)磷酸化水平的影响。方法培养原代大鼠睾丸支持细胞,将细胞分为空白对照组、空载体组、重组质粒组。将pEGFP-C1-Cox7a2重组质粒转染支持细胞24h后,收集细胞上清液,采用酶联免疫法,测定转铁蛋白(TF)、白细胞介素-1(interleukin-1, IL-1)β、白细胞介素-6(IL-6)含量。Western blot检测ERK的磷酸化水平。结果重组质粒组TF为(14.11±0.45)pmol/mL,显著低于空白对照组(24.3±0.64)pmol/mL和空载体组(25.16±0.42)pmol/mL(P<0.01)。重组质粒组IL-1

  11. FGF signalling regulates bone growth through autophagy.

    Science.gov (United States)

    Cinque, Laura; Forrester, Alison; Bartolomeo, Rosa; Svelto, Maria; Venditti, Rossella; Montefusco, Sandro; Polishchuk, Elena; Nusco, Edoardo; Rossi, Antonio; Medina, Diego L; Polishchuk, Roman; De Matteis, Maria Antonietta; Settembre, Carmine

    2015-12-10

    Skeletal growth relies on both biosynthetic and catabolic processes. While the role of the former is clearly established, how the latter contributes to growth-promoting pathways is less understood. Macroautophagy, hereafter referred to as autophagy, is a catabolic process that plays a fundamental part in tissue homeostasis. We investigated the role of autophagy during bone growth, which is mediated by chondrocyte rate of proliferation, hypertrophic differentiation and extracellular matrix (ECM) deposition in growth plates. Here we show that autophagy is induced in growth-plate chondrocytes during post-natal development and regulates the secretion of type II collagen (Col2), the major component of cartilage ECM. Mice lacking the autophagy related gene 7 (Atg7) in chondrocytes experience endoplasmic reticulum storage of type II procollagen (PC2) and defective formation of the Col2 fibrillary network in the ECM. Surprisingly, post-natal induction of chondrocyte autophagy is mediated by the growth factor FGF18 through FGFR4 and JNK-dependent activation of the autophagy initiation complex VPS34-beclin-1. Autophagy is completely suppressed in growth plates from Fgf18(-/-) embryos, while Fgf18(+/-) heterozygous and Fgfr4(-/-) mice fail to induce autophagy during post-natal development and show decreased Col2 levels in the growth plate. Strikingly, the Fgf18(+/-) and Fgfr4(-/-) phenotypes can be rescued in vivo by pharmacological activation of autophagy, pointing to autophagy as a novel effector of FGF signalling in bone. These data demonstrate that autophagy is a developmentally regulated process necessary for bone growth, and identify FGF signalling as a crucial regulator of autophagy in chondrocytes. PMID:26595272

  12. Extracellular protein analysis of activated sludge and their functions in wastewater treatment plant by shotgun proteomics

    OpenAIRE

    Peng Zhang; Yu Shen; Jin-Song Guo; Chun Li; Han Wang; You-Peng Chen; Peng Yan; Ji-Xiang Yang; Fang Fang

    2015-01-01

    In this work, proteins in extracellular polymeric substances extracted from anaerobic, anoxic and aerobic sludges of wastewater treatment plant (WWTP) were analyzed to probe their origins and functions. Extracellular proteins in WWTP sludges were identified using shotgun proteomics, and 130, 108 and 114 proteins in anaerobic, anoxic and aerobic samples were classified, respectively. Most proteins originated from cell and cell part, and their most major molecular functions were catalytic activ...

  13. Antimicrobial activity of extracellular metabolites from antagonistic bacteria isolated from potato (Solanum phureja) crops

    OpenAIRE

    Sinar David Granada García; Antoni Rueda Lorza; Carlos Alberto Peláez

    2014-01-01

    Microorganisms for biological control are capable of producing active compounds that inhibit the development of phytopathogens, constituting a promising tool toob tain active principles that could replace synthetic pesticides. This study evaluatedtheability of severalpotentialbiocontrol microorganismsto produce active extracellular metabolites. In vitro antagonistic capability of 50 bacterial isolates from rhizospheric soils of "criolla" potato (Solanum phureja) was tested through dual cultur...

  14. Extracellular enzyme activities during cassava fermentation for 'fufu' production.

    Science.gov (United States)

    Oyewole, O B; Odunfa, S A

    1992-01-01

    Amylase and pectin methyl esterase activities increased rapidly during the early period of the fermentation of cassava for 'fufu' production, attaining their peak activities after 12 and 24h, respectively. Cellulase activity was lower and approximately constant for most of the fermentation period.

  15. Insulin signaling regulates mitochondrial function in pancreatic beta-cells.

    Directory of Open Access Journals (Sweden)

    Siming Liu

    Full Text Available Insulin/IGF-I signaling regulates the metabolism of most mammalian tissues including pancreatic islets. To dissect the mechanisms linking insulin signaling with mitochondrial function, we first identified a mitochondria-tethering complex in beta-cells that included glucokinase (GK, and the pro-apoptotic protein, BAD(S. Mitochondria isolated from beta-cells derived from beta-cell specific insulin receptor knockout (betaIRKO mice exhibited reduced BAD(S, GK and protein kinase A in the complex, and attenuated function. Similar alterations were evident in islets from patients with type 2 diabetes. Decreased mitochondrial GK activity in betaIRKOs could be explained, in part, by reduced expression and altered phosphorylation of BAD(S. The elevated phosphorylation of p70S6K and JNK1 was likely due to compensatory increase in IGF-1 receptor expression. Re-expression of insulin receptors in betaIRKO cells partially restored the stoichiometry of the complex and mitochondrial function. These data indicate that insulin signaling regulates mitochondrial function and have implications for beta-cell dysfunction in type 2 diabetes.

  16. PATHOGEN IMPACT ON THE ACTIVITY DYNAMICS OF POTATO SUSPENSION CELLS EXTRA-CELLULAR PEROXIDASE

    Directory of Open Access Journals (Sweden)

    Graskova I.A.

    2005-08-01

    Full Text Available Changes in the activity of extracellular peroxidases were measured in cell suspension cultures of potato infected by Clavibacter michiganensis subsp. sepedonicus (Spieck. et Kotth. Skapt et Burkh. The total extracellular peroxidases activity of the resistant potato variety was higher than that of the sensitive variety both before and after infection. The enzyme of the resistant variety had a рН optimum of 6.2, while that of the sensitive variety was 5.4. Extracellular peroxidases of the sensitive potato variety were activated 10 minutes after infection, and displayed highest activity 1.5-2 hours later. In the resistant variety, peroxidase activity rose sharply in the first minutes of infection, and second peak of activity occurred 1.5-2 hours later. The increase of extracellular peroxidases activity of the sensitive potato variety under pathogenesis is connected with the change of genome expression and synthesis of proteins. The increase of enzyme activity of resistant potato variety in the first moments of infection is not related to proteins synthesis and is apparently conditioned by the change of kinetic parameters.

  17. Periphytic photosynthetic stimulation of extracellular enzyme activity in aquatic microbial communities associated with decaying typha litter.

    Science.gov (United States)

    Francoeur, Steven N; Schaecher, Mark; Neely, Robert K; Kuehn, Kevin A

    2006-11-01

    We examined the effect of light on extracellular enzyme activities of periphytic/endogenous microbial assemblages associated with decomposing litter of an emergent macrophyte Typha angustifolia within a small inland wetland in southeastern Michigan. Standing-dead Typha leaf litter was collected, placed into floating wire mesh litter baskets, and submerged in a wetland pool. Enzyme saturation assays were conducted on three occasions following litter submergence (days 9, 28, and 44) to generate saturation curves for the individual enzymes tested and to examine potential differences in enzyme saturation kinetics during microbial colonization and development. Experimental light manipulations were conducted on two occasions during microbial development (days 10 and 29). Short-term (30 min) light exposure significantly increased extracellular beta-glucosidase activity of litter-associated microbial communities. Activities of beta-xylosidase and leucine-aminopeptidase were not stimulated, and stimulation of phosphatase activity was variable. The exact mechanism for increased enzyme activity remains unknown, but it may have been increased pH arising from periphytic algal photosynthesis. These results suggest that extracellular enzyme activity in microbial communities colonizing natural organic substrata may be influenced by light/photosynthesis, as has previously been demonstrated for periphyton communities grown on artificial, inert substrata. Thus, light/photosynthetic mediated stimulation of extracellular enzyme activities may be a common occurrence in microbial communities associated with natural decaying plant litter in wetlands and might engender diurnal patterns in other microbial decay processes (e.g., production, organic matter decomposition, and mineralization). PMID:17082997

  18. Detection of Extracellular Enzyme Activity in Penicillium using Chromogenic Media

    OpenAIRE

    Yoon, Ji Hwan; Hong, Seung Beom; Ko, Seung Ju; Kim, Seong Hwan

    2007-01-01

    A total of 106 Penicillium species were tested to examine their ability of degrading cellobiose, pectin and xylan. The activity of β-glucosidase was generally strong in all the Penicillium species tested. P. citrinum, P. charlesii, P. manginii and P. aurantiacum showed the higher ability of producing β-glucosidase than other tested species. Pectinase activity was detected in 24 Penicillium species. P. paracanescens, P. sizovae, P. sartoryi, P. chrysogenum, and P. claviforme showed strong pect...

  19. Extracellular Ca2+ is a danger signal activating the NLRP3 inflammasome through G protein-coupled calcium sensing receptors

    DEFF Research Database (Denmark)

    Rossol, Manuela; Pierer, Matthias; Raulien, Nora;

    2012-01-01

    Activation of the NLRP3 inflammasome enables monocytes and macrophages to release high levels of interleukin-1ß during inflammatory responses. Concentrations of extracellular calcium can increase at sites of infection, inflammation or cell activation. Here we show that increased extracellular cal......, and this effect was inhibited in GPRC6A(-/-) mice. Our results demonstrate that G-protein-coupled receptors can activate the inflammasome, and indicate that increased extracellular calcium has a role as a danger signal and amplifier of inflammation....

  20. Microtubules regulate GEF-H1 in response to extracellular matrix stiffness

    Science.gov (United States)

    Heck, Jessica N.; Ponik, Suzanne M.; Garcia-Mendoza, Maria G.; Pehlke, Carolyn A.; Inman, David R.; Eliceiri, Kevin W.; Keely, Patricia J.

    2012-01-01

    Breast epithelial cells sense the stiffness of the extracellular matrix through Rho-mediated contractility. In turn, matrix stiffness regulates RhoA activity. However, the upstream signaling mechanisms are poorly defined. Here we demonstrate that the Rho exchange factor GEF-H1 mediates RhoA activation in response to extracellular matrix stiffness. We demonstrate the novel finding that microtubule stability is diminished by a stiff three-dimensional (3D) extracellular matrix, which leads to the activation of GEF-H1. Surprisingly, activation of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway did not contribute to stiffness-induced GEF-H1 activation. Loss of GEF-H1 decreases cell contraction of and invasion through 3D matrices. These data support a model in which matrix stiffness regulates RhoA through microtubule destabilization and the subsequent release and activation of GEF-H1. PMID:22593214

  1. Diverse impact of acute and long-term extracellular proteolytic activity on plasticity of neuronal excitability

    Directory of Open Access Journals (Sweden)

    Tomasz eWójtowicz

    2015-08-01

    Full Text Available Learning and memory require alteration in number and strength of existing synaptic connections. Extracellular proteolysis within the synapses has been shown to play a pivotal role in synaptic plasticity by determining synapse structure, function, and number. Although synaptic plasticity of excitatory synapses is generally acknowledged to play a crucial role in formation of memory traces, some components of neural plasticity are reflected by nonsynaptic changes. Since information in neural networks is ultimately conveyed with action potentials, scaling of neuronal excitability could significantly enhance or dampen the outcome of dendritic integration, boost neuronal information storage capacity and ultimately learning. However, the underlying mechanism is poorly understood. With this regard, several lines of evidence and our most recent study support a view that activity of extracellular proteases might affect information processing in neuronal networks by affecting targets beyond synapses. Here we review the most recent studies addressing the impact of extracellular proteolysis on plasticity of neuronal excitability and discuss how enzymatic activity may alter input-output/transfer function of neurons, supporting cognitive processes. Interestingly, extracellular proteolysis may alter intrinsic neuronal excitability and excitation/inhibition balance both rapidly (time of minutes to hours and in long-term window. Moreover, it appears that by cleavage of extracellular matrix constituents, proteases may modulate function of ion channels or alter inhibitory drive and hence facilitate active participation of dendrites and axon initial segments in adjusting neuronal input/output function. Altogether, a picture emerges whereby both rapid and long-term extracellular proteolysis may influence some aspects of information processing in neurons, such as initiation of action potential, spike frequency adaptation, properties of action potential and dendritic

  2. Microbial respiration and extracellular enzyme activity in sediments from the Gulf of Mexico hypoxic zone

    Science.gov (United States)

    This study explores the relationship between sediment chemistry (TC, TN, TP) and microbial respiration (DHA) and extracellular enzyme activity (EEA) across the Gulf of Mexico (GOM) hypoxic zone. TC, TN, and TP were all positively correlated with each other (r=0.19-0.68). DHA was ...

  3. Na+ -K+ pump activity in rat peritoneal mast cells: inhibition by extracellular calcium

    DEFF Research Database (Denmark)

    Knudsen, Torben; Johansen, Torben

    1989-01-01

    of an enzyme, and it is mediated by the Na+ -K+ pump located in the plasma membrane. It is demonstrated that the activity of the Na+ -K+ pump mechanism is inhibited by low concentrations of extracellular calcium (0.1-1.2 mmol l-1). The possibility is discussed that calcium-deprivation may increase the pump...

  4. Strategy for improving extracellular lipolytic activities by a novel thermotolerant Staphylococcus sp. strain

    Directory of Open Access Journals (Sweden)

    Cherif Slim

    2011-11-01

    Full Text Available Abstract Background Extracellular bacterial lipases received much attention for their substrate specificity and their ability to function under extreme environments (pH, temperature.... Many staphylococci produced lipases which were released into the culture medium. Reports of extracellular thermostable lipases from Staphylococcus sp. and active in alkaline conditions are not previously described. Results This study focused on novel strategies to increase extracellular lipolytic enzyme production by a novel Staphylococcus sp. strain ESW. The microorganism needed neutral or alkaline pH values between 7.0 and 12.0 for growth. For pH values outside this range, cell growth seemed to be significantly inhibited. Staphylococcus sp. culture was able to grow within a wide temperature range (from 30 to 55°C. The presence of oils in the culture medium leaded to improvements in cells growth and lipolytic enzyme activity. On the other hand, although chemical surfactants leaded to an almost complete inhibition of growth and lipolytic enzyme production, their addition along the culture could affect the location of the enzyme. In addition, our results showed that this novel Staphylococcus sp. strain produced biosurfactants simultaneously with lipolytic activity, when soapstock (The main co-product of the vegetable oil refining industry, was used as the sole carbon source. Conclusion A simultaneous biosurfactant and extracellular lipolytic enzymes produced bacterial strain with potential application in soap stock treatment

  5. 鞘内注射γ-氨基丁酸转运体抑制剂NO-711在骨癌痛大鼠脊髓水平印制磷酸化细胞外信号周节激酶1/2的上调%Intrathecal administration of γ-aminobutyric acid transporter-1 inhibitor NO-711 inhibits the up-regulation of extracellular signal-regulated kinase in lumbar spinal cord of bone cancer pain in rats

    Institute of Scientific and Technical Information of China (English)

    朱珊珊; 谭珊珊; 曾因明

    2011-01-01

    Objective To explore the role of extracellular signal-regulated kirase(ERK)activation in the lumbar spinal cord induced by the tibial cancer pain in rats.Methods Part Ⅰ:Forty-eight SD female rats(160 g-200 g)were randomly divided into two groups(n=24).Group A(control group):intra-tibial injection of 5 p.l saline.Group B(model group):intra-tibial injection of 5 μl Walker-256 mammary gland carcinoma cells of rats(1×105/μl).Mechanical withdrawl threshold(MWT)of mechanical stimulus and ambulatory-evoked pain scores were recorded one day before operation and on 1,3,5,7,10,14,21 d after operation respectively.Expressionof phosphorylated ERK1/2(p-ERK1/2)were detected on 7,14,21 d after operation respectively.Part Ⅱ:Sixty female Sprague-Dawley rats(160 g-200 g)were randomly divided into five groups(n=12):sham +NS group(group S);bone cancer pain+NS(group N1);bone cancer pain +NO-711 10 μg(group N2);bone cancer pain+ NO-711 20 μg(group N3);bone cancer pain+ NO-711 40 μg(group N4).On the 14th day after intra-tibial injection of 5 μ.l Walker-256 mammary gland carcinoma cells(post-treatment)NO-711 or normal saline were intrathecally administered thrice(once a day).MWT of mechanical stimulus and ambulatory-evoked pain scores were recorded at the time of 30 min,1,2,4,8,12,24 h after last injection.Test the p-ERK1/2 expression in the spinal cord by western blot analysis at 4 hours after last injection.Results Part Ⅰ:From the 7th day after operation,there were great significance differences at MWT(8.02±0.30)、ambulatory-evoked pain scores(0.88±0.22)and the expression of p-ERK1/2 compared with pre-operation in group B.MWT were significantly decreased while ambulatory-evoked pain scores and the expression of p-ERK1/2 were significanty increased in group B as compared to group A from the 7th day postoperation.Part Ⅱ:After and before i.t.administration in group N 1 the MWT and scores of ambulatory-evoked pain showed no statistical significance.Received with

  6. Extracellular enzymatic activities and physiological profiles of yeasts colonizing fruit trees.

    Science.gov (United States)

    Molnárová, Jana; Vadkertiová, Renáta; Stratilová, Eva

    2014-07-01

    Yeasts form a significant and diverse part of the phyllosphere microbiota. Some yeasts that inhabit plants have been found to exhibit extracellular enzymatic activities. The aim of the present study was to investigate the ability of yeasts isolated from leaves, fruits, and blossoms of fruit trees cultivated in Southwest Slovakia to produce extracellular enzymes, and to discover whether the yeasts originating from these plant organs differ from each other in their physiological properties. In total, 92 strains belonging to 29 different species were tested for: extracellular protease, β-glucosidase, lipase, and polygalacturonase activities; fermentation abilities; the assimilation of xylose, saccharose and alcohols (methanol, ethanol, glycerol); and for growth in a medium with 33% glucose. The black yeast Aureobasidium pullulans showed the largest spectrum of activities of all the species tested. Almost 70% of the strains tested demonstrated some enzymatic activity, and more than 90% utilized one of the carbon compounds tested. Intraspecies variations were found for the species of the genera Cryptococcus and Pseudozyma. Interspecies differences of strains exhibiting some enzymatic activities and utilizing alcohols were also noted. The largest proportion of the yeasts exhibited β-glucosidase activity and assimilated alcohols independently of their origin. The highest number of strains positive for all activities tested was found among the yeasts associated with leaves. Yeasts isolated from blossoms assimilated saccharose and D-xylose the most frequently of all the yeasts tested. The majority of the fruit-inhabiting yeasts grew in the medium with higher osmotic pressure.

  7. Extracellular-regulated kinase 2 is activated by the enhancement of hinge flexibility.

    OpenAIRE

    Sours, Kevin M.; Xiao,Yao; Ahn, Natalie G.

    2014-01-01

    Protein motions underlie conformational and entropic contributions to enzyme catalysis; however, relatively little is known about the ways in which this occurs. Studies of the mitogen-activated protein kinase ERK2 (extracellular-regulated protein kinase 2) by hydrogen-exchange mass spectrometry suggest that activation enhances backbone flexibility at the linker between N- and C-terminal domains while altering nucleotide binding mode. Here, we address the hypothesis that enhanced backbone flex...

  8. Bacterial community composition and extracellular enzyme activity in temperate streambed sediment during drying and rewetting.

    Directory of Open Access Journals (Sweden)

    Elisabeth Pohlon

    Full Text Available Droughts are among the most important disturbance events for stream ecosystems; they not only affect stream hydrology but also the stream biota. Although desiccation of streams is common in Mediterranean regions, phases of dryness in headwaters have been observed more often and for longer periods in extended temperate regions, including Central Europe, reflecting global climate change and enhanced water withdrawal. The effects of desiccation and rewetting on the bacterial community composition and extracellular enzyme activity, a key process in the carbon flow of streams and rivers, were investigated in a typical Central European stream, the Breitenbach (Hesse, Germany. Wet streambed sediment is an important habitat in streams. It was sampled and exposed in the laboratory to different drying scenarios (fast, intermediate, slow for 13 weeks, followed by rewetting of the sediment from the fast drying scenario via a sediment core perfusion technique for 2 weeks. Bacterial community structure was analyzed using CARD-FISH and TGGE, and extracellular enzyme activity was assessed using fluorogenic model substrates. During desiccation the bacterial community composition shifted toward composition in soil, exhibiting increasing proportions of Actinobacteria and Alphaproteobacteria and decreasing proportions of Bacteroidetes and Betaproteobacteria. Simultaneously the activities of extracellular enzymes decreased, most pronounced with aminopeptidases and less pronounced with enzymes involved in the degradation of polymeric carbohydrates. After rewetting, the general ecosystem functioning, with respect to extracellular enzyme activity, recovered after 10 to 14 days. However, the bacterial community composition had not yet achieved its original composition as in unaffected sediments within this time. Thus, whether the bacterial community eventually recovers completely after these events remains unknown. Perhaps this community undergoes permanent changes

  9. Spectral representation: analyzing single-unit activity in extracellularly recorded neuronal data without spike sorting

    OpenAIRE

    Luczak, Artur; Narayanan, Nandakumar S.

    2005-01-01

    One step in the conventional analysis of extracellularly recorded neuronal data is spike sorting, which separates electrical signal into action potentials from different neurons. Because spike sorting involves human judgment, it can be subjective and time intensive, particularly for large sets of neurons. Here we propose a simple, automated way to construct alternative representations of neuronal activity, called spectral representation (SR). In this approach, neuronal spikes are mapped to a ...

  10. Evaluation of viability and proliferative activity of human urothelial cells cultured onto xenogenic tissue-engineered extracellular matrices.

    LENUS (Irish Health Repository)

    Davis, Niall F

    2011-04-01

    To evaluate the viability and proliferative activity of human urothelial cells (HUCs) cultured on tissue-engineered extracellular matrix scaffolds and to assess the potential of extracellular matrixes to support the growth of HUCs in their expected in vivo urine environment.

  11. Intra- and extracellular activities of dicloxacillin against Staphylococcus aureus in vivo and in vitro.

    Science.gov (United States)

    Sandberg, Anne; Jensen, Klaus Skovbo; Baudoux, Pierre; Van Bambeke, Françoise; Tulkens, Paul M; Frimodt-Møller, Niels

    2010-06-01

    Antibiotic treatment of Staphylococcus aureus infections is often problematic due to the slow response and recurrences. The intracellular persistence of the staphylococci offers a plausible explanation for the treatment difficulties because of the impaired intracellular efficacies of the antibiotics. The intra- and extracellular time- and concentration-kill relationships were examined in vitro with THP-1 cells and in vivo by use of a mouse peritonitis model. The in vivo model was further used to estimate the most predictive pharmacokinetic/pharmacodynamic (PK/PD) indices (the ratio of the maximum concentration of drug in plasma/MIC, the ratio of the area under the concentration-time curve/MIC, or the cumulative percentage of a 24-h period that the free [f] drug concentration exceeded the MIC under steady-state pharmacokinetic conditions [fT(MIC)]) for dicloxacillin (DCX) intra- and extracellularly. In general, DCX was found to have similar intracellular activities, regardless of the model used. Both models showed (i) the relative maximal efficacy (1-log-unit reduction in the numbers of CFU) of DCX intracellularly and (ii) the equal relative potency of DCX intra- and extracellularly, with the MIC being a good indicator of the overall response in both situations. Discordant results, based on data obtained different times after dosing, were obtained from the two models when the extracellular activity of DCX was measured, in which the in vitro model showed a considerable reduction in the number of CFU from that in the original inoculum (3-log-unit decrease in the number of CFU after 24 h), whereas the extracellular CFU reduction achieved in vivo after 4 h did not exceed 1 log unit. Multiple dosing of DCX in vivo revealed increased extra- and intracellular efficacies (2.5 log and 2 log units of reduction in the numbers of CFU after 24 h, respectively), confirming that DCX is a highly active antistaphylococcal antibiotic. PK/PD analysis revealed that fT(MIC) is the index

  12. NOTCH SIGNALING REGULATES MOUSE AND HUMAN TH17 DIFFERENTIATION

    OpenAIRE

    Keerthivasan, Shilpa; Suleiman, Reem; Lawlor, Rebecca; Roderick, Justine; Bates, Tonya; Minter, Lisa; Anguita, Juan; Juncadella, Ignacio; Nickoloff, Brian J; Le Poole, I. Caroline; Miele, Lucio; Osborne, Barbara A.

    2011-01-01

    T helper17 (Th17) cells are known to play a critical role in adaptive immune responses to several important extracellular pathogens. Additionally, Th17 cells are implicated in the pathogenesis of several autoimmune and inflammatory disorders as well as in cancer. Therefore, it is essential to understand the mechanisms that regulate Th17 differentiation. Notch signaling is known to be important at several stages of T cell development and differentiation. Here we report that Notch1 is activated...

  13. Cytokinin signaling regulates pavement cell morphogenesis in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Hongjiang Li; Tongda Xu; Deshu Lin; Mingzhang Wen; Mingtang Xie; Jér(o)me Duclercq; Agnieszka Bielach

    2013-01-01

    The puzzle piece-shaped Arabidopsis leaf pavement cells (PCs) with interdigitated lobes and indents is a good model system to investigate the mechanisms that coordinate cell polarity and shape formation within a tissue.Auxin has been shown to coordinate the interdigitation by activating ROP GTPase-dependent signaling pathways.To identify additional components or mechanisms,we screened for mutants with abnormal PC morphogenesis and found that cytokinin signaling regulates the PC interdigitation pattern.Reduction in cytokinin accumulation and defects in cytokinin signaling (such as in ARR7-over-expressing lines,the ahk3cre1 cytokinin receptor mutant,and the ahp12345 cytokinin signaling mutant) enhanced PC interdigitation,whereas over-production of cytokinin and over-activation of cytokinin signaling in an ARR20 over-expression line delayed or abolished PC interdigitation throughout the cotyledon.Genetic and biochemical analyses suggest that cytokinin signaling acts upstream of ROPs to suppress the formation of interdigitated pattern.Our results provide novel mechanistic understanding of the pathways controlling PC shape and uncover a new role for cytokinin signaling in cell morphogenesis.

  14. Effects of Recurring Droughts on Extracellular Enzyme Activity in Mountain Grassland

    Science.gov (United States)

    Fuchslueger, L.; Bahn, M.; Kienzl, S.; Hofhansl, F.; Schnecker, J.; Richter, A.

    2015-12-01

    Water availability is a key factor for biogeochemical processes and determines microbial activity and functioning, and thereby organic matter decomposition in soils by affecting the osmotic potential, soil pore connectivity, substrate diffusion and nutrient availability. Low water availability during drought periods therefore directly affects microbial activity. Recurring drought periods likely induce shifts in microbial structure that might be reflected in altered responses of microbial turnover of organic matter by extracellular enzymes. To study this we measured a set of potential extracellular enzyme activity rates (cellobiohydrolase CBH; leucine-amino-peptidase LAP; phosphatase PHOS; phenoloxidase POX), in grassland soils that were exposed to extreme experimental droughts during the growing seasons of up to five subsequent years. During the first drought period after eight weeks of rain exclusion all measured potential enzyme activities were significantly decreased. In parallel, soil extractable organic carbon and nitrogen concentrations increased and microbial community structure, determined by phospholipid fatty acid analysis, changed. In soils that were exposed to two and three drought periods only PHOS decreased. After four years of drought again CBH, PHOS and POX decreased, while LAP was unaffected; after five years of drought PHOS and POX decreased and CBH and LAP remained stable. Thus, our results suggest that recurring extreme drought events can cause different responses of extracellular enzyme activities and that the responses change over time. We will discuss whether and to what degree these changes were related to shifts in microbial community composition. However, independent of whether a solitary or a recurrent drought was imposed, in cases when enzyme activity rates were altered during drought, they quickly recovered after rewetting. Overall, our data suggest that microbial functioning in mountain grassland is sensitive to drought, but highly

  15. NaCl-activated extracellular proteinase from Virgibacillus sp. SK37 isolated from fish sauce fermentation.

    Science.gov (United States)

    Sinsuwan, S; Rodtong, S; Yongsawatdigul, J

    2007-06-01

    Virgibacillus sp. SK37 exhibited high extracellular proteolytic activity in skim milk broth containing 10% NaCl. Optimum conditions of the crude proteinase were at pH 8.0 and 65 degrees C. The proteinase was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) and preferably hydrolyzed Suc-Ala-Ala-Pro-Phe-AMC, suggesting the serine proteinase with a subtilisin-like characteristic. Proteolytic activity increased with NaCl concentration up to 20%. Ca(2+) activated the enzyme activity but reduced enzyme stability at 65 degrees C. Several proteinases with dominant molecular mass (MW) of 81, 67, 63, 50, 38, and 18 kDa were detected on native-polyacrylamide gel electrophoresis (native-PAGE) activity staining in the absence and presence of 25% NaCl. These results demonstrated that Virgibacillus sp. SK37 produced salt-activated extracellular proteinases. Virgibacillus sp. SK37 could be a promising strain for starter culture development used in fish sauce fermentation. PMID:17995713

  16. The progesterone-induced enhancement of object recognition memory consolidation involves activation of the extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin (mTOR) pathways in the dorsal hippocampus

    OpenAIRE

    Orr, Patrick T.; Rubin, Amanda J.; Fan, Lu; Kent, Brianne A.; Frick, Karyn M.

    2012-01-01

    Although much recent work has elucidated the biochemical mechanisms underlying the modulation of memory by 17β-estradiol, little is known about the signaling events through which progesterone (P) regulates memory. We recently demonstrated that immediate post-training infusion of P into the dorsal hippocampus enhances object recognition memory consolidation in young ovariectomized female mice (Orr et al., 2009). The goal of the present study was to identify the biochemical alterations that mig...

  17. Aurora B is regulated by the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway and is a valuable potential target in melanoma cells.

    Science.gov (United States)

    Bonet, Caroline; Giuliano, Sandy; Ohanna, Mickaël; Bille, Karine; Allegra, Maryline; Lacour, Jean-Philippe; Bahadoran, Philippe; Rocchi, Stéphane; Ballotti, Robert; Bertolotto, Corine

    2012-08-24

    Metastatic melanoma is a deadly skin cancer and is resistant to almost all existing treatment. Vemurafenib, which targets the BRAFV600E mutation, is one of the drugs that improves patient outcome, but the patients next develop secondary resistance and a return to cancer. Thus, new therapeutic strategies are needed to treat melanomas and to increase the duration of v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitor response. The ERK pathway controls cell proliferation, and Aurora B plays a pivotal role in cell division. Here, we confirm that Aurora B is highly expressed in metastatic melanoma cells and that Aurora B inhibition triggers both senescence-like phenotypes and cell death in melanoma cells. Furthermore, we show that the BRAF/ERK axis controls Aurora B expression at the transcriptional level, likely through the transcription factor FOXM1. Our results provide insight into the mechanism of Aurora B regulation and the first molecular basis of Aurora B regulation in melanoma cells. The inhibition of Aurora B expression that we observed in vemurafenib-sensitive melanoma cells was rescued in cells resistant to this drug. Consistently, these latter cells remain sensitive to the effect of the Aurora B inhibitor. Noteworthy, wild-type BRAF melanoma cells are also sensitive to Aurora B inhibition. Collectively, our findings, showing that Aurora B is a potential target in melanoma cells, particularly in those vemurafenib-resistant, may open new avenues to improve the treatment of metastatic melanoma.

  18. Response to epidermal growth factor receptor inhibitors in non-small cell lung cancer cells : Limited antiproliferative effects and absence of apoptosis associated with persistent activity of extracellular signal-regulated kinase or Akt kinase pathways

    NARCIS (Netherlands)

    Janmaat, ML; Kruyt, FAE; Rodriguez, JA; Giaccone, G

    2003-01-01

    The epidermal growth factor receptor (EGFR) is an important novel target for anticancer therapy. In this study, we examined the molecular mechanisms that underlie the antitumor effects of the anti-EGFR monoclonal antibody C225 (Cetuximab) and the selective EGFR tyrosine kinase inhibitor ZD1839 (Ires

  19. Intracellular Transactivation of Epidermal Growth Factor Receptor by alpha(1A)-Adrenoceptor Is Mediated by Phosphatidylinositol 3-Kinase Independently of Activation of Extracellular Signal Regulated Kinases 1/2 and Serine-Threonine Kinases in Chinese Hamster Ovary Cells

    NARCIS (Netherlands)

    Ulu, Nadir; Henning, Robert H.; Guner, Sahika; Zoto, Teuta; Duman-Dalkilic, Basak; Duin, Marry; Gurdal, Hakan

    2013-01-01

    Transactivation of epidermal growth factor receptor (EGFR) by alpha(1)-adrenoceptor (alpha(1)-AR) is implicated in contraction and hypertrophy of vascular smooth muscle (VSM). We examine whether all alpha(1)-AR subtypes transactivate EGFR and explore the mechanism of transactivation. Chinese hamster

  20. A preliminary study on estimating extra-cellular nitrate reductase activities in estuarine systems

    Directory of Open Access Journals (Sweden)

    Pant H. K.

    2009-07-01

    Full Text Available Enzymes catalyzing ammonium (NH4+/nitrate (NO3– into nitrous oxide (N2O/molecular nitrogen (N2, play critical roles in water quality management. The objective of this paper was to investigate the role of extra-cellular enzymes in cycling of nitrogen (N in aquatic systems. It appears that N in estuaries, salt marshes, etc., does not stay long enough to be available for uptake, thus, creating N limited conditions. This study showed that indigenous extra-cellular nitrate reductase along with others involved in N transformations in the waters/sediments of estuarine systems can cause complete removal of NH4+ and NO3– from the waters and available NH4+ and NO3– from the sediments. These results indicate that due to high extra-cellular nitrate reductase and other enzymes associated with N transformations in sediments/waters, substantial amounts of NH4+ and NO3– can be quickly lost from the systems as N2O and/or nitric oxide (NO, in turn, creating N limited conditions in estuarine systems. Such high activities of indigenous nitrate reductase and others are useful in removing readily bioavailable N from the systems, thereby avoidance of eutrophic conditions. However, they might contribute in increasing the N2O, a potent greenhouse gas with global warming potential (GWP of 296, in the atmosphere.

  1. The modulation of platelet adhesion and activation by chitosan through plasma and extracellular matrix proteins.

    Science.gov (United States)

    Lord, Megan S; Cheng, Bill; McCarthy, Simon J; Jung, MoonSun; Whitelock, John M

    2011-10-01

    Chitosan has been shown to promote initial wound closure events to prevent blood loss. Platelet adhesion and activation are crucial early events in these processes after traumatic bleeding leading to thrombus formation. Platelet adhesion to chitosan was found to be enhanced in the presence of adsorbed plasma and extracellular matrix proteins and was found to be primarily mediated by α(IIb)β(3) integrins, while α(2)β(1) integrins were found to be involved in platelet adhesion to collagen and perlecan. Platelets were found to be activated by chitosan, as shown by an increase in the expression of α(IIb)β(3) integrins and P-selectin, while the extent of activation was modulated by the presence of proteins including perlecan and fibrinogen. Collagen-coated chitosan was found to activate platelets to the same extent as either chitosan or collagen alone. These data support the role of plasma and extracellular matrix proteins in promoting chitosan mediated platelet adhesion and activation supporting the hypothesis that chitosan promotes wound healing via these interactions.

  2. Interspecific nematode signals regulate dispersal behavior.

    Directory of Open Access Journals (Sweden)

    Fatma Kaplan

    Full Text Available BACKGROUND: Dispersal is an important nematode behavior. Upon crowding or food depletion, the free living bacteriovorus nematode Caenorhabditis elegans produces stress resistant dispersal larvae, called dauer, which are analogous to second stage juveniles (J2 of plant parasitic Meloidogyne spp. and infective juveniles (IJs of entomopathogenic nematodes (EPN, e.g., Steinernema feltiae. Regulation of dispersal behavior has not been thoroughly investigated for C. elegans or any other nematode species. Based on the fact that ascarosides regulate entry in dauer stage as well as multiple behaviors in C. elegans adults including mating, avoidance and aggregation, we hypothesized that ascarosides might also be involved in regulation of dispersal behavior in C. elegans and for other nematodes such as IJ of phylogenetically related EPNs. METHODOLOGY/PRINCIPAL FINDINGS: Liquid chromatography-mass spectrometry analysis of C. elegans dauer conditioned media, which shows strong dispersing activity, revealed four known ascarosides (ascr#2, ascr#3, ascr#8, icas#9. A synthetic blend of these ascarosides at physiologically relevant concentrations dispersed C. elegans dauer in the presence of food and also caused dispersion of IJs of S. feltiae and J2s of plant parasitic Meloidogyne spp. Assay guided fractionation revealed structural analogs as major active components of the S. feltiae (ascr#9 and C. elegans (ascr#2 dispersal blends. Further analysis revealed ascr#9 in all Steinernema spp. and Heterorhabditis spp. infected insect host cadavers. CONCLUSIONS/SIGNIFICANCE: Ascaroside blends represent evolutionarily conserved, fundamentally important communication systems for nematodes from diverse habitats, and thus may provide sustainable means for control of parasitic nematodes.

  3. Thioredoxin is involved in endothelial cell extracellular transglutaminase 2 activation mediated by celiac disease patient IgA.

    Directory of Open Access Journals (Sweden)

    Cristina Antonella Nadalutti

    Full Text Available PURPOSE: To investigate the role of thioredoxin (TRX, a novel regulator of extracellular transglutaminase 2 (TG2, in celiac patients IgA (CD IgA mediated TG2 enzymatic activation. METHODS: TG2 enzymatic activity was evaluated in endothelial cells (HUVECs under different experimental conditions by ELISA and Western blotting. Extracellular TG2 expression was studied by ELISA and immunofluorescence. TRX was analysed by Western blotting and ELISA. Serum immunoglobulins class A from healthy subjects (H IgA were used as controls. Extracellular TG2 enzymatic activity was inhibited by R281. PX12, a TRX inhibitor, was also employed in the present study. RESULTS: We have found that in HUVECs CD IgA is able to induce the activation of extracellular TG2 in a dose-dependent manner. Particularly, we noted that the extracellular modulation of TG2 activity mediated by CD IgA occurred only under reducing conditions, also needed to maintain antibody binding. Furthermore, CD IgA-treated HUVECs were characterized by a slightly augmented TG2 surface expression which was independent from extracellular TG2 activation. We also observed that HUVECs cultured in the presence of CD IgA evinced decreased TRX surface expression, coupled with increased secretion of the protein into the culture medium. Intriguingly, inhibition of TRX after CD IgA treatment was able to overcome most of the CD IgA-mediated effects including the TG2 extracellular transamidase activity. CONCLUSIONS: Altogether our findings suggest that in endothelial cells CD IgA mediate the constitutive activation of extracellular TG2 by a mechanism involving the redox sensor protein TRX.

  4. Transforming Growth Factor-β-Activated Kinase 1 Is Required for Human FcγRIIIb-Induced Neutrophil Extracellular Trap Formation.

    Science.gov (United States)

    Alemán, Omar Rafael; Mora, Nancy; Cortes-Vieyra, Ricarda; Uribe-Querol, Eileen; Rosales, Carlos

    2016-01-01

    Neutrophils (PMNs) are the most abundant leukocytes in the blood. PMN migrates from the circulation to sites of infection where they are responsible for antimicrobial functions. PMN uses phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. Several stimuli, including bacteria, fungi, and parasites, and some pharmacological compounds, such as Phorbol 12-myristate 13-acetate (PMA), are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. Recently, it was reported that FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. Direct cross-linking of FcγRIIA or integrins did not promote NET formation. FcγRIIIb-induced NET formation presented different kinetics from PMA-induced NET formation, suggesting differences in signaling. Because FcγRIIIb also induces a strong activation of extracellular signal-regulated kinase (ERK) and nuclear factor Elk-1, and the transforming growth factor-β-activated kinase 1 (TAK1) has recently been implicated in ERK signaling, in the present report, we explored the role of TAK1 in the signaling pathway activated by FcγRIIIb leading to NET formation. FcγRIIIb was stimulated by specific monoclonal antibodies, and NET formation was evaluated in the presence or absence of pharmacological inhibitors. The antibiotic LL Z1640-2, a selective inhibitor of TAK1 prevented FcγRIIIb-induced, but not PMA-induced NET formation. Both PMA and FcγRIIIb cross-linking induced phosphorylation of ERK. But, LL Z1640-2 only inhibited the FcγRIIIb-mediated activation of ERK. Also, only FcγRIIIb, similarly to transforming growth factor-β-induced TAK1 phosphorylation. A MEK (ERK kinase)-specific inhibitor was able to prevent ERK phosphorylation induced by both PMA and FcγRIIIb. These data show for the first time that FcγRIIIb cross-linking activates TAK1, and that this kinase is required for triggering the MEK/ERK signaling pathway to NETosis

  5. The diversity, extracellular enzymatic activities and photoprotective compounds of yeasts isolated in Antarctica

    Directory of Open Access Journals (Sweden)

    Aline B. M Vaz

    2011-09-01

    Full Text Available The diversity of yeasts collected from different sites in Antarctica (Admiralty Bay, King George Island and Port Foster Bay and Deception Island and their ability to produce extracellular enzymes and mycosporines were studied. Samples were collected during the austral summer season, between November 2006 and January 2007, from the rhizosphere of Deschampsia antarctica, ornithogenic (penguin guano soil, soil, marine and lake sediments, marine water and freshwater from lakes. A total of 89 isolates belonging to the following genera were recovered: Bensingtonia, Candida, Cryptococcus, Debaryomyces, Dioszegia, Exophiala, Filobasidium, Issatchenkia (Pichia, Kodamaea, Leucosporidium, Leucosporidiella, Metschnikowia, Nadsonia, Pichia, Rhodotorula, and Sporidiobolus, and the yeast-like fungi Aureobasidium, Leuconeurospora and Microglossum. Cryptococcus victoriae was the most frequently identified species. Several species isolated in our study have been previously reported to be Antarctic psychophilic yeasts, including Cr. antarcticus, Cr. victoriae, Dioszegia hungarica and Leucosporidium scottii. The cosmopolitan yeast species A. pullulans, C. zeylanoides, D. hansenii, I. orientalis, K. ohmeri, P. guilliermondii, Rh. mucilaginosa, and S. salmonicolor were also isolated. Five possible new species were identified. Sixty percent of the yeasts had at least one detectable extracellular enzymatic activity. Cryptococcus antarcticus, D. aurantiaca, D. crocea, D. hungarica, Dioszegia sp., E. xenobiotica, Rh. glaciales, Rh. laryngis, Microglossum sp. 1 and Microglossum sp. 2 produced mycosporines. Of the yeast isolates, 41.7% produced pigments and/or mycosporines and could be considered adapted to survive in Antarctica. Most of the yeasts had extracellular enzymatic activities at 4ºC and 20ºC, indicating that they could be metabolically active in the sampled substrates.

  6. Phytophthora infestans has a plethora of phospholipase D enzymes including a subclass that has extracellular activity.

    Directory of Open Access Journals (Sweden)

    Harold J G Meijer

    Full Text Available In eukaryotes phospholipase D (PLD is involved in many cellular processes. Currently little is known about PLDs in oomycetes. Here we report that the oomycete plant pathogen Phytophthora infestans has a large repertoire of PLDs divided over six subfamilies: PXPH-PLD, PXTM-PLD, TM-PLD, PLD-likes, and type A and B sPLD-likes. Since the latter have signal peptides we developed a method using metabolically labelled phospholipids to monitor if P. infestans secretes PLD. In extracellular medium of ten P. infestans strains PLD activity was detected as demonstrated by the production of phosphatidic acid and the PLD specific marker phosphatidylalcohol.

  7. Functions and behaviors of activated sludge extracellular polymeric substances (EPS): a promising environmental interest

    Institute of Scientific and Technical Information of China (English)

    TIAN Yu; ZHENG Lei; SUN De-zhi

    2006-01-01

    Extracellular polymeric substances (EPS) are the predominant constituents of activated sludge and represent up to 80% of the mass of activated sludge. They play a crucial role in the flocculation, settling and dewatering of activated sludge. Furthermore,EPS also show great efficiency in binding heavy metals. So EPS are key factors influencing reduction in sludge volume and mass, as well as activity and utilization of sludge. EPS are of considerable environmental interest and hundreds of articles on EPS have been published abroad, while information on EPS in China is limited. In this paper, results of over 60 publications related to constituents and characteristics of EPS and their influences on flocculation, settling and dewatering of sludge are compiled and analyzed.Metal-binding ability of EPS is also discussed, together with a brief consideration of possible research interests in the future.

  8. Growth and extracellular phosphatase activity of arbuscular mycorrhizal hyphae as influenced by soil organic matter

    DEFF Research Database (Denmark)

    Joner, E.J.; Jakobsen, I.

    1995-01-01

    length density was twice as high in soil with added straw compared to the two other treatments. Mycorrhizal colonization resulted in lower activity of acid phosphatase in the HC for two out of three treatments. Alkaline phosphatase activity was only decreased by mycorrhiza in soil without organic matter...... additions. In soil with added clover alkaline phosphatase activity increased due to the presence of mycorrhizal hyphae. We suggest that mycorrhizas may influence the exudation of acid phosphatase by roots. Hyphae of G. invermaium did apparently not excrete extracellular phosphatases, but their presence may......Two experiments were set up to investigate the influence of soil organic matter on growth of arbuscular mycorrhizal (AM) hyphae and concurrent changes in soil inorganic P, organic P and phosphatase activity. A sandy loam soil was kept for 14 months under two regimes (outdoor where surplus...

  9. Effect of Emodin on the Cell Proliferation and Extracellular Signal - regulated Kinase in Hepatic Stellate Cell-T6%大黄素对肝星状细胞增殖及细胞外信号调节激酶的影响

    Institute of Scientific and Technical Information of China (English)

    喻剑华; 黄兆胜; 赵珍东

    2012-01-01

    Objectives: To explore the effect of emodin on the cell proliferation and extracellular signal — regulate kinase in hepatic stellate cell -T6. Methods:The immortalized rat hepatic stellate cell line HSC -T6 was commonly cultured after revival ,and was allocated into normal control group and treated with different concentrations emodin. The cell proliferation was assessed via MTT assay,and cell cycle was analyzed by flow cytometry,and the expression of ERK1 mRNA was measured by semi - quantitative RT - PCR, and the expression of ERK, by immunohistochemistry. Results: After intervention of different concentration emodin(the extreme concentrations are 20μmol/mL,40μmol/mL,60μmol/mL respectively) ,the inhibition rates of proliferation increased significantly(compared with the normal group,P <0. 05, P < 0.01) and had dose -dependent manner. In cell cycle progression,emodin can inhibit cell cycle and the cell at G0/G1 phase increase obviously but descend at S phase, and G2/M phase decrease when emodin concentration is at 40 — 60μmol/mL(compared with the normal group,P<0.05). After exposure to emodin,the expressions of ERK1 mRNA are obviously downregulated,and there is statistical significance compared with the normal group(P<0.05,P<0.01). Im-munohistiochemical results showed that the position cells ERK, positive expression reduced obviously and the strength decrease. Conclusion: Emodin can inhibit the proliferation of HSC -T6,depress cell cycle at G0/G1 phase and restrain the expressions of ERK, and ERK, mRNA in HSC - T6.%目的:观察大黄素对HSC - T6细胞增殖及细胞外信号激酶(ERK1)表达的影响.方法:将培养细胞分成正常组和大黄素不同浓度干预组(大黄素终浓度分别为20μmol/L、40μmol/L、60μmol/L),采用MTT法观察大黄素对HSC - T6增殖的影响;采用流式细胞仪观察各组细胞周期的变化;采用反转录聚合酶链式反应(RT -PCR)半定量法观察ERK1mRNA的表达;采用

  10. Extracellular matrix is a source of mitogenically active platelet-derived growth factor.

    Science.gov (United States)

    Field, S L; Khachigian, L M; Sleigh, M J; Yang, G; Vandermark, S E; Hogg, P J; Chesterman, C N

    1996-08-01

    Platelet-derived growth factor (PDGF) is a chemotactic and mitogenic agent for fibroblasts and smooth muscle cells and plays a key role in the development of atherosclerotic lesions. PDGF is produced by a number of normal and transformed cell types and occurs as homo- or heterodimers of A and B polypeptide chains. Using Chinese hamster ovary (CHO) cells transfected with various forms of PDGF, we have previously shown that PDGF A(s) (short splice version) is secreted, PDGF A(l) (long splice version) predominantly extracellular matrix-associated, and PDGF B divided between medium, cells, and matrix. In the present study we have demonstrated the mitogenic activity of matrix-localized PDGF in artificial and more physiologically relevant models by culturing Balb/c-3T3 cells (3T3), human foreskin fibroblasts (HFF), and rabbit aortic smooth muscle cells (SMC) on extracellular matrix (ECM) laid down by PDGF-expressing CHO cells and human umbilical vein endothelial cells (HUVEC). These cells responded to the local growth stimulus of PDGF-containing CHO ECM and HUVEC ECM. We showed that 3T3 cells required proteolytic activity to utilize matrix-localized PDGF, as aprotinin and epsilon-ACA inhibited growth and 3T3 cells were shown to possess plasminogen activator activity. HFF and SMC did not appear to require proteolytic activity (including metalloproteinase and serine protease activity) as a prerequisite for mitogenesis but were able to access immobilized PDGF by contact with the matrix. An understanding of the mechanisms whereby the utilization of stored PDGF is controlled in situations of excessive cellular proliferation will aid in the development of therapy for these conditions. PMID:8707868

  11. Conservative Mechanisms of Extracellular Trap Formation by Annelida Eisenia andrei: Serine Protease Activity Requirement.

    Science.gov (United States)

    Homa, Joanna; Ortmann, Weronika; Kolaczkowska, Elzbieta

    2016-01-01

    Formation of extracellular traps (ETs) capturing and immobilizing pathogens is now a well-established defense mechanism added to the repertoire of vertebrate phagocytes. These ETs are composed of extracellular DNA (extDNA), histones and antimicrobial proteins. Formation of mouse and human ETs depends on enzymes (i) facilitating decondensation of chromatin by citrullination of histones, and (ii) serine proteases degrading histones. In invertebrates, initial reports revealed existence of ETs composed of extDNA and histones, and here we document for the first time that also coelomocytes, immunocompetent cells of an earthworm Eisenia andrei, cast ETs which successfully trap bacteria in a reactive oxygen species (ROS)-dependent and -independent manner. Importantly, the formation of ETs was observed not only when coelomocytes were studied ex vivo, but also in vivo, directly in the earthworm coelom. These ETs were composed of extDNA, heat shock proteins (HSP27) and H3 histones. Furthermore, the formation of E. andrei ETs depended on activity of serine proteases, including elastase-like activity. Moreover, ETs interconnected and hold together aggregating coelomocytes, a processes proceeding encapsulation. In conclusion, the study confirms ET formation by earthworms, and unravels mechanisms leading to ET formation and encapsulation in invertebrates. PMID:27416067

  12. Cellular contractility and extracellular matrix stiffness regulate matrix metalloproteinase activity in pancreatic cancer cells.

    Science.gov (United States)

    Haage, Amanda; Schneider, Ian C

    2014-08-01

    The pathogenesis of cancer is often driven by local invasion and metastasis. Recently, mechanical properties of the tumor microenvironment have been identified as potent regulators of invasion and metastasis, while matrix metalloproteinases (MMPs) are classically known as significant enhancers of cancer cell migration and invasion. Here we have been able to sensitively measure MMP activity changes in response to specific extracellular matrix (ECM) environments and cell contractility states. Cells of a pancreatic cancer cell line, Panc-1, up-regulate MMP activities between 3- and 10-fold with increased cell contractility. Conversely, they down-regulate MMP activities when contractility is blocked to levels seen with pan-MMP activity inhibitors. Similar, albeit attenuated, responses are seen in other pancreatic cancer cell lines, BxPC-3 and AsPC-1. In addition, MMP activity was modulated by substrate stiffness, collagen gel concentration, and the degree of collagen cross-linking, when cells were plated on collagen gels ranging from 0.5 to 5 mg/ml that span the physiological range of substrate stiffness (50-2000 Pa). Panc-1 cells showed enhanced MMP activity on stiffer substrates, whereas BxPC-3 and AsPC-1 cells showed diminished MMP activity. In addition, eliminating heparan sulfate proteoglycans using heparinase completely abrogated the mechanical induction of MMP activity. These results demonstrate the first functional link between MMP activity, contractility, and ECM stiffness and provide an explanation as to why stiffer environments result in enhanced cell migration and invasion.

  13. Extracellular polysaccharides produced by Ganoderma formosanum stimulate macrophage activation via multiple pattern-recognition receptors

    Directory of Open Access Journals (Sweden)

    Wang Cheng-Li

    2012-08-01

    Full Text Available Abstract Background The fungus of Ganoderma is a traditional medicine in Asia with a variety of pharmacological functions including anti-cancer activities. We have purified an extracellular heteropolysaccharide fraction, PS-F2, from the submerged mycelia culture of G. formosanum and shown that PS-F2 exhibits immunostimulatory activities. In this study, we investigated the molecular mechanisms of immunostimulation by PS-F2. Results PS-F2-stimulated TNF-α production in macrophages was significantly reduced in the presence of blocking antibodies for Dectin-1 and complement receptor 3 (CR3, laminarin, or piceatannol (a spleen tyrosine kinase inhibitor, suggesting that PS-F2 recognition by macrophages is mediated by Dectin-1 and CR3 receptors. In addition, the stimulatory effect of PS-F2 was attenuated in the bone marrow-derived macrophages from C3H/HeJ mice which lack functional Toll-like receptor 4 (TLR4. PS-F2 stimulation triggered the phosphorylation of mitogen-activated protein kinases JNK, p38, and ERK, as well as the nuclear translocation of NF-κB, which all played essential roles in activating TNF-α expression. Conclusions Our results indicate that the extracellular polysaccharides produced by G. formosanum stimulate macrophages via the engagement of multiple pattern-recognition receptors including Dectin-1, CR3 and TLR4, resulting in the activation of Syk, JNK, p38, ERK, and NK-κB and the production of TNF-α.

  14. Interaction with Shc prevents aberrant Erk activation in the absence of extracellular stimuli

    KAUST Repository

    Suen, KinMan

    2013-05-01

    Control mechanisms that prevent aberrant signaling are necessary to maintain cellular homeostasis. We describe a new mechanism by which the adaptor protein Shc directly binds the MAP kinase Erk, thus preventing its activation in the absence of extracellular stimuli. The Shc-Erk complex restricts Erk nuclear translocation, restraining Erk-dependent transcription of genes, including those responsible for oncogenic growth. The complex forms through unique binding sites on both the Shc PTB domain and the N-terminal lobe of Erk. Upon receptor tyrosine kinase stimulation, a conformational change within Shc - induced through interaction with the phosphorylated receptor - releases Erk, allowing it to fulfill its role in signaling. Thus, in addition to its established role in promoting MAP kinase signaling in stimulated cells, Shc negatively regulates Erk activation in the absence of growth factors and thus could be considered a tumor suppressor in human cells. © 2013 Nature America, Inc. All rights reserved.

  15. Enhancement of sludge reduction and methane production by removing extracellular polymeric substances from waste activated sludge.

    Science.gov (United States)

    Nguyen, Minh Tuan; Mohd Yasin, Nazlina Haiza; Miyazaki, Toshiki; Maeda, Toshinari

    2014-12-01

    The management of waste activated sludge (WAS) recycling is a concern that affects the development of the future low-carbon society, particularly sludge reduction and biomass utilization. In this study, we investigated the effect of removing extracellular polymeric substances (EPS), which play important roles in the adhesion and flocculation of WAS, on increased sludge disintegration, thereby enhancing sludge reduction and methane production by anaerobic digestion. EPS removal from WAS by ethylenediaminetetraacetic acid (EDTA) significantly enhanced sludge reduction, i.e., 49 ± 5% compared with 27 ± 1% of the control at the end the digestion process. Methane production was also improved in WAS without EPS by 8881 ± 109 CH4 μmol g(-1) dry-weight of sludge. Microbial activity was determined by denaturing gradient gel electrophoresis and real-time polymerase chain reaction, which showed that the hydrolysis and acetogenesis stages were enhanced by pretreatment with 2% EDTA, with a larger methanogenic community and better methane production.

  16. Nematocidal activity of extracellular enzymes produced by the nematophagous fungus Duddingtonia flagrans on cyathostomin infective larvae.

    Science.gov (United States)

    Braga, Fabio Ribeiro; Soares, Filippe Elias Freitas; Giuberti, Thais Zanotti; Lopes, Aline Del Carmen Garcias; Lacerda, Tracy; Ayupe, Tiago de Hollanda; Queiroz, Paula Viana; Gouveia, Angélica de Souza; Pinheiro, Larissa; Araújo, Andreia Luíza; Queiroz, José Humberto; Araújo, Jackson Victor

    2015-09-15

    Duddingtonia flagrans produces chitinases, however, optimization of the production of these enzymes still needs to be explored, and its nematocidal activity should still be the subject of studies. The objective of the present study was to optimize chitinase production, and evaluate the nematocidal activity of extracellular enzymes produced by the nematophagous fungus D. flagrans on cyathostomin infective larvae. An isolate from D. flagrans (AC001) was used in this study. For the production of enzymes (protease and chitinase), two different culture media were inoculated with AC001 conidia. Both enzymes were purified. The statistical Plackett-Burman factorial design was used to investigate some variables and their effect on the production of chitinases by D. flagrans. After that, the design central composite (CCD) was used in order to determine the optimum levels and investigate the interactions of these variables previously observed. Only two variables (moisture and incubation time), in the evaluated levels, had a significant effect (pemployability for this chitinase. PMID:26319197

  17. cAMP/PKA/CREB信号通路及相关调控蛋白PDE-4和ERK对学习记忆的影响%Influence of Learning and Memory on the Expression of Relevant Controlling Protein PDE-4 and Extracellular Signal Regulating Kinase

    Institute of Scientific and Technical Information of China (English)

    杨夏

    2011-01-01

    In recent years,a lot of learning and memory tests which have been done with animals are all prompted that cAMP / PKA / CREB signaling pathway and the proteins are related to the process of learning and memory. PKA phosphorylates and activates cAMP response element hinding protein( CREB ). The latter is an important nucleoprotein , and it regulates the gene transcription of a promoter that has cAMP. It has heen confirmed that PDE-4 and ERK are cAMP /PKA/CREB signaling pathway regulatory protein. The cAMP/PKA/CREB signaling pathway and its regulated protein : PDE-4 and ERK. and the relationship among them, and explore its impact on learning and memory were reviewed in this article.%近年来在动物身上进行了大量的学习记忆实验,均提示cAMP/PKA/CREB信号通路中的各蛋白均与学习记忆过程有关.环磷酸腺苷(cAMP)激活蛋白激酶A磷酸化并激活cAMP反应单元结合蛋白(CREB),后者是一种重要的核蛋白,其调节启动子中具有cAMP反应单元(CRE)的基因转录,这种核转录因子具有调节包括学习记忆在内的广泛的生物学功能.已有研究证实,PDE4和ERK为cAMP/PKA/CREB信号通路的调节蛋白.现对cAMP/PKA/CREB信号通路中的各蛋白及其调控蛋白PDE-4和ERK进行研究,阐述着三者之间的关系,并探讨其对学习记忆的影响.

  18. Antimicrobial activity of extracellular metabolites from antagonistic bacteria isolated from potato (Solanum phureja crops

    Directory of Open Access Journals (Sweden)

    Sinar David Granada García

    2014-09-01

    Full Text Available Microorganisms for biological control are capable of producing active compounds that inhibit the development of phytopathogens, constituting a promising tool toob tain active principles that could replace synthetic pesticides. This study evaluatedtheability of severalpotentialbiocontrol microorganismsto produce active extracellular metabolites. In vitro antagonistic capability of 50 bacterial isolates from rhizospheric soils of "criolla" potato (Solanum phureja was tested through dual culture in this plant with different plant pathogenic fungi and bacteria. Isolates that showed significantly higher antagonistic activity were fermented in liquid media and crude extracts from the supernatants had their biological activities assessed by optical density techniques. Inhibitory effecton tested pathogens was observed for concentrations between 0.5% and 1% of crude extracts. There was a correlation between the antimicrobial activity of extracts and the use of nutrient-rich media in bacteria fermentation. Using a bioguided method, a peptidic compound, active against Fusarium oxysporum, was obtained from the 7ANT04 strain (Pyrobaculum sp.. Analysis by nuclear magnetic resonance and liquid chromatography coupled to mass detector evidenced an 11-amino acid compound. Bioinformatic software using raw mass data confirmed the presence of a cyclic peptide conformed by 11 mostly non-standard amino acids.

  19. Extracellular Microvesicles from Astrocytes Contain Functional Glutamate Transporters: Regulation by Protein Kinase C and Cell Activation

    Directory of Open Access Journals (Sweden)

    Romain-Daniel eGosselin

    2013-12-01

    Full Text Available Glutamate transport through astrocytic excitatory amino-acid transporters (EAAT-1 and EAAT-2 is paramount for neural homeostasis. EAAT-1 has been reported in secreted extracellular microvesicles (eMV, such as exosomes and because the Protein Kinase C (PKC family controls the sub-cellular distribution of EAATs, we have explored whether PKCs drive EAATs into eMV. Using rat primary astrocytes, confocal immunofluorescence and ultracentrifugation on sucrose gradient we here report that PKC activation by phorbol myristate acetate (PMA reorganizes EAAT-1 distribution and reduces functional [3H]-aspartate reuptake. Western-blots show that EAAT-1 is present in eMV from astrocyte conditioned medium, together with NaK ATPase and glutamine synthetase all being further increased after PMA treatment. However, nanoparticle tracking analysis reveals that PKC activation did not change particle concentration. Functional analysis indicates that eMV have the capacity to reuptake [3H]-aspartate. In vivo, we demonstrate that spinal astrocytic reaction induced by peripheral nerve lesion (spared nerve injury, SNI is associated with a phosphorylation of PKC δ together with a shift of EAAT distribution ipsilaterally. Ex vivo, spinal explants from SNI rats release eMV with an increased content of NaK ATPase, EAAT-1 and EAAT-2. These data indicate PKC and cell activation as important regulators of EAAT-1 incorporation in eMV, and raise the possibility that microvesicular EAAT-1 may exert extracellular functions. Beyond a putative role in neuropathic pain, this phenomenon may be important for understanding neural homeostasis and a wide range of neurological diseases associated with astrocytic reaction as well as non-neurological diseases linked to eMV release.

  20. Proteolysis of decellularized extracellular matrices results in loss of fibronectin and cell binding activity.

    Science.gov (United States)

    Ramanathan, Anand; Karuri, Nancy

    2015-04-01

    Excessive inflammation in the chronic wound bed is believed to result in increased fibronectin (FN) proteolysis and poor tissue repair. However, FN fragments can prime the immune response and result in higher protease levels. The reciprocity between FN proteolysis and inflammation makes it challenging to determine the specific contribution of FN proteolysis in the extracellular matrix (ECM) on tissue responses. We studied the impact of proteolysis of decellularized extracellular matrices (dECMs) obtained from NIH 3T3 mouse fibroblasts on FN level and activity. The dECMs were treated with α chymotrypsin and proteolysis was stopped at different time points. The protease solution was obtained, the remaining dECM was scrapped and examined by immunoblotting and Bicinchoninic Acid assays. Fibronectin was 9.4 ± 1.8% of the total protein content in the dECM but was more susceptible to proteolysis. After 15 min of protease treatment there was a 67.6% and 11.1% decrease in FN and total protein, respectively, in the dECMs. Fibronectin fragments were present both in the proteolysis solution and in the dECM. Cell adhesion, spreading and actin extensions on dECMs decreased with increasing proteolysis time. Interestingly, the solutions obtained after proteolysis of the dECMs supported cell adhesion and spreading in a time dependent manner, thus demonstrating the presence of FN cell binding activity in the protease solution of dECMs. This study demonstrates the susceptibility of FN in the ECM to proteolysis and the resulting loss of cell adhesion due to the decrease of FN activity and places weight on bioengineering strategies to stabilize FN against proteolysis.

  1. Purification and characterization of an extracellular protease from Penicillium chrysogenum Pg222 active against meat proteins.

    Science.gov (United States)

    Benito, María J; Rodríguez, Mar; Núñez, Félix; Asensio, Miguel A; Bermúdez, María E; Córdoba, Juan J

    2002-07-01

    An extracellular protease from Penicillium chrysogenum (Pg222) isolated from dry-cured ham has been purified. The purification procedure involved several steps: ammonium sulfate precipitation, ion-exchange chromatography, filtration, and separation by high-performance liquid chromatography. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and gel filtration, the purified fraction showed a molecular mass of about 35 kDa. The hydrolytic properties of the purified enzyme (EPg222) on extracted pork myofibrillar proteins under several conditions were evaluated by SDS-PAGE. EPg222 showed activity in the range of 10 to 60 degrees C in temperature, 0 to 3 M NaCl, and pH 5 to 7, with maximum activity at pH 6, 45 degrees C, and 0.25 M NaCl. Under these conditions the enzyme was most active against tropomyosin, actin, and myosin. EPg222 showed collagenolytic activity but did not hydrolyze myoglobin. EPg222 showed higher activity than other proteolytic enzymes like papain, trypsin, and Aspergillus oryzae protease. The N-terminal amino acid sequence was determined and was found to be Glu-Asn-Pro-Leu-Gln-Pro-Asn-Ala-Pro-Ser-Trp. This partial amino acid sequence revealed a 55% homology with serine proteases from Penicillium citrinum. The activity of this novel protease may be of interest in ripening and generating the flavor of dry-cured meat products. PMID:12089038

  2. Neutrophil extracellular traps that are not degraded in systemic lupus erythematosus activate complement exacerbating the disease.

    Science.gov (United States)

    Leffler, Jonatan; Martin, Myriam; Gullstrand, Birgitta; Tydén, Helena; Lood, Christian; Truedsson, Lennart; Bengtsson, Anders A; Blom, Anna M

    2012-04-01

    Ongoing inflammation including activation of the complement system is a hallmark of systemic lupus erythematosus (SLE). Antimicrobial neutrophil extracellular traps (NETs) are composed of secreted chromatin that may act as a source of autoantigens typical for SLE. In this study, we investigated how complement interacts with NETs and how NET degradation is affected by complement in SLE patients. We found that sera from a subset of patients with active SLE had a reduced ability to degrade in vitro-generated NETs, which was mostly restored when these patients were in remission. Patients that failed to degrade NETs had a more active disease and they also displayed lower levels of complement proteins C4 and C3 in blood. We discovered that NETs activated complement in vitro and that deposited C1q inhibited NET degradation including a direct inhibition of DNase-I by C1q. Complement deposition on NETs may facilitate autoantibody production, and indeed, Abs against NETs and NET epitopes were more pronounced in patients with impaired ability to degrade NETs. NET-bound autoantibodies inhibited degradation but also further increased C1q deposition, potentially exacerbating the disease. Thus, NETs are a potent complement activator, and this interaction may play an important role in SLE. Targeting complement with inhibitors or by removing complement activators such as NETs could be beneficial for patients with SLE.

  3. Extracellular-regulated kinase 2 is activated by the enhancement of hinge flexibility.

    Science.gov (United States)

    Sours, Kevin M; Xiao, Yao; Ahn, Natalie G

    2014-05-01

    Protein motions underlie conformational and entropic contributions to enzyme catalysis; however, relatively little is known about the ways in which this occurs. Studies of the mitogen-activated protein kinase ERK2 (extracellular-regulated protein kinase 2) by hydrogen-exchange mass spectrometry suggest that activation enhances backbone flexibility at the linker between N- and C-terminal domains while altering nucleotide binding mode. Here, we address the hypothesis that enhanced backbone flexibility within the hinge region facilitates kinase activation. We show that hinge mutations enhancing flexibility promote changes in the nucleotide binding mode consistent with domain movement, without requiring phosphorylation. They also lead to the activation of monophosphorylated ERK2, a form that is normally inactive. The hinge mutations bypass the need for pTyr but not pThr, suggesting that Tyr phosphorylation controls hinge motions. In agreement, monophosphorylation of pTyr enhances both hinge flexibility and nucleotide binding mode, measured by hydrogen-exchange mass spectrometry. Our findings demonstrate that regulated protein motions underlie kinase activation. Our working model is that constraints to domain movement in ERK2 are overcome by phosphorylation at pTyr, which increases hinge dynamics to promote the active conformation of the catalytic site.

  4. TGF-β-activated kinase 1 (TAK1 signaling regulates TGF-β-induced WNT-5A expression in airway smooth muscle cells via Sp1 and β-catenin.

    Directory of Open Access Journals (Sweden)

    Kuldeep Kumawat

    Full Text Available WNT-5A, a key player in embryonic development and post-natal homeostasis, has been associated with a myriad of pathological conditions including malignant, fibroproliferative and inflammatory disorders. Previously, we have identified WNT-5A as a transcriptional target of TGF-β in airway smooth muscle cells and demonstrated its function as a mediator of airway remodeling. Here, we investigated the molecular mechanisms underlying TGF-β-induced WNT-5A expression. We show that TGF-β-activated kinase 1 (TAK1 is a critical mediator of WNT-5A expression as its pharmacological inhibition or siRNA-mediated silencing reduced TGF-β induction of WNT-5A. Furthermore, we show that TAK1 engages p38 and c-Jun N-terminal kinase (JNK signaling which redundantly participates in WNT-5A induction as only simultaneous, but not individual, inhibition of p38 and JNK suppressed TGF-β-induced WNT-5A expression. Remarkably, we demonstrate a central role of β-catenin in TGF-β-induced WNT-5A expression. Regulated by TAK1, β-catenin is required for WNT-5A induction as its silencing repressed WNT-5A expression whereas a constitutively active mutant augmented basal WNT-5A abundance. Furthermore, we identify Sp1 as the transcription factor for WNT-5A and demonstrate its interaction with β-catenin. We discover that Sp1 is recruited to the WNT-5A promoter in a TGF-β-induced and TAK1-regulated manner. Collectively, our findings describe a TAK1-dependent, β-catenin- and Sp1-mediated signaling cascade activated downstream of TGF-β which regulates WNT-5A induction.

  5. The extracellular signal-regulated kinase (ERK) pathway: a potential therapeutic target in hypertension

    OpenAIRE

    Roberts, Richard

    2012-01-01

    Richard E RobertsSchool of Biomedical Sciences, University of Nottingham, Nottingham, United KingdomAbstract: Hypertension is a risk factor for myocardial infarction, stroke, renal failure, heart failure, and peripheral vascular disease. One feature of hypertension is a hyperresponsiveness to contractile agents, and inhibition of vasoconstriction forms the basis of some of the treatments for hypertension. Hypertension is also associated with an increase in the growth and proliferation of vasc...

  6. Notch信号调节外周T细胞的活化、增殖与分化%Notch signaling regulates activation, proliferation and differentiation of peripheral T cells

    Institute of Scientific and Technical Information of China (English)

    唐晓燕; 季晓辉

    2008-01-01

    The differentiation of naive T cells to effector/memory T cells is regulated by a variety of factors. The recent advance of the contribution of Notch signaling in this differentiation step has provided a new path for better understanding the acquisition or persistence of the effector function of mature T cells. A growing body of literature indicates that the Notch pathway can influence the development of T cells in central immune organs. It is now clear that Notch' s ability to regulate cell-fate choices extends into the peripheral immune system, where the activation of the Notch signaling pathway can profoundly alter cytokine production in both CD4+ and CD8+T cells. In this review, we summarized the emerging and, in some points, conflicting evi-dences for Notch signaling on mature T cell activation, proliferation and differentiation. Although the effect of Notch ligation on CD4 + T cell cytokine production varies significantly from one report to another, it is clear that the Notch pathway is an important regulator of T cell activity. Specifically, the available data demonstrated that APCs utilize the Notch pathway to instruct T cell differentiation programs.%初始T细胞分化为效应T和记忆T细胞受到多种因素调节.最近在Notch信号途径的研究进展显示它也参于T细胞的活化与分化.大量研究已经表明Notch信号途径可以影响T细胞在中枢免疫器官的发育,现在关于它调节外周T细胞的分化状态也积累不少证据,Notch信号活化之后能够改变CD4+和CD8+T细胞分泌细胞因子的特点.以下着重介绍Notch信号参于调节外周T细胞的活化、增殖和分化的最新资料,尽管不同的研究者所得实验结果有冲突之处,但已经提示Notch信号在T细胞外周发育中的重要意义,特别重要的是抗原递呈细胞(APC)可以通过Notch信号途径调节T细胞的分化.

  7. Molecular characterization of a signal-regulated kinase homolog from Echinococcus granulosus

    Institute of Scientific and Technical Information of China (English)

    LI Jing; ZHANG Chuan-shan; L(U) Guo-dong; WANG Jun-hua; WEN Hao; YAN Gen-qiang; WEI Xu-fa; LIN Ren-yong

    2011-01-01

    Background Cystic echinococcosis due to Echinococcus granulosus (E. granulosus) is one of the most important chronic helminthic diseases, especially in sheep/cattle-raising regions. The larval stage of the parasite forms a cyst that grows in the liver, lung, or other organs ofthe host. To ensure a long life in the host tissues, the parasite establishes complex inter-cellular communication systems between its host to allow its differentiation toward each larval stage.Recent studies have reported that this communication is associated with the extracellular signal-regulated kinase (ERK)mitogen-activated protein kinase cascade in helminth parasites, and in particular that these protein kinases might serve as effective targets for a novel chemotherapy for cystic echinococcosis. The aim of the present study investigated the biological function of a novel ERK ortholog from E. granulosus, EgERK.Methods DNA encoding EgERK was isolated from protoscolices of E. granulosus and analyzed using the LA Taq polymerase chain reaction (PCR) approach and bioinformatics. Reverse transcription PCR (RT-PCR) was used to determine the transcription level of the gene at two different larval tissues. Western blotting was used to detect levels of EgERK protein. The expression profile of EgERK in protoscolices was examined by immunofluorescence.Results We cloned the entire Egerk genomic locus from E. granulosus. In addition, two alternatively spliced transcripts of Egerk, Egerk-A, and Egerk-B were identified. Egerk-A was found to constitutively expressed at the transcriptional and protein levels in two different larval tissues (cyst membranes and protoscolices). Egerk-A was expressed in the tegumental structures, hooklets, and suckers and in the tissue surrounding the rostellum of E. granulosus protoscolices.Conclusions We have cloned the genomic DNA of a novel ERK ortholog from E. granulosus, EgERK (GenBank ID HQ585923), and found that it is constitutively expressed in cyst membrane and

  8. Conductin/axin2 and Wnt signalling regulates centrosome cohesion

    OpenAIRE

    Hadjihannas, Michel V; Brückner, Martina; Behrens, Jürgen

    2010-01-01

    Wnt signalling regulates centrosome cohesion. Work by the Behrens group shows that conductin/axin2, a negative regulator of β-catenin, localizes to centrosomes by binding to the centriole-associated component C-Nap1. Conductin/axin2 promotes centrosome cohesion by phosphorylating β-catenin at centrosomes and the authors propose a model for the regulation of centrosome separation by conductin and Wnt signalling.

  9. Leukotriene B(4) BLT receptor signaling regulates the level and stability of cyclooxygenase-2 (COX-2) mRNA through restricted activation of Ras/Raf/ERK/p42 AUF1 pathway.

    Science.gov (United States)

    Zhai, Beibei; Yang, Huiqing; Mancini, Arturo; He, QingWen; Antoniou, John; Di Battista, John A

    2010-07-30

    Recent studies suggest that active resolution of the inflammatory response in animal models of arthritis may involve leukotriene B(4) (LTB(4))-dependent stimulation of "intermediate" prostaglandin production, which in turn favors the synthesis of "downstream" anti-inflammatory and pro-resolving lipoxins, resolvins, and protectins. We explored a putative mechanism involving LTB(4)-dependent control of cyclooxygenase-2 (COX-2) expression, the rate-limiting step in inflammatory prostaglandin biosynthesis. Indeed, LTB(4) potently up-regulated/stabilized interleukin-1beta-induced COX-2 mRNA and protein expression under conditions of COX-2 inhibitor-dependent blockade of PGE(2) release in human synovial fibroblasts (EC(50) = 16.5 + or - 1.7 nm for mRNA; 19 + or - 2.4 nm for protein, n = 4). The latter response was pertussis toxin-sensitive, and semi-quantitative reverse transcription-PCR confirmed the quantitative predominance of the BLT2 receptor. Transfection experiments, using human COX-2 promoter plasmids and chimeric luciferase-COX-2 mRNA 3'-untranslated region (3'-UTR) reporter constructs, revealed that LTB(4) exerted its stabilizing effect at the post-transcriptional level through a 116-bp adenylate/uridylate-rich sequence in the proximal region of the COX-2 3'-UTR. Using luciferase-COX-2 mRNA 3'-UTR reporter constructs and Ras/c-Raf expression and mutant constructs, we showed that the Ras/c-Raf/MEK1/2/ERK1/2 signaling pathway mediated LTB(4)-dependent COX-2 mRNA stabilization. Knockdown experiments with specific short hairpin RNAs confirmed that LTB(4) stabilization of COX-2 mRNA was apparently mediated through the RNA-binding protein, p42 AUF1. The nuclear export of p42 AUF1 was driven by c-Raf/MEK1/2/ERK1/2 signaling and sensitive to leptomycin B treatment, suggesting a CRM1-dependent mechanism. We conclude that LTB(4) may support the resolution phase of the inflammatory response by stabilizing COX-2, ensuring a reservoir of ambient pro-resolution lipid

  10. Comprehensive measurement of respiratory activity in permeabilized cells using extracellular flux analysis.

    Science.gov (United States)

    Salabei, Joshua K; Gibb, Andrew A; Hill, Bradford G

    2014-02-01

    Extracellular flux (XF) analysis has become a mainstream method for measuring mitochondrial function in cells and tissues. Although this technique is commonly used to measure bioenergetics in intact cells, we outline here a detailed XF protocol for measuring respiration in permeabilized cells. Cells are permeabilized using saponin (SAP), digitonin (DIG) or recombinant perfringolysin O (rPFO) (XF-plasma membrane permeabilizer (PMP) reagent), and they are provided with specific substrates to measure complex I- or complex II-mediated respiratory activity, complex III+IV respiratory activity or complex IV activity. Medium- and long-chain acylcarnitines or glutamine may also be provided for measuring fatty acid (FA) oxidation or glutamine oxidation, respectively. This protocol uses a minimal number of cells compared with other protocols and does not require isolation of mitochondria. The results are highly reproducible, and mitochondria remain well coupled. Collectively, this protocol provides comprehensive and detailed information regarding mitochondrial activity and efficiency, and, after preparative steps, it takes 6-8 h to complete. PMID:24457333

  11. Monitoring the excitability of neocortical efferent neurons to direct activation by extracellular current pulses.

    Science.gov (United States)

    Swadlow, H A

    1992-08-01

    1. Extracellular action potentials were recorded from antidromically activated efferent neurons in visual, somatosensory, and motor cortex of the awake rabbit using low-impedance metal microelectrodes. Efferent neurons were also activated by current pulses delivered near the soma [juxtasomal current pulses (JSCPs)] through the recording microelectrode. Action potentials generated by JSCPs were not directly observed (because of the stimulus artifact), but were inferred with the use of a collision paradigm. Efferent populations studied include callosal neurons [CC (n = 80)], ipsilateral corticocortical neurons [C-IC (n = 21)], corticothalamic neurons of layer 6 [CF-6 (n = 57)], and descending corticofugal neurons of layer 5 [CF-5, corticotectal neurons of the visual cortex (n = 48)]. 2. Most CC neurons (45/46) and all C-IC (8/8) and CF-6 neurons (39/39) were directly activated by JSCPs at near-threshold intensities. Some CF-5 neurons (9/38), however, showed evidence of indirect activation. All efferent classes had similar current thresholds (means 1.85-2.10 microA) to direct activation by JSCPs, and thresholds were inversely related to extracellular spike amplitude. For each neuron, the range of JSCP intensities that generated response probabilities of between 0.2 and 0.8 was measured, and this "range of uncertainty" was significantly greater in CF-5 neurons (mean 32.7% of threshold) than in CC (mean 19.0%) or CF-6 (mean 20.4%) neurons. 3. Several factors indicate that the threshold of efferent neurons to JSCPs is very sensitive to excitatory and inhibitory synaptic inputs. Iontophoretic applications of gamma-aminobutyric acid (GABA) increased the threshold to JSCPs, and glutamate reduced the threshold. Electrical stimulation of afferent pathways at intensities just below threshold for eliciting action potentials resulted in a dramatic decrease in JSCP threshold. This initial short-latency threshold decrease was specific to stimulation of particular afferent pathways

  12. 降钙素基因相关肽调控细胞外信号调节激酶减轻高体积分数氧对胎鼠肺泡Ⅱ型上皮细胞的损伤作用%Damage - Reduced Effects of Calcitonin Gene - Related Peptide on Hyperoxia - Exposed Type Ⅱ Alveolar Epithelial Cell Mediated by Extracellular Signal -Regulated Kinase

    Institute of Scientific and Technical Information of China (English)

    付红敏; 李利; 汤春辉; 皇甫春荣; 米弘瑛; 李献珍; 方芳; 许峰

    2012-01-01

    Objective To explore the effects of calcitonin gene - related peptide( CGRF) on type II alveolar epithelial cell( AEC II ) exposed to hyperoxia and whether the mechanism is mediated by extracellular signal - regulated kinase ( ERK) pathway. Methods AEC II were isolated from 21 d fetal rat lung and grew for 12 h to attach. Then AEC II were randomly divided into six groups;air group,CGRP/air group,CGRP8 -37/air group,hyperoxia group,CGRP/O2 and CGRP8 - 37/O2 group. Air or hyperoxia environment was achieved by exposing AEC1I into 210 mL · L-1oxygen or 850 mL · L-1 oxygen for 18 h. CGRP group or CGRP8 - 37 group was carried out by adding 10-1 mol · L-1 CGRP or both CGRP and CGRP8 -37(10-1 mol · L-1) ,a receptor antagonist against CGRP,into medium before cultured in air or 850 mL · I-1 oxygen. Lactate dehydrogenase (LDH) ,alkaline phosphatase (AKP) and malondialdehyde (MDA) were measured by immune tur-bidimetry and reactive oxygen species( ROS) by flow cytometry. Immunofluorescence microscopy was used to analyze the expression of surfactant protein C( SP - C) and Western blot was taken to detect the content of p - ERK1/2. Results The levels of MDA,LDH,AKP,ROS and p-ERKl/2 were markedly increased in hyperoxia group than those in air group [(2. 29 ±0.10) μmol · L-1 vs (1.06±0.14) μmol · L-1, (58.79 ±5.01) U ·L-1 vs (25.92 ±3.68) U · L-1,(24.63 ±2.92) U · L-1 vs (10. 34 ±1.78) U · L-1,47.74 ±3.35 vs 25.96 ±5.04, 1.21 ±0.06 vs 0.45 ±0.05 ,P, <0.01] .whereas expression of SP -C was decreased in hyperoxia group compared with air group (22.75 ±3.31 vs 43. 50 ± 4.42 ). Levels of MDA, LDH, AKP and ROS were reduced with an elevated expression of p - ERK1 /2 and SP - C in CGRP/O2 group compared with those in hyperoxia group and CGRP8 - 37/O2 group (Pa < 0. 01). There were no significant differences about the levels of MDA,LDH,AKP,ROS and SP- C among three groups cultured in air condition. The expression of p - ERK1/2 in CGRP/air group was also higher than

  13. Intracellular modulation, extracellular disposal and serum increase of MiR-150 mark lymphocyte activation.

    Directory of Open Access Journals (Sweden)

    Paola de Candia

    Full Text Available Activated lymphocytes release nano-sized vesicles (exosomes containing microRNAs that can be monitored in the bloodstream. We asked whether elicitation of immune responses is followed by release of lymphocyte-specific microRNAs. We found that, upon activation in vitro, human and mouse lymphocytes down-modulate intracellular miR-150 and accumulate it in exosomes. In vivo, miR-150 levels increased significantly in serum of humans immunized with flu vaccines and in mice immunized with ovalbumin, and this increase correlated with elevation of antibody titers. Immunization of immune-deficient mice, lacking MHCII, resulted neither in antibody production nor in elevation of circulating miR-150. This study provides proof of concept that serum microRNAs can be detected, with minimally invasive procedure, as biomarkers of vaccination and more in general of adaptive immune responses. Furthermore, the prompt reduction of intracellular level of miR-150, a key regulator of mRNAs critical for lymphocyte differentiation and functions, linked to its release in the external milieu suggests that the selective extracellular disposal of microRNAs can be a rapid way to regulate gene expression during lymphocyte activation.

  14. Allocation of extracellular enzymatic activity in relation to litter composition, N deposition, and mass loss

    Science.gov (United States)

    Sinsabaugh, R. L.; Carreiro, M.M.; Repert, D.A.

    2002-01-01

    Decomposition of plant material is a complex process that requires interaction among a diversity of microorganisms whose presence and activity is subject to regulation by a wide range of environmental factors. Analysis of extracellular enzyme activity (EEA) provides a way to relate the functional organization of microdecomposer communities to environmental variables. In this study, we examined EEA in relation to litter composition and nitrogen deposition. Mesh bags containing senescent leaves of Quercus borealis (red oak), Acer rubrum (red maple) and Cornus florida (flowering dogwood) were placed on forest floor plots in southeastern New York. One-third of the plots were sprayed monthly with distilled water. The other plots were sprayed monthly with NH4NO3 solution at dose rates equivalent to 2 or 8 g N m-2 y-1. Mass loss, litter composition, fungal mass, and the activities of eight enzymes were measured on 13 dates for each litter type. Dogwood was followed for one year, maple for two, oak for three, For each litter type and treatment, enzymatic turnover activities were calculated from regressions of LN (%mass remaining) vs. cumulative activity. The decomposition of dogwood litter was more efficient than that of maple and oak. Maple litter had the lowest fungal mass and required the most enzymatic work to decompose, even though its mass loss rate was twice that of oak. Across litter types, N amendment reduced apparent enzymatic efficiencies and shifted EEA away from N acquisition and toward P acquisition, and away from polyphenol oxidation and toward polysaccharide hydrolysis. The effect of these shifts on decomposition rate varied with litter composition: dogwood was stimulated, oak was inhibited and maple showed mixed effects. The results show that relatively small shifts in the activity of one or two critical enzymes can significantly alter decomposition rates.

  15. Differential Requirement of the Extracellular Domain in Activation of Class B G Protein-coupled Receptors.

    Science.gov (United States)

    Zhao, Li-Hua; Yin, Yanting; Yang, Dehua; Liu, Bo; Hou, Li; Wang, Xiaoxi; Pal, Kuntal; Jiang, Yi; Feng, Yang; Cai, Xiaoqing; Dai, Antao; Liu, Mingyao; Wang, Ming-Wei; Melcher, Karsten; Xu, H Eric

    2016-07-15

    G protein-coupled receptors (GPCRs) from the secretin-like (class B) family are key players in hormonal homeostasis and are important drug targets for the treatment of metabolic disorders and neuronal diseases. They consist of a large N-terminal extracellular domain (ECD) and a transmembrane domain (TMD) with the GPCR signature of seven transmembrane helices. Class B GPCRs are activated by peptide hormones with their C termini bound to the receptor ECD and their N termini bound to the TMD. It is thought that the ECD functions as an affinity trap to bind and localize the hormone to the receptor. This in turn would allow the hormone N terminus to insert into the TMD and induce conformational changes of the TMD to activate downstream signaling. In contrast to this prevailing model, we demonstrate that human class B GPCRs vary widely in their requirement of the ECD for activation. In one group, represented by corticotrophin-releasing factor receptor 1 (CRF1R), parathyroid hormone receptor (PTH1R), and pituitary adenylate cyclase activating polypeptide type 1 receptor (PAC1R), the ECD requirement for high affinity hormone binding can be bypassed by induced proximity and mass action effects, whereas in the other group, represented by glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R), the ECD is required for signaling even when the hormone is covalently linked to the TMD. Furthermore, the activation of GLP-1R by small molecules that interact with the intracellular side of the receptor is dependent on the presence of its ECD, suggesting a direct role of the ECD in GLP-1R activation. PMID:27226600

  16. Extracellular targeting of an active endoxylanase by a TolB negative mutant of Gluconobacter oxydans.

    Science.gov (United States)

    Kosciow, Konrad; Domin, Claudia; Schweiger, Paul; Deppenmeier, Uwe

    2016-07-01

    Gluconobacter (G.) oxydans strains have great industrial potential due to their ability to incompletely oxidize a wide range of carbohydrates. But there is one major limitation preventing their full production potential. Hydrolysis of polysaccharides is not possible because extracellular hydrolases are not encoded in the genome of Gluconobacter species. Therefore, as a first step for the generation of exoenzyme producing G. oxydans, a leaky outer membrane mutant was created by deleting the TolB encoding gene gox1687. As a second step the xynA gene encoding an endo-1,4-β-xylanase from Bacillus subtilis was expressed in G. oxydans ΔtolB. More than 70 % of the total XynA activity (0.91 mmol h(-1) l culture(-1)) was detected in the culture supernatant of the TolB mutant and only 10 % of endoxylanase activity was observed in the supernatant of G. oxydans xynA. These results showed that a G. oxydans strain with an increased substrate spectrum that is able to use the renewable polysaccharide xylan as a substrate to produce the prebiotic compounds xylobiose and xylooligosaccharides was generated. This is the first report about the combination of the process of incomplete oxidation with the degradation of renewable organic materials from plants for the production of value-added products. PMID:27097633

  17. Aryl hydrocarbon receptor activation impairs extracellular matrix remodeling during zebra fish fin regeneration.

    Science.gov (United States)

    Andreasen, Eric A; Mathew, Lijoy K; Löhr, Christiane V; Hasson, Rachelle; Tanguay, Robert L

    2007-01-01

    Adult zebra fish completely regenerate their caudal (tail) fin following partial amputation. Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibits this regenerative process. Proper regulation of transcription, innervation, vascularization, and extracellular matrix (ECM) composition is essential for complete fin regeneration. Previous microarray studies suggest that genes involved in ECM regulation are misexpressed following activation of the aryl hydrocarbon receptor. To investigate whether TCDD blocks regeneration by impairing ECM remodeling, male zebra fish were i.p. injected with 50 ng/g TCDD or vehicle, and caudal fins were amputated. By 3 days postamputation (dpa), the vascular network in the regenerating fin of TCDD-exposed fish was disorganized compared to vehicle-exposed animals. Furthermore, immunohistochemical staining revealed that axonal outgrowth was impacted by TCDD as early as 3 dpa. Histological analysis demonstrated that TCDD exposure leads to an accumulation of collagen at the end of the fin ray just distal to the amputation site by 3 dpa. Mature lepidotrichial-forming cells (fin ray-forming cells) were not observed in the fins of TCDD-treated fish. The capacity to metabolize ECM was also altered by TCDD exposure. Quantitative real-time PCR studies revealed that the aryl hydrocarbon pathway is active and that matrix-remodeling genes are expressed in the regenerate following TCDD exposure.

  18. Vibrio cholerae hemagglutinin(HA)/protease: An extracellular metalloprotease with multiple pathogenic activities.

    Science.gov (United States)

    Benitez, Jorge A; Silva, Anisia J

    2016-06-01

    Vibrio cholerae of serogroup O1 and O139, the etiological agent of the diarrheal disease cholera, expresses the extracellular Zn-dependent metalloprotease hemagglutinin (HA)/protease also reported as vibriolysin. This enzyme is also produced by non-O1/O139 (non-cholera) strains that cause mild, sporadic illness (i.e. gastroenteritis, wound or ear infections). Orthologs of HA/protease are present in other members of the Vibrionaceae family pathogenic to humans and fish. HA/protease belongs to the M4 neutral peptidase family and displays significant amino acid sequence homology to Pseudomonas aeruginosa elastase (LasB) and Bacillus thermoproteolyticus thermolysin. It exhibits a broad range of potentially pathogenic activities in cell culture and animal models. These activities range from the covalent modification of other toxins, the degradation of the protective mucus barrier and disruption of intestinal tight junctions. Here we review (i) the structure and regulation of HA/protease expression, (ii) its interaction with other toxins and the intestinal mucosa and (iii) discuss the possible role(s) of HA/protease in the pathogenesis of cholera. PMID:26952544

  19. Molecular evaluation of extracellular activity of medicinal herb Clinacanthus nutans against herpes simplex virus type-2.

    Science.gov (United States)

    Vachirayonstien, Thaveechai; Promkhatkaew, Duanthanorm; Bunjob, Malee; Chueyprom, Asawachai; Chavalittumrong, Pranee; Sawanpanyalert, Pathom

    2010-02-01

    Clinacanthus nutans (Burm. f.) Lindau (C. nutans), a medicinal herb belonging to the family Acanthaceae, has traditionally been used in herpes simplex virus (HSV) treatment in Thailand. Clinical trials have indicated that topical preparations produced from its extracts were effective in HSV-2 treatment. However, there is no clear evidence of the mechanism of action or a molecular target of C. nutans. In this study, the extracellular activity of C. nutans extracts against HSV-2 infected on HEp-2 cells was investigated in terms of its molecular aspects. HSV-2 was treated with the extracts and adsorped into the HEp-2 cells. After infection, HSV-2 DNA quantities in the infected cells were assessed and compared by the quantitative dot blot hybridisation technique. The results showed that treating the viruses with either less or more highly purified extracts before infection resulted in great reductions of viral infectivity. Further investigation was performed by Western blot analysis to determine the activities of the extracts on the viral proteins. At least eight viral proteins of the infected cell proteins (ICP) and some structural proteins, including 146, 125, 78, 69, 55, 44, 40 and 20 KDa proteins, were depleted and reduced gradually with higher and lower concentrated herb extracts, respectively. These suggest that the C. nutans extracts highly inactivated or inhibited HSV-2 before infection. PMID:20140802

  20. Augmented expression of urokinase plasminogen activator and extracellular matrix proteins associates with multiple myeloma progression.

    Science.gov (United States)

    Khan, Rehan; Gupta, Nidhi; Kumar, Raman; Sharma, Manoj; Kumar, Lalit; Sharma, Alpana

    2014-06-01

    Multiple myeloma (MM) represents a B cell malignancy, characterized by a monoclonal proliferation of malignant plasma cells. Interactions between tumor cells and extracellular matrix (ECM) are of importance for tumor invasion and metastasis. Protein levels of urokinase plasminogen activator (uPA) and fibulin 1, nidogen and laminin in plasma and serum respectively and mRNA levels of these molecules in peripheral blood mononuclear cells were determined in 80 subjects by using ELISA and quantitative PCR and data was analyzed with severity of disease. Pearson correlation was determined to observe interrelationship between different molecules. A statistical significant increase for ECM proteins (laminin, nidogen and fibulin 1) and uPA at circulatory level as well as at mRNA level was observed compared to healthy controls. The levels of these molecules in serum might be utilized as a marker of active disease. Significant positive correlation of all ECM proteins with uPA was found and data also correlates with severity of disease. Strong association found between ECM proteins and uPA in this study supports that there might be interplay between these molecules which can be targeted. This study on these molecules may help to gain insight into processes of growth, spread, and clinical behavior of MM.

  1. Extracellular ATP activates MAPK and ROS signaling during injury response in the fungus Trichoderma atroviride

    Directory of Open Access Journals (Sweden)

    Elizabeth eMedina-Castellanos

    2014-11-01

    Full Text Available The response to mechanical damage is crucial for the survival of multicellular organisms, enabling their adaptation to hostile environments. Trichoderma atroviride, a filamentous fungus of great importance in the biological control of plant diseases, responds to mechanical damage by activating regenerative processes and asexual reproduction (conidiation. During this response, reactive oxygen species (ROS are produced by the NADPH oxidase (Nox1/NoxR complex. To understand the underlying early signaling events, we evaluated molecules such as extracellular ATP (eATP and Ca2+ that are known to trigger wound-induced responses in plants and animals. Concretely, we investigated the activation of mitogen-activated protein kinase (MAPK pathways by eATP, Ca2+ and ROS. Indeed, application of exogenous ATP and Ca2+ triggered conidiation. Furthermore, eATP promoted the Nox1-dependent production of ROS and activated a MAPK pathway. Mutants in the MAPK-encoding genes tmk1 and tmk3 were affected in wound-induced conidiation, and phosphorylation of both Tmk1 and Tmk3 was triggered by eATP. We conclude that in this fungus, eATP acts as a damage-associated molecular pattern (DAMP. Our data indicate the existence of an eATP receptor and suggest that in fungi, eATP triggers pathways that converge to regulate asexual reproduction genes that are required for injury-induced conidiation. By contrast, Ca2+ is more likely to act as a downstream second messenger. The early steps of mechanical damage response in T. atroviride share conserved elements with those known from plants and animals.

  2. Phosphotidylserine exposure and neutrophil extracellular traps enhance procoagulant activity in patients with inflammatory bowel disease.

    Science.gov (United States)

    He, Zhangxiu; Si, Yu; Jiang, Tao; Ma, Ruishuang; Zhang, Yan; Cao, Muhua; Li, Tao; Yao, Zhipeng; Zhao, Lu; Fang, Shaohong; Yu, Bo; Dong, Zengxiang; Thatte, Hemant S; Bi, Yayan; Kou, Junjie; Yang, Shufen; Piao, Daxun; Hao, Lirong; Zhou, Jin; Shi, Jialan

    2016-04-01

    Inflammatory bowel disease (IBD)-associated thromboembolic event often lacks precise aetiology. The aim of this study was to investigate the contribution of phosphatidylserine (PS) exposure and neutrophil extracellular traps (NETs) towards the hypercoagulable state in IBD. We demonstrated that the levels of PS exposed MPs and the sources of MP-origin, platelets, erythrocytes, leukocytes and cultured endothelial cells (ECs) were higher in IBD groups than in healthy controls using flow cytometry and confocal microscopy. Wright-Giemsa and immunofluorescence staining demonstrated that the elevated NETs were released by activated IBD neutrophils or by control neutrophils treated with IBD sera obtained from patients with the active disease. MPs and MP-origin cells in IBD groups, especially in active stage, markedly shortened coagulation time and had increased levels of fibrin, thrombin and FXa production as assessed by coagulation function assays. Importantly, we found that on stimulated ECs, PS rich membranes provided binding sites for FXa and FVa, promoting fibrin formation while TNF blockage or IgG depletion attenuated this effect. Treatment of control neutrophils with TNF and isolated IgG from PR3-ANCA-positive active IBD patients also resulted in the release of NETs. Blockade of PS with lactadherin prolonged coagulation time, decreased fibrin formation to control levels, and inhibited the procoagulant enzymes production in the MPs and MP-origin cells. NET cleavage by DNase I partly decreased PCA in IBD or stimulated neutrophils. Our study reveals a previously unrecognised link between hypercoagulable state and PS exposure or NETs, and may further explain the epidemiological association of thrombosis within IBD patients. PMID:26660948

  3. 豚鼠前庭上皮胰岛素样生长因子1及其受体与细胞外信号调节激酶1/2表达在庆大霉素损伤后的变化%Expression changes of insulin-like growth factor-1 and its receptor as well as extracellular signal regulated kinase 1/2 in the vestibular epithelium of guinea pigs following gentamycin toxicity

    Institute of Scientific and Technical Information of China (English)

    盛宏申; 何跃; 徐光; 孙景豫; 王锦玲

    2007-01-01

    平表达,庆大霉素损伤后停药1 d其表达最强,之后逐渐下降,但停药后21 d其表达仍高于对照组.各组间胰岛素样生长因子1及其受体表达灰度值比较差异明显(F=51.8,45.7,P<0.05).二者变化规律基本一致.④对照组豚鼠细胞外信号调节激酶1/2有低水平表达.庆大霉素损伤后其表达逐渐增强,停药后7 d表达最强,之后逐渐下降,停药后21 d其表达仍高于对照组.各组间细胞外信号调节激酶1/2表达比较差异明显(F=103.7,106.4,P<0.05).二者变化规律相近.结论:庆大霉素损伤后豚鼠前庭上皮胰岛素样生长因子1及其受体,细胞外信号调节激酶1/2表达增加.胰岛素样生长因子1可能是内源性的促有丝分裂剂,通过旁分泌或自分泌的方式在豚鼠前庭毛细胞修复的早期发挥重要作用.细胞外信号调节激酶1/2可能在庆大霉素损伤后豚鼠前庭毛细胞自发修复中发挥重要的信号转导作用.%BACKGROUND:Insulin-like growth factor-1 (IGF-1) is an important mitogen and an indispensable regulator during normal hair cell development. Extracellular signal regulated kinase 1/2 (ERK1/2) is also expressed in mammalian vestibular organs.OBJECTIVE: To investigate the changes of the expression and the distribution of IGF-1, IGF-1 receptor (IGF-1R) and ERK1/2 in guinea pig's vestibular epithelium following gentamycin injury.DESIGN: A randomly controlled study.SETTING: Department of Otorhinolaryngology, General Hospital of Chengdu Military Area Command of Chinese PLA.MATERIALS: Twenty healthy and adult guinea pigs, weighing 300 to 350 g, provided by the Experimental Animal Center,Fourth Military Medical University of Chinese PLA, were randomly divided into four experimental groups and a control group with 4 in each group.METHODS: The experiment was performed at Department of Otorhinolaryngology Research Laboratory of Xijing Hospital,Fourth Military Medical University of Chinese PLA from January 2002 to May 2002. In the experimental

  4. Extracellular enzymatic activities of cold-adapted bacteria from polar oceans and effect of temperature and salinity on cell growth

    Institute of Scientific and Technical Information of China (English)

    Zeng Yinxin; Yu Yong; Chen Bo; Li Huirong

    2004-01-01

    The potential of 324 bacteria isolated from different habitats in polar oceans to produce a variety of extracellular enzymatic activities at low temperature was investigated. By plate assay, lipase, protease, amylase, gelatinase, agarase, chitinase or cellulase were detected. Lipases were generally present by bacteria living in polar oceans. Protease-producing bacteria held the second highest proportion in culturable isolates. Strains producing amylase kept a relative stable proportion of around 30% in different polar marine habitats. All 50 Arctic sea-ice bacteria producing proteases were cold-adapted strains, however, only 20% were psychrophilic. 98% of them could grow at 3% NaCl, and 56% could grow without NaCl. On the other hand, 98% of these sea-ice bacteria produced extracellular proteases with optimum temperature at or higher than 35℃, well above the upper temperature limit of cell growth. Extracellular enzymes including amylase, agarase, cellulase and lipase released by bacteria from seawater or sediment in polar oceans, most expressed maximum activities between 25 and 35℃. Among extracellular enzymes released by bacterial strain BSw20308, protease expressed maximum activity at 40℃, higher than 35℃ of polysaccharide hydrolases and 25℃ of lipase.

  5. Increasing extracellular matrix collagen level and MMP activity induces cyst development in polycystic kidney disease

    Directory of Open Access Journals (Sweden)

    Liu Bin

    2012-09-01

    Full Text Available Abstract Background Polycystic Kidney Disease (PKD kidneys exhibit increased extracellular matrix (ECM collagen expression and metalloproteinases (MMPs activity. We investigated the role of these increases on cystic disease progression in PKD kidneys. Methods We examined the role of type I collagen (collagen I and membrane bound type 1 MMP (MT1-MMP on cyst development using both in vitro 3 dimensional (3D collagen gel culture and in vivo PCK rat model of PKD. Results We found that collagen concentration is critical in controlling the morphogenesis of MDCK cells cultured in 3D gels. MDCK cells did not form 3D structures at collagen I concentrations lower than 1 mg/ml but began forming tubules when the concentration reaches 1 mg/ml. Significantly, these cells began to form cyst when collagen I concentration reached to 1.2 mg/ml, and the ratios of cyst to tubule structures increased as the collagen I concentration increased. These cells exclusively formed cyst structures at a collagen I concentration of 1.8 mg/ml or higher. Overexpression of MT1-MMP in MDCK cells significantly induced cyst growth in 3D collagen gel culture. Conversely, inhibition of MMPs activity with doxycycline, a FDA approved pan-MMPs inhibitor, dramatically slowed cyst growth. More importantly, the treatment of PCK rats with doxycycline significantly decreased renal tubule cell proliferation and markedly inhibited the cystic disease progression. Conclusions Our data suggest that increased collagen expression and MMP activity in PKD kidneys may induce cyst formation and expansion. Our findings also suggest that MMPs may serve as a therapeutic target for the treatment of human PKD.

  6. Influence of Humic Acid Complexation with Metal Ions on Extracellular Electron Transfer Activity

    Science.gov (United States)

    Zhou, Shungui; Chen, Shanshan; Yuan, Yong; Lu, Qin

    2015-11-01

    Humic acids (HAs) can act as electron shuttles and mediate biogeochemical cycles, thereby influencing the transformation of nutrients and environmental pollutants. HAs commonly complex with metals in the environment, but few studies have focused on how these metals affect the roles of HAs in extracellular electron transfer (EET). In this study, HA-metal (HA-M) complexes (HA-Fe, HA-Cu, and HA-Al) were prepared and characterized. The electron shuttle capacities of HA-M complexes were experimentally evaluated through microbial Fe(III) reduction, biocurrent generation, and microbial azoreduction. The results show that the electron shuttle capacities of HAs were enhanced after complexation with Fe but were weakened when using Cu or Al. Density functional theory calculations were performed to explore the structural geometry of the HA-M complexes and revealed the best binding sites of the HAs to metals and the varied charge transfer rate constants (k). The EET activity of the HA-M complexes were in the order HA-Fe > HA-Cu > HA-Al. These findings have important implications for biogeochemical redox processes given the ubiquitous nature of both HAs and various metals in the environment.

  7. Modeling the development of biofilm density including active bacteria, inert biomass, and extracellular polymeric substances.

    Science.gov (United States)

    Laspidou, Chrysi S; Rittmann, Bruce E

    2004-01-01

    We present the unified multi-component cellular automaton (UMCCA) model, which predicts quantitatively the development of the biofilm's composite density for three biofilm components: active bacteria, inert or dead biomass, and extracellular polymeric substances. The model also describes the concentrations of three soluble organic components (soluble substrate and two types of soluble microbial products) and oxygen. The UMCCA model is a hybrid discrete-differential mathematical model and introduces the novel feature of biofilm consolidation. Our hypothesis is that the fluid over the biofilm creates pressures and vibrations that cause the biofilm to consolidate, or pack itself to a higher density over time. Each biofilm compartment in the model output consolidates to a different degree that depends on the age of its biomass. The UMCCA model also adds a cellular automaton algorithm that identifies the path of least resistance and directly moves excess biomass along that path, thereby ensuring that the excess biomass is distributed efficiently. A companion paper illustrates the trends that the UMCCA model is able to represent and shows a comparison with experimental results. PMID:15276752

  8. Inhibition of cerebrovascular raf activation attenuates cerebral blood flow and prevents upregulation of contractile receptors after subarachnoid hemorrhage

    DEFF Research Database (Denmark)

    Ansar, Saema; Maddahi, Aida; Edvinsson, Lars

    2011-01-01

    of mitogen-activated protein kinase (MAPK) of the extracellular signal-regulated kinase (ERK)1/2 signal pathway. We hypothesize that SAH initiates cerebrovascular ERK1/2 activation, resulting in receptor upregulation. The raf inhibitor will inhibit the molecular events upstream ERK1/2 and may provide...

  9. Conserved charged amino acid residues in the extracellular region of sodium/iodide symporter are critical for iodide transport activity

    Directory of Open Access Journals (Sweden)

    Liang Ji-An

    2010-11-01

    Full Text Available Abstract Background Sodium/iodide symporter (NIS mediates the active transport and accumulation of iodide from the blood into the thyroid gland. His-226 located in the extracellular region of NIS has been demonstrated to be critical for iodide transport in our previous study. The conserved charged amino acid residues in the extracellular region of NIS were therefore characterized in this study. Methods Fourteen charged residues (Arg-9, Glu-79, Arg-82, Lys-86, Asp-163, His-226, Arg-228, Asp-233, Asp-237, Arg-239, Arg-241, Asp-311, Asp-322, and Asp-331 were replaced by alanine. Iodide uptake abilities of mutants were evaluated by steady-state and kinetic analysis. The three-dimensional comparative protein structure of NIS was further modeled using sodium/glucose transporter as the reference protein. Results All the NIS mutants were expressed normally in the cells and targeted correctly to the plasma membrane. However, these mutants, except R9A, displayed severe defects on the iodide uptake. Further kinetic analysis revealed that mutations at conserved positively charged amino acid residues in the extracellular region of NIS led to decrease NIS-mediated iodide uptake activity by reducing the maximal rate of iodide transport, while mutations at conserved negatively charged residues led to decrease iodide transport by increasing dissociation between NIS mutants and iodide. Conclusions This is the first report characterizing thoroughly the functional significance of conserved charged amino acid residues in the extracellular region of NIS. Our data suggested that conserved charged amino acid residues, except Arg-9, in the extracellular region of NIS were critical for iodide transport.

  10. Neutrophil extracellular traps downregulate lipopolysaccharide-induced activation of monocyte-derived dendritic cells.

    Science.gov (United States)

    Barrientos, Lorena; Bignon, Alexandre; Gueguen, Claire; de Chaisemartin, Luc; Gorges, Roseline; Sandré, Catherine; Mascarell, Laurent; Balabanian, Karl; Kerdine-Römer, Saadia; Pallardy, Marc; Marin-Esteban, Viviana; Chollet-Martin, Sylvie

    2014-12-01

    Polymorphonuclear neutrophils (PMN) play a central role in inflammation and participate in its control, notably by modulating dendritic cell (DC) functions via soluble mediators or cell-cell contacts. Neutrophil extracellular traps (NETs) released by PMN could play a role in this context. To evaluate NET effects on DC maturation, we developed a model based on monocyte-derived DC (moDC) and calibrated NETs isolated from fresh human PMN. We found that isolated NETs alone had no discernable effect on moDC. In contrast, they downregulated LPS-induced moDC maturation, as shown by decreased surface expression of HLA-DR, CD80, CD83, and CD86, and by downregulated cytokine production (TNF-α, IL-6, IL-12, IL-23), with no increase in the expression of tolerogenic DC genes. Moreover, the presence of NETs during moDC maturation diminished the capacity of these moDC to induce T lymphocyte proliferation in both autologous and allogeneic conditions, and modulated CD4(+) T lymphocyte polarization by promoting the production of Th2 cytokines (IL-5 and IL-13) and reducing that of Th1 and Th17 cytokines (IFN-γ and IL-17). Interestingly, the expression and activities of the lymphoid chemokine receptors CCR7 and CXCR4 on moDC were not altered when moDC matured in the presence of NETs. Together, these findings reveal a new role for NETs in adaptive immune responses, modulating some moDC functions and thereby participating in the control of inflammation.

  11. Neuroinflammation and Depression: Microglia Activation, Extracellular Microvesicles and microRNA Dysregulation.

    Science.gov (United States)

    Brites, Dora; Fernandes, Adelaide

    2015-01-01

    Patients with chronic inflammation are often associated with the emergence of depression symptoms, while diagnosed depressed patients show increased levels of circulating cytokines. Further studies revealed the activation of the brain immune cell microglia in depressed patients with a greater magnitude in individuals that committed suicide, indicating a crucial role for neuroinflammation in depression brain pathogenesis. Rapid advances in the understanding of microglial and astrocytic neurobiology were obtained in the past 15-20 years. Indeed, recent data reveal that microglia play an important role in managing neuronal cell death, neurogenesis, and synaptic interactions, besides their involvement in immune-response generating cytokines. The communication between microglia and neurons is essential to synchronize these diverse functions with brain activity. Evidence is accumulating that secreted extracellular vesicles (EVs), comprising ectosomes and exosomes with a size ranging from 0.1-1 μm, are key players in intercellular signaling. These EVs may carry specific proteins, mRNAs and microRNAs (miRNAs). Transfer of exosomes to neurons was shown to be mediated by oligodendrocytes, microglia and astrocytes that may either be supportive to neurons, or instead disseminate the disease. Interestingly, several recent reports have identified changes in miRNAs in depressed patients, which target not only crucial pathways associated with synaptic plasticity, learning and memory but also the production of neurotrophic factors and immune cell modulation. In this article, we discuss the role of neuroinflammation in the emergence of depression, namely dynamic alterations in the status of microglia response to stimulation, and how their activation phenotypes may have an etiological role in neurodegeneneration, in particular in depressive-like behavior. We will overview the involvement of miRNAs, exosomes, ectosomes and microglia in regulating critical pathways associated with

  12. Neuroinflammation and depression: microglia activation, extracellular microvesicles and microRNA dysregulation

    Directory of Open Access Journals (Sweden)

    Dora eBrites

    2015-12-01

    Full Text Available Patients with chronic inflammation are often associated with the emergence of depression symptoms, while diagnosed depressed patients show increased levels of circulating cytokines. Further studies revealed the activation of the brain immune cell microglia in depressed patients with a greater magnitude in individuals that committed suicide, indicating a crucial role for neuroinflammation in depression brain pathogenesis. Rapid advances in the understanding of microglial and astrocytic neurobiology were obtained in the past fifteen to twenty years. Indeed, recent data reveal that microglia play an important role in managing neuronal cell death, neurogenesis, and synaptic interactions, besides their involvement in immune-response generating cytokines. The communication between microglia and neurons is essential to synchronize these diverse functions with brain activity. Evidence is accumulating that secreted extracellular vesicles (EVs, comprising ectosomes and exosomes with a size ranging from 0.1 to 1 μm, are key players in intercellular signaling. These EVs may carry specific proteins, mRNAs and microRNAs (miRNAs. Transfer of exosomes to neurons was shown to be mediated by oligodendrocytes, microglia and astrocytes that may either be supportive to neurons, or instead disseminate the disease. Interestingly, several recent reports have identified changes in miRNAs in depressed patients, which target not only crucial pathways associated with synaptic plasticity, learning and memory but also the production of neurotrophic factors and immune cell modulation. In this article, we discuss the role of neuroinflammation in the emergence of depression, namely dynamic alterations in the status of microglia response to stimulation, and how their activation phenotypes may have an etiological role in neurodegeneneration, in particular in depressive-like behavior. We will overview the involvement of miRNAs, exosomes, ectosomes and microglia in regulating

  13. Increased extracellular dopamine and 5-hydroxytryptamine levels contribute to enhanced subthalamic nucleus neural activity during exhausting exercise

    OpenAIRE

    Hu, Y.; Liu, X.; D. QIAO

    2015-01-01

    The purpose of the study was to explore the mechanism underlying the enhanced subthalamic nucleus (STN) neural activity during exhausting exercise from the perspective of monoamine neurotransmitters and changes of their corresponding receptors. Rats were randomly divided into microdialysis and immunohistochemistry study groups. For microdialysis study, extracellular fluid of the STN was continuously collected with a microdialysis probe before, during and 90 min after one bout of exhausting ex...

  14. The effect of environmental conditions on extracellular protease activity in controlled fermentations of Aspergillus niger

    NARCIS (Netherlands)

    Braaksma, M.; Smilde, A.K.; Werf, M.J. van der; Punt, P.J.

    2009-01-01

    Proteolytic degradation by host proteases is one of the key issues in the application of filamentous fungi for non-fungal protein production. In this study the influence of several environmental factors on the production of extracellular proteases of Aspergillus niger was investigated systematically

  15. 细胞外信号调节蛋白激酶5在慢性氟中毒大鼠脑组织中的表达及其与大鼠记忆力改变的关系%Correlation between expression of extracellular signal-regulated protein kinase 5 in rat brain and changed capacity of learning and memory of rats with chronic fluorosis

    Institute of Scientific and Technical Information of China (English)

    邱志伟; 刘艳洁; 官志忠

    2016-01-01

    Objective Through observation of the expression and activity of extracellular regulated protein kinase 5 (ERK5) and its relationship with the learning and memory ability in rats with chronic fluorosis,to further study the pathogenesis of chronic fluorosis in nervous system.Methods Thirty SD rats were divided into 3 groups according to body weight by means of a random number table (10 rats in each group,half male and half female).The rats in control group were fed with free drinking tap water containing less than 0.5 mg/L fluoride (NaF);the rats in low fluoride group with 10.0 mg/L fluoride;the rat in high dose fluoride group with 50.0 mg/L fluoride.After 6months of experiment,rat brain tissue was took,mRNA expression level of ERK5 was detected by real-time fluorescence quantitative PCR (real-time PCR),protein expression level and activity of ERK5 were detected by Western blotting;the learning and memory ability of rats with chronic fluorosis were detected by Morris water maze test.Results The rat in groups exposed to fluoride exhibited different degrees of dental fluorosis and the fluoride content in urine of rats increased gradually with increase of fluoride doses (F =164.10,P < 0.05).The protein levels of phosphor-ERK5 in the control group,low fluoride group and high fluoride group were 0.13 ± 0.03,0.29 ± 0.10and 0.43 ±0.17,respectively,the difference was statistically significant (F=11.96,P< 0.05),and low fluoride group and high fluoride group were higher than control group (all P < 0.05).The total protein levels of ERK5 in control group,low fluoride group and high fluoride group were 0.32 ± 0.11,0.37 ± 0.13 and 0.49 ± 0.16,respectively,the difference was statistically significant (F =3.45,P < 0.05),and high fluoride group was higher than control group (P < 0.05).The expression of ERK5 mRNA in rat brains between groups was not significantly different (F =0.81,P > 0.05).The second,third,and forth days of directional navigation experiment

  16. Increased extracellular dopamine and 5-hydroxytryptamine levels contribute to enhanced subthalamic nucleus neural activity during exhausting exercise.

    Science.gov (United States)

    Hu, Y; Liu, X; Qiao, D

    2015-09-01

    The purpose of the study was to explore the mechanism underlying the enhanced subthalamic nucleus (STN) neural activity during exhausting exercise from the perspective of monoamine neurotransmitters and changes of their corresponding receptors. Rats were randomly divided into microdialysis and immunohistochemistry study groups. For microdialysis study, extracellular fluid of the STN was continuously collected with a microdialysis probe before, during and 90 min after one bout of exhausting exercise. Dopamine (DA) and 5-hydroxytryptamine (5-HT) levels were subsequently detected with high-performance liquid chromatography (HPLC). For immunohistochemistry study, the expression of DRD2 and HT2C receptors in the STN, before, immediately after and 90 min after exhaustion was detected through immunohistochemistry technique. Microdialysis study results showed that the extracellular DA and 5-HT neurotransmitters increased significantly throughout the procedure of exhausting exercise and the recovery period (Prat STN immediately after exhausting exercise and at the time point of 90 min after exhaustion were both higher than those of the rest condition, but the difference was not significant (P>0.05). Our results suggest that the increased extracellular DA and 5-HT in the STN might be one important factor leading to the enhanced STN neural activity and the development of fatigue during exhausting exercise. This study may essentially offer useful evidence for better understanding of the mechanism of the central type of exercise-induced fatigue. PMID:26424920

  17. Microbial respiration and kinetics of extracellular enzymes activities through rhizosphere and detritusphere at agricultural site

    Science.gov (United States)

    Löppmann, Sebastian; Blagodatskaya, Evgenia; Kuzyakov, Yakov

    2014-05-01

    detritivore communities in the soil. The kinetics (Km and Vmax) of four extracellular hydrolytic enzymes responsible for C- and phosphorous-cycle (β-glucosidase, β-xylosidase, β-cellobiohydrolase and acid phosphatase), microbial biomass, basal respiration (BR) and substrate-induced respiration (SIR) were measured in rhizosphere, detritusphere and control from 0 - 10 and 10 - 20 cm. The metabolic quotient (qCO2) was calculated as specific indicator for efficiency of microbial substrate utilization. We observed clear differences in enzymes activities at low and high concentrations of substrate. At substrate saturation enzyme activity rates of were significantly higher in rooted plots compared to litter amended plots, whereas at lower concentration no treatment effect could be found. The BR, SIR and qCO2 values were significantly higher at 0 - 10 cm of the planted treatment compared to litter and control plots, revealing a significantly higher respiration at lower efficiency of microbial substrate utilization in the rhizosphere. The Michaelis-Menten constant (Km) decreased with depth, especially for β-glucosidase, acid phosphatase and β-xylosidase, indicating higher substrate affinity of microorganisms in deeper soil and therefore different enzyme systems functioning. The substrate affinity factor (Vmax/Km) increased 2-fold with depth for various enzymes, reflecting a switch of predominantly occurring microbial strategies. Vmax/Km ratio indicated relative domination of zymogenous microbial communities (r-strategists) in 0 - 10 cm depth as compared with 10 - 20 cm depth where the K-strategists dominated.

  18. Wnt signaling regulates multipolar-to-bipolar transition of migrating neurons in the cerebral cortex.

    Science.gov (United States)

    Boitard, Michael; Bocchi, Riccardo; Egervari, Kristof; Petrenko, Volodymyr; Viale, Beatrice; Gremaud, Stéphane; Zgraggen, Eloisa; Salmon, Patrick; Kiss, Jozsef Z

    2015-03-01

    The precise timing of pyramidal cell migration from the ventricular germinal zone to the cortical plate is essential for establishing cortical layers, and migration errors can lead to neurodevelopmental disorders underlying psychiatric and neurological diseases. Here, we report that Wnt canonical as well as non-canonical signaling is active in pyramidal precursors during radial migration. We demonstrate using constitutive and conditional genetic strategies that transient downregulation of canonical Wnt/β-catenin signaling during the multipolar stage plays a critical role in polarizing and orienting cells for radial migration. In addition, we show that reduced canonical Wnt signaling is triggered cell autonomously by time-dependent expression of Wnt5A and activation of non-canonical signaling. We identify ephrin-B1 as a canonical Wnt-signaling-regulated target in control of the multipolar-to-bipolar switch. These findings highlight the critical role of Wnt signaling activity in neuronal positioning during cortical development. PMID:25732825

  19. Activated ras Prevents Downregulation of Bcl-XL Triggered by Detachment from the Extracellular Matrix

    OpenAIRE

    Rosen, Kirill; RAK, Janusz; Leung, Thomas; Dean, Nicholas M.; Kerbel, Robert S.; Filmus, Jorge

    2000-01-01

    Detachment of epithelial cells from the extracellular matrix (ECM) results in a form of apoptosis often referred to as anoikis. Transformation of intestinal epithelial cells by oncogenic ras leads to resistance to anoikis, and this resistance is required for the full manifestation of the malignant phenotype. Previously, we demonstrated that ras-induced inhibition of anoikis in intestinal epithelial cells results, in part, from the ras-induced constitutive downregulation of Bak, a pro-apoptoti...

  20. Intra- and Extracellular Activities of Dicloxacillin against Staphylococcus aureus In Vivo and In Vitro▿

    OpenAIRE

    Sandberg, Anne; Jensen, Klaus Skovbo; Baudoux, Pierre; Van Bambeke, Françoise; Tulkens, Paul M.; Frimodt-Møller, Niels

    2010-01-01

    Antibiotic treatment of Staphylococcus aureus infections is often problematic due to the slow response and recurrences. The intracellular persistence of the staphylococci offers a plausible explanation for the treatment difficulties because of the impaired intracellular efficacies of the antibiotics. The intra- and extracellular time- and concentration-kill relationships were examined in vitro with THP-1 cells and in vivo by use of a mouse peritonitis model. The in vivo model was further used...

  1. Flax Fiber Hydrophobic Extract Inhibits Human Skin Cells Inflammation and Causes Remodeling of Extracellular Matrix and Wound Closure Activation.

    Science.gov (United States)

    Styrczewska, Monika; Kostyn, Anna; Kulma, Anna; Majkowska-Skrobek, Grazyna; Augustyniak, Daria; Prescha, Anna; Czuj, Tadeusz; Szopa, Jan

    2015-01-01

    Inflammation is the basis of many diseases, with chronic wounds amongst them, limiting cell proliferation and tissue regeneration. Our previous preclinical study of flax fiber applied as a wound dressing and analysis of its components impact on the fibroblast transcriptome suggested flax fiber hydrophobic extract use as an anti-inflammatory and wound healing preparation. The extract contains cannabidiol (CBD), phytosterols, and unsaturated fatty acids, showing great promise in wound healing. In in vitro proliferation and wound closure tests the extract activated cell migration and proliferation. The activity of matrix metalloproteinases in skin cells was increased, suggesting activation of extracellular components remodeling. The expression of cytokines was diminished by the extract in a cannabidiol-dependent manner, but β-sitosterol can act synergistically with CBD in inflammation inhibition. Extracellular matrix related genes were also analyzed, considering their importance in further stages of wound healing. The extract activated skin cell matrix remodeling, but the changes were only partially cannabidiol- and β-sitosterol-dependent. The possible role of fatty acids also present in the extract is suggested. The study shows the hydrophobic flax fiber components as wound healing activators, with anti-inflammatory cannabidiol acting in synergy with sterols, and migration and proliferation promoting agents, some of which still require experimental identification.

  2. Impact of warming and drying on microbial activity in subarctic tundra soils: inferences from patterns in extracellular enzyme activity

    Science.gov (United States)

    Schade, J. D.; Natali, S.; Spawn, S.; Sistla, S.; Schuur, E. A. G.

    2014-12-01

    Permafrost contains a large pool of carbon that has accumulated for thousands of years, and remains frozen in organic form. As climate warms, permafrost thaw will increase rates of microbial breakdown of old soil organic matter (SOM), accelerating release of carbon to the atmosphere. Higher rates of microbial decomposition may also release reactive nitrogen, which may increase plant production and carbon fixation. The net effect on atmospheric carbon, and the strength of climate feedback, depends on the balance between direct and indirect effects of increased microbial activity, which depends on changes in soil conditions and microbial responses to them. In particular, soil moisture and availability of C and N for microbes strongly influence soil respiration and primary production. Current understanding of changes in these factors as climate warms is limited. We present results from analysis of soil extracellular enzyme activities (EEA) from a long-term warming and drying experiment in subarctic Alaskan tundra (the CiPEHR experiment) as an indicator of changes in soil microbial activity and relative availability of C and N for microbes. We collected soil samples from control (C), warming (W), and warming + drying (WD) treatments and used fluorometric methods to estimate EEA in shallow (0-5 cm) and deep (5-15) soils. We measured soil moisture, SOM, and C:N, and plant tissue C:N as an indicator of N availability. Activity of N-acquiring enzymes was higher in WD soils at both depths. Carbon EEA in W soils was lower in surface, but higher in deeper soils. We also found significantly lower soil C:N in both W and WD in deeper soils, where C:N was generally lower than surface. In general, EEA results suggest drying leads to increased C availability relative to N. This may be due to lower soil moisture leading to greater aeration of soils in WD plots relative to W plots, which may be saturated due to significant land subsidence. Greater aeration may increase efficiency of

  3. Appetitive Cue-Evoked ERK Signaling in the Nucleus Accumbens Requires NMDA and D1 Dopamine Receptor Activation and Regulates CREB Phosphorylation

    Science.gov (United States)

    Kirschmann, Erin K. Z.; Mauna, Jocelyn C.; Willis, Cory M.; Foster, Rebecca L.; Chipman, Amanda M.; Thiels, Edda

    2014-01-01

    Conditioned stimuli (CS) can modulate reward-seeking behavior. This modulatory effect can be maladaptive and has been implicated in excessive reward seeking and relapse to drug addiction. We previously demonstrated that exposure to an appetitive CS causes an increase in the activation of extracellular signal-regulated kinase (ERK) and cyclic-AMP…

  4. Extracellular cold active lipase from the psychrotrophic Halomonas sp. BRI 8 isolated from the Antarctic sea water

    OpenAIRE

    Vipra Vijay Jadhav; Swanandi Suresh Pote; Amit Yadav; Shouche, Yogesh S.; Rama Kaustubh Bhadekar

    2013-01-01

    An extracellular cold active lipase-producing psychrotrophic bacterium (BRI 8) was isolated from the Antarctic sea water sample. The 16s rRNA sequence study revealed that the isolate belongs to the genus Halomonas (929 bp). The present paper reports optimization of fermentation conditions for production of lipase (EC 3.1.1.3) from Halomonas sp. BRI 8. Highest lipase production was observed in the medium containing olive oil and peptone. The optimum pH and temperature for enzyme catalysis were...

  5. Active regions' setting of the extracellular ligand-binding domain of human interleukin-6 receptor

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The reliable three dimensional (3-D) structure of the extracellular ligand-binding domain (V106-P322) of human interleukin-6 receptor (hIL-6R) has been constructed by means of computer-guided homology modeling techniques using the crystal structure of the extracellular ligand-binding region (K52-L251) of human growth hormone receptor (hGHR) as templet. The space location of some key residues which influence the combination ability between the receptor and the ligand has been observed and the effects of point mutagenesis of the four conservative cysteine residues on the space conformation are analyzed. The results show that the space conformation of the side-chain carboxyl of E305 plays a key role in the ligand-binding ability. Furthermore, the space conformation of the side-chain carboxyl of E305 is very important for the electrostatic potential complementarity between hIL-6R and hIL-6 according to the docking method.

  6. Investigating the fate of activated sludge extracellular proteins in sludge digestion using sodium dodecyl sulfate polyacrylamide gel electrophoresis.

    Science.gov (United States)

    Park, Chul; Helm, Richard F; Novak, John T

    2008-12-01

    The fate of activated sludge extracellular proteins in sludge digestion was investigated using three different cation-associated extraction methods and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Extraction methods used were the cation exchange resin (CER) method for extracting calcium (Ca2+) and magnesium (Mg2+), sulfide extraction for removing iron, and base treatment (pH 10.5) for dissolving aluminum. Extracellular polymeric substances extracted were then subjected to SDS-PAGE, and the resultant protein profiles were examined before and after sludge digestion. The SDS-PAGE results showed that three methods led to different SDS-PAGE profiles for both undigested and digested sludges. The results further revealed that CER-extracted proteins remained mainly undegraded in anaerobic digestion, but were degraded in aerobic digestion. While the fate of sulfide- and base-extracted proteins was not clear for aerobic digestion, their changes in anaerobic digestion were elucidated. Most sulfide-extracted proteins were removed by anaerobic digestion, while the increase in protein band intensity and diversity was observed for base-extracted proteins. These results suggest that activated sludge flocs contain different fractions of proteins that are distinguishable by their association with certain cations and that each fraction undergoes different fates in anaerobic and aerobic digestion. The proteins that were resistant to degradation and generated during anaerobic digestion were identified by liquid chromatography tandem mass spectrometry. Protein identification results and their putative roles in activated sludge and anaerobic digestion are discussed in this study. PMID:19146099

  7. High extracellular Ca2+ stimulates Ca2+-activated Cl- currents in frog parathyroid cells through the mediation of arachidonic acid cascade.

    Directory of Open Access Journals (Sweden)

    Yukio Okada

    Full Text Available Elevation of extracellular Ca(2+ concentration induces intracellular Ca(2+ signaling in parathyroid cells. The response is due to stimulation of the phospholipase C/Ca(2+ pathways, but the direct mechanism responsible for the rise of intracellular Ca(2+ concentration has remained elusive. Here, we describe the electrophysiological property associated with intracellular Ca(2+ signaling in frog parathyroid cells and show that Ca(2+-activated Cl(- channels are activated by intracellular Ca(2+ increase through an inositol 1,4,5-trisphophate (IP(3-independent pathway. High extracellular Ca(2+ induced an outwardly-rectifying conductance in a dose-dependent manner (EC(50 ∼6 mM. The conductance was composed of an instantaneous time-independent component and a slowly activating time-dependent component and displayed a deactivating inward tail current. Extracellular Ca(2+-induced and Ca(2+ dialysis-induced currents reversed at the equilibrium potential of Cl(- and were inhibited by niflumic acid (a specific blocker of Ca(2+-activated Cl(- channel. Gramicidin-perforated whole-cell recording displayed the shift of the reversal potential in extracellular Ca(2+-induced current, suggesting the change of intracellular Cl(- concentration in a few minutes. Extracellular Ca(2+-induced currents displayed a moderate dependency on guanosine triphosphate (GTP. All blockers for phospholipase C, diacylglycerol (DAG lipase, monoacylglycerol (MAG lipase and lipoxygenase inhibited extracellular Ca(2+-induced current. IP(3 dialysis failed to induce conductance increase, but 2-arachidonoylglycerol (2-AG, arachidonic acid and 12S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12(S-HPETE dialysis increased the conductance identical to extracellular Ca(2+-induced conductance. These results indicate that high extracellular Ca(2+ raises intracellular Ca(2+ concentration through the DAG lipase/lipoxygenase pathway, resulting in the activation of Cl(- conductance.

  8. Enhanced airway smooth muscle cell thromboxane receptor signaling via activation of JNK MAPK and extracellular calcium influx

    DEFF Research Database (Denmark)

    Lei, Ying; Cao, Yongxiao; Zhang, Yaping;

    2011-01-01

    airway smooth muscle cells by using an organ culture model and a set of selective pharmacological inhibitors for mitogen-activated protein kinase (MAPK) and calcium signal pathways. Western-blot, immunohistochemistry, myograph and a selective TP receptor agonist U46619 were used for examining TP receptor...... signal proteins and function. Organ culture of rat bronchial segments for up to 48 h induces a time-dependently increased airway contractile response to U46619. This indicates that organ culture increases TP receptor signaling in the airway smooth muscle cells. The enhanced bronchial contraction was...... attenuated by the inhibition of c-Jun N-terminal kinase (JNK) MAPK activity, chelation of extracellular calcium and calcium channel blocker nifedipine, suggesting that JNK MAPK activity and elevated intracellular calcium level are required for the TP receptor signaling. In conclusion, airway smooth muscle...

  9. Cell-free extracellular enzymatic activity is linked to seasonal temperature changes: a case study in the Baltic Sea

    Science.gov (United States)

    Baltar, Federico; Legrand, Catherine; Pinhassi, Jarone

    2016-05-01

    Extracellular enzymatic activities (EEAs) are a crucial step in the degradation of organic matter. Dissolved (cell-free) extracellular enzymes in seawater can make up a significant contribution of the bulk EEA. However, the factors controlling the proportion of dissolved EEA in the marine environment remain unknown. Here we studied the seasonal changes in the proportion of dissolved relative to total EEA (of alkaline phosphatase (APase), β-glucosidase (BGase), and leucine aminopeptidase (LAPase)), in the Baltic Sea for 18 months. The proportion of dissolved EEA ranged between 37 and 100, 0 and 100, and 34 and 100 % for APase, BGase, and LAPase, respectively. A consistent seasonal pattern in the proportion of dissolved EEA was found among all the studied enzymes, with values up to 100 % during winter and hydrolysis rates from microbial dynamics in cold waters. This implies that under cold conditions, cell-free enzymes can contribute to substrate availability at large distances from the producing cell, increasing the dissociation between the hydrolysis of organic compounds and the actual microbes producing the enzymes. This might also suggest a potential effect of global warming on the hydrolysis of organic matter via a reduction of the contribution of cell-free enzymes to the bulk hydrolytic activity.

  10. Response of Nodularia spumigena to pCO2 – Part 2: Exudation and extracellular enzyme activities

    Directory of Open Access Journals (Sweden)

    M. Nausch

    2012-04-01

    Full Text Available The filamentous and diazotrophic cyanobacterium Nodularia spumigena plays a major role in the productivity of the Baltic Sea as it forms extensive blooms regularly. Under phosphorus limiting conditions Nodularia spumigena has a high enzyme affinity for dissolved organic phosphorus (DOP by production and release of alkaline phosphatase. Additionally, it is able to degrade proteinaceous compounds by expressing the extracellular enzyme leucine aminopeptidase. As atmospheric CO2 concentrations are increasing, we expect marine phytoplankton to experience changes in several environmental parameters including pH, temperature, and nutrient availability. The aim of this study was to investigate the combined effect of CO2-induced changes in seawater carbonate chemistry and of phosphate deficiency on the exudation of organic matter, and its subsequent recycling by extracellular enzymes in a Nodularia spumigena culture. Batch cultures of Nodularia spumigena were grown for 15 days aerated with three different pCO2 levels corresponding to values from glacial periods to future values projected for the year 2100. Extracellular enzyme activities as well as changes in organic and inorganic compound concentrations were monitored. CO2 treatment–related effects were identified for cyanobacterial growth, which in turn was influencing exudation and recycling of organic matter by extracellular enzymes. Biomass production was increased by 56.5% and 90.7% in the medium and high pCO2 treatment, respectively, compared to the low pCO2 treatment and simultaneously increasing exudation. During the growth phase significantly more mucinous substances accumulated in the high pCO2 treatment reaching 363 μg Gum Xanthan eq l−1 compared to 269 μg Gum Xanthan eq l−1 in the low pCO2 treatment. However, cell-specific rates did not change. After phosphate depletion, the acquisition of P from DOP by alkaline phosphatase was significantly enhanced. Alkaline phosphatase activities

  11. Participation of Mitogen-activated Protein Kinase in Luteinizing Hormone-induced Differential Regulation of Steroidogenesis and Steroidogenic Gene Expression in Mural and Cumulus Granulosa Cells of Mouse Preovulatory Follicles

    DEFF Research Database (Denmark)

    Su, You-Qiang; Nyegaard, Mette; Overgaard, Michael Toft;

    2006-01-01

    was to investigate whether these processes that commonly occur in mural granulosa cells (MGCs) also occur in cumulus cells, and whether they are mediated by the mitogen-activated protein kinase (MAPK), specifically MAPK3/1 (also commonly known as extracellular signal-regulated kinase 1&2, ERK1/2). The standard...

  12. Role of mitogen- activated protein kinase in myocardial hypertrophy%丝裂原活化蛋白激酶信号途径在心肌肥厚中的作用进展

    Institute of Scientific and Technical Information of China (English)

    黄朝阳; 朱建华

    2005-01-01

    Myocardial hypertrophy is an independent risk factor for cardiac events. Mitogen-activated protein kinases(MAPK), including extracellular signal-regulated kinases, C-jun N-terminal kinases and P38-MAPK, are the common intracellular pathway of transducing hypertrophic signs. All three MAPK subfamilies play an important role in development of myocardial hypertrophy.

  13. Extracellular gentamicin reduces the activity of connexin hemichannels and interferes with purinergic Ca2+ signaling in HeLa cells

    Science.gov (United States)

    Figueroa, Vania A.; Retamal, Mauricio A.; Cea, Luis A.; Salas, José D.; Vargas, Aníbal A.; Verdugo, Christian A.; Jara, Oscar; Martínez, Agustín D.; Sáez, Juan C.

    2014-01-01

    Gap junction channels (GJCs) and hemichannels (HCs) are composed of protein subunits termed connexins (Cxs) and are permeable to ions and small molecules. In most organs, GJCs communicate the cytoplasm of adjacent cells, while HCs communicate the intra and extracellular compartments. In this way, both channel types coordinate physiological responses of cell communities. Cx mutations explain several genetic diseases, including about 50% of autosomal recessive non-syndromic hearing loss. However, the possible involvement of Cxs in the etiology of acquired hearing loss remains virtually unknown. Factors that induce post-lingual hearing loss are diverse, exposure to gentamicin an aminoglycoside antibiotic, being the most common. Gentamicin has been proposed to block GJCs, but its effect on HCs remains unknown. In this work, the effect of gentamicin on the functional state of HCs was studied and its effect on GJCs was reevaluated in HeLa cells stably transfected with Cxs. We focused on Cx26 because it is the main Cx expressed in the cochlea of mammals where it participates in purinergic signaling pathways. We found that gentamicin applied extracellularly reduces the activity of HCs, while dye transfer across GJCs was not affected. HCs were also blocked by streptomycin, another aminoglycoside antibiotic. Gentamicin also reduced the adenosine triphosphate release and the HC-dependent oscillations of cytosolic free-Ca2+ signal. Moreover, gentamicin drastically reduced the Cx26 HC-mediated membrane currents in Xenopus laevis oocytes. Therefore, the extracellular gentamicin-induced inhibition of Cx HCs may adversely affect autocrine and paracrine signaling, including the purinergic one, which might partially explain its ototoxic effects. PMID:25237294

  14. Effects of Dihydroartemisinin and Artemether on the Growth, Chlorophyll Fluorescence, and Extracellular Alkaline Phosphatase Activity of the Cyanobacterium Microcystis aeruginosa

    Science.gov (United States)

    Wang, Shoubing; Xu, Ziran

    2016-01-01

    Increased eutrophication in the recent years has resulted in considerable research focus on identification of methods for preventing cyanobacterial blooms that are rapid and efficient. The objectives of this study were to investigate the effects of dihydroartemisinin and artemether on the growth of Microcystis aeruginosa and to elucidate its mode of action. Variations in cell density, chlorophyll a, soluble protein, malondialdehyde, extracellular alkaline phosphatase activity (APA), and chlorophyll fluorescence parameters (Fv/Fm, ΦPSII, ETR, rapid light curves, fast chlorophyll fluorescence curves on fluorescence intensity, and relative variable fluorescence) were evaluated by lab-cultured experiments. Our results demonstrated that both dihydroartemisinin and artemether inhibited the growth of M.aeruginosa by impairing the photosynthetic center in photosystem II and reducing extracellular APA, with a higher sensitivity exhibited toward artemether. The inhibitory effects of dihydroartemisinin on M.aeruginosa increased with concentration, and the maximum growth inhibitory rate was 42.17% at 24 mg·L-1 after 120h exposure, whereas it was 55.72% at 6 mg·L-1 artemetherafter 120h exposure. Moreover, the chlorophyll fluorescence was significantly inhibited (p<0.05) after 120h exposure to 12 and 24 mg·L-1 dihydroartemisinin. Furthermore, after 120h exposure to 6 mg·L-1 artemether, Fv/Fm, ΦPSII, ETR and rETRmax showed a significant decrease (p<0.01) from initial values of 0.490, 0.516, 17.333, and 104.800, respectively, to 0. One-way analysis of variance showed that 6 mg·L-1 artemether and 24 mg·L-1 dihydroartemisinin had significant inhibitory effects on extracellular APA (p<0.01). The results of this study would be useful to further studies to validate the feasibility of dihydroartemisinin and artemether treatment to inhibit overall cyanobacterial growth in water bodies, before this can be put into practice. PMID:27755566

  15. Diversity and extracellular enzymatic activities of yeasts isolated from King George Island, the sub-Antarctic region

    Directory of Open Access Journals (Sweden)

    Carrasco Mario

    2012-11-01

    Full Text Available Abstract Background Antarctica has been successfully colonized by microorganisms despite presenting adverse conditions for life such as low temperatures, high solar radiation, low nutrient availability and dryness. Although these “cold-loving” microorganisms are recognized as primarily responsible for nutrient and organic matter recycling/mineralization, the yeasts, in particular, remain poorly characterized and understood. The aim of this work was to study the yeast microbiota in soil and water samples collected on King George Island. Results A high number of yeast isolates was obtained from 34 soil and 14 water samples. Molecular analyses based on rDNA sequences revealed 22 yeast species belonging to 12 genera, with Mrakia and Cryptococcus genera containing the highest species diversity. The species Sporidiobolus salmonicolor was by far the most ubiquitous, being identified in 24 isolates from 13 different samples. Most of the yeasts were psychrotolerant and ranged widely in their ability to assimilate carbon sources (consuming from 1 to 27 of the 29 carbon sources tested. All species displayed at least 1 of the 8 extracellular enzyme activities tested. Lipase, amylase and esterase activity dominated, while chitinase and xylanase were less common. Two yeasts identified as Leuconeurospora sp. and Dioszegia fristingensis displayed 6 enzyme activities. Conclusions A high diversity of yeasts was isolated in this work including undescribed species and species not previously isolated from the Antarctic region, including Wickerhamomyces anomalus, which has not been isolated from cold regions in general. The diversity of extracellular enzyme activities, and hence the variety of compounds that the yeasts may degrade or transform, suggests an important nutrient recycling role of microorganisms in this region. These yeasts are of potential use in industrial applications requiring high enzyme activities at low temperatures.

  16. Factor H-related protein 5 interacts with pentraxin 3 and the extracellular matrix and modulates complement activation.

    Science.gov (United States)

    Csincsi, Ádám I; Kopp, Anne; Zöldi, Miklós; Bánlaki, Zsófia; Uzonyi, Barbara; Hebecker, Mario; Caesar, Joseph J E; Pickering, Matthew C; Daigo, Kenji; Hamakubo, Takao; Lea, Susan M; Goicoechea de Jorge, Elena; Józsi, Mihály

    2015-05-15

    The physiological roles of the factor H (FH)-related proteins are controversial and poorly understood. Based on genetic studies, FH-related protein 5 (CFHR5) is implicated in glomerular diseases, such as atypical hemolytic uremic syndrome, dense deposit disease, and CFHR5 nephropathy. CFHR5 was also identified in glomerular immune deposits at the protein level. For CFHR5, weak complement regulatory activity and competition for C3b binding with the plasma complement inhibitor FH have been reported, but its function remains elusive. In this study, we identify pentraxin 3 (PTX3) as a novel ligand of CFHR5. Binding of native CFHR5 to PTX3 was detected in human plasma and the interaction was characterized using recombinant proteins. The binding of PTX3 to CFHR5 is of ∼2-fold higher affinity compared with that of FH. CFHR5 dose-dependently inhibited FH binding to PTX3 and also to the monomeric, denatured form of the short pentraxin C-reactive protein. Binding of PTX3 to CFHR5 resulted in increased C1q binding. Additionally, CFHR5 bound to extracellular matrix in vitro in a dose-dependent manner and competed with FH for binding. Altogether, CFHR5 reduced FH binding and its cofactor activity on pentraxins and the extracellular matrix, while at the same time allowed for enhanced C1q binding. Furthermore, CFHR5 allowed formation of the alternative pathway C3 convertase and supported complement activation. Thus, CFHR5 may locally enhance complement activation via interference with the complement-inhibiting function of FH, by enhancement of C1q binding, and by activating complement, thereby contributing to glomerular disease.

  17. Soluble FGFR4 extracellular domain inhibits FGF19-induced activation of FGFR4 signaling and prevents nonalcoholic fatty liver disease

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Qiang [State Key Laboratory of Stress Cell Biology, School of Life Sciences, Xiamen University, Xiamen (China); The First Affiliated Hospital of Xiamen University, Xiamen (China); Jiang, Yuan; An, Yuan; Zhao, Na; Zhao, Yang [State Key Laboratory of Stress Cell Biology, School of Life Sciences, Xiamen University, Xiamen (China); Yu, Chundong, E-mail: cdyu@xmu.edu.cn [State Key Laboratory of Stress Cell Biology, School of Life Sciences, Xiamen University, Xiamen (China)

    2011-06-17

    Highlights: {yields} Soluble FGFR4 extracellular domain (FGFR4-ECD) was effectively expressed. {yields} FGFR4-ECD inhibited FGF19-induced activation of FGFR4 signaling. {yields} FGFR4-ECD reduced palmitic acid-induced steatosis of HepG2 cells. {yields} FGFR4-ECD reduced tetracycline-induced fatty liver in mice. {yields} FGFR4-ECD partially restored tetracycline-repressed PPAR{alpha} expression. -- Abstract: Fibroblast growth factor receptor 4 (FGFR4) is a transmembrane tyrosine kinase receptor that plays a crucial role in the regulation of hepatic bile acid and lipid metabolism. FGFR4 underlies high-fat diet-induced hepatic steatosis, suggesting that inhibition of FGFR4 activation may be an effective way to prevent or treat nonalcoholic fatty liver disease (NAFLD). To determine whether neutralization of FGFR4 ligands by soluble FGFR4 extracellular domain (FGFR4-ECD) can inhibit the activation of FGFR4, we constructed FGFR4-ECD expression vector and showed that FGFR4-ECD was effectively expressed in cells and secreted into culture medium. FGFR4-ECD inhibited FGF19-induced activation of FGFR4 signaling and reduced steatosis of HepG2 induced by palmitic acid in vitro. Furthermore, in a tetracycline-induced fatty liver model, expression of FGFR4-ECD in mouse liver reduced the accumulation of hepatic lipids and partially restored the expression of peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}), which promotes the mitochondrial fatty acid beta-oxidation but is repressed by tetracycline. Taken together, these results demonstrate that FGFR4-ECD can block FGFR4 signaling and prevent hepatic steatosis, highlighting the potential value of inhibition of FGFR4 signaling as a method for therapeutic intervention against NAFLD.

  18. Ras-Mek-Erk signaling regulates Nf1 heterozygous neointima formation.

    Science.gov (United States)

    Stansfield, Brian K; Bessler, Waylan K; Mali, Raghuveer; Mund, Julie A; Downing, Brandon D; Kapur, Reuben; Ingram, David A

    2014-01-01

    Neurofibromatosis type 1 (NF1) results from mutations in the NF1 tumor-suppressor gene, which encodes neurofibromin, a negative regulator of diverse Ras signaling cascades. Arterial stenosis is a nonneoplastic manifestation of NF1 that predisposes some patients to debilitating morbidity and sudden death. Recent murine studies demonstrate that Nf1 heterozygosity (Nf1(+/-)) in monocytes/macrophages significantly enhances intimal proliferation after arterial injury. However, the downstream Ras effector pathway responsible for this phenotype is unknown. Based on in vitro assays demonstrating enhanced extracellular signal-related kinase (Erk) signaling in Nf1(+/-) macrophages and vascular smooth muscle cells and in vivo evidence of Erk amplification without alteration of phosphatidylinositol 3-kinase signaling in Nf1(+/-) neointimas, we tested the hypothesis that Ras-Erk signaling regulates intimal proliferation in a murine model of NF1 arterial stenosis. By using a well-established in vivo model of inflammatory cell migration and standard cell culture, neurofibromin-deficient macrophages demonstrate enhanced sensitivity to growth factor stimulation in vivo and in vitro, which is significantly diminished in the presence of PD0325901, a specific inhibitor of Ras-Erk signaling in phase 2 clinical trials for cancer. After carotid artery injury, Nf1(+/-) mice demonstrated increased intimal proliferation compared with wild-type mice. Daily administration of PD0325901 significantly reduced Nf1(+/-) neointima formation to levels of wild-type mice. These studies identify the Ras-Erk pathway in neurofibromin-deficient macrophages as the aberrant pathway responsible for enhanced neointima formation. PMID:24211110

  19. Nature of extracellular signal that triggers RhoA/ROCK activation for the basal internal anal sphincter tone in humans.

    Science.gov (United States)

    Rattan, Satish; Singh, Jagmohan; Kumar, Sumit; Phillips, Benjamin

    2015-06-01

    The extracellular signal that triggers activation of rho-associated kinase (RhoA/ROCK), the major molecular determinant of basal internal anal sphincter (IAS) smooth muscle tone, is not known. Using human IAS tissues, we identified the presence of the biosynthetic machineries for angiotensin II (ANG II), thromboxane A2 (TXA2), and prostaglandin F2α (PGF2α). These end products of the renin-angiotensin system (RAS) (ANG II) and arachidonic acid (TXA2 and PGF2α) pathways and their effects in human IAS vs. rectal smooth muscle (RSM) were studied. A multipronged approach utilizing immunocytochemistry, Western blot analyses, and force measurements was implemented. Additionally, in a systematic analysis of the effects of respective inhibitors along different steps of biosynthesis and those of antagonists, their end products were evaluated either individually or in combination. To further describe the molecular mechanism for the IAS tone via these pathways, we monitored RhoA/ROCK activation and its signal transduction cascade. Data showed characteristically higher expression of biosynthetic machineries of RAS and AA pathways in the IAS compared with the RSM. Additionally, specific inhibition of the arachidonic acid (AA) pathway caused ~80% decrease in the IAS tone, whereas that of RAS lead to ~20% decrease. Signal transduction studies revealed that the end products of both AA and RAS pathways cause increase in the IAS tone via activation of RhoA/ROCK. Both AA and RAS (via the release of their end products TXA2, PGF2α, and ANG II, respectively), provide extracellular signals which activate RhoA/ROCK for the maintenance of the basal tone in human IAS.

  20. Structural characterization and anti-aging activity of a novel extracellular polysaccharide from fungus Phellinus sp. in a mammalian system.

    Science.gov (United States)

    Ma, Xiao-Kui; Guo, Dan Dan; Peterson, Eric Charles; Dun, Ying; Li, Dan Yang

    2016-08-10

    Little is known about the chemical structure of purified extracellular polysaccharides from Phellinus sp., a fungal species with known medicinal properties. A combination of IR spectroscopy, methylation analysis and NMR were performed for the structural analysis of a purified extracellular polysaccharide derived from Phellinus sp. culture, denoted as SHP-1, along with an evaluation of the anti-aging effect in vivo of the polysaccharide supplementation. The structure of SHP-1 was established, with a backbone composed of →2,4)-α-d-glucopyranose-(1→ and →2)-β-d-mannopyranose-(1→ and two terminal glucopyranose branches. Biochemical analysis from mammalian animal experiments demonstrated that SHP-1 possesses the ability to enhance antioxidant enzyme activities, such as catalase (CAT) and superoxide dismutase (SOD) activities, Trolox equivalent antioxidant capacity (TEAC) in serum of d-galactose-aged mice, while reducing lipofuscin levels, another indicator of cell aging, indicating a potential association with anti-aging activities in a dose dependent manner. This compound had a favourable influence on immune organ indices, and a marked amelioration ability of histopathological hepatic lesions such as necrosis, karyolysis and reduced inflammation and apoptosis in mouse hepatocytes. These results suggest that SHP-1 has strong antioxidant activities and a significant protective effect against oxidative stress or hepatotoxicity induced by d-galactose in mice and it could be developed as a food ingredient or a pharmaceutical to prevent many age-associated diseases such as major depressive disorder and hepatotoxicity. To our knowledge, this is the first report on the antioxidant effects of a novel purified exopolysaccharide derived from Phellinus sp. PMID:27405813

  1. Apoptosis signal-regulating kinase 1 mediates denbinobin-induced apoptosis in human lung adenocarcinoma cells

    Directory of Open Access Journals (Sweden)

    Pan Shiow-Lin

    2009-05-01

    Full Text Available Abstract In the present study, we explore the role of apoptosis signal-regulating kinase 1 (ASK1 in denbinobin-induced apoptosis in human lung adenocarcinoma (A549 cells. Denbinobin-induced cell apoptosis was attenuated by an ASK1 dominant-negative mutant (ASK1DN, two antioxidants (N-acetyl-L-cysteine (NAC and glutathione (GSH, a c-Jun N-terminal kinase (JNK inhibitor (SP600125, and an activator protein-1 (AP-1 inhibitor (curcumin. Treatment of A549 cells with denbinobin caused increases in ASK1 activity and reactive oxygen species (ROS production, and these effects were inhibited by NAC and GSH. Stimulation of A549 cells with denbinobin caused JNK activation; this effect was markedly inhibited by NAC, GSH, and ASK1DN. Denbinobin induced c-Jun phosphorylation, the formation of an AP-1-specific DNA-protein complex, and Bim expression. Bim knockdown using a bim short interfering RNA strategy also reduced denbinobin-induced A549 cell apoptosis. The denbinobin-mediated increases in c-Jun phosphorylation and Bim expression were inhibited by NAC, GSH, SP600125, ASK1DN, JNK1DN, and JNK2DN. These results suggest that denbinobin might activate ASK1 through ROS production to cause JNK/AP-1 activation, which in turn induces Bim expression, and ultimately results in A549 cell apoptosis.

  2. Aryl Hydrocarbon Receptor Activation by TCDD Modulates Expression of Extracellular Matrix Remodeling Genes during Experimental Liver Fibrosis

    Science.gov (United States)

    Lamb, Cheri L.; Cholico, Giovan N.; Perkins, Daniel E.; Fewkes, Michael T.; Oxford, Julia Thom; Lujan, Trevor J.; Morrill, Erica E.

    2016-01-01

    The aryl hydrocarbon receptor (AhR) is a soluble, ligand-activated transcription factor that mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Increasing evidence implicates the AhR in regulating extracellular matrix (ECM) homeostasis. We recently reported that TCDD increased necroinflammation and myofibroblast activation during liver injury elicited by carbon tetrachloride (CCl4). However, TCDD did not increase collagen deposition or exacerbate fibrosis in CCl4-treated mice, which raises the possibility that TCDD may enhance ECM turnover. The goal of this study was to determine how TCDD impacts ECM remodeling gene expression in the liver. Male C57BL/6 mice were treated for 8 weeks with 0.5 mL/kg CCl4, and TCDD (20 μg/kg) was administered during the last two weeks. Results indicate that TCDD increased mRNA levels of procollagen types I, III, IV, and VI and the collagen processing molecules HSP47 and lysyl oxidase. TCDD also increased gelatinase activity and mRNA levels of matrix metalloproteinase- (MMP-) 3, MMP-8, MMP-9, and MMP-13. Furthermore, TCDD modulated expression of genes in the plasminogen activator/plasmin system, which regulates MMP activation, and it also increased TIMP1 gene expression. These findings support the notion that AhR activation by TCDD dysregulates ECM remodeling gene expression and may facilitate ECM metabolism despite increased liver injury.

  3. DAMPs-activated neutrophil extracellular trap exacerbates sterile inflammatory liver injury

    Science.gov (United States)

    Huang, Hai; Tohme, Samer; Al-Khafaji, Ahmed B; Tai, Sheng; Loughran, Patricia; Chen, Li; Wang, Shu; Kim, Jiyun; Billiar, Timothy; Wang, Yanming; Tsung, Allan

    2015-01-01

    Innate immunity plays a crucial role in the response to sterile inflammation such as liver ischemia/reperfusion (I/R) injury. The initiation of liver I/R injury results in the release of damage associated molecular patterns (DAMPs), which trigger innate immune and inflammatory cascade via pattern recognition receptors. Neutrophils are recruited to the liver after I/R and contribute to the organ damage, innate immune and inflammatory responses. Formation of neutrophil extracellular trap (NET) has been recently found in response to various stimuli. However, the role of NETs during liver I/R injury remains unknown. We show that NETs form in the sinusoids of ischemic liver lobes in vivo. This was associated with increased NET markers, serum level of myeloperoxidase (MPO)-DNA complexes and tissue level of citrullinated-histone H3 compared to control mice. Treatment with peptidyl-arginine-deiminase (PAD) 4 inhibitor or DNase I significantly protected hepatocytes and reduced inflammation after liver I/R as evidenced by inhibition of NET formation, indicating the pathophysiological role of NETs in liver I/R injury. In vitro, NETs increase hepatocyte death and induce Kupffer cells to release proinflammatory cytokines. DAMPs, such as HMGB1 and histones, released by injured hepatocytes stimulate NET formation through Toll-like receptor (TLR4)- and TLR9-MyD88 signaling pathways. After neutrophil depletion in mice, the adoptive transfer of TLR4 knockout (KO) or TLR9 KO neutrophils confers significant protection from liver I/R injury with significant decrease in NET formation. In addition, we found inhibition of NET formation by PAD4 inhibitor or DNase I reduces HMGB1 and histone-mediated liver I/R injury. Conclusion DAMPs released during liver I/R promotes NET formation through TLRs signaling pathway. Development of NETs subsequently exacerbates organ damage and initiates inflammatory responses during liver I/R. PMID:25855125

  4. Secretion and Reversible Assembly of Extracellular-like Matrix by Enzyme-Active Colloidosome-Based Protocells.

    Science.gov (United States)

    Akkarachaneeyakorn, Khrongkhwan; Li, Mei; Davis, Sean A; Mann, Stephen

    2016-03-29

    The secretion and reversible assembly of an extracellular-like matrix by enzyme-active inorganic protocells (colloidosomes) is described. Addition of N-fluorenyl-methoxycarbonyl-tyrosine-(O)-phosphate to an aqueous suspension of alkaline phosphatase-containing colloidosomes results in molecular uptake and dephosphorylation to produce a time-dependent sequence of supramolecular hydrogel motifs (outer membrane wall, cytoskeletal-like interior and extra-protocellular matrix) that are integrated and remodelled within the microcapsule architecture and surrounding environment. Heat-induced disassembly of the extra-protocellular matrix followed by cooling produces colloidosomes with a densely packed hydrogel interior. These procedures are exploited for the fabrication of nested colloidosomes with spatially delineated regions of hydrogelation. PMID:26981922

  5. Characterization of nucleoside triphosphate diphosphohydrolase activity in Trichomonas gallinae and the influence of penicillin and streptomycin in extracellular nucleotide hydrolysis.

    Science.gov (United States)

    Borges, Fernanda Pires; de Brum Vieira, Patrícia; Wiltuschnig, Renata C M; Tasca, Tiana; De Carli, Geraldo Attilio; Bonan, Carla Denise

    2008-06-01

    Here we described an nucleoside triphosphate diphosphohydrolase (NTPDase) activity in living trophozoites of Trichomonas gallinae. The enzyme hydrolyzes a variety of purine and pyrimidine nucleoside di- and triphosphates in an optimum pH range of 6.0-8.0. This enzyme activity was activated by high concentrations of divalent cations, such as calcium and magnesium. Contaminant activities were ruled out because the enzyme was not inhibited by classical inhibitors of ATPases (ouabain, 5.0 mM sodium azide, oligomycin) and alkaline phosphatases (levamisole). A significant inhibition of ATP hydrolysis (38%) was observed in the presence of 20 mM sodium azide. Sodium orthovanadate inhibited ATP and ADP hydrolysis (24% and 78%), respectively. The apparent K(M) (Michaelis constant) values were 667.62+/-13 microM for ATP and 125+/-5.3 microM for ADP. V(max) (maximum velocity) values were 0.44+/-0.007 nmol Pi min(-1) per 10(6) trichomonads and 0.91+/-0.12 nmol Pi min(-1) per 10(6) trichomonads for ATP and ADP, respectively. Moreover, we showed a marked decrease in ATP, ADP and AMP hydrolysis when the parasites were grown in the presence of penicillin and streptomycin. The existence of an NTPDase activity in T. gallinae may be involved in pathogenicity, protecting the parasite from the cytolytic effects of the extracellular nucleotides. PMID:18422631

  6. Oxidized LDL induced extracellular trap formation in human neutrophils via TLR-PKC-IRAK-MAPK and NADPH-oxidase activation.

    Science.gov (United States)

    Awasthi, Deepika; Nagarkoti, Sheela; Kumar, Amit; Dubey, Megha; Singh, Abhishek Kumar; Pathak, Priya; Chandra, Tulika; Barthwal, Manoj Kumar; Dikshit, Madhu

    2016-04-01

    Neutrophil extracellular traps (NETs) formation was initially linked with host defence and extracellular killing of pathogens. However, recent studies have highlighted their inflammatory potential. Oxidized low density lipoprotein (oxLDL) has been implicated as an independent risk factor in various acute or chronic inflammatory diseases including systemic inflammatory response syndrome (SIRS). In the present study we investigated effect of oxLDL on NETs formation and elucidated the underlying signalling mechanism. Treatment of oxLDL to adhered PMNs led to a time and concentration dependent ROS generation and NETs formation. OxLDL induced free radical formation and NETs release were significantly prevented in presence of NADPH oxidase (NOX) inhibitors suggesting role of NOX activation in oxLDL induced NETs release. Blocking of both toll like receptor (TLR)-2 and 6 significantly reduced oxLDL induced NETs formation indicating requirement of both the receptors. We further identified Protein kinase C (PKC), Interleukin-1 receptor associated kinase (IRAKs), mitogen-activated protein kinase (MAPK) pathway as downstream intracellular signalling mediators involved in oxLDL induced NETs formation. OxLDL components such as oxidized phospholipids (lysophosphatidylcholine (LPC) and oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (oxPAPC)) were most potent NETs inducers and might be crucial for oxLDL mediating NETs release. Other components like, oxysterols, malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) were however less potent as compared to oxidized phospholipids. This study thus demonstrates for the first time that treatment of human PMNs with oxLDL or its various oxidized phopholipid component mediated NETs release, implying their role in the pathogenesis of inflammatory diseases such as SIRS. PMID:26774674

  7. Extracellular cold active lipase from the psychrotrophic Halomonas sp. BRI 8 isolated from the Antarctic sea water

    Directory of Open Access Journals (Sweden)

    Vipra Vijay Jadhav

    2013-12-01

    Full Text Available An extracellular cold active lipase-producing psychrotrophic bacterium (BRI 8 was isolated from the Antarctic sea water sample. The 16s rRNA sequence study revealed that the isolate belongs to the genus Halomonas (929 bp. The present paper reports optimization of fermentation conditions for production of lipase (EC 3.1.1.3 from Halomonas sp. BRI 8. Highest lipase production was observed in the medium containing olive oil and peptone. The optimum pH and temperature for enzyme catalysis were 7.0 and 10°C respectively. The enzyme was relatively more stable in acidic pH range and retained 50% activity when incubated at 30°C for 1 h. The enzyme was stable in various organic solvents and showed more than 100% activity in presence of isoamyl alcohol. Significant enzyme activity was also observed in the presence of metal ions and detergents. The molecular mass of partially purified lipase was found to be around 66 kD.

  8. AMPK Activation by Metformin Suppresses Abnormal Extracellular Matrix Remodeling in Adipose Tissue and Ameliorates Insulin Resistance in Obesity.

    Science.gov (United States)

    Luo, Ting; Nocon, Allison; Fry, Jessica; Sherban, Alex; Rui, Xianliang; Jiang, Bingbing; Xu, X Julia; Han, Jingyan; Yan, Yun; Yang, Qin; Li, Qifu; Zang, Mengwei

    2016-08-01

    Fibrosis is emerging as a hallmark of metabolically dysregulated white adipose tissue (WAT) in obesity. Although adipose tissue fibrosis impairs adipocyte plasticity, little is known about how aberrant extracellular matrix (ECM) remodeling of WAT is initiated during the development of obesity. Here we show that treatment with the antidiabetic drug metformin inhibits excessive ECM deposition in WAT of ob/ob mice and mice with diet-induced obesity, as evidenced by decreased collagen deposition surrounding adipocytes and expression of fibrotic genes including the collagen cross-linking regulator LOX Inhibition of interstitial fibrosis by metformin is likely attributable to the activation of AMPK and the suppression of transforming growth factor-β1 (TGF-β1)/Smad3 signaling, leading to enhanced systemic insulin sensitivity. The ability of metformin to repress TGF-β1-induced fibrogenesis is abolished by the dominant negative AMPK in primary cells from the stromal vascular fraction. TGF-β1-induced insulin resistance is suppressed by AMPK agonists and the constitutively active AMPK in 3T3L1 adipocytes. In omental fat depots of obese humans, interstitial fibrosis is also associated with AMPK inactivation, TGF-β1/Smad3 induction, aberrant ECM production, myofibroblast activation, and adipocyte apoptosis. Collectively, integrated AMPK activation and TGF-β1/Smad3 inhibition may provide a potential therapeutic approach to maintain ECM flexibility and combat chronically uncontrolled adipose tissue expansion in obesity. PMID:27207538

  9. Wnt Signaling Regulates Multipolar-to-Bipolar Transition of Migrating Neurons in the Cerebral Cortex

    Directory of Open Access Journals (Sweden)

    Michael Boitard

    2015-03-01

    Full Text Available The precise timing of pyramidal cell migration from the ventricular germinal zone to the cortical plate is essential for establishing cortical layers, and migration errors can lead to neurodevelopmental disorders underlying psychiatric and neurological diseases. Here, we report that Wnt canonical as well as non-canonical signaling is active in pyramidal precursors during radial migration. We demonstrate using constitutive and conditional genetic strategies that transient downregulation of canonical Wnt/β-catenin signaling during the multipolar stage plays a critical role in polarizing and orienting cells for radial migration. In addition, we show that reduced canonical Wnt signaling is triggered cell autonomously by time-dependent expression of Wnt5A and activation of non-canonical signaling. We identify ephrin-B1 as a canonical Wnt-signaling-regulated target in control of the multipolar-to-bipolar switch. These findings highlight the critical role of Wnt signaling activity in neuronal positioning during cortical development.

  10. Extracellular acidification activates ovarian cancer G-protein-coupled receptor 1 and GPR4 homologs of zebra fish

    Energy Technology Data Exchange (ETDEWEB)

    Mochimaru, Yuta [Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571 (Japan); Azuma, Morio [Laboratory of Regulatory Biology, Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama 930-8555 (Japan); Oshima, Natsuki; Ichijo, Yuta; Satou, Kazuhiro [Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571 (Japan); Matsuda, Kouhei [Laboratory of Regulatory Biology, Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama 930-8555 (Japan); Asaoka, Yoichi; Nishina, Hiroshi [Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510 (Japan); Nakakura, Takashi [Department of Anatomy, Graduate School of Medicine, Teikyo University, 2-11-1 Kaga Itabashi-Ku, Tokyo 173-8605 (Japan); Mogi, Chihiro; Sato, Koichi; Okajima, Fumikazu [Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512 (Japan); Tomura, Hideaki, E-mail: tomurah@meiji.ac.jp [Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571 (Japan)

    2015-02-20

    Mammalian ovarian G-protein-coupled receptor 1 (OGR1) and GPR4 are identified as a proton-sensing G-protein-coupled receptor coupling to multiple intracellular signaling pathways. In the present study, we examined whether zebra fish OGR1 and GPR4 homologs (zOGR1 and zGPR4) could sense protons and activate the multiple intracellular signaling pathways and, if so, whether the similar positions of histidine residue, which is critical for sensing protons in mammalian OGR and GPR4, also play a role to sense protons and activate the multiple signaling pathways in the zebra fish receptors. We found that extracellular acidic pH stimulated CRE-, SRE-, and NFAT-promoter activities in zOGR1 overexpressed cells and stimulated CRE- and SRE- but not NFAT-promoter activities in zGPR4 overexpressed cells. The substitution of histidine residues at the 12th, 15th, 162th, and 264th positions from the N-terminal of zOGR1 with phenylalanine attenuated the proton-induced SRE-promoter activities. The mutation of the histidine residue at the 78th but not the 84th position from the N-terminal of zGPR4 to phenylalanine attenuated the proton-induced SRE-promoter activities. These results suggest that zOGR1 and zGPR4 are also proton-sensing G-protein-coupled receptors, and the receptor activation mechanisms may be similar to those of the mammalian receptors. - Highlights: • Zebra fish OGR1 and GPR4 homologs (zOGR1, zGPR4) are proton-sensing receptors. • The signaling pathways activated by zOGR1 and zGPR4 are different. • Histidine residues critical for sensing protons are conserved.

  11. Active conformation of the erythropoietin receptor: random and cysteine-scanning mutagenesis of the extracellular juxtamembrane and transmembrane domains.

    Science.gov (United States)

    Lu, Xiaohui; Gross, Alec W; Lodish, Harvey F

    2006-03-17

    In the absence of erythropoietin (Epo) cell surface Epo receptors (EpoR) are dimeric; dimerization is mediated mainly by the transmembrane domain. Binding of Epo changes the orientation of the two receptor subunits. This conformational change is transmitted through the juxtamembrane and transmembrane domains, leading to activation of JAK2 kinase and induction of proliferation and survival signals. To define the active EpoR conformation(s) we screened libraries of EpoRs with random mutations in the transmembrane domain and identified several point mutations that activate the EpoR in the absence of ligand, including changes of either of the first two transmembrane domain residues (Leu(226) and Ile(227)) to cysteine. Following this discovery, we performed cysteine-scanning mutagenesis in the EpoR juxtamembrane and transmembrane domains. Many mutants formed disulfide-linked receptor dimers, but only EpoR dimers linked by cysteines at positions 223, 226, or 227 activated EpoR signal transduction pathways and supported proliferation of Ba/F3 cells in the absence of cytokines. These data suggest that activation of dimeric EpoR by Epo binding is achieved by reorienting the EpoR transmembrane and the connected cytosolic domains and that certain disulfide-bonded dimers represent the activated dimeric conformation of the EpoR, constitutively activating downstream signaling. Based on our data and the previously determined structure of Epo bound to a dimer of the EpoR extracellular domain, we present a model of the active and inactive conformations of the Epo receptor.

  12. A Transcriptional Mechanism Integrating Inputs from Extracellular Signals to Activate Hippocampal Stem Cells

    OpenAIRE

    Andersen, Jimena; Urbán, Noelia; Achimastou, Angeliki; Ito, Ayako; Simic, Milesa; Ullom, Kristy; Martynoga, Ben; Lebel, Mélanie; Göritz, Christian; Frisén, Jonas; Nakafuku, Masato; Guillemot, François

    2014-01-01

    Summary The activity of adult stem cells is regulated by signals emanating from the surrounding tissue. Many niche signals have been identified, but it is unclear how they influence the choice of stem cells to remain quiescent or divide. Here we show that when stem cells of the adult hippocampus receive activating signals, they first induce the expression of the transcription factor Ascl1 and only subsequently exit quiescence. Moreover, lowering Ascl1 expression reduces the proliferation rate...

  13. Production, characterization, gene cloning, and nematocidal activity of the extracellular protease from Stenotrophomonas maltophilia N4.

    Science.gov (United States)

    Jankiewicz, Urszula; Larkowska, Ewa; Swiontek Brzezinska, Maria

    2016-06-01

    A rhizosphere strain of the bacterium Stenotrophomonas maltophilia N4 secretes the serine protease PN4, whose molecular mass is approximately 42 kDa. The optimal temperature for the enzyme activity of the 11-fold purified protein was 50°C and the optimal pH was 10.5. The activity of the enzyme was strongly inhibited by specific serine protease inhibitors, which allowed for its classification as an alkaline serine protease family. Ca(2+) ions stimulated the activity of the protease PN4, while Mg(2+) ions stabilized its activity, and Zn(2+) and Cd(2+) ions strongly inhibited its activity. The enzyme has broad substrate specificity. For example, it is able to hydrolyse casein, keratin, albumin, haemoglobin, and gelatin, as well as the insoluble modified substrates azure keratin and azocoll. The gene that encodes the 1740 bp precursor form of the enzyme (accession number: LC031815) was cloned. We then deduced that its amino acid sequence includes the region of the conserved domain of the S8 family of peptidases as well as the catalytic triad Asp/His/Ser. The bacterial culture fluid as well as the purified protease PN4 demonstrated biocidal activity with regard to the nematodes Caenorhabditis elegans and Panagrellus spp. PMID:26896861

  14. Extracellular Polymeric Substances in Activated Sludge Flocs: Extraction, Identification, and Investigation of Their Link with Cations and Fate in Sludge Digestion

    OpenAIRE

    Park, Chul

    2007-01-01

    Extracellular polymeric substances (EPS) in activated sludge are known to account for the flocculent nature of activated sludge. Extensive studies over the last few decades have attempted to extract and characterize activated sludge EPS, but a lack of agreement between studies has also been quite common. The molecular makeup of EPS has, however, remained nearly unexplored, leaving their identity, function, and fate over various stages in the activated sludge system mainly unknown. In spite o...

  15. Chelation of Membrane-Bound Cations by Extracellular DNA Activates the Type VI Secretion System in Pseudomonas aeruginosa.

    Science.gov (United States)

    Wilton, Mike; Wong, Megan J Q; Tang, Le; Liang, Xiaoye; Moore, Richard; Parkins, Michael D; Lewenza, Shawn; Dong, Tao G

    2016-08-01

    Pseudomonas aeruginosa employs its type VI secretion system (T6SS) as a highly effective and tightly regulated weapon to deliver toxic molecules to target cells. T6SS-secreted proteins of P. aeruginosa can be detected in the sputum of cystic fibrosis (CF) patients, who typically present a chronic and polymicrobial lung infection. However, the mechanism of T6SS activation in the CF lung is not fully understood. Here we demonstrate that extracellular DNA (eDNA), abundant within the CF airways, stimulates the dynamics of the H1-T6SS cluster apparatus in Pseudomonas aeruginosa PAO1. Addition of Mg(2+) or DNase with eDNA abolished such activation, while treatment with EDTA mimicked the eDNA effect, suggesting that the eDNA-mediated effect is due to chelation of outer membrane-bound cations. DNA-activated H1-T6SS enables P. aeruginosa to nonselectively attack neighboring species regardless of whether or not it was provoked. Because of the importance of the T6SS in interspecies interactions and the prevalence of eDNA in the environments that P. aeruginosa inhabits, our report reveals an important adaptation strategy that likely contributes to the competitive fitness of P. aeruginosa in polymicrobial communities. PMID:27271742

  16. Chelation of Membrane-Bound Cations by Extracellular DNA Activates the Type VI Secretion System in Pseudomonas aeruginosa.

    Science.gov (United States)

    Wilton, Mike; Wong, Megan J Q; Tang, Le; Liang, Xiaoye; Moore, Richard; Parkins, Michael D; Lewenza, Shawn; Dong, Tao G

    2016-08-01

    Pseudomonas aeruginosa employs its type VI secretion system (T6SS) as a highly effective and tightly regulated weapon to deliver toxic molecules to target cells. T6SS-secreted proteins of P. aeruginosa can be detected in the sputum of cystic fibrosis (CF) patients, who typically present a chronic and polymicrobial lung infection. However, the mechanism of T6SS activation in the CF lung is not fully understood. Here we demonstrate that extracellular DNA (eDNA), abundant within the CF airways, stimulates the dynamics of the H1-T6SS cluster apparatus in Pseudomonas aeruginosa PAO1. Addition of Mg(2+) or DNase with eDNA abolished such activation, while treatment with EDTA mimicked the eDNA effect, suggesting that the eDNA-mediated effect is due to chelation of outer membrane-bound cations. DNA-activated H1-T6SS enables P. aeruginosa to nonselectively attack neighboring species regardless of whether or not it was provoked. Because of the importance of the T6SS in interspecies interactions and the prevalence of eDNA in the environments that P. aeruginosa inhabits, our report reveals an important adaptation strategy that likely contributes to the competitive fitness of P. aeruginosa in polymicrobial communities.

  17. 2,4,6-Trichlorophenol mediated increases in extracellular peroxidase activity in three species of Lemnaceae.

    Science.gov (United States)

    Biswas, Dilip K; Scannell, Gillian; Akhmetov, Nurlan; Fitzpatrick, Dara; Jansen, Marcel A K

    2010-11-01

    Chlorinated phenols, or chlorophenols, are persistent priority pollutants that are widespread in the environment. Class III peroxidases are well-characterised plant enzymes that can catalyse the oxidative dechlorination of chlorophenols. Expression of these enzymes by plants is commonly associated with plant stress, therefore limiting scope for phytoremediation. In this study, we have quantitatively compared peroxidase activity and phytotoxicity as a function of 2,4,6-trichlorophenol (TCP) concentration in three species of Lemnaceae; Lemna minor, Lemna gibba and Landoltia punctata. Effects of TCP on the growth rates of the three species differed considerably with L. punctata being the most tolerant species. TCP also affected photosynthetic parameters, causing a decrease in open photosystem II reaction centres (qP) and, in L. punctata only, a decrease in non-photochemical quenching (qN). In parallel, TCP exposure resulted in increased peroxidase activity in all three species. Peroxidase activity in L. minor and L. gibba displayed an inverse relationship with biomass accumulation, i.e. the more growth reduction the more peroxidase activity. In contrast, induction of peroxidase activity in L. punctata was bi-phasic, with a TCP-induced activity peak at concentrations that had no major effect on growth, and further induction under phytotoxic concentrations. The mechanism by which L. punctata recognises and responds to low concentrations of an anthropogenic compound, in the absence of wide-ranging stress, remains enigmatic. However, we conclude that this "window" of peroxidase production in the absence of major growth inhibition offers potential for the development of sustainable, peroxidise-mediated phytoremediation systems. PMID:20810175

  18. Pneumococcal hydrogen peroxide-induced stress signaling regulates inflammatory genes.

    Science.gov (United States)

    Loose, Maria; Hudel, Martina; Zimmer, Klaus-Peter; Garcia, Ernesto; Hammerschmidt, Sven; Lucas, Rudolf; Chakraborty, Trinad; Pillich, Helena

    2015-01-15

    Microbial infections can induce aberrant responses in cellular stress pathways, leading to translational attenuation, metabolic restriction, and activation of oxidative stress, with detrimental effects on cell survival. Here we show that infection of human airway epithelial cells with Streptococcus pneumoniae leads to induction of endoplasmic reticulum (ER) and oxidative stress, activation of mitogen-associated protein kinase (MAPK) signaling pathways, and regulation of their respective target genes. We identify pneumococcal H2O2 as the causative agent for these responses, as both catalase-treated and pyruvate oxidase-deficient bacteria lacked these activities. Pneumococcal H2O2 induced nuclear NF-κB translocation and transcription of proinflammatory cytokines. Inhibition of translational arrest and ER stress by salubrinal or of MAPK signaling pathways attenuate cytokine transcription. These results provide strong evidence for the notion that inhibition of translation is an important host pathway in monitoring harmful pathogen-associated activities, thereby enabling differentiation between pathogenic and nonpathogenic bacteria. PMID:25183769

  19. Extracellular engagement of ADAM12 induces clusters of invadopodia with localized ectodomain shedding activity

    DEFF Research Database (Denmark)

    Albrechtsen, Reidar; Hansen, Dorte Stautz; Sanjay, Archana;

    2011-01-01

    -Src interaction site in the ADAM12 cytoplasmic domain, but was independent of the catalytic activity of ADAM12. Caveolin-1 and transmembrane protease MMP14/MT1-MMP were both present in the ADAM12-induced clusters of invadopodia, and cholesterol depletion prevented their formation, suggesting that lipid-raft...

  20. Carbon and Nitrogen Sources Influence Tricalcium Phosphate Solubilization and Extracellular Phosphatase Activity by Talaromyces flavus.

    Science.gov (United States)

    Stefanoni Rubio, P J; Godoy, M S; Della Mónica, I F; Pettinari, M J; Godeas, A M; Scervino, J M

    2016-01-01

    The aim of this work was to study phosphate (P) solubilization (and the processes involved in this event) by Talaromyces flavus (BAFC 3125) as a function of carbon and/or nitrogen sources. P solubilization was evaluated in NBRIP media supplemented with different carbon (glucose, sorbitol, sucrose, and fructose) and nitrogen (L-asparagine, urea, ammonium sulfate (AS), and ammonium nitrate (AN) combinations. The highest P solubilization was related to the highest organic acid production (especially gluconic acid) and pH drop for those treatments where glucose was present. Also P solubilization was higher when an inorganic nitrogen source was supplemented to the media when compared to an organic one. Although not being present an organic P source, phosphatase activity was observed. This shows that P mineralization and P solubilization can occur simultaneously, and that P mineralization is not induced by the enzyme substrate. The combination that showed highest P solubilization was for AN-glucose. The highest acid phosphatase activity was for AS-fructose, while for alkaline phosphatase were for AS-fructose and AN-fructose. Acid phosphatase activity was higher than alkaline. P solubilization and phosphatase activity (acid and alkaline) were influenced by the different carbon-nitrogen combinations. A better understanding of phosphate-solubilizing fungi could bring a better use of soil P.

  1. Extracellular acidification synergizes with PDGF to stimulate migration of mouse embryo fibroblasts through activation of p38MAPK with a PTX-sensitive manner

    International Nuclear Information System (INIS)

    The elucidation of the functional mechanisms of extracellular acidification stimulating intracellular signaling pathway is of great importance for developing new targets of treatment for solid tumors, and inflammatory disorders characterized by extracellular acidification. In the present study, we focus on the regulation of extracellular acidification on intracellular signaling pathways in mouse embryo fibroblasts (MEFs). We found extracellular acidification was at least partly involved in stimulating p38MAPK pathway through PTX-sensitive behavior to enhance cell migration in the presence or absence of platelet-derived growth factor (PDGF). Statistical analysis showed that the actions of extracellular acidic pH and PDGF on inducing enhancement of cell migration were not an additive effect. However, we also found extracellular acidic pH did inhibit the viability and proliferation of MEFs, suggesting that extracellular acidification stimulates cell migration probably through proton-sensing mechanisms within MEFs. Using OGR1-, GPR4-, and TDAG8-gene knock out technology, and real-time qPCR, we found known proton-sensing G protein-coupled receptors (GPCRs), transient receptor potential vanilloid subtype 1 (TRPV1), and acid-sensing ion channels (ASICs) were unlikely to be involved in the regulation of acidification on cell migration. In conclusion, our present study validates that extracellular acidification stimulates chemotactic migration of MEFs through activation of p38MAPK with a PTX-sensitive mechanism either by itself, or synergistically with PDGF, which was not regulated by the known proton-sensing GPCRs, TRPV1, or ASICs. Our results suggested that others proton-sensing GPCRs or ion channels might exist in MEFs, which mediates cell migration induced by extracellular acidification in the presence or absence of PDGF. - Highlights: • Acidic pH and PDGF synergize to stimulate MEFs migration via Gi/p38MAPK pathway. • Extracellular acidification inhibits the

  2. Extracellular acidification synergizes with PDGF to stimulate migration of mouse embryo fibroblasts through activation of p38MAPK with a PTX-sensitive manner

    Energy Technology Data Exchange (ETDEWEB)

    An, Caiyan [Department of Biochemistry and Molecular Biology, College of Life Sciences, Inner Mongolia University, Hohhot, Inner Mongolia (China); Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi (Japan); Clinical Medicine Research Center of the Affiliated Hospital, Inner Mongolia Medical University, Hohhot, Inner Mongolia (China); Sato, Koichi [Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi (Japan); Wu, Taoya; Bao, Muqiri; Bao, Liang [Department of Biochemistry and Molecular Biology, College of Life Sciences, Inner Mongolia University, Hohhot, Inner Mongolia (China); Tobo, Masayuki [Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi (Japan); Damirin, Alatangaole, E-mail: bigaole@imu.edu.cn [Department of Biochemistry and Molecular Biology, College of Life Sciences, Inner Mongolia University, Hohhot, Inner Mongolia (China)

    2015-05-01

    The elucidation of the functional mechanisms of extracellular acidification stimulating intracellular signaling pathway is of great importance for developing new targets of treatment for solid tumors, and inflammatory disorders characterized by extracellular acidification. In the present study, we focus on the regulation of extracellular acidification on intracellular signaling pathways in mouse embryo fibroblasts (MEFs). We found extracellular acidification was at least partly involved in stimulating p38MAPK pathway through PTX-sensitive behavior to enhance cell migration in the presence or absence of platelet-derived growth factor (PDGF). Statistical analysis showed that the actions of extracellular acidic pH and PDGF on inducing enhancement of cell migration were not an additive effect. However, we also found extracellular acidic pH did inhibit the viability and proliferation of MEFs, suggesting that extracellular acidification stimulates cell migration probably through proton-sensing mechanisms within MEFs. Using OGR1-, GPR4-, and TDAG8-gene knock out technology, and real-time qPCR, we found known proton-sensing G protein-coupled receptors (GPCRs), transient receptor potential vanilloid subtype 1 (TRPV1), and acid-sensing ion channels (ASICs) were unlikely to be involved in the regulation of acidification on cell migration. In conclusion, our present study validates that extracellular acidification stimulates chemotactic migration of MEFs through activation of p38MAPK with a PTX-sensitive mechanism either by itself, or synergistically with PDGF, which was not regulated by the known proton-sensing GPCRs, TRPV1, or ASICs. Our results suggested that others proton-sensing GPCRs or ion channels might exist in MEFs, which mediates cell migration induced by extracellular acidification in the presence or absence of PDGF. - Highlights: • Acidic pH and PDGF synergize to stimulate MEFs migration via Gi/p38MAPK pathway. • Extracellular acidification inhibits the

  3. Membrane–initiated estradiol signaling regulating sexual receptivity

    Directory of Open Access Journals (Sweden)

    Paul E Micevych

    2011-09-01

    Full Text Available Estradiol has profound actions on the structure and function of the nervous system. In addition to nuclear actions that directly modulate gene expression, the idea that estradiol can rapidly activate cell signaling by binding to membrane estrogen receptors (mERs has emerged. Even the regulation of sexual receptivity, an action previously thought to be completely regulated by nuclear ERs, has been shown to have a membrane-initiated estradiol signaling (MIES component. This highlighted the question of the nature of mERs. Several candidates have been proposed, ERα, ERβ, ER-X, GPR30 (G protein coupled estrogen receptor; GPER, and a receptor activated by a diphenylacrylamide compound, STX. Although each of these receptors has been shown to be active in specific assays, we present evidence for and against their participation in sexual receptivity by acting in the lordosis-regulating circuit. The initial MIES that activates the circuit is in the arcuate nucleus of the hypothalamus (ARH. Using both activation of μ-opioid receptors (MOR in the medial preoptic nucleus and lordosis behavior, we document that both ERα and the STX receptor participate in the required MIES. ERα and the STX receptor activation of cell signaling are dependent on the transactivation of type 1 metabotropic glutamate receptors (mGluR1a that augment progesterone synthesis in astrocytes and protein kinase C (PKC in ARH neurons. While estradiol-induced sexual receptivity does not depend on neuroprogesterone, proceptive behaviors do. Moreover, the ERα and the STX receptor activation of medial preoptic MORs and augmentation of lordosis were sensitive to mGluR1a blockade. These observations suggest a common mechanism through which mERs are coupled to intracellular signaling cascades, not just in regulating reproduction, but in actions throughout the neuraxis including the cortex, hippocampus, striatum and DRGs.

  4. Decreased phosphorylation of protein kinase B and extracellular signal-regulated kinase in neutrophils from patients with myelodysplasia

    NARCIS (Netherlands)

    Fuhler, GM; Drayer, AL; Vellenga, E

    2003-01-01

    Neutrophils from patients with myelodysplastic syndrome (MDS) show a disturbed differentiation pattern and are generally dysfunctional. To study these defects in more detail, we investigated reactive-oxygen species (ROS) production and F-actin polymerization in neutrophils from MDS patients and heal

  5. Extracellular signal-regulated kinases control expression of G protein-coupled receptor kinase 2 (GRK2)

    DEFF Research Database (Denmark)

    Theilade, Juliane; Lerche Hansen, Jakob; Haunsø, Stig;

    2002-01-01

    G protein-coupled receptor kinase 2 (GRK2) phosphorylates G protein-coupled receptors resulting in uncoupling from G proteins. Receptors modulate GRK2 expression, however the mechanistic basis for this effect is largely unknown. Here we report a novel mechanism by which receptors use...

  6. Light induces Fos expression via extracellular signal-regulated kinases 1/2 in melanopsin-expressing PC12 cells

    DEFF Research Database (Denmark)

    Moldrup, Marie-Louise Bülow; Georg, Birgitte; Falktoft, Birgitte;

    2010-01-01

    The photopigment melanopsin is expressed in a subtype of mammalian ganglion cells in the retina that project to the circadian clock in the hypothalamic suprachiasmatic nucleus to mediate non-visual light information. Melanopsin renders these retinal ganglion cells intrinsically photosensitive and...

  7. Methodological interference of biochar in the determination of extracellular enzyme activities in composting samples

    OpenAIRE

    K. Jindo; Matsumoto, K.; C. García Izquierdo; Sonoki, T.; Sanchez-Monedero, M. A.

    2014-01-01

    Biochar application has received increasing attention as a means to trap recalcitrant carbon and enhance soil fertility. Hydrolytic enzymatic assays, such as β-glucosidase and phosphatase activities, are used for the assessment of soil quality and composting process, which are based on use of p-nitrophenol (PNP) derivatives as substrate. However, sorption capacity of biochar can interfere with colorimetric determination of the hydrolysed PNP, either by the sorption of the su...

  8. Methodological interference of biochar in the determination of extracellular enzyme activities in composting samples

    OpenAIRE

    K. Jindo; Matsumoto, K.; C. García Izquierdo; Sonoki, T.; Sanchez-Monedero, M. A.

    2014-01-01

    Biochar application has received increasing attention as a means to trap recalcitrant carbon and enhance soil fertility. Hydrolytic enzymatic assays, such as β-glucosidase and phosphatase activities, are used for the assessment of soil quality and composting process, which are based on use of p-nitrophenol (PNP) derivatives as substrate. However, sorption capacity of biochar can interfere colorimetric determination of the hydrolysed PNP, either by the sorpti...

  9. Catalytic activity of zymomonas mobilis extracellular "levan-levansucrase" complex in sucrose medium.

    Science.gov (United States)

    Bekers, M; Upite, D; Kaminska, E; Laukevics, J; Ionina, R; Vigants, A

    2003-01-01

    The fructan biosynthesis by ethanol sedimented "levan-levansucrase" complex from Zymomonas mobilis fermentation broth as well as purified levansucrase was investigated. The fructooligosaccharide (FOS) producing activity of "levan-levamsucrase" sediment was investigated in 55% sucrose syrup at 45 degrees C. It was shown that FOS in the syrup were presented by 1-kestose, 6-kestose, neokestose and nystose. The increase of gluconic acid concentration was observed in the reaction mixture during the incubation suggesting about presence of glucose/fructose oxidoreductase in "levan-levansucrase" sediment. The influence of ethanol, glycerol and NaCl on levan and fructooligosaccharide formation by "levan-levansucrase" complex and purified levansucrase was studied and the changes in the ratio between different activities of levansucrase (sucrose hydrolysis, levan biosynthesis and FOS formation) were observed. Ethanol increases the FOS biosynthesis part in total activity of purified levansucrase. The technology of the production of prebiotics containing food product--fructan syrup by "levan-levansucrase " sediment as biocatalyst was developed. PMID:15296187

  10. In vitro activity of artemisone and artemisinin derivatives against extracellular and intracellular Helicobacter pylori.

    Science.gov (United States)

    Sisto, Francesca; Scaltrito, Maria Maddalena; Masia, Carla; Bonomi, Arianna; Coccè, Valentina; Marano, Giuseppe; Haynes, Richard K; Miani, Alessandro; Farronato, Giampietro; Taramelli, Donatella

    2016-07-01

    The in vitro activity of the new artemisinin derivative artemisone as well as other molecules of the same class against Helicobacter pylori and their effects when combined with standard antibiotics were evaluated. Since H. pylori can be internalised into gastric epithelial cells, the effects of artemisinin, dihydroartemisinin and artemisone against intracellular H. pylori were also investigated. Bacteriostatic [minimum inhibitory concentration (MIC)] and bactericidal [minimum bactericidal concentration (MBC)] activities were assessed against 24 clinical strains of H. pylori with different antibiotics susceptibilities. Artemisone showed MIC50 and MIC90 values of 0.25 mg/L and 0.5 mg/L, respectively, and an MBC50 value of 0.5 mg/L. Artemisone was synergistic with amoxicillin in 60% of strains, with clarithromycin in 40% and with metronidazole in 20%. There was no interaction between artemisone and omeprazole or bismuth citrate. Against intracellular H. pylori, only dihydroartemisinin at 2× MIC caused a 1 log10 CFU decrease after 18 h and 24 h of incubation. This is the first demonstration in vitro of the activity of artemisinin derivatives against intracellular H. pylori and indicates that artemisone has the potential to be efficacious for the treatment of H. pylori infection, especially in combination with antibiotics. PMID:27216383

  11. N-linked glycosylation of protease-activated receptor-1 at extracellular loop 2 regulates G-protein signaling bias.

    Science.gov (United States)

    Soto, Antonio G; Smith, Thomas H; Chen, Buxin; Bhattacharya, Supriyo; Cordova, Isabel Canto; Kenakin, Terry; Vaidehi, Nagarajan; Trejo, JoAnn

    2015-07-01

    Protease-activated receptor-1 (PAR1) is a G-protein-coupled receptor (GPCR) for the coagulant protease thrombin. Similar to other GPCRs, PAR1 is promiscuous and couples to multiple heterotrimeric G-protein subtypes in the same cell and promotes diverse cellular responses. The molecular mechanism by which activation of a given GPCR with the same ligand permits coupling to multiple G-protein subtypes is unclear. Here, we report that N-linked glycosylation of PAR1 at extracellular loop 2 (ECL2) controls G12/13 versus Gq coupling specificity in response to thrombin stimulation. A PAR1 mutant deficient in glycosylation at ECL2 was more effective at stimulating Gq-mediated phosphoinositide signaling compared with glycosylated wildtype receptor. In contrast, wildtype PAR1 displayed a greater efficacy at G12/13-dependent RhoA activation compared with mutant receptor lacking glycosylation at ECL2. Endogenous PAR1 rendered deficient in glycosylation using tunicamycin, a glycoprotein synthesis inhibitor, also exhibited increased PI signaling and diminished RhoA activation opposite to native receptor. Remarkably, PAR1 wildtype and glycosylation-deficient mutant were equally effective at coupling to Gi and β-arrestin-1. Consistent with preferential G12/13 coupling, thrombin-stimulated PAR1 wildtype strongly induced RhoA-mediated stress fiber formation compared with mutant receptor. In striking contrast, glycosylation-deficient PAR1 was more effective at increasing cellular proliferation, associated with Gq signaling, than wildtype receptor. These studies suggest that N-linked glycosylation at ECL2 contributes to the stabilization of an active PAR1 state that preferentially couples to G12/13 versus Gq and defines a previously unidentified function for N-linked glycosylation of GPCRs in regulating G-protein signaling bias. PMID:26100877

  12. Exposure of activated sludge to nanosilver and silver ion: Inhibitory effects and binding to the fractions of extracellular polymeric substances.

    Science.gov (United States)

    Geyik, Ayse Gul; Çeçen, Ferhan

    2016-07-01

    The main aim of the study was to determine the inhibitory effects of Ag(+) and AgNP (commercial and synthesized) on activated sludge by using respirometry. Along with this aim, also the changes taking place in extracellular polymeric substances (EPS) were studied. Additionally, the binding of Ag(+) or AgNP to the different fractions in EPS was assessed using voltammetry. Synthesized AgNP led to an obvious inhibition whereas commercial AgNP had no effect on activated sludge. For Ag(+) and AgNP, IC50 values were found between 2.3-3.0mg/L and 3.2-11.1mg/L, respectively. Thus, AgNP was less inhibitory than silver ion, since the release of free silver from AgNP was very small. The protein and carbohydrate content of EPS generally increased when Ag(+) was added. Both tightly- and loosely bound fractions in EPS could bind Ag(+) and AgNP. Silver binding capacity of EPS was seen to depend on the molecular weight of proteins. PMID:27060244

  13. Methodological interference of biochar in the determination of extracellular enzyme activities in composting samples

    Directory of Open Access Journals (Sweden)

    K. Jindo

    2014-03-01

    Full Text Available Biochar application has received increasing attention as a means to trap recalcitrant carbon and enhance soil fertility. Hydrolytic enzymatic assays, such as β-glucosidase and phosphatase activities, are used for the assessment of soil quality and composting process, which are based on use of p-nitrophenol (PNP derivatives as substrate. However, sorption capacity of biochar can interfere colorimetric determination of the hydrolysed PNP, either by the sorption of the substrate or the reaction-product of hydrolysis into biochar surface. The aim of the present work is to study the biochar sorption capacity for PNP in biochar-blended composting mixtures in order to assess its impact on the estimation of the colorimetric-based enzymatic assays. A retention test was conducted by adding a solution of known amounts of PNP in universal buffer solution (pH = 5, 6.5 and 11, corresponding to the β-glucosidase, acid and alkaline phosphatase activity assays, respectively, in samples taken at the initial stage and after maturation stage from 4 different composting piles (two manure composting piles (PM: poultry manure, CM: cow manure and two other similar piles containing 10% of additional biochar (PM + B, CM + B. The results show that biochar blended composts (PM + B, CM + B generally exhibited low enzymatic activities, compared to manure compost without biochar (PM, CM. In terms of the difference between the initial and maturation stage of composting process, the PNP retention in biochar was shown more clearly at maturation stage, caused by an enlarged proportion of biochar inside compost mixture after the selective degradation of easily decomposable organic matter. The retention of PNP was more pronounced at low pH (5 and 6.5 than at high pH (11, 3 reflecting on pH dependency of sorption 49 capacity of biochar and/or PNP 50 solubility.

  14. Methodological interference of biochar in the determination of extracellular enzyme activities in composting samples

    Science.gov (United States)

    Jindo, K.; Matsumoto, K.; García Izquierdo, C.; Sonoki, T.; Sanchez-Monedero, M. A.

    2014-07-01

    Biochar application has received increasing attention as a means to trap recalcitrant carbon and enhance soil fertility. Hydrolytic enzymatic assays, such as β-glucosidase and phosphatase activities, are used for the assessment of soil quality and composting process, which are based on use of p-nitrophenol (PNP) derivatives as substrate. However, sorption capacity of biochar can interfere with colorimetric determination of the hydrolysed PNP, either by the sorption of the substrate or the reaction product of hydrolysis into biochar surface. The aim of the present work is to study the biochar sorption capacity for PNP in biochar-blended composting mixtures in order to assess its impact on the estimation of the colorimetric-based enzymatic assays. A retention test was conducted by adding a solution of known amounts of PNP in universal buffer solution (pH = 5, 6.5 and 11, corresponding to the β-glucosidase, acid and alkaline phosphatase activity assays, respectively), in samples taken at the initial stage and after maturation stage from four different composting piles (two manure composting piles; PM: poultry manure, CM: cow manure and two other similar piles containing 10% of additional biochar (PM + B, CM + B)). The results show that biochar-blended composts (PM + B, CM + B) generally exhibited low enzymatic activities, compared to manure compost without biochar (PM, CM). In terms of the difference between the initial and maturation stage of composting process, the PNP retention in biochar was shown higher at maturation stage, caused most probably by an enlarged proportion of biochar inside compost mixture after the selective degradation of easily decomposable organic matter. TThe retention of PNP on biochar was influenced by pH dependency of sorption capacity of biochar and/or PNP solubility, since PNP was more efficiently retained by biochar at low pH values (5 and 6.5) than at high pH values (11).

  15. In vitro and comparative study on the extracellular enzyme activity of molds isolated from keratomycosis and soil

    Science.gov (United States)

    Mythili, Arumugam; Babu Singh, Yendranbam Randhir; Priya, Ravindran; Shafeeq Hassan, Anamangadan; Manikandan, Palanisamy; Panneerselvam, Kanesan; Narendran, Venkatapathy; Shobana, Coimbatore Subramanian

    2014-01-01

    AIM To isolate and identify the molds involved in mycotic keratitis; to isolate corresponding species from soil samples; to compare the extracellular enzyme activity indices of the molds isolated from keratitis cases and the corresponding soil isolates. METHODS The specimens were collected from the target patients attending the microbiology laboratory of tertiary eye hospital in Coimbatore, Tamilnadu state, India. The isolates were subjected for identification based on the growth on solid media, direct microscopy and lacto phenol cotton blue wet mount preparation. Extracellular enzymes such as lipase, deoxyribonuclease (DNase), α-amylase, protease, cellulase and pectinase produced by the fungal isolates were screened on solid media supplemented with the corresponding substrates. Based on growth and zone diameter, the enzyme activity indices were calculated and were compared with that of the soil fungal isolates. RESULTS A total of 108 clinical samples were collected from a tertiary eye care hospital and out of which 60 fungal isolates were obtained. Among these, Fusarium spp. (n=30), non sporulating molds (n=9), Aspergillus flavus (n=6), Bipolaris spp. (n=6), Exserohilum spp. (n=4), Curvularia spp. (n=3), Alternaria spp. (n=1) and Exophiala spp. (n=1) were identified and designated as FS1-30, NSM1-9, AF1-6, BS1-6, ES1-4, CS1-3, AS1 and EX1, respectively. For comparative analysis, soil samples were also collected from which, one isolate of each Fusarium spp., Aspergillus flavus, Bipolaris spp., Exserohilum spp., and Curvularia spp., respectively were selected. Highest lipase activity was seen in corneal isolate NSM2 (EAI= 2.14). The DNase activity was higher in NSM9 (EAI=1.88). In case of protease, Fusarium spp. (FS9) had prominent enzyme activity index of 1.38; α-amylase activity was also superior in corneal isolate FS13 with EAI of 1.63 when compared to other isolates. The enzyme activity index for cellulase was also noted to be higher in corneal isolates i

  16. In vitro and comparative study on the extracellular enzyme activity of molds isolated from keratomycosis and soil

    Institute of Scientific and Technical Information of China (English)

    Arumugam; Mythili; Yendranbam; Randhir; Babu; Singh; Ravindran; Priya; Anamangadan; Shafeeq; Hassan; Palanisamy; Manikandan; Kanesan; Panneerselvam; Venkatapathy; Narendran; Coimbatore; Subramanian; Shobana

    2014-01-01

    AIM:To isolate and identify the molds involved in mycotic keratitis; to isolate corresponding species from soil samples; to compare the extracellular enzyme activity indices of the molds isolated from keratitis cases and the corresponding soil isolates.METHODS:The specimens were collected from the target patients attending the microbiology laboratory of tertiary eye hospital in Coimbatore, Tamilnadu state,India. The isolates were subjected for identification based on the growth on solid media, direct microscopy and lacto phenol cotton blue wet mount preparation.Extracellular enzymes such as lipase, deoxyribonuclease(DNase), α-amylase, protease, cellulase and pectinase produced by the fungal isolates were screened on solid media supplemented with the corresponding substrates.Based on growth and zone diameter, the enzyme activity indices were calculated and were compared with that of the soil fungal isolates.RESULTS:A total of 108 clinical samples were collected from a tertiary eye care hospital and out of which 60 fungal isolates were obtained. Among these,Fusarium spp.(n =30), non sporulating molds(n =9),Aspergillus flavus(n =6), Bipolaris spp.(n =6),Exserohilum spp.(n =4), Curvularia spp.(n =3),Alternaria spp.(n =1) and Exophiala spp.(n =1) were identified and designated as FS1-30, NSM1-9, AF1-6,BS1-6, ES1-4, CS1-3, AS1 and EX1, respectively. For comparative analysis, soil samples were also collected from which, one isolate of each Fusarium spp.,Aspergillus flavus, Bipolaris spp., Exserohilum spp., and Curvularia spp., respectively were selected. Highest lipase activity was seen in corneal isolate NSM2(EAI= 2.14).The DNase activity was higher in NSM9(EAI =1.88). In case of protease, Fusarium spp.(FS9) had prominent enzyme activity index of 1.38; α-amylase activity was also superior in corneal isolate FS13 with EAI of 1.63 when compared to other isolates. The enzyme activity index for cellulase was also noted to be higher in corneal isolates i.e. NSM7 with EAI of

  17. The cellular distribution of extracellular superoxide dismutase in macrophages is altered by cellular activation but unaffected by the natural occurring R213G substitution

    DEFF Research Database (Denmark)

    Gottfredsen, Randi Heidemann; Goldstrohm, David; Hartney, John;

    2014-01-01

    and associated with the cell surface via the extracellular matrix (ECM)-binding region. Upon cellular activation induced by lipopolysaccharide, EC-SOD is relocated and detected both in the cell culture medium and in lipid raft structures. Although the secreted material presented a significantly reduced ligand...

  18. Hedgehog signaling regulates telomerase reverse transcriptase in human cancer cells.

    Directory of Open Access Journals (Sweden)

    Tapati Mazumdar

    Full Text Available The Hedgehog (HH signaling pathway is critical for normal embryonic development, tissue patterning and cell differentiation. Aberrant HH signaling is involved in multiple human cancers. HH signaling involves a multi-protein cascade activating the GLI proteins that transcriptionally regulate HH target genes. We have previously reported that HH signaling is essential for human colon cancer cell survival and inhibition of this signal induces DNA damage and extensive cell death. Here we report that the HH/GLI axis regulates human telomerase reverse transcriptase (hTERT, which determines the replication potential of cancer cells. Suppression of GLI1/GLI2 functions by a C-terminus truncated GLI3 repressor mutant (GLI3R, or by GANT61, a pharmacological inhibitor of GLI1/GLI2, reduced hTERT protein expression in human colon cancer, prostate cancer and Glioblastoma multiforme (GBM cell lines. Expression of an N-terminus deleted constitutively active mutant of GLI2 (GLI2ΔN increased hTERT mRNA and protein expression and hTERT promoter driven luciferase activity in human colon cancer cells while GANT61 inhibited hTERT mRNA expression and hTERT promoter driven luciferase activity. Chromatin immunoprecipitation with GLI1 or GLI2 antibodies precipitated fragments of the hTERT promoter in human colon cancer cells, which was reduced upon exposure to GANT61. In contrast, expression of GLI1 or GLI2ΔN in non-malignant 293T cells failed to alter the levels of hTERT mRNA and protein, or hTERT promoter driven luciferase activity. Further, expression of GLI2ΔN increased the telomerase enzyme activity, which was reduced by GANT61 administration in human colon cancer, prostate cancer, and GBM cells. These results identify hTERT as a direct target of the HH signaling pathway, and reveal a previously unknown role of the HH/GLI axis in regulating the replication potential of cancer cells. These findings are of significance in understanding the important regulatory

  19. Extracellular thermostable proteolytic activity of the milk spoilage bacterium Pseudomonas fluorescens PS19 on bovine caseins.

    Science.gov (United States)

    Stuknytė, M; Decimo, M; Colzani, M; Silvetti, T; Brasca, M; Cattaneo, S; Aldini, G; De Noni, I

    2016-06-01

    We studied the thermostable proteolytic activity of Pseudomonas fluorescens PS19 isolated from raw bovine milk. The heat-treated cell-free supernatant (HT-CFS) contained a thermostable protease of approximately 45 kDa, as revealed by casein zymography. We assigned this enzyme to P. fluorescens AprX metalloprotease (UniProtKB Acc. No. C9WKP6). After concentration by ultrafiltration at 10 kDa, the HT-CFS showed 2 other thermostable proteolytic bands on zymogram, with molecular masses of approximately 15 and 25 kDa. The former resulted a fragment of the AprX protease, whereas the 25-kDa protease was not homologous to any known protein of Pseudomonas spp. Subsequently, we assessed the proteolytic activity of the HT-CFS on bovine αS-, β-, and κ-casein during in vitro incubation at 7 or 22°C. By means of ultra-performance liquid chromatography-tandem mass spectrometry we identified the released peptides (n=591). Some of them resisted proteolysis during the whole incubation period at both incubation temperatures and, therefore, they could be assumed as indicators of the proteolytic action of P. fluorescens PS19 on bovine caseins. PMID:26995139

  20. Paradoxical signaling regulates structural plasticity in dendritic spines.

    Science.gov (United States)

    Rangamani, Padmini; Levy, Michael G; Khan, Shahid; Oster, George

    2016-09-01

    Transient spine enlargement (3- to 5-min timescale) is an important event associated with the structural plasticity of dendritic spines. Many of the molecular mechanisms associated with transient spine enlargement have been identified experimentally. Here, we use a systems biology approach to construct a mathematical model of biochemical signaling and actin-mediated transient spine expansion in response to calcium influx caused by NMDA receptor activation. We have identified that a key feature of this signaling network is the paradoxical signaling loop. Paradoxical components act bifunctionally in signaling networks, and their role is to control both the activation and the inhibition of a desired response function (protein activity or spine volume). Using ordinary differential equation (ODE)-based modeling, we show that the dynamics of different regulators of transient spine expansion, including calmodulin-dependent protein kinase II (CaMKII), RhoA, and Cdc42, and the spine volume can be described using paradoxical signaling loops. Our model is able to capture the experimentally observed dynamics of transient spine volume. Furthermore, we show that actin remodeling events provide a robustness to spine volume dynamics. We also generate experimentally testable predictions about the role of different components and parameters of the network on spine dynamics. PMID:27551076

  1. Intra- and Extracellular Activities of Trimethoprim-Sulfamethoxazole against Susceptible and Multidrug-Resistant Mycobacterium tuberculosis

    Science.gov (United States)

    Schön, T.; Simonsson, U. S. H.; Bruchfeld, J.; Larsson, M.; Juréen, P.; Sturegård, E.; Giske, C. G.; Ängeby, K.

    2014-01-01

    We investigated the activity of trimethoprim-sulfamethoxazole (SXT) against Mycobacterium tuberculosis, the pathogen that causes tuberculosis (TB). The MIC distribution of SXT was 0.125/2.4 to 2/38 mg/liter for the 100 isolates tested, including multi- and extensively drug-resistant isolates (MDR/XDR-TB), whereas the intracellular MIC90 of sulfamethoxazole (SMX) for the pansusceptible strain H37Rv was 76 mg/liter. In an exploratory analysis using a ratio of the unbound area under the concentration-time curve from 0 to 24 h over MIC (fAUC0–24/MIC) using ≥25 as a potential target, the cumulative fraction response was ≥90% at doses of ≥2,400 mg of SMX. SXT is a potential treatment option for MDR/XDR-TB. PMID:25246405

  2. Extracellular acid block and acid-enhanced inactivation of the Ca2+-activated cation channel TRPM5 involve residues in the S3-S4 and S5-S6 extracellular domains.

    Science.gov (United States)

    Liu, Dan; Zhang, Zheng; Liman, Emily R

    2005-05-27

    TRPM5, a member of the superfamily of transient receptor potential ion channels, is essential for the detection of bitter, sweet, and amino acid tastes. In heterologous cell types it forms a nonselective cation channel that is activated by intracellular Ca(2+). TRPM5 is likely to be part of the taste transduction cascade, and regulators of TRPM5 are likely to affect taste sensation. In this report we show that TRPM5, but not the related channel TRPM4b, is potently blocked by extracellular acidification. External acidification has two effects, a fast reversible block of the current (IC(50) pH = 6.2) and a slower irreversible enhancement of current inactivation. Mutation of a single Glu residue in the S3-S4 linker and a His residue in the pore region each reduced sensitivity of TRPM5 currents to fast acid block (IC(50) pH = 5.8 for both), and the double mutant was nearly insensitive to acidic pH (IC(50) pH = 5.0). Prolonged exposure to acidic pH enhanced inactivation of TRPM5 currents, and mutant channels that were less sensitive to acid block were also less sensitive to acid-enhanced inactivation, suggesting an intimate association between the two processes. These processes are, however, distinct because the pore mutant H896N, which has normal sensitivity to acid block, shows significant recovery from acid-enhanced inactivation. These data show that extracellular acidification acts through specific residues on TRPM5 to block conduction through two distinct but related mechanisms and suggest a possible interaction between extracellular pH and activation and adaptation of bitter, sweet, and amino acid taste transduction.

  3. Mitogen activated protein kinase signaling pathways participate in the active principle region of Buyang Huanwu decoction-induced differentiation of bone marrow mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Jinghui Zheng; Jian Liang; Xin Deng; Xiaofeng Chen; Fasheng Wu; Xiaofang Zhao; Yuan Luo; Lei Fu; Zuling Jiang

    2012-01-01

    Our preliminary studies confirmed that an active principle region of Buyang Huanwu decoction, comprising alkaloid, polysaccharide, aglycon, glucoside and volatile oil, can induce bone marrow mesenchymal stem cell differentiation into neurons. Mitogen-activated protein kinase signaling was identified as one of the key pathways underlying this differentiation process. The present study shows phosphorylated extracellular signal-regulated protein kinase and phosphorylated p38 protein expression was increased after differentiation. Cellular signaling pathway blocking agents, PD98059 and SB203580, inhibited extracellular signal-regulated protein kinase and p38 in mitogen-activated protein kinase signaling pathways respectively. mRNA and protein expression of the neuronal marker, neuron specific enolase, and neural stem cell marker, nestin, were decreased in bone marrow mesenchymal stem cells after treatment with the active principle region of Buyang Huanwu decoction. Experimental findings indicate that, extracellular signal-regulated protein kinase and p38 in mitogen-activated protein kinase signaling pathways participate in bone marrow mesenchymal stem cell differentiation into neuron-like cells, induced by the active principle region of Buyang Huanwu decoction.

  4. Green Synthesis and Antibacterial Activities of Silver Nanoparticles Using Extracellular Laccase of Lentinus edodes

    Directory of Open Access Journals (Sweden)

    Agbaje LATEEF

    2015-12-01

    Full Text Available This study reports the multi-step mutagenesis of Lentinus edodes towards optimization of the production of laccase and novel application of laccase in the biosynthesis of silver nanoparticles (AgNPs which could be used to develop an eco-friendly method for the rapid biosynthesis of AgNPs. The wild strain of L. edodes was subjected to UV irradiation at 254 nm and the resultant viable mutant was further treated with acridine orange, a chemical mutagen. The strains were evaluated for the production of laccase and the crude laccase of the UV mutant (UV10 was used for the green synthesis of AgNPs. The particles were characterized by UV-Visible spectroscopy, Fourier transform infrared (FTIR spectroscopy and scanning electron microscopy (SEM. Laccase activities of wild, UV10 and UV10ACR8 strains of L. edodes were obtained as 2.6, 10.6 and 2.8 U/ml/min respectively after 7 days of fermentation, showing laccase yield improvement of 4.08-fold for UV10 mutant. UV-Visible spectroscopy indicated the formation of AgNPs at absorption band of 430 nm. FTIR result indicated that proteins were responsible for AgNP synthesis, while SEM analysis confirmed the formation of walnut-shaped nanoparticles with size range of 50-100 nm. The biosynthesized nanoparticles revealed effective inhibition against clinical isolates of Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae. To the best of the authors’ knowledge, this result represents the first report on the biosynthesis of AgNPs using L. edodes metabolite. The report adds to the growing relevance of L. edodes as potential industrially viable organism, used for diverse biotechnological applications.

  5. 5-Azacytidine Induces Cardiac Differentiation of Human Umbilical Cord-Derived Mesenchymal Stem Cells by Activating Extracellular Regulated Kinase

    Science.gov (United States)

    Qian, Qian; Qian, Hui; Zhang, Xu; Zhu, Wei; Yan, Yongmin; Ye, Shengqin; Peng, Xiujuan; Li, Wei; Xu, Zhe; Sun, Lingyun

    2012-01-01

    5-Azacytidine (5-Aza) induces differentiation of mesenchymal stem cells (MSCs) into cardiomyocytes. However, the underlying mechanisms are not well understood. Our previous work showed that 5-Aza induces human bone marrow-derived MSCs to differentiate into cardiomyocytes. Here, we demonstrated that 5-Aza induced cardiac differentiation of human umbilical cord-derived MSCs (hucMSCs) and explored the potential signaling pathway. Our results showed that hucMSCs had cardiomyocyte phenotypes after 5-Aza treatment. In addition, myogenic cells differentiated from hucMSCs were positive for mRNA and protein of desmin, β-myosin heavy chain, cardiac troponin T, A-type natriuretic peptide, and Nkx2.5. Human diploid lung fibroblasts treated with 5-Aza expressed no cardiac-specific genes. 5-Aza did not induce hucMSCs to differentiate into osteoblasts. Further study revealed that 5-Aza treatment activated extracellular signal related kinases (ERK) in hucMSCs, but protein kinase C showed no response to 5-Aza administration. U0126, a specific inhibitor of ERK, could inhibit 5-Aza-induced expression of cardiac-specific genes and proteins in hucMSCs. Increased phosphorylation of signal transducers and activators of transcription 3, and up-regulation of myocyte enhancer-binding factor-2c and myogenic differentiation antigen in 5-Aza-treated hucMSCs were also suppressed by U0126. Taken together, these results suggested that sustained activation of ERK by 5-Aza contributed to the induction of the differentiation of hucMSCs into cardiomyocytes in vitro. PMID:21476855

  6. Improving ice nucleation activity of zein film through layer-by-layer deposition of extracellular ice nucleators.

    Science.gov (United States)

    Shi, Ke; Yu, Hailong; Lee, Tung-Ching; Huang, Qingrong

    2013-11-13

    Zein protein has been of scientific interest in the development of biodegradable functional food packaging. This study aimed at developing a novel zein-based biopolymer film with ice nucleation activity through layer-by-layer deposition of biogenic ice nucleators, that is, extracellular ice nucleators (ECINs) isolated from Erwinia herbicola , onto zein film surface. The adsorption behaviors and mechanisms were investigated using quartz crystal microbalance with dissipation monitoring (QCM-D). On unmodified zein surface, the highest ECINs adsorption occurred at pH 5.0; on UV/ozone treated zein surface followed by deposition of poly(diallyldimethylammonium chloride) (PDADMAC) layer, the optimum condition for ECINs adsorption occurred at pH 7.0 and I 0.05 M, where the amount of ECINs adsorbed was also higher than that on unmodified zein surface. QCM-D analyses further revealed a two-step adsorption process on unmodified zein surfaces, compared to a one-step adsorption process on PDADMAC-modified zein surface. Also, significantly, in order to quantify the ice nucleation activity of ECINs-coated zein films, an empirical method was developed to correlate the number of ice nucleators with the ice nucleation temperature measured by differential scanning calorimetry. Calculated using this empirical method, the highest ice nucleation activity of ECINs on ECINs-modified zein film reached 64.1 units/mm(2), which was able to elevate the ice nucleation temperature of distilled water from -15.5 °C to -7.3 °C. PMID:24106783

  7. Extracellular biosynthesis of silver nanoparticles using Bacillus sp. GP-23 and evaluation of their antifungal activity towards Fusarium oxysporum

    Science.gov (United States)

    Gopinath, V.; Velusamy, P.

    2013-04-01

    In last few decades nanoparticles have attracted and emerged as a field in biomedical research due to their incredible applications. The current research was focused on extracellular synthesis of silver nanoparticles (AgNPs) using cell free culture supernatant of strain GP-23. It was found that the strain GP-23 belonged to Bacillus species by 16S rRNA sequence analysis. Biosynthesis of AgNPs was achieved by addition of culture supernatant with aqueous silver nitrate solution, after 24 h it turned to brown color solution with a peak at 420 nm corresponding to the Plasmon absorbance of AgNPs by UV-Vis Spectroscopy. The nanoparticles were characterized by FTIR, XRD, HRTEM, EDX and AFM. The synthesized nanoparticles were found to be spherical in shape with size in the range of 7-21 nm. It was stable in aqueous solution for five months period of storage at room temperature under dark condition. The biosynthesized AgNPs exhibited strong antifungal activity against plant pathogenic fungus, Fusarium oxysporum at the concentration of 8 μg ml-1. The results suggest that the synthesized AgNPs act as an effective antifungal agent/fungicide.

  8. Extracellular polymeric substances, microbial activity and microbial community of biofilm and suspended sludge at different divalent cadmium concentrations.

    Science.gov (United States)

    Wang, Zichao; Gao, Mengchun; Wei, Junfeng; Ma, Kedong; Zhang, Jing; Yang, Yusuo; Yu, Shuping

    2016-04-01

    The differences between biofilm and suspended sludge (S-sludge) in extracellular polymeric substances (EPS), microbial activity, and microbial community in an anoxic-aerobic sequencing batch biofilm reactor (SBBR) at different concentrations of divalent cadmium (Cd(II)) were investigated. As the increase of Cd(II) concentration from 0 to 50mgL(-1), the specific ammonium oxidation rate (SAOR), specific nitrite oxidation rate (SNOR), and specific nitrate reduction rate (SNRR) of biofilm decreased from 4.85, 5.22 and 45mgNg(-1) VSSh(-1) to 1.54, 2.38 and 26mgNg(-1)VSSh(-1), respectively, and the SAOR, SNOR and SNRR of S-sludge decreased from 4.80, 5.02 and 34mgNg(-1)VSSh(-1) to 1.46, 2.20 and 17mgNg(-1)VSSh(-1), respectively. Biofilm had higher protein (PN) content in EPS than S-sludge. Contrast to S-sludge, biofilm could provide Nitrobacter vulgaris, beta proteobacterium INBAF015, and Pseudoxanthomonas mexicana with the favorable conditions of growth and reproduction. PMID:26829529

  9. Rapid Biosynthesis of Gold Nanoparticles by the Extracellular Secretion of Bacillus niabensis 45: Characterization and Antibiofilm Activity

    Directory of Open Access Journals (Sweden)

    Yumei Li

    2016-01-01

    Full Text Available The present study demonstrated that the extracellular biosynthesis of gold nanoparticles (GNPs using B. niabensis 45 may be mediated by a cyclic peptide (P2. The molecular weight of P2 was determined to be about 1122 Da by MALDI-TOF-MS and ESI-MS. A novel protocol for rapid biosynthesis of GNPs using P2 was developed. The results showed that GNP synthesis could be completed in a wide range of temperatures (40–100°C and pH (6.0–10.0 within few minutes when 9 mL of P2 (2 mg/mL and 1 mL of HAuCl4 solution (2 mM were mixed together. The synthesized GNPs were further characterized. Energy dispersive spectroscopy (EDS and X-ray diffraction (XRD analysis confirmed the presence of elemental gold and crystalline structure of the GNPs, respectively. Scanning electron microscopy (SEM and transmission electron microscopy (TEM revealed the formation of spherical metallic GNPs. The size distribution of GNPs calculated using ImageJ software was found to be 10–20 nm. And these GNPs showed excellent antibiofilm activity against Pseudomonas aeruginosa PAO1 and Staphylococcus aureus ATCC25923. The results revealed microbial cyclic peptides could be used as synthesis of GNPs which had potent antibiofilm potential.

  10. Extracellular synthesis of silver nanoparticles by novel Pseudomonas veronii AS41G inhabiting Annona squamosa L. and their bactericidal activity

    Science.gov (United States)

    Baker, Syed; Mohan Kumar, K.; Santosh, P.; Rakshith, D.; Satish, S.

    2015-02-01

    In present investigation extracellular synthesis of silver nanoparticles were synthesized using cell free supernatant of Pseudomonas veronii AS41G isolated from Annona squamosa L. The bacterium significantly reduced silver nitrate to generate silver nanoparticles which was characterized with hyphenated techniques. Synthesis of silver nanoparticles preliminary confirmed by UV-Visible spectrophotometry with the intense peak at 410 nm, Further FTIR analysis revealed the possible role of biomolecules in the supernatant responsible for mediating the nanoparticles formation. The XRD spectra exhibited the characteristic Bragg peaks of 1 0 0, 1 1 1, 2 0 0, and 2 2 0 facets of the face centred cubic symmetry of nanoparticles suggesting that these nanoparticles were crystalline in nature. TEM microgram showed polydispersity of nanoparticles with size ranging from 5 to 50 nm. Synthesized silver nanoparticles showed antibacterial activity against human and environmental pathogens including MRSA. The study enlightens the role of biosynthesized silver nanoparticles as an emerging alternative for drug resistant microorganisms. The obtained results are promising enough to pave the environmentally benign nanoparticle synthesis processes without use of any toxic chemicals and also envision the emerging role of endophytes towards synthesis of nanoparticles. With scanty reports available on P.veronii species, a new role has been reported in this study which will be very valuable for future researchers working on it.

  11. TGF-β Signaling Regulates the Differentiation of Motile Cilia

    Directory of Open Access Journals (Sweden)

    Janos Tözser

    2015-05-01

    Full Text Available The cilium is a small cellular organelle with motility- and/or sensory-related functions that plays a crucial role during developmental and homeostatic processes. Although many molecules or signal transduction pathways that control cilia assembly have been reported, the mechanisms of ciliary length control have remained enigmatic. Here, we report that Smad2-dependent transforming growth factor β (TGF-β signaling impacts the length of motile cilia at the Xenopus left-right (LR organizer, the gastrocoel roof plate (GRP, as well as at the neural tube and the epidermis. Blocking TGF-β signaling resulted in the absence of the transition zone protein B9D1/MSKR-1 from cilia in multi-ciliated cells (MCCs of the epidermis. Interestingly, this TGF-β activity is not mediated by Mcidas, Foxj1, and RFX2, the known major regulators of ciliogenesis. These data indicate that TGF-β signaling is crucial for the function of the transition zone, which in turn may affect the regulation of cilia length.

  12. Development of ERK Activity Sensor, an in vitro, FRET-based sensor of Extracellular Regulated Kinase activity

    Directory of Open Access Journals (Sweden)

    Alberola-Ila José

    2005-07-01

    Full Text Available Abstract Background Study of ERK activation has thus far relied on biochemical assays that are limited to the use of phospho-specific antibodies and radioactivity in vitro, and analysis of whole cell populations in vivo. As with many systems, fluorescence resonance energy transfer (FRET can be utilized to make highly sensitive detectors of molecular activity. Here we introduce FRET-based ERK Activity Sensors, which utilize variants of Enhanced Green Fluorescent Protein fused by an ERK-specific peptide linker to detect ERK2 activity. Results ERK Activity Sensors display varying changes in FRET upon phosphorylation by active ERK2 in vitro depending on the composition of ERK-specific peptide linker sequences derived from known in vivo ERK targets, Ets1 and Elk1. Analysis of point mutations reveals specific residues involved in ERK binding and phosphorylation of ERK Activity Sensor 3. ERK2 also shows high in vitro specificity for these sensors over two other major MAP Kinases, p38 and pSAPK/JNK. Conclusion EAS's are a convenient, non-radioactive alternative to study ERK dynamics in vitro. They can be utilized to study ERK activity in real-time. This new technology can be applied to studying ERK kinetics in vitro, analysis of ERK activity in whole cell extracts, and high-throughput screening technologies.

  13. Lipid rafts as major platforms for signaling regulation in cancer.

    Science.gov (United States)

    Mollinedo, Faustino; Gajate, Consuelo

    2015-01-01

    Cell signaling does not apparently occur randomly over the cell surface, but it seems to be integrated very often into cholesterol-rich membrane domains, termed lipid rafts. Membrane lipid rafts are highly ordered membrane domains that are enriched in cholesterol, sphingolipids and gangliosides, and behave as major modulators of membrane geometry, lateral movement of molecules, traffic and signal transduction. Because the lipid and protein composition of membrane rafts differs from that of the surrounding membrane, they provide an additional level of compartmentalization, serving as sorting platforms and hubs for signal transduction proteins. A wide number of signal transduction processes related to cell adhesion, migration, as well as to cell survival and proliferation, which play major roles in cancer development and progression, are dependent on lipid rafts. Despite lipid rafts harbor mainly critical survival signaling pathways, including insulin-like growth factor I (IGF-I)/phosphatidylinositol 3-kinase (PI3K)/Akt signaling, recent evidence suggests that these membrane domains can also house death receptor-mediated apoptotic signaling. Recruitment of this death receptor signaling pathway in membrane rafts can be pharmacologically modulated, thus opening up the possibility to regulate cell demise with a therapeutic use. The synthetic ether phospholipid edelfosine shows a high affinity for cholesterol and accumulates in lipid rafts in a number of malignant hematological cells, leading to an efficient in vitro and in vivo antitumor activity by inducing translocation of death receptors and downstream signaling molecules to these membrane domains. Additional antitumor drugs have also been shown to act, at least in part, by recruiting death receptors in lipid rafts. The partition of death receptors together with downstream apoptotic signaling molecules in membrane rafts has led us to postulate the concept of a special liquid-ordered membrane platform coined as

  14. Lipid rafts as major platforms for signaling regulation in cancer.

    Science.gov (United States)

    Mollinedo, Faustino; Gajate, Consuelo

    2015-01-01

    Cell signaling does not apparently occur randomly over the cell surface, but it seems to be integrated very often into cholesterol-rich membrane domains, termed lipid rafts. Membrane lipid rafts are highly ordered membrane domains that are enriched in cholesterol, sphingolipids and gangliosides, and behave as major modulators of membrane geometry, lateral movement of molecules, traffic and signal transduction. Because the lipid and protein composition of membrane rafts differs from that of the surrounding membrane, they provide an additional level of compartmentalization, serving as sorting platforms and hubs for signal transduction proteins. A wide number of signal transduction processes related to cell adhesion, migration, as well as to cell survival and proliferation, which play major roles in cancer development and progression, are dependent on lipid rafts. Despite lipid rafts harbor mainly critical survival signaling pathways, including insulin-like growth factor I (IGF-I)/phosphatidylinositol 3-kinase (PI3K)/Akt signaling, recent evidence suggests that these membrane domains can also house death receptor-mediated apoptotic signaling. Recruitment of this death receptor signaling pathway in membrane rafts can be pharmacologically modulated, thus opening up the possibility to regulate cell demise with a therapeutic use. The synthetic ether phospholipid edelfosine shows a high affinity for cholesterol and accumulates in lipid rafts in a number of malignant hematological cells, leading to an efficient in vitro and in vivo antitumor activity by inducing translocation of death receptors and downstream signaling molecules to these membrane domains. Additional antitumor drugs have also been shown to act, at least in part, by recruiting death receptors in lipid rafts. The partition of death receptors together with downstream apoptotic signaling molecules in membrane rafts has led us to postulate the concept of a special liquid-ordered membrane platform coined as

  15. Facilitated extracellular electron transfer of Shewanella loihica PV-4 by antimony-doped tin oxide nanoparticles as active microelectrodes

    Science.gov (United States)

    Zhang, Xiaojian; Liu, Huan; Wang, Jinrong; Ren, Guangyuan; Xie, Beizhen; Liu, Hong; Zhu, Ying; Jiang, Lei

    2015-11-01

    Dissimilatory metal reducing bacteria are capable of extracellular electron transfer (EET) to insoluble metal oxides as external electron acceptors for their anaerobic respiration, which is recognized as an important energy-conversion process in natural and engineered environments, such as in mineral cycling, bioremediation, and microbial fuel/electrolysis cells. However, the low EET efficiency remains one of the major bottlenecks for its practical application. We report firstly that the microbial current generated by Shewanella loihica PV-4 (S. loihica PV-4) could be greatly improved that is up to ca. 115 fold, by adding antimony-doped tin oxide (ATO) nanoparticles in the electrochemical reactor. The results demonstrate that the biocompatible, electrically conductive ATO nanoparticles acted as active microelectrodes could facilitate the formation of a cells/ATO composite biofilm and the reduction of the outer membrane c-type cytochromes (OM c-Cyts) that are beneficial for the electron transfer from cells to electrode. Meanwhile, a synergistic effect between the participation of OM c-Cyts and the accelerated EET mediated by cell-secreted flavins may play an important role for the enhanced current generation in the presence of ATO nanoparticles. Moreover, it is worth noting that the TCA cycle in S. loihica PV-4 cells is activated by adding ATO nanoparticles, even if the potential is poised at +0.2 V, thereby also improving the EET process. The results presented here may provide a simple and effective strategy to boost the EET of S. loihica PV-4 cells, which is conducive to providing potential applications in bioelectrochemical systems.Dissimilatory metal reducing bacteria are capable of extracellular electron transfer (EET) to insoluble metal oxides as external electron acceptors for their anaerobic respiration, which is recognized as an important energy-conversion process in natural and engineered environments, such as in mineral cycling, bioremediation, and

  16. The effects of three commonly used extraction methods on the redox properties of extracellular polymeric substances from activated sludge.

    Science.gov (United States)

    Lu, Qin; Chang, Ming; Yu, Zhen; Zhou, Shungui

    2015-01-01

    Recently, the redox properties of extracellular polymeric substances (EPS) have attracted the attention of scientists due to their associated environmental significance, such as organic pollutant (e.g. nitroaromatics and substituted nitrobenzenes) degradation and heavy metal (e.g. Cr(VI) and U(VI)) detoxification. Although the separation of EPS from bacterial cells is more often the first step in studies on EPS, and studies have demonstrated that extraction procedures can influence the sorption properties of EPS, few attempts have been made to investigate how separation methods affect the redox properties of the obtained EPS. In this study, three common extraction approaches, that is, centrifugation, formaldehyde+NaOH and ethylene diamine tetra-acetic acid (EDTA), were employed to extract EPS from activated sludge, and the obtained EPS were evaluated for their redox properties using electrochemical means, including cyclic voltammetry and chronoamperometry. In addition, spectroscopic techniques were utilized to explore the structural characteristics and composition of EPS. The results indicated that EPS extracted by EDTA clearly displayed reversible oxidation-reduction peaks in cyclic voltammograms and significantly higher electron-accepting capacity compared with EPS extracted using the other two approaches. Fourier transform infrared spectra and three-dimensional excitation-emission matrix spectra suggested that the EPS extracted with EDTA presented better redox properties because of the effective and efficient extraction of the humic substances, which are important components of the EPS of activated sludge. Therefore, extraction method has an impact on the composition and redox properties of EPS and should be chosen according to research purpose and EPS source.

  17. Inhibitor of apoptosis signal-regulating kinase 1 protects against acetaminophen-induced liver injury

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Yuchao; Ramachandran, Anup [Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Breckenridge, David G.; Liles, John T. [Department of Biology, Gilead Sciences, Inc., Foster City, CA (United States); Lebofsky, Margitta [Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Farhood, Anwar [Department of Pathology, St. David' s North Austin Medical Center, Austin, TX 78756 (United States); Jaeschke, Hartmut, E-mail: hjaeschke@kumc.edu [Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States)

    2015-07-01

    Metabolic activation and oxidant stress are key events in the pathophysiology of acetaminophen (APAP) hepatotoxicity. The initial mitochondrial oxidative stress triggered by protein adduct formation is amplified by c-jun-N-terminal kinase (JNK), resulting in mitochondrial dysfunction and ultimately cell necrosis. Apoptosis signal-regulating kinase 1 (ASK1) is considered the link between oxidant stress and JNK activation. The objective of the current study was to assess the efficacy and mechanism of action of the small-molecule ASK1 inhibitor GS-459679 in a murine model of APAP hepatotoxicity. APAP (300 mg/kg) caused extensive glutathione depletion, JNK activation and translocation to the mitochondria, oxidant stress and liver injury as indicated by plasma ALT activities and area of necrosis over a 24 h observation period. Pretreatment with 30 mg/kg of GS-459679 almost completely prevented JNK activation, oxidant stress and injury without affecting the metabolic activation of APAP. To evaluate the therapeutic potential of GS-459679, mice were treated with APAP and then with the inhibitor. Given 1.5 h after APAP, GS-459679 was still protective, which was paralleled by reduced JNK activation and p-JNK translocation to mitochondria. However, GS-459679 treatment was not more effective than N-acetylcysteine, and the combination of GS-459679 and N-acetylcysteine exhibited similar efficacy as N-acetylcysteine monotherapy, suggesting that GS-459769 and N-acetylcysteine affect the same pathway. Importantly, inhibition of ASK1 did not impair liver regeneration as indicated by PCNA staining. In conclusion, the ASK1 inhibitor GS-459679 protected against APAP toxicity by attenuating JNK activation and oxidant stress in mice and may have therapeutic potential for APAP overdose patients. - Highlights: • Two ASK1 inhibitors protected against acetaminophen-induced liver injury. • The ASK1 inhibitors protect when used as pre- or post-treatment. • Protection by ASK1 inhibitor is

  18. IKKα/CHUK regulates extracellular matrix remodeling independent of its kinase activity to facilitate articular chondrocyte differentiation.

    Directory of Open Access Journals (Sweden)

    Eleonora Olivotto

    Full Text Available BACKGROUND: The non-canonical NF-κB activating kinase IKKα, encoded by CHUK (conserved-helix-loop-helix-ubiquitous-kinase, has been reported to modulate pro- or anti- inflammatory responses, cellular survival and cellular differentiation. Here, we have investigated the mechanism of action of IKKα as a novel effector of human and murine chondrocyte extracellular matrix (ECM homeostasis and differentiation towards hypertrophy. METHODOLOGY/PRINCIPAL FINDINGS: IKKα expression was ablated in primary human osteoarthritic (OA chondrocytes and in immature murine articular chondrocytes (iMACs derived from IKKα(f/f:CreERT2 mice by retroviral-mediated stable shRNA transduction and Cre recombinase-dependent Lox P site recombination, respectively. MMP-10 was identified as a major target of IKKα in chondrocytes by mRNA profiling, quantitative RT-PCR analysis, immunohistochemistry and immunoblotting. ECM integrity, as assessed by type II collagen (COL2 deposition and the lack of MMP-dependent COL2 degradation products, was enhanced by IKKα ablation in mice. MMP-13 and total collagenase activities were significantly reduced, while TIMP-3 (tissue inhibitor of metalloproteinase-3 protein levels were enhanced in IKKα-deficient chondrocytes. IKKα deficiency suppressed chondrocyte differentiation, as shown by the quantitative inhibition of.Alizarin red staining and the reduced expression of multiple chondrocyte differentiation effectors, including Runx2, Col10a1 and Vegfa,. Importantly, the differentiation of IKKα-deficient chondrocytes was rescued by a kinase-dead IKKα protein mutant. CONCLUSIONS/SIGNIFICANCE: IKKα acts independent of its kinase activity to help drive chondrocyte differentiation towards a hypertrophic-like state. IKKα positively modulates ECM remodeling via multiple downstream targets (including MMP-10 and TIMP-3 at the mRNA and post-transcriptional levels, respectively to maintain maximal MMP-13 activity, which is required for ECM

  19. Extra-cellular matrix proteins induce matrix metalloproteinase-1 (MMP-1 activity and increase airway smooth muscle contraction in asthma.

    Directory of Open Access Journals (Sweden)

    Natasha K Rogers

    Full Text Available Airway remodelling describes the histopathological changes leading to fixed airway obstruction in patients with asthma and includes extra-cellular matrix (ECM deposition. Matrix metalloproteinase-1 (MMP-1 is present in remodelled airways but its relationship with ECM proteins and the resulting functional consequences are unknown. We used airway smooth muscle cells (ASM and bronchial biopsies from control donors and patients with asthma to examine the regulation of MMP-1 by ECM in ASM cells and the effect of MMP-1 on ASM contraction. Collagen-I and tenascin-C induced MMP-1 protein expression, which for tenascin-C, was greater in asthma derived ASM cells. Tenascin-C induced MMP-1 expression was dependent on ERK1/2, JNK and p38 MAPK activation and attenuated by function blocking antibodies against the β1 and β3 integrin subunits. Tenascin-C and MMP-1 were not expressed in normal airways but co-localised in the ASM bundles and reticular basement membrane of patients with asthma. Further, ECM from asthma derived ASM cells stimulated MMP-1 expression to a greater degree than ECM from normal ASM. Bradykinin induced contraction of ASM cells seeded in 3D collagen gels was reduced by the MMP inhibitor ilomastat and by siRNA knockdown of MMP-1. In summary, the induction of MMP-1 in ASM cells by tenascin-C occurs in part via integrin mediated MAPK signalling. MMP-1 and tenascin-C are co-localised in the smooth muscle bundles of patients with asthma where this interaction may contribute to enhanced airway contraction. Our findings suggest that ECM changes in airway remodelling via MMP-1 could contribute to an environment promoting greater airway narrowing in response to broncho-constrictor stimuli and worsening asthma symptoms.

  20. A second disulfide bridge from the N-terminal domain to extracellular loop 2 dampens receptor activity in GPR39

    DEFF Research Database (Denmark)

    Storjohann, Laura; Holst, Birgitte; Schwartz, Thue W

    2008-01-01

    . Disruption of the nonconserved disulfide bridge by mutagenesis led to an increase in the Zn (2+) potency. This phenotype, with an approximate 10-fold increase in agonist potency and a slight increase in E max, was mimicked by treatment of the wild-type receptor with TCEP at low concentrations, which had no......A highly conserved feature across all families of 7TM receptors is a disulfide bridge between a Cys residue located at the extracellular end of transmembrane segment III (TM-III) and one in extracellular loop 2 (ECL-2). The zinc sensor GPR39 contains four Cys residues in the extracellular domains....... By using mutagenesis, treatment with the reducing agent TCEP, and a labeling procedure for free sulfhydryl groups, we identify the pairing of these Cys residues in two disulfide bridges: the prototypical bridge between Cys (108) in TM-III and Cys (210) in ECL-2 and a second disulfide bridge...

  1. Effect of dialyser membranes on extracellular and intracellular granulocyte and monocyte activation in ex vivo pyrogen-free conditions.

    Science.gov (United States)

    Mahiout, A; Courtney, J M

    1994-10-01

    This study examined effects of blood-contacting materials on the monocyte reaction following the first contact of human blood with hollow fibre dialyser membranes under pyrogen-free conditions. Membrane materials were the unchanged regenerated cellulose, the synthetic polysulphone (PS), a positively charged diethylaminoethyl cellulose (DEAE-C), the negatively charged carboxymethyl cellulose (CMC) and acrylonitrile copolymer (AN). The experimental system involved perfusion with human fresh venous blood through different modules containing the materials in the form of hollow fibre membranes. Extracellular and intracellular aspects of blood reactions after the first contact with the materials were investigated in Ficoll-separated granulocytes and peripheral blood mononuclear cells. Investigations were done by release reactions of platelet activating factor (PAF), oxygen radical (O2-), leukotriene B4, prostaglandin E2 (PGE2) and cytokines (IL-1 beta, TNF-alpha, IL-6). The intracellular activation of peripheral blood mononuclear cells was done by mRNA transcription of IL-1 beta, TNF-alpha, IL-6, IL-8 and beta 2-microglobulin (beta 2-MG). From the set of parameters, release reactions were only measurable for PAF, PGE2 and O2- if a second stimulus (phorbol myristate acetate, lipopolysaccharide, zymosan and calcium ionophore) was used after blood-membrane interaction. Although the extent of the release reaction was weak, negatively charged membranes were, in general, more active. All dialysers exhibited the same increase in beta 2-MG mRNA transcription, suggesting that all blood-contacting membranes initiate the gene expression of beta 2-MG at the same level. TNF-alpha, IL-6, IL-1 beta and IL-8 mRNAs were demonstrated in the AN and CMC membranes rather than the other materials, which exhibit a lower transcription than the tubing set. As has been found, an enhanced generation of PGE2 for both CMC and AN membranes supports, therefore, the concept of an effect of the negative

  2. Lung epithelial cell-derived extracellular vesicles activate macrophage-mediated inflammatory responses via ROCK1 pathway.

    Science.gov (United States)

    Moon, H-G; Cao, Y; Yang, J; Lee, J H; Choi, H S; Jin, Y

    2015-01-01

    Despite decades of research, the pathogenesis of acute respiratory distress syndrome (ARDS) remains poorly understood, thus impeding the development of effective treatment. Diffuse alveolar damage (DAD) and lung epithelial cell death are prominent features of ARDS. Lung epithelial cells are the first line of defense after inhaled stimuli, such as in the case of hyperoxia. We hypothesized that lung epithelial cells release 'messenger' or signaling molecules to adjacent or distant macrophages, thereby initiating or propagating inflammatory responses after noxious insult. We found that, after hyperoxia, a large amount of extracellular vesicles (EVs) were generated and released into bronchoalveolar lavage fluid (BALF). These hyperoxia-induced EVs were mainly derived from live lung epithelial cells as the result of hyperoxia-associated endoplasmic reticulum (ER) stress. These EVs were remarkably different from epithelial 'apoptotic bodies', as reflected by the significantly smaller size and differentially expressed protein markers. These EVs fall mainly in the size range of the exosomes and smaller microvesicles (MVs) (50-120 nm). The commonly featured protein markers of apoptotic bodies were not found in these EVs. Treating alveolar macrophages with hyperoxia-induced, epithelial cell-derived EVs led to an increased secretion of pro-inflammatory cytokines and macrophage inflammatory protein 2 (MIP-2). Robustly increased macrophage and neutrophil influx was found in the lung tissue of the mice intranasally treated with hyperoxia-induced EVs. It was determined that EV-encapsulated caspase-3 was largely responsible for the alveolar macrophage activation via the ROCK1 pathway. Caspase-3-deficient EVs induced less cytokine/MIP-2 release, reduced cell counts in BALF, less neutrophil infiltration and less inflammation in lung parenchyma, both in vitro and in vivo. Furthermore, the serum circulating EVs were increased and mainly derived from lung epithelial cells after

  3. Extracellular activation of Wnt signaling through epigenetic dysregulation of Wnt inhibitory factor-1 (Wif-1) is associated with pathogenesis of adrenocortical tumor

    OpenAIRE

    Mitsui, Yozo; Yasumoto, Hiroaki; Nagami, Taichi; Hiraki, Miho; Arichi, Naoko; Ishikawa, Noriyoshi; Araki, Asuka; Maruyama, Riruke; Tanaka, Yuichiro; Dahiya, Rajvir; Shiina, Hiroaki

    2014-01-01

    Wnt/β-catenin signaling is considered to be an essential regulator of adrenocortical oncogenesis. Wnt inhibitory factor-1 (Wif-1), an extracellular regulator of Wnt signaling, is frequently down-regulated by hypermethylation of the promoter CpG. We investigated epigenetic regulation of Wif-1 and its association with adrenocortical (AC) tumor pathogenesis in light of Wnt activation. The AC tumors showed a high prevalence of Wif-1 promoter methylation and low prevalence of Wif-1 mRNA transcript...

  4. Estradiol-induced object memory consolidation in middle-aged female mice requires dorsal hippocampal ERK and PI3K activation

    OpenAIRE

    Fan, Lu; Zhao, Zaorui; Orr, Patrick T.; Chambers, Cassie H.; Michael C. Lewis; Frick, Karyn M.

    2010-01-01

    We previously demonstrated that dorsal hippocampal extracellular signal-regulated kinase (ERK) activation is necessary for 17β-estradiol (E2) to enhance novel object recognition in young ovariectomized mice (Fernandez et al., 2008). Here, we asked whether E2 has similar memory-enhancing effects in middle-aged and aged ovariectomized mice, and whether these effects depend on ERK and phosphatidylinositol 3-kinase (PI3K)/Akt activation. We first demonstrated that intracerebroventricular (ICV) E2...

  5. Extracellular calcium sensing and extracellular calcium signaling

    Science.gov (United States)

    Brown, E. M.; MacLeod, R. J.; O'Malley, B. W. (Principal Investigator)

    2001-01-01

    The cloning of a G protein-coupled extracellular Ca(2+) (Ca(o)(2+))-sensing receptor (CaR) has elucidated the molecular basis for many of the previously recognized effects of Ca(o)(2+) on tissues that maintain systemic Ca(o)(2+) homeostasis, especially parathyroid chief cells and several cells in the kidney. The availability of the cloned CaR enabled the development of DNA and antibody probes for identifying the CaR's mRNA and protein, respectively, within these and other tissues. It also permitted the identification of human diseases resulting from inactivating or activating mutations of the CaR gene and the subsequent generation of mice with targeted disruption of the CaR gene. The characteristic alterations in parathyroid and renal function in these patients and in the mice with "knockout" of the CaR gene have provided valuable information on the CaR's physiological roles in these tissues participating in mineral ion homeostasis. Nevertheless, relatively little is known about how the CaR regulates other tissues involved in systemic Ca(o)(2+) homeostasis, particularly bone and intestine. Moreover, there is evidence that additional Ca(o)(2+) sensors may exist in bone cells that mediate some or even all of the known effects of Ca(o)(2+) on these cells. Even more remains to be learned about the CaR's function in the rapidly growing list of cells that express it but are uninvolved in systemic Ca(o)(2+) metabolism. Available data suggest that the receptor serves numerous roles outside of systemic mineral ion homeostasis, ranging from the regulation of hormonal secretion and the activities of various ion channels to the longer term control of gene expression, programmed cell death (apoptosis), and cellular proliferation. In some cases, the CaR on these "nonhomeostatic" cells responds to local changes in Ca(o)(2+) taking place within compartments of the extracellular fluid (ECF) that communicate with the outside environment (e.g., the gastrointestinal tract). In others

  6. Atmospheric CO2 and soil extracellular enzyme activity: A meta-analysis and CO2 gradient experiment

    Science.gov (United States)

    Rising atmospheric CO2 concentrations may alter carbon and nutrient cycling and microbial processes in terrestrial ecosystems. One of the primary ways that microbes interact with soil organic matter is through the production of extracellular enzymes, which break down large, complex organic molecules...

  7. The Extracellular Protease Matrix Metalloproteinase-9 Is Activated by Inhibitory Avoidance Learning and Required for Long-Term Memory

    Science.gov (United States)

    Nagy, Vanja; Bozdagi, Ozlem; Huntley, George W.

    2007-01-01

    Matrix metalloproteinases (MMPs) are a family of extracellularly acting proteolytic enzymes with well-recognized roles in plasticity and remodeling of synaptic circuits during brain development and following brain injury. However, it is now becoming increasingly apparent that MMPs also function in normal, nonpathological synaptic plasticity of the…

  8. Extracellular zinc stimulates a calcium-activated chloride conductance through mobilisation of intracellular calcium in renal inner medullary collecting duct cells.

    Science.gov (United States)

    Linley, J E; Simmons, N L; Gray, M A

    2007-01-01

    We have used the perforated patch clamp and fura-2 fluorescence techniques to study the effect of extracellular Zn(2+) on whole-cell Ca(2+)-activated Cl(-) currents (I (CLCA)) in mouse inner medullary collecting duct cells (mIMCD-3). I (CLCA) was spontaneously active in 74% of cells under basal conditions and displayed time and voltage-independent kinetics and an outwardly rectifying current/voltage relationship (I/V). Addition of zinc chloride (10-400 microM) to the bathing solution resulted in a dose-dependent increase in I (CLCA) with little change in Cl(-) selectivity or biophysical characteristics, whereas gadolinium chloride (30 microM) and lanthanum chloride (100 microM) had no significant effect on the whole-cell current. Using fura-2-loaded mIMCD-3 cells, extracellular Zn(2+) (400 microM) stimulated an increase in intracellular Ca(2+) to an elevated plateau. The Zn(2+)-stimulated [Ca(2+)](i) increase was inhibited by thapsigargin (200 nM), the IP(3) receptor antagonist 2-aminoethoxydiphenyl borate (10 microM) and removal of bath Ca(2+). Pre-exposure to Zn(2+) (400 microM) markedly attenuated the ATP (100 microM)-stimulated [Ca(2+)](i) increase. These data are consistent with the hypothesis that extracellular Zn(2+) stimulates an increase in [Ca(2+)](i) by a release of calcium from thapsigargin/IP(3) sensitive stores. A possible physiological role for a divalent metal ion receptor, distinct from the extracellular Ca(2+)-sensing receptor, in IMCD cells is discussed. PMID:17021797

  9. Salvia miltiorrhiza Induces Tonic Contraction of the Lower Esophageal Sphincter in Rats via Activation of Extracellular Ca2+ Influx

    Directory of Open Access Journals (Sweden)

    Ching-Chung Tsai

    2015-08-01

    Full Text Available Up to 40% of patients with gastroesophageal reflux disease (GERD suffer from proton pump inhibitor refractory GERD but clinically the medications to strengthen the lower esophageal sphincter (LES to avoid irritating reflux are few in number. This study aimed to examine whether Salvia miltiorrhiza (SM extracts induce tonic contraction of rat LES ex vivo and elucidate the underlying mechanisms. To investigate the mechanism underlying the SM extract-induced contractile effects, rats were pretreated with atropine (a muscarinic receptor antagonist, tetrodotoxin (a sodium channel blocker, nifedipine (a calcium channel blocker, and Ca2+-free Krebs-Henseleit solution with ethylene glycol tetraacetic acid (EGTA, followed by administration of cumulative dosages of SM extracts. SM extracts induced dose-related tonic contraction of the LES, which was unaffected by tetrodotoxin, atropine, or nifedipine. However, the SM extract-induced LES contraction was significantly inhibited by Ca2+-free Krebs-Henseleit solution with EGTA. Next, SM extracts significantly induce extracellular Ca2+ entry into primary LES cells in addition to intracellular Ca2+ release and in a dose-response manner. Confocal fluorescence microscopy showed that the SM extracts consistently induced significant extracellular Ca2+ influx into primary LES cells in a time-dependent manner. In conclusion, SM extracts could induce tonic contraction of LES mainly through the extracellular Ca2+ influx pathway.

  10. Receptor tyrosine kinase signaling regulates replication of the peste des petits ruminants virus.

    Science.gov (United States)

    Chaudhary, K; Chaubey, K K; Singh, S V; Kumar, N

    2015-03-01

    In this study, we found out that blocking the receptor tyrosine kinase (RTK) signaling in Vero cells by tryphostin AG879 impairs the in vitro replication of the peste des petits ruminants virus (PPRV). A reduced virus replication in Trk1-knockdown (siRNA) Vero cells confirmed the essential role of RTK in the virus replication, in particular a specific regulation of viral RNA synthesis. These data represent the first evidence that the RTK signaling regulates replication of a morbillivirus. PMID:25790054

  11. Activation of P-glycoprotein (Pgp)-mediated drug efflux by extracellular acidosis: in vivo imaging with {sup 68}Ga-labelled PET tracer

    Energy Technology Data Exchange (ETDEWEB)

    Thews, Oliver; Dillenburg, Wolfgang [University Medicine Mainz, Institute of Physiology and Pathophysiology, Mainz (Germany); Fellner, Marco; Roesch, Frank [University of Mainz, Institute of Nuclear Chemistry, Mainz (Germany); Buchholz, Hans-Georg; Bausbacher, Nicole; Schreckenberger, Mathias [University Medicine Mainz, Department of Nuclear Medicine, Mainz (Germany)

    2010-10-15

    In vitro it has been shown that the functional activity of P-glycoprotein (Pgp), an important drug transporter responsible for multidrug resistance, can be strongly increased by extracellular acidosis. Here mitogen-activated protein kinases (MAPK) (p38, ERK1/2) seem to play an important role for signal transduction. However, it is unclear whether these effects are also relevant in vivo. With the newly developed PET tracer Schiff base-based {sup 68}Ga-MFL6.MZ the functional Pgp activity was visualized under acidic conditions and during inhibition of MAPKs non-invasively by means of microPET in rat tumours. Tumours were acidified either by inspiratory hypoxia (8% O{sub 2}) or by injection of lactic acid. Inhibitors of the MAPK were injected intratumourally. With increasing tumour volume the tumour pH changed from 7.0 to 6.7 and simultaneously the Pgp activity increased almost linearly. When the tumour was acidified by direct lactic acid injection the PET tracer uptake was reduced by 20% indicating a higher transport rate out of the cells. Changing the inspiratory O{sub 2} fraction to 8% dynamically led to a reduction of extracellular pH and in parallel to a decrease of tracer concentration. While inhibition of the p38 pathway reduced the Pgp transport rate, inhibition of ERK1/2 had practically no impact. An acidic extracellular environment significantly stimulates the Pgp activity. The p38 MAPK pathway plays an important role for Pgp regulation in vivo, whereas ERK1/2 is of minor importance. From these results new strategies for overcoming multidrug resistance (e.g. reducing tumour acidosis, inhibition of p38) may be developed. (orig.)

  12. Rapid fluctuations in extracellular brain glucose levels induced by natural arousing stimuli and intravenous cocaine: fueling the brain during neural activation

    Science.gov (United States)

    Lenoir, Magalie

    2012-01-01

    Glucose, a primary energetic substrate for neural activity, is continuously influenced by two opposing forces that tend to either decrease its extracellular levels due to enhanced utilization in neural cells or increase its levels due to entry from peripheral circulation via enhanced cerebral blood flow. How this balance is maintained under physiological conditions and changed during neural activation remains unclear. To clarify this issue, enzyme-based glucose sensors coupled with high-speed amperometry were used in freely moving rats to evaluate fluctuations in extracellular glucose levels induced by brief audio stimulus, tail pinch (TP), social interaction with another rat (SI), and intravenous cocaine (1 mg/kg). Measurements were performed in nucleus accumbens (NAcc) and substantia nigra pars reticulata (SNr), which drastically differ in neuronal activity. In NAcc, where most cells are powerfully excited after salient stimulation, glucose levels rapidly (latency 2–6 s) increased (30–70 μM or 6–14% over baseline) by all stimuli; the increase differed in magnitude and duration for each stimulus. In SNr, where most cells are transiently inhibited by salient stimuli, TP, SI, and cocaine induced a biphasic glucose response, with the initial decrease (−20–40 μM or 5–10% below baseline) followed by a reboundlike increase. The critical role of neuronal activity in mediating the initial glucose response was confirmed by monitoring glucose currents after local microinjections of glutamate (GLU) or procaine (PRO). While intra-NAcc injection of GLU transiently increased glucose levels in this structure, intra-SNr PRO injection resulted in rapid, transient decreases in SNr glucose. Therefore, extracellular glucose levels in the brain change very rapidly after physiological and pharmacological stimulation, the response is structure specific, and the pattern of neuronal activity appears to be a critical factor determining direction and magnitude of physiological

  13. Disruption of parathyroid hormone and parathyroid hormone-related peptide receptor phosphorylation prolongs ERK1/2 MAPK activation and enhances c-fos expression

    OpenAIRE

    Tawfeek, Hesham A.; Abou-Samra, Abdul B.

    2012-01-01

    Previous studies have demonstrated that parathyroid hormone (PTH) binding to the PTH/PTH-related peptide receptor (PPR) stimulates G protein coupling, receptor phosphorylation, β-arrestin translocation, and internalization of the ligand/receptor complex. The extracellular signal-regulated mitogen-activated protein kinases 1/2 (ERK1/2 MAPK) are downstream effectors of PPR. In the current study, we investigated the role of PPR phosphorylation in the PTH regulation of the ERK1/2 MAPK pathway. Sh...

  14. Analysis of a second bacteriophage hyaluronidase gene from Streptococcus pyogenes: evidence for a third hyaluronidase involved in extracellular enzymatic activity.

    OpenAIRE

    Hynes, W L; Hancock, L; Ferretti, J J

    1995-01-01

    The hyaluronidase gene (hylP2) from a second group A streptococcal bacteriophage was isolated from ATCC T-type-22 hyaluronidase-producing strain 10403, a strain known to produce increased amounts of extracellular hyaluronidase. Sequence analysis of hylP2 and alignment with the previously described bacteriophage hyaluronidase gene (hylP) showed a high degree of similarity; however, hylP2 had deletions of regions specifying 34 amino acids. Twenty-eight of the deleted amino acids were in a regio...

  15. The effect of acute and long-term physical activity on extracellular matrix and serglycin in human skeletal muscle

    OpenAIRE

    Hjorth, Marit; Norheim, Frode; Meen, Astri Jeanette; Pourteymour, Shirin; Lee, Sindre; Holen, Torgeir; Jensen, Jørgen; Birkeland, Kåre I; Martinov, Vladimir Nikolkaev; Langleite, Torgrim Mikal; Eckardt, Kristin; Drevon, Christian A.; Kolset, Svein Olav

    2015-01-01

    Remodeling of extracellular matrix (ECM), including regulation of proteoglycans in skeletal muscle can be important for physiological adaptation to exercise. To investigate the effects of acute and long-term exercise on the expression of ECM-related genes and proteoglycans in particular, 26 middle-aged, sedentary men underwent a 12 weeks supervised endurance and strength training intervention and two acute, 45 min bicycle tests (70% VO2max), one at baseline and one after 12 weeks of training....

  16. Tsukushi modulates Xnr2, FGF and BMP signaling: regulation of Xenopus germ layer formation.

    Directory of Open Access Journals (Sweden)

    Samantha A Morris

    Full Text Available BACKGROUND: Cell-cell communication is essential in tissue patterning. In early amphibian development, mesoderm is formed in the blastula-stage embryo through inductive interactions in which vegetal cells act on overlying equatorial cells. Members of the TGF-beta family such as activin B, Vg1, derrière and Xenopus nodal-related proteins (Xnrs are candidate mesoderm inducing factors, with further activity to induce endoderm of the vegetal region. TGF-beta-like ligands, including BMP, are also responsible for patterning of germ layers. In addition, FGF signaling is essential for mesoderm formation whereas FGF signal inhibition has been implicated in endoderm induction. Clearly, several signaling pathways are coordinated to produce an appropriate developmental output; although intracellular crosstalk is known to integrate multiple pathways, relatively little is known about extracellular coordination. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that Xenopus Tsukushi (X-TSK, a member of the secreted small leucine rich repeat proteoglycan (SLRP family, is expressed in ectoderm, endoderm, and the organizer during early development. We have previously reported that X-TSK binds to and inhibits BMP signaling in cooperation with chordin. We now demonstrate two novel interactions: X-TSK binds to and inhibits signaling by FGF8b, in addition to binding to and enhancement of Xnr2 signaling. This signal integration by X-TSK at the extracellular level has an important role in germ layer formation and patterning. Vegetally localized X-TSK potentiates endoderm formation through coordination of BMP, FGF and Xnr2 signaling. In contrast, X-TSK inhibition of FGF-MAPK signaling blocks ventrolateral mesoderm formation, while BMP inhibition enhances organizer formation. These actions of X-TSK are reliant upon its expression in endoderm and dorsal mesoderm, with relative exclusion from ventrolateral mesoderm, in a pattern shaped by FGF signals. CONCLUSIONS

  17. Characterization of a heat-resistant extracellular protease from Pseudomonas fluorescens 07A shows that low temperature treatments are more effective in deactivating its proteolytic activity.

    Science.gov (United States)

    Alves, Maura P; Salgado, Rafael L; Eller, Monique R; Vidigal, Pedro Marcus P; Fernandes de Carvalho, Antonio

    2016-10-01

    This work discusses the biological and biochemical characterization of an extracellular protease produced by Pseudomonas fluorescens. The enzyme has a molecular weight of 49.486 kDa and hydrolyzes gelatin, casein, and azocasein, but not BSA. Its maximum activity is found at 37°C and pH 7.5, but it retained almost 70% activity at pH 10.0. It was shown to be a metalloprotease inhibited by Cu(2+), Ni(2+), Zn(2+), Hg(2+), Fe(2+), and Mg(2+), but induced by Mn(2+). After incubation at 100°C for 5min, the enzyme presented over 40% activity, but only 14 to 30% when submitted to milder heat treatments. This behavior may cause significant problems under conditions commonly used for the processing and storage of milk and dairy products, particularly UHT milk. A specific peptide sequenced by mass spectrometer analysis allowed the identification of gene that encodes this extracellular protease in the genome of Pseudomonas fluorescens 07A strain. The enzyme has 477 AA and highly conserved Ca(2+)- and Zn(2+)-binding domains, indicating that Ca(2+), the main ion in milk, is also a cofactor. This work contributes to the understanding of the biochemical aspects of enzyme activity and associates them with its sequence and structure. These findings are essential for the full understanding and control of these enzymes and the technological problems they cause in the dairy industry. PMID:27497896

  18. Involvement of mitogen-activated protein kinase pathways in N-methyl-D-aspartate-induced excitotoxicity

    Institute of Scientific and Technical Information of China (English)

    Xiaorong Yang; Ping Sun; Huaping Qin; Rui Wang; Ye Wang; Ruihong Shi; Xin Zhao; Ce Zhang

    2011-01-01

    Previous studies have shown that mitogen-activated protein kinase (MAPK) signaling pathways are involved in N-methyl-D-aspartate (NMDA)-mediated excitotoxicity. However, a systematic observation or analysis of the role of these various MAPK pathways in excitotoxicity processes does not exist. The present study further evaluated the role and contribution of three MAPK pathways extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 MAPK in an NMDA-mediated excitotoxicity model using MAPK-specific inhibitor. Results demonstrated that c-Jun N-terminal kinase inhibitor SP600125 and/or p38 MAPK inhibitor SB203580 inhibited NMDA-induced reduction in cell viability, as well as reduced NMDA-induced lactate dehydrogenase leakage and reactive oxygen species production. However, PD98059, an inhibitor of extracellular signal-regulated kinase, did not influence this model. Results demonstrated an involvement of c-Jun N-terminal kinase and p38 MAPK, but not extracellular signal-regulated kinase, in NMDA-mediated excitotoxicity in cortical neurons.

  19. Conformational constraining of inactive and active States of a seven transmembrane receptor by metal ion site engineering in the extracellular end of transmembrane segment V

    DEFF Research Database (Denmark)

    Rosenkilde, Mette M; David, Ralf; Oerlecke, Ilka;

    2006-01-01

    The extracellular part of transmembrane segment V (TM-V) is expected to be involved in the activation process of 7TM receptors, but its role is far from clear. Here, we study the highly constitutively active CXC-chemokine receptor encoded by human herpesvirus 8 (ORF74-HHV8), in which a metal ion...... site was introduced at the extracellular end of TM-V by substitution of two arginines at positions V:01 and V:05 with histidines [R208H; R212H]. The metal ion site conferred high-potency inverse agonist properties (EC(50), 1.7 microM) to Zn(II) in addition to agonist and allosteric enhancing properties....... The activating properties of Zn(II) were not due to a metal ion site between the ligand and the receptor because CXCL1/GROalpha analogs in which the putative metal-ion binding residues had been substituted-[H19A] and [H34A]-acted like wild-type CXCL1/GROalpha. Based on the complex action of Zn...

  20. Cloning and heterologous expression of the ftfCNC-2(1) gene from Weissella confusa MBFCNC-2(1) as an extracellular active fructansucrase in Bacillus subtilis.

    Science.gov (United States)

    Malik, Amarila; Hapsari, Maria Tyas; Ohtsu, Iwao; Ishikawa, Shu; Takagi, Hiroshi

    2015-05-01

    Fructan-exopolysaccharides (fructan-EPS) (inulin and levan) and their oligosaccharides (fructooligosaccharides, FOS) have drawn considerable interest in the food and pharmaceutical industries. EPS-producing lactic acid bacteria have been reported to produce β-fructans (inulin and levan), as well as α-glucans, by the function of sucrase enzymes, i.e., fructansucrase and glucansucrase. A fructansucrase ftfCNC-2(1) gene from Weissella confusa strain MBFCNC-2(1) was previously cloned in Escherichia coli. In this study, we aimed to express the ftf[CNC-2(1)] gene in Bacillus subtilis to obtain the active form of the extracellular recombinant protein FTF[CNC-2(1)]. This cloning was achieved by inserting the gene in-fusion with the signal sequence of the B. subtilis subtilisin E. SDS-polyacrylamide gel electrophoresis analysis and in situ activity assay with Periodic Acid-Schiff staining revealed that the recombinant FTF[CNC-2(1)] was successfully expressed as an extracellular protein from B. subtilis DB403 in its active form, which was confirmed using sucrose and raffinose. PMID:25454699

  1. Biomimetic aggrecan reduces cartilage extracellular matrix from degradation and lowers catabolic activity in ex vivo and in vivo models.

    Science.gov (United States)

    Sharma, Shaili; Lee, Aeju; Choi, Kuiwon; Kim, Kwangmeyung; Youn, Inchan; Trippel, Stephen B; Panitch, Alyssa

    2013-09-01

    Aggrecan, a major macromolecule in cartilage, protects the extracellular matrix (ECM) from degradation during the progression of osteoarthritis (OA). However, aggrecan itself is also susceptible to proteolytic cleavage. Here, the use of a biomimetic proteoglycan (mAGC) is presented, which functionally mimics aggrecan but lacks the known cleavage sites, protecting the molecule from proteolytic degradation. The objective of this study is to test the efficacy of this molecule in ex vivo (human OA synovial fluid) and in vivo (Sprague-Dawley rats) osteoarthritic models. These results indicate that mAGC's may protect articular cartilage against the loss of key ECM components, and lower catabolic protein and gene expression in both models. This suppression of matrix degradation has the potential to provide a healthy environment for tissue repair.

  2. CUX1/Wnt signaling regulates Epithelial Mesenchymal Transition in EBV infected epithelial cells

    International Nuclear Information System (INIS)

    Idiopathic pulmonary fibrosis (IPF) is a refractory and lethal interstitial lung disease characterized by alveolar epithelial cells apoptosis, fibroblast proliferation and extra-cellular matrix protein deposition. EBV, localised to alveolar epithelial cells of pulmonary fibrosis patients is associated with a poor prognosis. A strategy based on microarray-differential gene expression analysis to identify molecular drivers of EBV-associated lung fibrosis was utilized. Alveolar epithelial cells were infected with EBV to identify genes whose expression was altered following TGFβ1-mediated lytic phase. EBV lytic reactivation by TGFβ1 drives a selective alteration in CUX1 variant (a) (NCBI accession number NM181552) expression, inducing activation of non-canonical Wnt pathway mediators, implicating it in Epithelial Mesenchymal Transition (EMT), the molecular event underpinning scar production in tissue fibrosis. The role of EBV in EMT can be attenuated by antiviral strategies and inhibition of Wnt signaling by using All-Trans Retinoic Acids (ATRA). Activation of non-canonical Wnt signaling pathway by EBV in epithelial cells suggests a novel mechanism of EMT via CUX1 signaling. These data present a framework for further description of the link between infectious agents and fibrosis, a significant disease burden.

  3. CUX1/Wnt signaling regulates Epithelial Mesenchymal Transition in EBV infected epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Malizia, Andrea P.; Lacey, Noreen [Clinical Research Centre, School of Medicine and Medical Science, University College Dublin. 21, Nelson Street. Dublin, 7. Ireland (Ireland); Walls, Dermot [School of Biotechnology, Dublin City University. Dublin, 9. Ireland (Ireland); Egan, Jim J. [Advanced Lung Disease and Lung Transplant Program, Mater Misericordiae University Hospital. 44, Eccles Street. Dublin, 7. Ireland (Ireland); Doran, Peter P., E-mail: peter.doran@ucd.ie [Clinical Research Centre, School of Medicine and Medical Science, University College Dublin. 21, Nelson Street. Dublin, 7. Ireland (Ireland)

    2009-07-01

    Idiopathic pulmonary fibrosis (IPF) is a refractory and lethal interstitial lung disease characterized by alveolar epithelial cells apoptosis, fibroblast proliferation and extra-cellular matrix protein deposition. EBV, localised to alveolar epithelial cells of pulmonary fibrosis patients is associated with a poor prognosis. A strategy based on microarray-differential gene expression analysis to identify molecular drivers of EBV-associated lung fibrosis was utilized. Alveolar epithelial cells were infected with EBV to identify genes whose expression was altered following TGF{beta}1-mediated lytic phase. EBV lytic reactivation by TGF{beta}1 drives a selective alteration in CUX1 variant (a) (NCBI accession number NM{sub 1}81552) expression, inducing activation of non-canonical Wnt pathway mediators, implicating it in Epithelial Mesenchymal Transition (EMT), the molecular event underpinning scar production in tissue fibrosis. The role of EBV in EMT can be attenuated by antiviral strategies and inhibition of Wnt signaling by using All-Trans Retinoic Acids (ATRA). Activation of non-canonical Wnt signaling pathway by EBV in epithelial cells suggests a novel mechanism of EMT via CUX1 signaling. These data present a framework for further description of the link between infectious agents and fibrosis, a significant disease burden.

  4. A new classification paradigm of extracellular polymeric substances (EPS) in activated sludge: separation and characterization of exopolymers between floc level and microcolony level.

    Science.gov (United States)

    Wang, Bin-Bin; Chang, Qing; Peng, Dang-Cong; Hou, Yin-Ping; Li, Hui-Juan; Pei, Li-Ying

    2014-11-01

    Extracellular polymeric substances (EPS) play a crucial role in the formation of activated sludge flocs. However, until now, the EPS are rather classified by the method used for extraction than by a theoretical consideration of their function and composition. In this paper, a new classification paradigm of EPS was proposed, which offered a novel approach to identify the role of EPS in the formation of activated sludge flocs. The current study gave an exploration to distinguish the EPS in the floc level (extra-microcolony polymers, EMPS) and in the microcolony level (extra-cellular polymers, ECPS). It was found that cation exchange resin treatment is efficient to disintegrate the flocs for EMPS extraction, however, inefficient to disaggregate the microcolonies for ECPS harvesting. A two-steps extraction strategy (cation exchange resin treatment followed by ultrasonication-high speed centrifugation treatment) was suggested to separate these two types of EPS in activated sludge flocs and the physicochemical characteristics of EMPS and ECPS were compared. The protein/polysaccharide ratio of ECPS was higher than that of EMPS and the molecular weight of proteins in EMPS and ECPS were found to be different. The ECPS contained higher molecular weight proteins and more hydrophobic substances than the EMPS contained. The result of excitation-emission matrix fluorescence spectroscopy analysis also showed that the EMPS and the ECPS have different fluorescent expressions and the components of EMPS were more diverse than that of ECPS. All results reported herein demonstrated that two different types of exopolymers exist in the activated sludge flocs and the inter-particle forces for aggregation of activated sludge flocs are not identical between the floc level and the microcolony level. It suggested that cation bridging interactions are more crucial in floc level flocculation, while the entanglement and hydrophobic interactions are more important in microcolony level cohesion.

  5. Egr-1 activation by cancer-derived extracellular vesicles promotes endothelial cell migration via ERK1/2 and JNK signaling pathways.

    Directory of Open Access Journals (Sweden)

    Yae Jin Yoon

    Full Text Available Various mammalian cells, including cancer cells, shed extracellular vesicles (EVs, also known as exosomes and microvesicles, into surrounding tissues. These EVs play roles in tumor growth and metastasis by promoting angiogenesis. However, the detailed mechanism of how cancer-derived EVs elicit endothelial cell activation remains unknown. Here, we provide evidence that early growth response-1 (Egr-1 activation in endothelial cells is involved in the angiogenic activity of colorectal cancer cell-derived EVs. Both RNA interference-mediated downregulation of Egr-1 and ERK1/2 or JNK inhibitor significantly blocked EV-mediated Egr-1 activation and endothelial cell migration. Furthermore, lipid raft-mediated endocytosis inhibitor effectively blocked endothelial Egr-1 activation and migration induced by cancer-derived EVs. Our results suggest that Egr-1 activation in endothelial cells may be a key mechanism involved in the angiogenic activity of cancer-derived EVs. These findings will improve our understanding regarding the proangiogenic activities of EVs in diverse pathological conditions including cancer, cardiovascular diseases, and neurodegenerative diseases.

  6. Cloning, expression, and characterization of a soluble calcium-activated nucleotidase, a human enzyme belonging to a new family of extracellular nucleotidases.

    Science.gov (United States)

    Smith, Thomas M; Hicks-Berger, Carrie A; Kim, Sunkyu; Kirley, Terence L

    2002-10-01

    The salivary apyrases of blood-feeding arthropods are nucleotide-hydrolyzing enzymes implicated in the inhibition of host platelet aggregation through the hydrolysis of extracellular adenosine diphosphate. A human cDNA homologous to the apyrase cDNA of the blood-feeding bed bug was identified, revealing an open reading frame encoding a 371-amino acid protein. A cleavable signal peptide generates a secreted protein of 333 residues with a predicted core molecular mass of 37,193 Da. Expression in COS-1 cells produced a secreted apyrase in the cell media. The ADPase and ATPase activities were dependent upon calcium, with a pH optimum between pH 6.2 and 7.2. Interestingly, the preferred substrate was not ADP, as might be expected for an enzyme modulating platelet aggregation, but rather UDP, followed by GDP, UTP, GTP, ADP, and ATP. The nucleotidase did not hydrolyze nucleoside monophosphates. Size-exclusion chromatography and Western blot analysis revealed a molecular mass of approximately 34-37 kDa. Treatment of the enzyme with peptide N-glycosidase F indicated that the protein is glycosylated. Northern analysis identified the transcript in a range of human tissues, including testis, placenta, prostate, and lung. No traditional apyrase-conserved regions or nucleotide-binding domains were identified in this human enzyme, indicating membership in a new family of extracellular nucleotidases. PMID:12234496

  7. The tissue plasminogen activator (tPA)/plasmin extracellular proteolytic system regulates seizure-induced hippocampal mossy fiber outgrowth through a proteoglycan substrate.

    Science.gov (United States)

    Wu, Y P; Siao, C J; Lu, W; Sung, T C; Frohman, M A; Milev, P; Bugge, T H; Degen, J L; Levine, J M; Margolis, R U; Tsirka, S E

    2000-03-20

    Short seizure episodes are associated with remodeling of neuronal connections. One region where such reorganization occurs is the hippocampus, and in particular, the mossy fiber pathway. Using genetic and pharmacological approaches, we show here a critical role in vivo for tissue plasminogen activator (tPA), an extracellular protease that converts plasminogen to plasmin, to induce mossy fiber sprouting. We identify DSD-1-PG/phosphacan, an extracellular matrix component associated with neurite reorganization, as a physiological target of plasmin. Mice lacking tPA displayed decreased mossy fiber outgrowth and an aberrant band at the border of the supragranular region of the dentate gyrus that coincides with the deposition of unprocessed DSD-1-PG/phosphacan and excessive Timm-positive, mossy fiber termini. Plasminogen-deficient mice also exhibit the laminar band and DSD- 1-PG/phosphacan deposition, but mossy fiber outgrowth through the supragranular region is normal. These results demonstrate that tPA functions acutely, both through and independently of plasmin, to mediate mossy fiber reorganization.

  8. Mesenchymal Transition of High-Grade Breast Carcinomas Depends on Extracellular Matrix Control of Myeloid Suppressor Cell Activity.

    Science.gov (United States)

    Sangaletti, Sabina; Tripodo, Claudio; Santangelo, Alessandra; Castioni, Nadia; Portararo, Paola; Gulino, Alessandro; Botti, Laura; Parenza, Mariella; Cappetti, Barbara; Orlandi, Rosaria; Tagliabue, Elda; Chiodoni, Claudia; Colombo, Mario P

    2016-09-27

    The extracellular matrix (ECM) contributes to the biological and clinical heterogeneity of breast cancer, and different prognostic groups can be identified according to specific ECM signatures. In high-grade, but not low-grade, tumors, an ECM signature characterized by high SPARC expression (ECM3) identifies tumors with increased epithelial-to-mesenchymal transition (EMT), reduced treatment response, and poor prognosis. To better understand how this ECM3 signature is contributing to tumorigenesis, we expressed SPARC in isogenic cell lines and found that SPARC overexpression in tumor cells reduces their growth rate and induces EMT. SPARC expression also results in the formation of a highly immunosuppressive microenvironment, composed by infiltrating T regulatory cells, mast cells, and myeloid-derived suppressor cells (MDSCs). The ability of SPARC to induce EMT depended on the localization and suppressive function of myeloid cells, and inhibition of the suppressive function MDSCs by administration of aminobisphosphonates could revert EMT, rendering SPARC-overexpressing tumor cells sensitive to Doxil. We conclude that that SPARC is regulating the interplay between MDSCs and the ECM to drive the induction of EMT in tumor cells.

  9. Analysis of extracellular RNA by digital PCR

    Directory of Open Access Journals (Sweden)

    Kenji eTakahashi

    2014-06-01

    Full Text Available The transfer of extracellular RNA is emerging as an important mechanism for intracellular communication. The ability for the transfer of functionally active RNA molecules from one cell to another within vesicles such as exosomes enables a cell to modulate cellular signaling and biological processes within recipient cells. The study of extracellular RNA requires sensitive methods for the detection of these molecules. In this methods article, we will describe protocols for the detection of such extracellular RNA using sensitive detection technologies such as digital PCR. These protocols should be valuable to researchers interested in the role and contribution of extracellular RNA to tumor cell biology.

  10. Extracellular mtDNA activates NF-κB via toll-like receptor 9 and induces cell death in cardiomyocytes.

    Science.gov (United States)

    Bliksøen, Marte; Mariero, Lars Henrik; Torp, May Kristin; Baysa, Anton; Ytrehus, Kirsti; Haugen, Fred; Seljeflot, Ingebjørg; Vaage, Jarle; Valen, Guro; Stensløkken, Kåre-Olav

    2016-07-01

    Acute myocardial infarction (AMI) causes sterile inflammation, which exacerbates tissue injury. Elevated levels of circulating mitochondrial DNA (mtDNA) have been associated with AMI. We hypothesized that mtDNA triggers an innate immune response via TLR9 and NF-κB activation, causing cardiomyocyte injury. Murine cardiomyocytes express TLR9 mRNA and protein and were able to internalize fluorescently labeled mouse mtDNA. Incubation of human embryonic kidney cells with serum from AMI patients containing naturally elevated levels of mtDNA induced TLR9-dependent NF-κB activity. This effect was mimicked by isolated mtDNA. mtDNA activated NF-κB in reporter mice both in vivo and in isolated cardiomyocytes. Moreover, incubation of isolated cardiomyocytes with mtDNA induced cell death after 4 and 24 h. Laser confocal microscopy showed that incubation of cardiomyocytes with mtDNA accelerated mitochondrial depolarization induced by reactive oxygen species. In contrast to mtDNA, isolated total DNA did not activate NF-κB nor induce cell death. In conclusion, mtDNA can induce TLR9-dependent NF-κB activation in reporter cells and activate NF-κB in cardiomyocytes. In cardiomyocytes, mtDNA causes mitochondrial dysfunction and death. Endogenous mtDNA in the extracellular space is a danger signal with direct detrimental effects on cardiomyocytes. PMID:27164906

  11. The matrix-binding domain of microfibril-associated glycoprotein-1 targets active connective tissue growth factor to a fibroblast-produced extracellular matrix.

    Science.gov (United States)

    Weinbaum, Justin S; Tranquillo, Robert T; Mecham, Robert P

    2010-11-10

    It is advantageous to use biomaterials in tissue engineering that stimulate extracellular matrix (ECM) production by the cellular component. Connective tissue growth factor (CTGF) stimulates type I collagen (COL1A1) transcription, but is functionally limited as a free molecule. Using a matrix-binding domain (MBD) from microfibril-associated glycoprotein-1, the fusion protein MBD-CTGF was targeted to the ECM and tested for COL1A1 transcriptional activation. MBD-CTGF produced by the ECM-synthesizing fibroblasts, or provided exogenously, localized to the elastic fiber ECM. MBD-CTGF, but not CTGF alone, led to a two-fold enhancement of COL1A1 expression. This study introduces a targeting technology that can be used to elevate collagen transcription in engineered tissues and thereby improve tissue mechanics.

  12. Novel anti-bacterial activities of β-defensin 1 in human platelets: suppression of pathogen growth and signaling of neutrophil extracellular trap formation.

    Directory of Open Access Journals (Sweden)

    Bjoern F Kraemer

    2011-11-01

    Full Text Available Human β-defensins (hBD are antimicrobial peptides that curb microbial activity. Although hBD's are primarily expressed by epithelial cells, we show that human platelets express hBD-1 that has both predicted and novel antibacterial activities. We observed that activated platelets surround Staphylococcus aureus (S. aureus, forcing the pathogens into clusters that have a reduced growth rate compared to S. aureus alone. Given the microbicidal activity of β-defensins, we determined whether hBD family members were present in platelets and found mRNA and protein for hBD-1. We also established that hBD-1 protein resided in extragranular cytoplasmic compartments of platelets. Consistent with this localization pattern, agonists that elicit granular secretion by platelets did not readily induce hBD-1 release. Nevertheless, platelets released hBD-1 when they were stimulated by α-toxin, a S. aureus product that permeabilizes target cells. Platelet-derived hBD-1 significantly impaired the growth of clinical strains of S. aureus. hBD-1 also induced robust neutrophil extracellular trap (NET formation by target polymorphonuclear leukocytes (PMNs, which is a novel antimicrobial function of β-defensins that was not previously identified. Taken together, these data demonstrate that hBD-1 is a previously-unrecognized component of platelets that displays classic antimicrobial activity and, in addition, signals PMNs to extrude DNA lattices that capture and kill bacteria.

  13. Genetic interaction of two abscisic acid signaling regulators, HY5 and FIERY1, in mediating lateral root formation

    KAUST Repository

    Chen, Hao

    2011-01-01

    Root architecture is continuously shaped in a manner that helps plants to better adapt to the environment. Gene regulation at the transcriptional or post-transcriptional levels largely controls this environmental response. Recently, RNA silencing has emerged as an important player in gene regulation and is involved in many aspects of plant development, including lateral root formation. In a recent study, we found that FIERY1, a bifunctional abiotic stress and abscisic acid (ABA) signaling regulator and an endogenous RNA silencing suppressor, mediates auxin response during lateral root formation in Arabidopsis. We proposed that FRY1 regulates lateral root development through its activity on adenosine 3,5-bisphosphate (PAP), a strong inhibitor of exoribonucleases (XRNs). Interestingly, some of the phenotypes of fry1, such as enhanced response to light in repressing hypocotyl elongation and hypersensitivity to ABA in lateral root growth, are opposite to those of another light- and ABA-signaling mutant, hy5. Here we analyzed the hy5 fry1 double mutant for root and hypocotyl growth. We found that the hy5 mutation can suppress the enhanced light sensitivity in fry1 hypocotyl elongation and restore the lateral root formation. The genetic interaction between HY5 and FRY1 indicates that HY5 and FRY1 may act in overlapping pathways that mediate light signaling and lateral root development. © 2011 Landes Bioscience.

  14. Modulation of constitutive activity and signaling bias of the ghrelin receptor by conformational constraint in the second extracellular loop

    DEFF Research Database (Denmark)

    Mokrosinski, Jacek; Frimurer, Thomas M; Sivertsen, Bjoern;

    2012-01-01

    . Moreover, the constitutive activity of the receptor was inhibited by Zn(2+) binding in an engineered metal-ion site stabilizing an a-helical conformation of this loop segment. It is concluded that the high constitutive activity of the ghrelin receptor is dependent upon flexibility in the C-terminal segment...

  15. Interactions of Marsh Orchid (Dactylorhiza spp.) and Soil Microorganisms in Relation to Extracellular Enzyme Activities in a Peat Soil

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The nature of the interactions between microbes and roots of plants in a peaty soil were studied in a laboratorybased experiment by measuring activities of β-glucosidase, phosphatase, N-acetylglucosaminidase, and arylsulphatase. The experiment was based on control (autoclaved), bacteria-inoculated, and plant (transplanted with Dactylorhiza) treatments,and samples were collected over 4 sampling intervals. Higher enzyme activities were associated with the bacteria-inoculated treatment, suggesting that soil enzyme activities are mainly of microbial origin. For example, β-glucosidase activity varied between 25-30 μmol g-1 min-1 in the bacteria-inoculated samples whilst the activity of the control ranged between 4-12μmol g-1 min-1. A similar pattern was found for all other enzymes.At the end of the incubation, the microcosms were destructively sampled and the enzyme activities determined in bulk soil, rhizospheric soil, and on the root surface. Detailed measurement in different fractions of the peat indicated that higher activities were found in rhizosphere. However, the higher activities ofβ-glucosidase, N-acetylglucosaminidase, and arylsulphatase appeared to be associated with bacterial proliferation on the root surface, whilst a larger proportion of phosphatase appeared to be released from root surface.

  16. High activity and low temperature optima of extracellular enzymes in Arctic sediments: implications for carbon cycling by heterotrophic microbial communities

    DEFF Research Database (Denmark)

    Arnosti, C.; Jørgensen, BB

    2003-01-01

    (chondroitin sulfate, fucoidan, xylan and pullulan) to determine the temperature-activity responses of hydrolysis of a related class of compounds. All 4 enzyme activities showed similarly low temperature optima in the range of 15 to 18degreesC. These temperature optima are considerably lower than most previous...

  17. Enzymes in Action: An Interactive Activity Designed to Highlight Positive Attributes of Extracellular Enzymes Synthesized by Microbes

    Directory of Open Access Journals (Sweden)

    Rachel M.C. Gillespie

    2014-07-01

    Full Text Available Microbial activities are widely exploited in the manufacture of valuable products. However, the many beneficial uses of microorganisms are often overshadowed by negative associations with disease and decay. This article describes an interactive activity aimed at school-aged children and members of the public, which introduces the concept of microbial enzymes and ultimately illustrates how the industrial uses of microbes have a positive impact on everyday life. Participants are guided through a simple chemical assay which allows them to use a hands-on approach to reveal bacterial enzymes at work. This activity is safe and economical to run and is suitable for use in both the classroom and external learning environments. Also included are supplemental educational resources to support the demonstration and suggestions for extensions to the activity described, which enable further exploration of the topic. This activity has been tested by more than 2000 people at public engagement events and has received much positive feedback.

  18. Differential decolorization of textile dyes in mixtures and the joint effect of laccase and cellobiose dehydrogenase activities present in extracellular extracts from Funalia trogii.

    Science.gov (United States)

    Tilli, Silvia; Ciullini, Ilaria; Scozzafava, Andrea; Briganti, Fabrizio

    2011-10-10

    The largest part of the bio-decolorization investigations have been performed to date on a single dye without exploring the behavior in complex mixtures as the real dyeing baths. Therefore, mixtures of dyes belonging to azo and anthraquinonic classes, chosen among the most utilized in textile wool dyeing, were employed for comparative enzymatic decolorization studies using the extracellular extracts from the white rot fungus Funalia trogii, to understand how the concomitant presence of more than one dye could influence their degradation course and yield. Fungal extracts containing laccase activity only were capable to partially decolorize dyes mixtures from the different classes analyzed. The deconvolution of the decolorization with time allowed to monitor the degradation of the single dyes in the mixtures evidencing a time dependent differential decolorization not observed for the singles alone. Some dyes in the blend were in fact decolorized only when the most easily converted dyes were largely transformed. These experiments would allow to help the dyeing factories in the selection of the most readily degraded dyes. Since F. trogii grown on different media and activators shows diverse levels of expression of the redox enzymes laccase and cellobiose dehydrogenase (CDH), the dyes mixtures recalcitrant to decolorization by laccase activity alone, were subjected to the combined action of extracts containing laccase and CDH. The use of CDH, in support to the activity of laccase, resulted in substantial decolorization increases (>84%) for all the refractory dyes mixtures.

  19. Extracellular superoxide dismutase ameliorates house dust mite-induced atopic dermatitis-like skin inflammation and inhibits mast cell activation in mice.

    Science.gov (United States)

    Lee, Yun Sang; Choi, Jung-Hye; Lee, Ji-Hyun; Lee, Han-Woong; Lee, Weontae; Kim, Woo Taek; Kim, Tae-Yoon

    2016-08-01

    Extracellular superoxide dismutase (EC-SOD) is an enzyme that catalyses the dismutation of superoxide anions. It has multiple functions, such as reactive oxygen species scavenging, anti-angiogenic, anti-inflammatory, antichemotatic and antitumor activities. Recently, we demonstrated that EC-SOD inhibits ovalbumin-induced allergic airway inflammation in mice. However, the anti-allergic effect of EC-SOD on skin tissue and the role of EC-SOD in mast cells, which are important for allergic responses, have not been well studied. In this study, we investigated whether EC-SOD can alleviate atopic dermatitis in mice and inhibit mast cell activation. Treatment with human recombinant EC-SOD ameliorated house dust mite-induced atopic dermatitis in mice. Furthermore, the levels of pro-allergic cytokine gene expression and histamine release increased in EC-SOD KO mast cells and decreased in EC-SOD overexpressing mast cells, suggesting that EC-SOD inhibits mast cell activation. Consistently, a passive cutaneous anaphylaxis experiment showed more blood leakage from EC-SOD KO mouse ear skin, implying that the lack of EC-SOD increases allergic responses. These results suggest that EC-SOD inhibits mast cell activation and atopic dermatitis and that the loss of EC-SOD causes more severe allergic responses, implying that EC-SOD might be a good drug candidate for treatment of allergic disorders, such as atopic dermatitis. PMID:27061078

  20. Particle-size fractions-dependent extracellular enzyme activity in sediments and implications for resource allocation in a subtropical mangrove ecosystem

    Directory of Open Access Journals (Sweden)

    L. Luo

    2015-01-01

    Full Text Available The distribution of extracellular enzyme activities in particle-size fractions of sediments was investigated in a subtropical mangrove ecosystem. Five enzymes involved in carbon (C, nitrogen (N, and phosphorus (P cycling were analyzed in the sand, silt, and clay of sediments. Among these fractions, the highest activities of phenol oxidase (PHO, β-D glucosidase (GLU, and N-acetyl-glucosiminidase (NAG were found in sand, and greater than bulk sediments of both intertidal zone (IZ and mangrove forest (MG. This result implied that sand fractions might protect selective enzymes through the adsorption without affecting their activities. Additionally, the enzyme-based resource allocation in various particle-size fractions demonstrated that nutirents availability varied with different particle-size fractions and only sand fraction of MG with highest total C showed high N and P availability among fractions. Besides, the analysis between elemental contents and enzymes activities in particle-size fractions suggested that enzymes could monitor the changes of nutrients availability and be good indicators of ecosystem responses to environmental changes. Thus, these results provided a means to assess the availability of different nutrients (C, N, and P during decomposition of sediment organic matter (SOM, and thus helping to better manage the subtropical mangrove ecosystems to sequester C into SOM.

  1. Inhibition of PKC-dependent extracellular Ca2+ entry contributes to the depression of contractile activity in long-term pressure-overloaded endothelium-denuded rat aortas

    International Nuclear Information System (INIS)

    We examined the contractile responsiveness of rat thoracic aortas under pressure overload after long-term suprarenal abdominal aortic coarctation (lt-Srac). Endothelium-dependent angiotensin II (ANG II) type 2 receptor (AT2R)-mediated depression of contractions to ANG II has been reported in short-term (1 week) pressure-overloaded rat aortas. Contractility was evaluated in the aortic rings of rats subjected to lt-Srac or sham surgery (Sham) for 8 weeks. ANG I and II levels and AT2R protein expression in the aortas of lt-Srac and Sham rats were also evaluated. lt-Srac attenuated the contractions of ANG II and phenylephrine in the aortas in an endothelium-independent manner. However, lt-Srac did not influence the transient contractions induced in endothelium-denuded aortic rings by ANG II, phenylephrine, or caffeine in Ca2+-free medium or the subsequent tonic constrictions induced by the addition of Ca2+ in the absence of agonists. Thus, the contractions induced by Ca2+ release from intracellular stores and Ca2+ influx through stored-operated channels were not inhibited in the aortas of lt-Srac rats. Potassium-elicited contractions in endothelium-denuded aortic rings of lt-Srac rats remained unaltered compared with control tissues. Consequently, the contractile depression observed in aortic tissues of lt-Srac rats cannot be explained by direct inhibition of voltage-operated Ca2+ channels. Interestingly, 12-O-tetradecanoylphorbol-13-acetate-induced contractions in endothelium-denuded aortic rings of lt-Srac rats were depressed in the presence but not in the absence of extracellular Ca2+. Neither levels of angiotensins nor of AT2R were modified in the aortas after lt-Srac. The results suggest that, in rat thoracic aortas, lt-Srac selectively inhibited protein kinase C-mediated activation of contraction that is dependent on extracellular Ca2+ entry

  2. Catalase, but not MnSOD, inhibits glucose deprivation-activated ASK1-MEK-MAPK signal transduction pathway and prevents relocalization of Daxx: hydrogen peroxide as a major second messenger of metabolic oxidative stress.

    Science.gov (United States)

    Song, Jae J; Lee, Yong J

    2003-10-01

    Overexpression of catalase, but not manganese superoxide dismutase (MnSOD), inhibited glucose deprivation-induced cytotoxicity and c-Jun N-terminal kinase 1 (JNK1) activation in human prostate adenocarcinoma DU-145 cells. Suppression of JNK1 activation by catalase overexpression resulted from inhibition of apoptosis signal-regulating kinase 1 (ASK1) activation by preventing dissociation of thioredoxin (TRX) from ASK1. Overexpression of catalase also inhibited relocalization of Daxx from the nucleus to the cytoplasm as well as association of Daxx with ASK1 during glucose deprivation. Taken together, hydrogen peroxide (H(2)O(2)) rather than superoxide anion (O(2) (*-)) acts as a second messenger of metabolic oxidative stress to activate the ASK1-MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)-mitogen-activated protein kinase (MAPK) signal transduction pathway.

  3. Priming Endothelial Cells With a Melanoma-Derived Extracellular Matrix Triggers the Activation of αvβ3/VEGFR2 Axis.

    Science.gov (United States)

    Helal-Neto, Edward; Brandão-Costa, Renata M; Saldanha-Gama, Roberta; Ribeiro-Pereira, Cristiane; Midlej, Victor; Benchimol, Marlene; Morandi, Verônica; Barja-Fidalgo, Christina

    2016-11-01

    The unique composition of tumor-produced extracellular matrix (ECM) can be a determining factor in changing the profile of endothelial cells in the tumor microenvironment. As the main receptor for ECM proteins, integrins can activate a series of signaling pathways related to cell adhesion, migration, and differentiation of endothelial cells that interact with ECM proteins. We studied the direct impact of the decellularized ECM produced by a highly metastatic human melanoma cell line (MV3) on the activation of endothelial cells and identified the intracellular signaling pathways associated with cell differentiation. Our data show that compared to the ECM derived from a human melanocyte cell line (NGM-ECM), ECM produced by a melanoma cell line (MV3-ECM) is considerably different in ultrastructural organization and composition and possesses a higher content of tenascin-C and laminin and a lower expression of fibronectin. When cultured directly on MV3-ECM, endothelial cells change morphology and show increased adhesion, migration, proliferation, and tubulogenesis. Interaction of endothelial cells with MV3-ECM induces the activation of integrin signaling, increasing FAK phosphorylation and its association with Src, which activates VEGFR2, potentiating the receptor response to VEGF. The blockage of αvβ3 integrin inhibited the FAK-Src association and VEGFR activation, thus reducing tubulogenesis. Together, our data suggest that the interaction of endothelial cells with the melanoma-ECM triggers integrin-dependent signaling, leading to Src pathway activation that may potentiate VEGFR2 activation and up-regulate angiogenesis. J. Cell. Physiol. 231: 2464-2473, 2016. © 2016 Wiley Periodicals, Inc. PMID:27420801

  4. Mitogen-Activated Protein Kinases and Hypoxic/Ischemic Nephropathy

    Directory of Open Access Journals (Sweden)

    Fengbao Luo

    2016-08-01

    Full Text Available Tissue hypoxia/ischemia is a pathological feature of many human disorders including stroke, myocardial infarction, hypoxic/ischemic nephropathy, as well as cancer. In the kidney, the combination of limited oxygen supply to the tissues and high oxygen demand is considered the main reason for the susceptibility of the kidney to hypoxic/ischemic injury. In recent years, increasing evidence has indicated that a reduction in renal oxygen tension/blood supply plays an important role in acute kidney injury, chronic kidney disease, and renal tumorigenesis. However, the underlying signaling mechanisms, whereby hypoxia alters cellular behaviors, remain poorly understood. Mitogen-activated protein kinases (MAPKs are key signal-transducing enzymes activated by a wide range of extracellular stimuli, including hypoxia/ischemia. There are four major family members of MAPKs: the extracellular signal-regulated kinases-1 and -2 (ERK1/2, the c-Jun N-terminal kinases (JNK, p38 MAPKs, and extracellular signal-regulated kinase-5 (ERK5/BMK1. Recent studies, including ours, suggest that these MAPKs are differentially involved in renal responses to hypoxic/ischemic stress. This review will discuss their changes in hypoxic/ischemic pathophysiology with acute kidney injury, chronic kidney diseases and renal carcinoma.

  5. Extracellular activation of Wnt signaling through epigenetic dysregulation of Wnt inhibitory factor-1 (Wif-1) is associated with pathogenesis of adrenocortical tumor.

    Science.gov (United States)

    Mitsui, Yozo; Yasumoto, Hiroaki; Nagami, Taichi; Hiraki, Miho; Arichi, Naoko; Ishikawa, Noriyoshi; Araki, Asuka; Maruyama, Riruke; Tanaka, Yuichiro; Dahiya, Rajvir; Shiina, Hiroaki

    2014-04-30

    Wnt/β-catenin signaling is considered to be an essential regulator of adrenocortical oncogenesis. Wnt inhibitory factor-1 (Wif-1), an extracellular regulator of Wnt signaling, is frequently down-regulated by hypermethylation of the promoter CpG. We investigated epigenetic regulation of Wif-1 and its association with adrenocortical (AC) tumor pathogenesis in light of Wnt activation. The AC tumors showed a high prevalence of Wif-1 promoter methylation and low prevalence of Wif-1 mRNA transcription as compared to the normal adrenal (NA) samples. Furthermore, a significant correlation was found between Wif-1 promoter methylation and mRNA transcription in the tumors. Either intracellular β-catenin accumulation or β-catenin mRNA transcription was significantly elevated in the AC tumors, which also showed an inverse correlation with Wif-1 mRNA transcription. Cyclin D1, a target gene of Wnt signaling, was also up-regulated in the AC tumors as compared with the NA samples. In addition, down-regulation of Wif-1 was correlated with increased cyclin D1 at both mRNA and protein levels. However, despite the proposed activation of Wnt signaling in AC tumors, only 2 of 20 with intracellular β-catenin accumulation showed β-catenin mutations. Thus, genetic alterations of β-catenin and epigenetics-related Wif-1 promoter hypermethylation may be important mechanisms underlying AC tumor formation though aberrant canonical Wnt/β-catenin signaling activation. PMID:24755523

  6. High Glucose-Induced Oxidative Stress Mediates Apoptosis and Extracellular Matrix Metabolic Imbalances Possibly via p38 MAPK Activation in Rat Nucleus Pulposus Cells

    Directory of Open Access Journals (Sweden)

    Xiaofei Cheng

    2016-01-01

    Full Text Available Objectives. To investigate whether high glucose-induced oxidative stress is implicated in apoptosis of rat nucleus pulposus cells (NPCs and abnormal expression of critical genes involved in the metabolic balance of extracellular matrix (ECM. Methods. NPCs were cultured with various concentrations of glucose to detect cell viability and apoptosis. Cells cultured with high glucose (25 mM were untreated or pretreated with N-acetylcysteine or a p38 MAPK inhibitor SB 202190. Reactive oxygen species (ROS production was evaluated. Activation of p38 MAPK was measured by Western blot. The expression of ECM metabolism-related genes, including type II collagen, aggrecan, SRY-related high-mobility-group box 9 (Sox-9, matrix metalloproteinase 3 (MMP-3, and tissue inhibitor of metalloproteinase 1 (TIMP-1, was analyzed by semiquantitative RT-PCR. Results. High glucose reduced viability of NPCs and induced apoptosis. High glucose resulted in increased ROS generation and p38 MAPK activation. In addition, it negatively regulated the expression of type II collagen, aggrecan, Sox-9, and TIMP-1 and positively regulated MMP-3 expression. These results were changed by pretreatment with N-acetylcysteine or SB 202190. Conclusions. High glucose might promote apoptosis of NPCs, trigger ECM catabolic pathways, and inhibit its anabolic activities, possibly through a p38 MAPK-dependent oxidative stress mechanism.

  7. High Glucose-Induced Oxidative Stress Mediates Apoptosis and Extracellular Matrix Metabolic Imbalances Possibly via p38 MAPK Activation in Rat Nucleus Pulposus Cells.

    Science.gov (United States)

    Cheng, Xiaofei; Ni, Bin; Zhang, Feng; Hu, Ying; Zhao, Jie

    2016-01-01

    Objectives. To investigate whether high glucose-induced oxidative stress is implicated in apoptosis of rat nucleus pulposus cells (NPCs) and abnormal expression of critical genes involved in the metabolic balance of extracellular matrix (ECM). Methods. NPCs were cultured with various concentrations of glucose to detect cell viability and apoptosis. Cells cultured with high glucose (25 mM) were untreated or pretreated with N-acetylcysteine or a p38 MAPK inhibitor SB 202190. Reactive oxygen species (ROS) production was evaluated. Activation of p38 MAPK was measured by Western blot. The expression of ECM metabolism-related genes, including type II collagen, aggrecan, SRY-related high-mobility-group box 9 (Sox-9), matrix metalloproteinase 3 (MMP-3), and tissue inhibitor of metalloproteinase 1 (TIMP-1), was analyzed by semiquantitative RT-PCR. Results. High glucose reduced viability of NPCs and induced apoptosis. High glucose resulted in increased ROS generation and p38 MAPK activation. In addition, it negatively regulated the expression of type II collagen, aggrecan, Sox-9, and TIMP-1 and positively regulated MMP-3 expression. These results were changed by pretreatment with N-acetylcysteine or SB 202190. Conclusions. High glucose might promote apoptosis of NPCs, trigger ECM catabolic pathways, and inhibit its anabolic activities, possibly through a p38 MAPK-dependent oxidative stress mechanism. PMID:27635402

  8. High Glucose-Induced Oxidative Stress Mediates Apoptosis and Extracellular Matrix Metabolic Imbalances Possibly via p38 MAPK Activation in Rat Nucleus Pulposus Cells

    Science.gov (United States)

    Cheng, Xiaofei; Ni, Bin; Zhang, Feng; Hu, Ying

    2016-01-01

    Objectives. To investigate whether high glucose-induced oxidative stress is implicated in apoptosis of rat nucleus pulposus cells (NPCs) and abnormal expression of critical genes involved in the metabolic balance of extracellular matrix (ECM). Methods. NPCs were cultured with various concentrations of glucose to detect cell viability and apoptosis. Cells cultured with high glucose (25 mM) were untreated or pretreated with N-acetylcysteine or a p38 MAPK inhibitor SB 202190. Reactive oxygen species (ROS) production was evaluated. Activation of p38 MAPK was measured by Western blot. The expression of ECM metabolism-related genes, including type II collagen, aggrecan, SRY-related high-mobility-group box 9 (Sox-9), matrix metalloproteinase 3 (MMP-3), and tissue inhibitor of metalloproteinase 1 (TIMP-1), was analyzed by semiquantitative RT-PCR. Results. High glucose reduced viability of NPCs and induced apoptosis. High glucose resulted in increased ROS generation and p38 MAPK activation. In addition, it negatively regulated the expression of type II collagen, aggrecan, Sox-9, and TIMP-1 and positively regulated MMP-3 expression. These results were changed by pretreatment with N-acetylcysteine or SB 202190. Conclusions. High glucose might promote apoptosis of NPCs, trigger ECM catabolic pathways, and inhibit its anabolic activities, possibly through a p38 MAPK-dependent oxidative stress mechanism.

  9. Extracellular histones induce tissue factor expression in vascular endothelial cells via TLR and activation of NF-κB and AP-1.

    Science.gov (United States)

    Yang, Xinyu; Li, Lin; Liu, Jin; Lv, Ben; Chen, Fangping

    2016-01-01

    Extracellular histones have been recognized recently as proinflammatory mediators; they are released from dying cells in response to inflammatory challenge, contributing to endothelial cell dysfunction, thrombin formation, organ failure, and death during sepsis. Clinical studies suggest that the plasma concentration of the histone-DNA complex is correlated with the severity of DIC and is a poor independent prognostic marker in sepsis. In addition, platelet activation stimulates thrombus formation. Whether histones contribute to procoagulant activity in other ways remains elusive. In this study, we confirmed that histones induce tissue factor (TF) expression in a concentration- and time-dependent manner in vascular endothelial cells (ECs) and macrophages. However, histones did not affect TF pathway inhibitor expression. Moreover, blocking the cell surface receptors TLR4 and TLR2 with specific neutralizing antibodies significantly reduced histone-induced TF expression. Furthermore, histones enhanced the nuclear translocation of NF-κB (c-Rel/p65) and AP-1 expression in a time-dependent manner in ECs. Mutating NF-κB and AP-1 significantly reduced histone-induced TF expression. Altogether, our experiments suggest that histone induces TF expression in ECs via cell surface receptors TLR4 and TLR2, simultaneously depending on the activation of the transcription factors NF-κB and AP-1.

  10. Extracellular ATP induces spikes in cytosolic free Ca(2+) but not in NADPH oxidase activity in neutrophils

    DEFF Research Database (Denmark)

    Brasen, Jens Christian; Olsen, Lars Folke; Hallett, Maurice B

    2011-01-01

    In order to establish whether non-mitochondrial oxidase activity in human neutrophils is tightly related to cytosolic Ca(2+) concentration, we simultaneously measured Ca(2+) oscillations induced by ATP and oxidant production in single adherent neutrophils using confocal microscopy. ATP induced fast...

  11. Extracellular ATP induces spikes in cytosolic free Ca2+ but not in NADH oxidase activity in neutrophils

    DEFF Research Database (Denmark)

    Brasen, Jens Christian; Olsen, Lars Folke; Hallett, Maurice B.

    2011-01-01

    In order to establish whether non-mitochondrial oxidase activity in human neutrophils is tightly related to cytosolic Ca2+ concentration, we simultaneously measured Ca2+ oscillations induced by ATP and oxidant production in single adherent neutrophils using confocal microscopy. ATP induced fast...

  12. Virulence testing and extracellular subtilisin-like (Pr1) and trypsin-like (Pr2) activity during propagule production of Paecilomyces fumosoroseus isolates from whiteflies (Homoptera: Aleyrodidae).

    Science.gov (United States)

    Castellanos-Moguel, Judith; González-Barajas, Margarita; Mier, Teresa; Reyes-Montes, María Del Rocío; Aranda, Eduardo; Toriello, Conchita

    2007-03-01

    To properly characterize several isolates of Paecilomyces fumosoroseus, a fungal entomopathogen of whiteflies (Homoptera: Aleyrodidae) and other insect pests for biocontrol purposes, virulence towards Trialeurodes vaporariorum, and subtilisin-like (Pr1) and trypsin-like (Pr2) protease activity during propagule production were investigated in monospore cultures (MCs). The virulence of three MCs towards second instar whiteflies was measured and expressed as lethal median concentration (LC50). Number and widthlength ratio of propagules (blastospores, hyphal bodies, short hyphae, submerged conidia) and extracellular proteolytic activity was determined simultaneously in liquid medium. Total protease activity was assayed with azocasein, Pr1 and Pr2 activity was determined with the substrates N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and N-Benzoyl-Phe-Val-Arg-pnitroanilide, respectively. Natural variability in virulence, propagule production and cuticle-degrading proteases among isolates was observed. Bioassays showed a LC50 of 1.1 x 1,000, 2.5 x 10,000 and 7.6 x 10,000 conidia/ml for MCs EH-506/3, EH-503/3 and EH-520/3, respectively, EH-506/3 being the most virulent isolate. Isolate EH-503/3 produced the highest yield of propagules (7.7 x 10000000 propagules/ml), followed by EH-520/3 with 6.4 x 10000000 and EH-506/3 with 1.0 x 10000000 propagules/ml. Subtilisin-like (Pr1) and trypsin-like (Pr2) activity was present in the three MCs. Subtilisin-like (Pr1) activity was highest (745.7 UPr1/ml at 120 h) in the most virulent isolate, EH-506/3, pointing at Pr1 as a phenotypic marker of virulence for P. fumosoroseus. EH-506/3 appears to be a good candidate for whitefly biocontrol due to its high virulence, Pr1 concentration and rapid transition to blastospores in submerged liquid medium.

  13. Decreased expression of the plasminogen activator inhibitor type 1 is involved in degradation of extracellular matrix surrounding cervical cancer stem cells.

    Science.gov (United States)

    Sato, Masakazu; Kawana, Kei; Adachi, Katsuyuki; Fujimoto, Asaha; Yoshida, Mitsuyo; Nakamura, Hiroe; Nishida, Haruka; Inoue, Tomoko; Taguchi, Ayumi; Takahashi, Juri; Kojima, Satoko; Yamashita, Aki; Tomio, Kensuke; Nagamatsu, Takeshi; Wada-Hiraike, Osamu; Oda, Katsutoshi; Osuga, Yutaka; Fujii, Tomoyuki

    2016-02-01

    The plasminogen activator (PA) system consists of plasminogen activator inhibitor type 1 (PAI-1), urokinase-type plasminogen activator and its receptor (uPA and uPAR). PAI-1 inhibits the activation of uPA (which converts plasminogen to plasmin), and is involved in cancer invasion and metastasis, by remodeling the extracellular matrix (ECM) through regulating plasmin. Cancer stem cells (CSCs) are a small subset of cells within tumors, and are thought to be involved in tumor recurrence and metastasis. Considering these facts, we investigated the relationship between PAI-1 and cervical CSCs. We used ALDH1 as a marker of cervical CSCs. First, we demonstrated that culturing ALDH1-high cells and ALDH-low cells on collagen IV-coted plates increased their expression of active PAI-1 (ELISA), and these increases were suggested to be at mRNA expression levels (RT-qPCR). Secondly, we demonstrated PAI-1 was indeed involved in the ECM maintenance. With gelatin zymography assays, we found that ALDH1-high cells and ALDH-low cells expressed pro-matrix metalloproteinase-2 (pro-MMP-2) irrespective of their coatings. With gelatinase/collagenase assay kit, we confirmed that collagenase activity was increased when ALDH1-low cells were exposed to TM5275, a small molecule inhibitor of PAI-1. Putting the data together, we hypothesized that cancer cells adhered to basal membrane secrete abundant PAI-1, on the other hand, cancer cells (especially CSCs rather than non-CSCs) distant from basal membrane secrete less PAI-1, which makes the ECM surrounding CSCs more susceptible to degradation. Our study could be an explanation of conflicting reports, where some researchers found negative impacts of PAI-1 expression on clinical outcomes and others not, by considering the concept of CSCs.

  14. Interleukin-1β regulates cell proliferation and activity of extracellular matrix remodelling enzymes in cultured primary pig heart cells

    International Nuclear Information System (INIS)

    Research highlights: → Levels of IL-1β are increased in the pig myocardium after infarction. → Cultured pig heart cells possess IL-1 receptors. → IL-1β increases cell proliferation of pig heart cells in-vitro. → IL-1β increases MMP-2 and MMP-9 activity in pig heart cells in-vitro. → IL-1β may be important for tissue remodelling events after myocardial infarction. -- Abstract: After myocardial infarction, elevated levels of interleukins (ILs) are found within the myocardial tissue and IL-1β is considered to play a major role in tissue remodelling events throughout the body. In the study presented, we have established a cell culture model of primary pig heart cells to evaluate the effects of different concentrations of IL-1β on cell proliferation as well as expression and activity of enzymes typically involved in tissue remodelling. Primary pig heart cell cultures were derived from three different animals and stimulated with recombinant pig IL-1β. RNA expression was detected by RT-PCR, protein levels were evaluated by Western blotting, activity of matrix metalloproteinases (MMPs) was quantified by gelatine zymography and cell proliferation was measured using colorimetric MTS assays. Pig heart cells express receptors for IL-1 and application of IL-1β resulted in a dose-dependent increase of cell proliferation (P < 0.05 vs. control; 100 ng/ml; 24 h). Gene expression of caspase-3 was increased by IL-1β (P < 0.05 vs. control; 100 ng/ml; 3 h), and pro-caspase-3 but not active caspase was detected in lysates of pig heart cells by Western blotting. MMP-2 gene expression as well as enzymatic activities of MMP-2 and MMP-9 were increased by IL-1β (P < 0.05 vs. control; 100 ng/ml; 3 h for gene expression, 48 and 72 h for enzymatic activities of MMP-2 and MMP-9, respectively). Our in vitro data suggest that IL-1β plays a major role in the events of tissue remodelling in the heart. Combined with our recently published in vivo data (Meybohm et al., PLoS One

  15. Impact of repeated dry-wet cycles on soil greenhouse gas emissions, extracellular enzyme activity and nutrient cycling in a temperate forest

    Science.gov (United States)

    Leitner, Sonja; Zimmermann, Michael; Bockholt, Jan; Schartner, Markus; Brugner, Paul; Holtermann, Christian; Zechmeister-Boltenstern, Sophie

    2014-05-01

    Climate change research predicts that both frequency and intensity of weather extremes such as long drought periods and heavy rainfall events will increase in mid Europe over the next decades. Soil moisture is one of the major factors controlling microbial soil processes, and it has been widely agreed that feedback effects between altered precipitation and changed soil fluxes of the greenhouse gases CO2, CH4 and N2O could intensify climate change. In a field experiment in an Austrian beech forest, we established a precipitation manipulation experiment, which will be conducted for 3 years. We use roofs to exclude rainfall from reaching the forest soil and simulate drought periods, and a sprinkler system to simulate heavy rainfall events. We applied repeated dry-wet cycles in two intensities: one treatment received 6 cycles of 1 month drought followed by 75mm irrigation within 2 hours, and a parallel treatment received 3 cycles of 2 months drought followed by 150mm irrigation within 3 hours. We took soil samples 1 day before, 1 day after and 1 week after rewetting events and analyzed them for soil nutrients and extracellular enzyme activities. Soil fluxes of CO2, N2O and CH4 were constantly monitored with an automated flux chamber system, and environmental parameters were recorded via dataloggers. In addition, we determined fluxes and nutrient concentrations of bulk precipitation, throughfall, stemflow, litter percolate and soil water. Next we plan to analyze soil microbial community composition via PLFAs to investigate microbial stress resistance and resilience, and we will use ultrasonication to measure soil aggregate stability and protection of soil organic matter in stressed and control plots. The results of the first year show that experimental rainfall manipulation has influenced soil extracellular enzymes. Potential phenoloxidase activity was significantly reduced in stressed treatments compared to control plots. All measured hydrolytic enzymes (cellulase

  16. Regulated Extracellular Choline Acetyltransferase Activity- The Plausible Missing Link of the Distant Action of Acetylcholine in the Cholinergic Anti-Inflammatory Pathway.

    Directory of Open Access Journals (Sweden)

    Swetha Vijayaraghavan

    Full Text Available Acetylcholine (ACh, the classical neurotransmitter, also affects a variety of nonexcitable cells, such as endothelia, microglia, astrocytes and lymphocytes in both the nervous system and secondary lymphoid organs. Most of these cells are very distant from cholinergic synapses. The action of ACh on these distant cells is unlikely to occur through diffusion, given that ACh is very short-lived in the presence of acetylcholinesterase (AChE and butyrylcholinesterase (BuChE, two extremely efficient ACh-degrading enzymes abundantly present in extracellular fluids. In this study, we show compelling evidence for presence of a high concentration and activity of the ACh-synthesizing enzyme, choline-acetyltransferase (ChAT in human cerebrospinal fluid (CSF and plasma. We show that ChAT levels are physiologically balanced to the levels of its counteracting enzymes, AChE and BuChE in the human plasma and CSF. Equilibrium analyses show that soluble ChAT maintains a steady-state ACh level in the presence of physiological levels of fully active ACh-degrading enzymes. We show that ChAT is secreted by cultured human-brain astrocytes, and that activated spleen lymphocytes release ChAT itself rather than ACh. We further report differential CSF levels of ChAT in relation to Alzheimer's disease risk genotypes, as well as in patients with multiple sclerosis, a chronic neuroinflammatory disease, compared to controls. Interestingly, soluble CSF ChAT levels show strong correlation with soluble complement factor levels, supporting a role in inflammatory regulation. This study provides a plausible explanation for the long-distance action of ACh through continuous renewal of ACh in extracellular fluids by the soluble ChAT and thereby maintenance of steady-state equilibrium between hydrolysis and synthesis of this ubiquitous cholinergic signal substance in the brain and peripheral compartments. These findings may have important implications for the role of cholinergic

  17. Metals content of Glossoscolex paulistus extracellular hemoglobin: Its peroxidase activity and the importance of these ions in the protein stability.

    Science.gov (United States)

    Caruso, Celia S; Biazin, Ezer; Carvalho, Francisco A O; Tabak, Marcel; Bachega, José F R

    2016-08-01

    In this work we investigate the presence of divalent cations bound to the Glossoscolex paulistus (HbGp) hemoglobin and their effect over the protein stability and the peroxidase (POD) activity. Atomic absorption studies show that the HbGp iron content is consistent with the presence of 144 ions per protein. Moreover, using iron as a reference, the content of calcium was estimated as 30±4 ions per protein, independently of the EDTA pre-treatment or not prior to the acidic treatment performed in the protein digestion. The zinc content was 14±2 ions in the absence of EDTA pre-treatment, and 3±1 ions per protein in the presence of EDTA pre-treatment, implying the presence of one zinc ion per protomer (1/12 of the whole molecule). Finally, the copper concentration is negligible. Different from the vertebrate hemoglobins, where the effectors are usually organic anions, the hexagonal bilayer hemoglobins have as effectors inorganic cations that increase the oxygen affinity and stabilize the structure. Previous studies have suggested that the presence of divalent cations, such as copper and zinc, is related to the different types of antioxidant enzymatic activities as the superoxide dismutase (SOD) activity shown by giant hemoglobin from Lumbricus terrestris (HbLt). Recently, studies on HbGp crystal structure have confirmed the presence of Zn(2+) and Ca(2+) binding sites. The Ca(2+) sites are similar as observed in the HbLt crystal structure. Otherwise, the Zn(2+) sites have no relation with those observed in Cu/Zn SODs. Our peroxidase assays with guaiacol confirm the POD activity and the effect of the zinc ions for HbGp. Our present results on HbGp metal content and their stability effects is the first step to understand the role of these cations in HbGp function in the future. PMID:27221949

  18. Activation of phagocytic cells by Staphylococcus epidermidis biofilms: effects of extracellular matrix proteins and the bacterial stress protein GroEL on netosis and MRP-14 release.

    Science.gov (United States)

    Dapunt, Ulrike; Gaida, Matthias M; Meyle, Eva; Prior, Birgit; Hänsch, Gertrud M

    2016-07-01

    The recognition and phagocytosis of free-swimming (planktonic) bacteria by polymorphonuclear neutrophils have been investigated in depth. However, less is known about the neutrophil response towards bacterial biofilms. Our previous work demonstrated that neutrophils recognize activating entities within the extracellular polymeric substance (EPS) of biofilms (the bacterial heat shock protein GroEL) and that this process does not require opsonization. Aim of this study was to evaluate the release of DNA by neutrophils in response to biofilms, as well as the release of the inflammatory cytokine MRP-14. Neutrophils were stimulated with Staphylococcus epidermidis biofilms, planktonic bacteria, extracted EPS and GroEL. Release of DNA and of MRP-14 was evaluated. Furthermore, tissue samples from patients suffering from biofilm infections were collected and evaluated by histology. MRP-14 concentration in blood samples was measured. We were able to show that biofilms, the EPS and GroEL induce DNA release. MRP-14 was only released after stimulation with EPS, not GroEL. Histology of tissue samples revealed MRP-14 positive cells in association with neutrophil infiltration and MRP-14 concentration was elevated in blood samples of patients suffering from biofilm infections. Our data demonstrate that neutrophil-activating entities are present in the EPS and that GroEL induces DNA release by neutrophils. PMID:27109773

  19. Composition analysis of fractions of extracellular polymeric substances from an activated sludge culture and identification of dominant forces affecting microbial aggregation

    Science.gov (United States)

    Guo, Xuan; Wang, Xu; Liu, Junxin

    2016-06-01

    Extracellular polymeric substances (EPS) appear to play a critical role in the formation of bioaggregates, such as sludge flocs, in activated sludge processes. Here, we systematically investigated the composition and chemical structure of various EPS fractions excreted from an activated sludge culture using multi-analysis techniques to examine the ability of the sludge to aggregate. Chemical analysis was used with a three-dimensional excitation emission matrix and Fourier transform infrared spectroscopy, applying inter-particle forces theory. The combined findings revealed that hydrophobic groups, especially protein-related N–H, were present in a greater proportion in tightly bound EPS (TB-EPS). This result, which explained the specificity of TB-EPS in the chemical structure, was consistent with data indicating that TB-EPS contained a large amount of protein-like substances (86.7 mg/g of mixed liquor volatile suspended solids, 39.7% of the total EPS). Subsequently, a novel experimental procedure was developed to pinpoint key inter-particle forces in sludge aggregation. The result revealed that hydrogen bonds are the predominant triggers that promote sludge aggregation. This comprehensive analysis indicated that hydrophobic proteins in TB-EPS are responsible for the critical role played by hydrogen bonds in sludge formation. Our findings highlight the need to elucidate the mechanisms of TB-EPS-mediated flocculation in future efforts.

  20. Optical ATP Biosensor for Extracellular ATP Measurement

    OpenAIRE

    Wang, C; Huang, C.-Y.C.; Lin, W-C

    2013-01-01

    Extracellular Adenosine-5′-triphosphate (ATP) is an important multi-functional molecule which can mediate numerous physiological activities by activating purinergic P2 receptors. The objective of this study was to develop a novel optical ATP sensor for in-situ extracellular ATP measurement in biological tissues. The optical ATP sensor was made by applying two layers of sol-gel coating to the end of an optical fiber probe end. The first layer contained ruthenium complex for sensing changes in ...

  1. Anti-Müllerian Hormone Signaling Regulates Epithelial Plasticity and Chemoresistance in Lung Cancer.

    Science.gov (United States)

    Beck, Tim N; Korobeynikov, Vladislav A; Kudinov, Alexander E; Georgopoulos, Rachel; Solanki, Nehal R; Andrews-Hoke, Magda; Kistner, Timothy M; Pépin, David; Donahoe, Patricia K; Nicolas, Emmanuelle; Einarson, Margret B; Zhou, Yan; Boumber, Yanis; Proia, David A; Serebriiskii, Ilya G; Golemis, Erica A

    2016-07-19

    Anti-Müllerian hormone (AMH) and its type II receptor AMHR2, both previously thought to primarily function in gonadal tissue, were unexpectedly identified as potent regulators of transforming growth factor (TGF-β)/bone morphogenetic protein (BMP) signaling and epithelial-mesenchymal transition (EMT) in lung cancer. AMH is a TGF-β/BMP superfamily member, and AMHR2 heterodimerizes with type I receptors (ALK2, ALK3) also used by the type II receptor for BMP (BMPR2). AMH signaling regulates expression of BMPR2, ALK2, and ALK3, supports protein kinase B-nuclear factor κB (AKT-NF-κB) and SMAD survival signaling, and influences BMP-dependent signaling in non-small cell lung cancer (NSCLC). AMH and AMHR2 are selectively expressed in epithelial versus mesenchymal cells, and loss of AMH/AMHR2 induces EMT. Independent induction of EMT reduces expression of AMH and AMHR2. Importantly, EMT associated with depletion of AMH or AMHR2 results in chemoresistance but sensitizes cells to the heat shock protein 90 (HSP90) inhibitor ganetespib. Recognition of this AMH/AMHR2 axis helps to further elucidate TGF-β/BMP resistance-associated signaling and suggests new strategies for therapeutic targeting of EMT. PMID:27396341

  2. Anti-Müllerian Hormone Signaling Regulates Epithelial Plasticity and Chemoresistance in Lung Cancer

    Directory of Open Access Journals (Sweden)

    Tim N. Beck

    2016-07-01

    Full Text Available Anti-Müllerian hormone (AMH and its type II receptor AMHR2, both previously thought to primarily function in gonadal tissue, were unexpectedly identified as potent regulators of transforming growth factor (TGF-β/bone morphogenetic protein (BMP signaling and epithelial-mesenchymal transition (EMT in lung cancer. AMH is a TGF-β/BMP superfamily member, and AMHR2 heterodimerizes with type I receptors (ALK2, ALK3 also used by the type II receptor for BMP (BMPR2. AMH signaling regulates expression of BMPR2, ALK2, and ALK3, supports protein kinase B-nuclear factor κB (AKT-NF-κB and SMAD survival signaling, and influences BMP-dependent signaling in non-small cell lung cancer (NSCLC. AMH and AMHR2 are selectively expressed in epithelial versus mesenchymal cells, and loss of AMH/AMHR2 induces EMT. Independent induction of EMT reduces expression of AMH and AMHR2. Importantly, EMT associated with depletion of AMH or AMHR2 results in chemoresistance but sensitizes cells to the heat shock protein 90 (HSP90 inhibitor ganetespib. Recognition of this AMH/AMHR2 axis helps to further elucidate TGF-β/BMP resistance-associated signaling and suggests new strategies for therapeutic targeting of EMT.

  3. Regulation of mitogen-activated protein kinase pathways by the plasma membrane Na+/H+ exchanger, NHE1

    DEFF Research Database (Denmark)

    Pedersen, Stine Helene Falsig; Darborg, Barbara Vasek; Rentsch, Maria Louise;

    2006-01-01

    The mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, play a major role in the regulation of pivotal cellular processes such as cell death/survival balance, cell cycle progression, and cell migration. MAPK...... activity is regulated by a three-tiered phosphorelay system, which is in turn regulated by a complex network of signaling events and scaffolding proteins. The ubiquitous plasma membrane Na(+)/H(+) exchanger NHE1 is activated by, and implicated in, the physiological/pathophysiological responses to many of...

  4. Early LPS-induced ERK activation in retinal pigment epithelium cells is dependent on PIP 2 -PLC.

    Science.gov (United States)

    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.

  5. Extracellular neural microstimulation may activate much larger regions than expected by simulations: a combined experimental and modeling study.

    Directory of Open Access Journals (Sweden)

    Sébastien Joucla

    Full Text Available Electrical stimulation of the central nervous system has been widely used for decades for either fundamental research purposes or clinical treatment applications. Yet, very little is known regarding the spatial extent of an electrical stimulation. If pioneering experimental studies reported that activation threshold currents (TCs increase with the square of the neuron-to-electrode distance over a few hundreds of microns, there is no evidence that this quadratic law remains valid for larger distances. Moreover, nowadays, numerical simulation approaches have supplanted experimental studies for estimating TCs. However, model predictions have not yet been validated directly with experiments within a common paradigm. Here, we present a direct comparison between experimental determination and modeling prediction of TCs up to distances of several millimeters. First, we combined patch-clamp recording and microelectrode array stimulation in whole embryonic mouse spinal cords to determine TCs. Experimental thresholds did not follow a quadratic law beyond 1 millimeter, but rather tended to remain constant for distances larger than 1 millimeter. We next built a combined finite element--compartment model of the same experimental paradigm to predict TCs. While theoretical TCs closely matched experimental TCs for distances <250 microns, they were highly overestimated for larger distances. This discrepancy remained even after modifications of the finite element model of the potential field, taking into account anisotropic, heterogeneous or dielectric properties of the tissue. In conclusion, these results show that quadratic evolution of TCs does not always hold for large distances between the electrode and the neuron and that classical models may underestimate volumes of tissue activated by electrical stimulation.

  6. Influence of extracellular pH on growth, viability, cell size, acidification activity, and intracellular pH of Lactococcus lactis in batch fermentations.

    Science.gov (United States)

    Hansen, Gunda; Johansen, Claus Lindvald; Marten, Gunvor; Wilmes, Jacqueline; Jespersen, Lene; Arneborg, Nils

    2016-07-01

    In this study, we investigated the influence of three extracellular pH (pHex) values (i.e., 5.5, 6.5, and 7.5) on the growth, viability, cell size, acidification activity in milk, and intracellular pH (pHi) of Lactococcus lactis subsp. lactis DGCC1212 during pH-controlled batch fermentations. A universal parameter (e.g., linked to pHi) for the description or prediction of viability, specific acidification activity, or growth behavior at a given pHex was not identified. We found viability as determined by flow cytometry to remain high during all growth phases and irrespectively of the pH set point. Furthermore, regardless of the pHex, the acidification activity per cell decreased over time which seemed to be linked to cell shrinkage. Flow cytometric pHi determination demonstrated an increase of the averaged pHi level for higher pH set points, while the pH gradient (pHi-pHex) and the extent of pHi heterogeneity decreased. Cells maintained positive pH gradients at a low pHex of 5.5 and even during substrate limitation at the more widely used pHex 6.5. Moreover, the strain proved able to grow despite small negative or even absent pH gradients at a high pHex of 7.5. The larger pHi heterogeneity at pHex 5.5 and 6.5 was associated with more stressful conditions resulting, e.g., from higher concentrations of non-dissociated lactic acid, while the low pHi heterogeneity at pHex 7.5 most probably corresponded to lower concentrations of non-dissociated lactic acid which facilitated the cells to reach the highest maximum active cell counts of the three pH set points. PMID:27020293

  7. Signals regulating myelination in peripheral nerves and the Schwann cell response to injury

    OpenAIRE

    Glenn, Thomas D.; William S Talbot

    2013-01-01

    In peripheral nerves, Schwann cells form myelin, which facilitates the rapid conduction of action potentials along axons in the vertebrate nervous system. Myelinating Schwann cells are derived from neural crest progenitors in a step-wise process that is regulated by extracellular signals and transcription factors. In addition to forming the myelin sheath, Schwann cells orchestrate much of the regenerative response that occurs after injury to peripheral nerves. In response to injury, myelinati...

  8. Extracellular biosynthesis of CdTe quantum dots by the fungus Fusarium oxysporum and their anti-bacterial activity

    Science.gov (United States)

    Syed, Asad; Ahmad, Absar

    2013-04-01

    The growing demand for semiconductor [quantum dots (Q-dots)] nanoparticles has fuelled significant research in developing strategies for their synthesis and characterization. They are extensively investigated by the chemical route; on the other hand, use of microbial sources for biosynthesis witnessed the highly stable, water dispersible nanoparticles formation. Here we report, for the first time, an efficient fungal-mediated synthesis of highly fluorescent CdTe quantum dots at ambient conditions by the fungus Fusarium oxysporum when reacted with a mixture of CdCl2 and TeCl4. Characterization of these biosynthesized nanoparticles was carried out by different techniques such as Ultraviolet-visible (UV-Vis) spectroscopy, Photoluminescence (PL), X-ray Diffraction (XRD), X-ray Photoelectron spectroscopy (XPS), Transmission Electron Microscopy (TEM) and Fourier Transformed Infrared Spectroscopy (FTIR) analysis. CdTe nanoparticles shows antibacterial activity against Gram positive and Gram negative bacteria. The fungal based fabrication provides an economical, green chemistry approach for production of highly fluorescent CdTe quantum dots.

  9. Extracellular matrix molecules and synaptic plasticity: immunomapping of intracellular and secreted Reelin in the adult rat brain.

    Science.gov (United States)

    Ramos-Moreno, Tania; Galazo, Maria J; Porrero, Cesar; Martínez-Cerdeño, Verónica; Clascá, Francisco

    2006-01-01

    Reelin, a large extracellular matrix glycoprotein, is secreted by several neuron populations in the developing and adult rodent brain. Secreted Reelin triggers a complex signaling pathway by binding lipoprotein and integrin membrane receptors in target cells. Reelin signaling regulates migration and dendritic growth in developing neurons, while it can modulate synaptic plasticity in adult neurons. To identify which adult neural circuits can be modulated by Reelin-mediated signaling, we systematically mapped the distribution of Reelin in adult rat brain using sensitive immunolabeling techniques. Results show that the distribution of intracellular and secreted Reelin is both very widespread and specific. Some interneuron and projection neuron populations in the cerebral cortex contain Reelin. Numerous striatal neurons are weakly immunoreactive for Reelin and these cells are preferentially located in striosomes. Some thalamic nuclei contain Reelin-immunoreactive cells. Double-immunolabeling for GABA and Reelin reveals that the Reelin-immunoreactive cells in the visual thalamus are the intrinsic thalamic interneurons. High local concentrations of extracellular Reelin selectively outline several dendrite spine-rich neuropils. Together with previous mRNA data, our observations suggest abundant axoplasmic transport and secretion in pathways such as the retino-collicular tract, the entorhino-hippocampal ('perforant') path, the lateral olfactory tract or the parallel fiber system of the cerebellum. A preferential secretion of Reelin in these neuropils is consistent with reports of rapid, activity-induced structural changes in adult brain circuits.

  10. IL-12 and IL-18 induce MAP kinase-dependent adhesion of T cells to extracellular matrix components.

    Science.gov (United States)

    Ariel, Amiram; Novick, Daniela; Rubinstein, Menachem; Dinarello, Charles A; Lider, Ofer; Hershkoviz, Rami

    2002-07-01

    Cytokines and chemokines play an essential role in recruiting leukocytes from the circulation to the peripheral sites of inflammation by modulating cellular interactions with endothelial cell ligands and extracellular matrix (ECM). Herein, we examined regulation of T cell adhesion to ECM ligands by two major proinflammatory cytokines, interleukin (IL)-12 and IL-18. IL-12 and IL-18 induced T cell adhesion to fibronectin (FN) and hyaluronic acid at low (pM) concentrations that were mediated by specific adhesion molecules expressed on the T cell surface, namely, beta(1) integrins and CD44, respectively. The induction of adhesion by IL-12 and IL-18 was inhibited by extracellular signal-regulated kinase and p38 mitogen-activated protein kinase inhibitors (PD098059 and SB203580, respectively). In contrast, IL-12- and IL-18-induced interferon-gamma (INF-gamma) secretion from T cells was inhibited by SB203580, but not by PD098059. It is interesting that low concentrations of IL-12 and IL-18 induced T cell adhesion to FN in a synergistic manner. Thus, in addition to the regulation of late inflammatory functions such as INF-gamma production, IL-12 and IL-18, alone or in combination, regulate early inflammatory events such as T cell adhesion to inflamed sites. PMID:12101280

  11. An extracellular material elaborated by Micrococcus sodonensis.

    Science.gov (United States)

    CAMPBELL, J N; EVANS, J B; PERRY, J J; NIVEN, C F

    1961-12-01

    Campbell, J. N. (American Meat Institute Foundation, Chicago, Ill.), James B. Evans Jerome J. Perry, and C. F. Niven, Jr. An extracellular material elaborated by Micrococcus sodonensis. J. Bacteriol. 82:828-837. 1961.-When actively growing in a yeast extract-tryptone broth, Micrococcus sodonensis produced rather large quantities of an extracellular material. On a dry weight basis, this material consisted of approximately 80% deoxyribonucleic acid (DNA), 6% ribonucleic acid, 13% protein, and a small quantity of a cell pigment which contained an associated iron porphyrinlike compound. The extracellular material was not produced in other complex or synthetic media. Removal of cells from the appropriate growth medium and suspension in distilled water resulted in the prevention of formation of the material if it had not already begun, or its immediate cessation in those cells which had been actively producing the material. Cells producing the extracellular material exhibited an increasingly higher concentration of intracellular DNA as incubation proceeded, whereas cells growing under conditions not supporting such production showed a decline in intracellular DNA as the culture aged. A similar increase of intracellular DNA, but not release of extracellular material, could be effected by addition of yeast extract or high levels of purine compounds to a medium which did not support the production of extracellular material. The base composition and ratios of both extracellular and intracellular DNA produced under all conditions tested were compared and found to be similar or identical, and to correspond to the classical Watson-Crick structure for DNA. PMID:13876041

  12. Foodomics study on the effects of extracellular production of hydrogen peroxide by rosemary polyphenols on the anti-proliferative activity of rosemary polyphenols against HT-29 cells.

    Science.gov (United States)

    Valdés, Alberto; García-Cañas, Virginia; Koçak, Engin; Simó, Carolina; Cifuentes, Alejandro

    2016-07-01

    A number of studies have demonstrated a strong association between the antioxidant properties of rosemary polyphenols and their chemoprotective activity. However, the prooxidant effects of rosemary polyphenols have been rarely reported. In this work, a foodomics study is performed to investigate the in vitro autooxidation of carnosic acid (CA), carnosol (CS) and a polyphenol-enriched rosemary extract (SC-RE) in cell culture conditions. The results revealed that rosemary polyphenols autooxidation in culture medium generated H2 O2 at different rates. Generated H2 O2 levels by SC-RE and CA, but not CS, were correlated with intracellular reactive oxygen species (ROS) generation in HT-29 cells, and were partially involved in their anti-proliferative effect in this cell line. These compounds also induced different effects on glutathione metabolism. Results also indicated that high extracellular H2 O2 concentrations, resulting of using high (45 μg/mL) SC-RE concentration in culture media, exerted some artifactual effects related with cell cycle, but they did not influence the expression of relevant molecular biomarkers of stress. PMID:26842614

  13. Activation of Yes-Associated Protein in Low-Grade Meningiomas Is Regulated by Merlin, Cell Density, and Extracellular Matrix Stiffness.

    Science.gov (United States)

    Tanahashi, Kuniaki; Natsume, Atsushi; Ohka, Fumiharu; Motomura, Kazuya; Alim, Adiljan; Tanaka, Ichidai; Senga, Takeshi; Harada, Ichiro; Fukuyama, Ryuichi; Sumiyoshi, Naoyuki; Sekido, Yoshitaka; Wakabayashi, Toshihiko

    2015-07-01

    The NF2 gene product Merlin is a protein containing ezrin, radixin, and moesin domains; it is a member of the 4.1 protein superfamily associated with the membrane cytoskeleton and also interacts with cell surface molecules. The mammalian Hippo cascade, a downstream signaling cascade of merlin, inactivates the Yes-associated protein (YAP). Yes-associated protein is activated by loss of the NF2 gene and functions as an oncogene in meningioma cells; however, the factors controlling YAP expression, phosphorylation, and subcellular localization in meningiomas have not been fully elucidated. Here, we demonstrate that merlin expression is heterogeneous in 1 NF2 gene-negative and 3 NF2 gene-positive World Health Organization grade I meningiomas. In the NF2 gene-positive meningiomas, regions with low levels of merlin (tumor rims) had greater numbers of cells with nuclear YAP versus regions with high merlin levels (tumor cores). Merlin expression and YAP phosphorylation were also affected by cell density in the IOMM-Lee and HKBMM human meningioma cell lines; nuclear localization of YAP was regulated by cell density and extracellular matrix (ECM) stiffness in IOMM-Lee cells. These results suggest that cell density and ECM stiffness may contribute to the heterogeneous loss of merlin and increased nuclear YAP expression in human meningiomas.

  14. Extracellular Matrix Biomarker, Fibulin-1, Is Closely Related to NT-proBNP and Soluble Urokinase Plasminogen Activator Receptor in Patients with Aortic Valve Stenosis (The SEAS Study)

    DEFF Research Database (Denmark)

    Kruger, Ruan; Rasmussen, Lars M; Argraves, William S;

    2014-01-01

    BACKGROUND: Fibulin-1, a circulating extracellular matrix glycoprotein, has been associated with arterial disease and elevated N-terminal prohormone B-type natriuretic peptide (NT-proBNP) in diabetes. Soluble urokinase plasminogen activator receptor (suPAR), a marker of inflammation, has been...... associated with subclinical atherosclerosis. Therefore, we aimed to explore the interplay between these biomarkers and mild to moderate aortic valve stenosis (AS). METHODS: In 374 patients with mild to moderate AS, we investigated the relationship of fibulin-1 with NT-proBNP, levels of suPAR and the degree...... of AS at baseline and after one and four years of treatment with Simvastatin 40 mg and Ezetimibe 10 mg or placebo. RESULTS: During treatment, fibulin-1 became more closely associated with NT-proBNP (βyear0 = 0.10, p = 0.08, βyear1 = 0.16, p = 0.005, βyear4 = 0.22, p

  15. Extracellular proteolysis in the adult murine brain.

    Science.gov (United States)

    Sappino, A P; Madani, R; Huarte, J; Belin, D; Kiss, J Z; Wohlwend, A; Vassalli, J D

    1993-08-01

    Plasminogen activators are important mediators of extracellular metabolism. In the nervous system, plasminogen activators are thought to be involved in the remodeling events required for cell migration during development and regeneration. We have now explored the expression of the plasminogen activator/plasmin system in the adult murine central nervous system. Tissue-type plasminogen activator is synthesized by neurons of most brain regions, while prominent tissue-type plasminogen activator-catalyzed proteolysis is restricted to discrete areas, in particular within the hippocampus and hypothalamus. Our observations indicate that tissue-type plasminogen activator-catalyzed proteolysis in neural tissues is not limited to ontogeny, but may also contribute to adult central nervous system physiology, for instance by influencing neuronal plasticity and synaptic reorganization. The identification of an extracellular proteolytic system active in the adult central nervous system may also help gain insights into the pathogeny of neurodegenerative disorders associated with extracellular protein deposition.

  16. Extracellular enzyme activities in a tropical mountain rainforest region of southern Ecuador affected by low soil P status and land-use change

    Science.gov (United States)

    Tischer, Alexander; Blagodatskaya, Evgenia; Ute, Hamer

    2014-05-01

    Little is known about the enzymatic response of microorganisms in soils having a low P status and being subjected to global change phenomena, such as forest disturbance and land-use change. Along a land-use sequence (natural forest - young pasture - old pasture - abandoned pasture - shrubland) in the Andes of southern Ecuador mineral topsoils of Cambisols / Umbrisols were investigated. We tested whether the activities of the six hydrolytic enzymes (cellobiohydrolase, β-glucosidase, N-acetylglucosaminidase, α-glucosidase, xylanase, acid phosphomonoesterase) were affected by nutrient status and land-use induced alterations in soil pH (pHH2O from 3.7 to 5.2), resource quantity and quality (e.g. a SOC:N:P ratio from 182:13:1 to 1050:38:1) and microbial community structure (as monitored by phospholipid fatty acids). Microbial production of acid phosphatase responded to the low P status of the sites by a higher investment in the acquisition of P compared to C. We determined three major drivers of enzyme activities: 1.) Microbial demand for P regulated the production of acid phosphatase, provided that N and C were available. At the natural forest site the two-fold higher specific activity of acid phosphatase pointed to a high microbial P-demand, whereas the production of acid phosphatase was constrained by the availability of N and DOC after pasture abandonment. 2.) Microbial biomass that was controlled by pH and resource availability (total soil N (organic and inorganic N), organic P (Bray-fraction)) was the main driver for cellobiohydrolase, β-glucosidase and N-acetylglucosaminidase activities. 3.) Substrate induction due to increased litter inputs of herbaceous plant species seemed to regulate α-glucosidase and xylanase activities during secondary succession. In contrast, alterations in the abundance of microbial groups affected the variation in extracellular enzyme activities only marginally. At the level of broadly defined microbial groups (PLFA), our results

  17. Changes to gonadotropin-releasing hormone (GnRH) receptor extracellular loops differentially affect GnRH analog binding and activation: evidence for distinct ligand-stabilized receptor conformations.

    Science.gov (United States)

    Pfleger, Kevin D G; Pawson, Adam J; Millar, Robert P

    2008-06-01

    GnRH and its structural variants bind to GnRH receptors from different species with different affinities and specificities. By investigating chimeric receptors that combine regions of mammalian and nonmammalian GnRH receptors, a greater understanding of how different domains influence ligand binding and receptor activation can be achieved. Using human-catfish and human-chicken chimeric receptors, we demonstrate the importance of extracellular loop conformation for ligand binding and agonist potency, providing further evidence for GnRH and GnRH II stabilization of distinct active receptor conformations. We demonstrate examples of GnRH receptor gain-of-function mutations that apparently improve agonist potency independently of affinity, implicating a role for extracellular loops in stabilizing the inactive receptor conformation. We also show that entire extracellular loop substitution can overcome the detrimental effects of localized mutations, thereby demonstrating the importance of considering the conformation of entire domains when drawing conclusions from point-mutation studies. Finally, we present evidence implicating the configuration of extracellular loops 2 and 3 in combination differentiating GnRH analog binding modes. Because there are two endogenous forms of GnRH ligand but only one functional form of full-length GnRH receptor in humans, understanding how GnRH and GnRH II can elicit distinct functional effects through the same receptor is likely to provide important insights into how these ligands can have differential effects in both physiological and pathological situations. PMID:18356273

  18. Mitogen-activated protein kinases mediate Mycobacterium tuberculosis–induced CD44 surface expression in monocytes

    Indian Academy of Sciences (India)

    Natarajan Palaniappan; S Anbalagan; Sujatha Narayanan

    2012-03-01

    CD44, an adhesion molecule, has been reported to be a binding site for Mycobacterium tuberculosis (M. tuberculosis) in macrophages and it also mediates mycobacterial phagocytosis, macrophage recruitment and protective immunity against pulmonary tuberculosis in vivo. However, the signalling pathways that are involved in M. tuberculosis–induced CD44 surface expression in monocytic cells are currently unknown. Exposure of THP-1 human monocytes to M. tuberculosis H37Rv and H37Ra induced distinct, time-dependent, phosphorylation of mitogen-activated protein kinase kinase-1, extracellular signal regulated kinase 1/2, mitogen-activated protein kinase kinase 3/6, p38 mitogen-activated protein kinase and c-jun N-terminal kinases. The strains also differed in their usage of CD14 and human leukocyte antigen-DR (HLA-DR) receptors in mediating mitogen-activated protein kinase activation. M. tuberculosis H37Rv strain induced lower CD44 surface expression and tumour necrosis factor-alpha levels, whereas H37Ra the reverse. Using highly specific inhibitors of mitogen-activated protein kinase kinase-1, p38 mitogen-activated protein kinase and c-jun N-terminal kinase, we report that inhibition of extracellular signal regulated kinase 1/2 and c-jun N-terminal kinases increases, but that inhibition of p38 mitogen-activated protein kinase decreases M. tuberculosis–induced CD44 surface expression in THP-1 human monocytes.

  19. Relationship between myocardial extracellular space expansion estimated with post-contrast T1 mapping MRI and left ventricular remodeling and neurohormonal activation in patients with dilated cardiomyopathy

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Ji Hyun; Son, Jung Woo; Chung, Hye Moon [Cardiology Division, Dept. of Internal Medicine, Yonsei University College of Medicine, Seoul (Korea, Republic of); and others

    2015-10-15

    Post-contrast T1 values are closely related to the degree of myocardial extracellular space expansion. We determined the relationship between post-contrast T1 values and left ventricular (LV) diastolic function, LV remodeling, and neurohormonal activation in patients with dilated cardiomyopathy (DCM). Fifty-nine patients with DCM (mean age, 55 ± 15 years; 41 males and 18 females) who underwent both 1.5T magnetic resonance imaging and echocardiography were enrolled. The post-contrast 10-minute T1 value was generated from inversion time scout images obtained using the Look-Locker inversion recovery sequence and a curve-fitting algorithm. The T1 sample volume was obtained from three interventricular septal points, and the mean T1 value was used for analysis. The N-Terminal pro-B-type natriuretic peptide (NT-proBNP) level was measured in 40 patients. The mean LV ejection fraction was 24 ± 9% and the post-T1 value was 254.5 ± 46.4 ms. The post-contrast T1 value was significantly correlated with systolic longitudinal septal velocity (s'), peak late diastolic velocity of the mitral annulus (a'), the diastolic elastance index (Ed, [E/e']/stroke volume), LV mass/volume ratio, LV end-diastolic wall stress, and LV end-systolic wall stress. In a multivariate analysis without NT-proBNP, T1 values were independently correlated with Ed (β = -0.351, p = 0.016) and the LV mass/volume ratio (β = 0.495, p = 0.001). When NT-proBNP was used in the analysis, NT-proBNP was independently correlated with the T1 values (β = -0.339, p = 0.017). Post-contrast T1 is closely related to LV remodeling, diastolic function, and neurohormonal activation in patients with DCM.

  20. Enhanced Proliferation of Porcine Bone Marrow Mesenchymal Stem Cells Induced by Extracellular Calcium is Associated with the Activation of the Calcium-Sensing Receptor and ERK Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Jingjing Ye

    2016-01-01

    Full Text Available Porcine bone marrow mesenchymal stem cells (pBMSCs have the potential for application in regenerative medicine. This study aims to investigate the effects of extracellular calcium (Ca2+o on pBMSCs proliferation and to explore the possible underlying mechanisms. The results demonstrated that 4 mM Ca2+o significantly promoted pBMSCs proliferation by reducing the G0/G1 phase cell percentage and by increasing the S phase cell proportion and the proliferation index of pBMSCs. Accordingly, Ca2+o stimulated the expression levels of proliferative genes such as cyclin A2, cyclin D1/3, cyclin E2, and PCNA and inhibited the expression of p21. In addition, Ca2+o resulted in a significant elevation of intracellular calcium and an increased ratio of p-ERK/ERK. However, inhibition of calcium-sensing receptor (CaSR by its antagonist NPS2143 abolished the aforementioned effects of Ca2+o. Moreover, Ca2+o-induced promotion of pBMSCs proliferation, the changes of proliferative genes expression levels, and the activation of ERK1/2 signaling pathway were effectively blocked by U0126, a selective ERK kinase inhibitor. In conclusion, our findings provided evidence that the enhanced pBMSCs proliferation in response to Ca2+o was associated with the activation of CaSR and ERK1/2 signaling pathway, which may be useful for the application of pBMSCs in future clinical studies aimed at tissue regeneration and repair.

  1. Interaction with extracellular matrix proteins influences Lsh/Ity/Bcg (candidate Nramp) gene regulation of macrophage priming/activation for tumour necrosis factor-alpha and nitrite release.

    Science.gov (United States)

    Formica, S; Roach, T I; Blackwell, J M

    1994-05-01

    The murine resistance gene Lsh/Ity/Bcg regulates activation of macrophages for tumour necrosis factor-alpha (TNF-alpha)-dependent production of nitric oxide mediating antimicrobial activity against Leishmania, Salmonella and Mycobacterium. As Lsh is differentially expressed in macrophages from different tissue sites, experiments were performed to determine whether interaction with extracellular matrix (ECM) proteins would influence the macrophage TNF-alpha response. Plating of bone marrow-derived macrophages onto purified fibrinogen or fibronectin-rich L929 cell-derived matrices, but not onto mannan, was itself sufficient to stimulate TNF-alpha release, with significantly higher levels released from congenic B10.L-Lshr compared to C57BL/10ScSn (Lshs) macrophages. Only macrophages plated onto fibrinogen also released measurable levels of nitrites, again higher in Lshr compared to Lshs macrophages. Addition of interferon-gamma (IFN-gamma), but not bacterial lipopolysaccharide or mycobacterial lipoarabinomannan, as a second signal enhanced the TNF-alpha and nitrite responses of macrophages plated onto fibrinogen, particularly in the Lshr macrophages. Interaction with fibrinogen and fibronectin also primed macrophages for an enhanced TNF-alpha response to leishmanial parasites, but this was only translated into enhanced nitrite responses in the presence of IFN-gamma. In these experiments, Lshr macrophages remained superior in their TNF-alpha responses throughout, but to a degree which reflected the magnitude of the difference observed on ECM alone. Hence, the specificity for the enhanced TNF-alpha responses of Lshr macrophages lay in their interaction with fibrinogen and fibronectin ECM, while a differential nitrite response was only observed with fibrinogen and/or IFN-gamma. The results are discussed in relation to the possible function of the recently cloned candidate gene Nramp, which has structural identity to eukaryote transporters and an N-terminal cytoplasmic

  2. Impact of fire, landscape position, aspect, and soil depth on microbial extracellular enzyme activities in the Jemez River Basin Critical Zone Observatory.

    Science.gov (United States)

    Fairbanks, D.; Murphy, M. A.; Frost, G.; Chorover, J.; Gallery, R. E.; Rich, V. I.

    2014-12-01

    Fire frequency and severity are increasing across the western US, and post-fire recovery and effects on critical zone structure are not fully understood. Resident microbiota (bacteria and fungi) transform the majority of carbon in ecosystems, and the structure of these communities influence seedling establishment and the trajectory of vegetative recovery as well as biogeochemical cycling. We surveyed changes in microbial composition and activity after wildfire to better understand soil microbial resilience and fire ecology. Specifically, we assessed potential extracellular enzyme activities in response to fire severity across landscape position and aspect. We sampled 18 days after containment of the June 2013 Thompson Ridge Fire in the Jemez River Basin Critical Zone Observatory, across a gradient of burn severities in a mixed-conifer zero order basin. We subsampled six depths through the surface soil profile and measured potential activities of seven hydrolytic enzymes using established fluorometric techniques. Four of these enzymes hydrolyze C-rich substrates (β-glucosidase [BG], β-D-cellubiosidase [CB], xylosidase [XYL], and α-glucosidase [AG], two hydrolyze N-rich substrates N-acetyl-β-glucosaminidase [NAG] and leucine aminopeptidase [LAP]), and one hydrolyzes a P-rich substrate (acid phosphatase [PHOS]). Results showed decreased activities with depth for BG, CB, and LAP. Significantly higher potential enzyme activity was observed for convergent sites relative to planar or divergent sites across all depths sampled. Additionally, we looked at shifts in enzyme nutrient acquisition ratios that correspond with resource limitations relative to microbial stoichiometric demands. Higher acquisition potential is interpreted as greater resource allocation towards nutrient acquisition. Results showed a variance in resource acquisition potential with depth for C relative to N, with greater resources being allocated towards acquiring C at shallower depth. Conversely

  3. Response of microbial extracellular enzyme activities and r- vs. K- selected microorganisms to elevated atmospheric CO2 depends on soil aggregate size

    Science.gov (United States)

    Dorodnikov, Maxim; Blagodatskaya, Evgenia; Blagodatskiy, Sergey; Kuzyakov, Yakov

    2014-05-01

    Increased belowground carbon (C) transfer by plant roots under elevated atmospheric CO2 and the contrasting environment in soil macro- and microaggregates could affect properties of the microbial community in the rhizosphere. We evaluated the effect of 5 years of elevated CO2 (550 ppm) on four extracellular enzymes: ß-glucosidase, chitinase, phosphatase, and sulfatase along with the contribution of fast- (r-strategists) and slow-growing microorganisms (K-strategists) in soil aggregates. We fractionated the bulk soil from the ambient and elevated CO2 treatments of FACE-Hohenheim (Stuttgart) into large macro- (>2 mm), small macro- (0.25-2.00 mm), and microaggregates (<0.25 mm) using a modified dry sieving. Microbial biomass (C-mic by SIR), the maximal specific growth rate (µ), growing microbial biomass (GMB) and lag-period (t-lag) were estimated by the kinetics of CO2 emission from bulk soil and aggregates amended with glucose and nutrients. In the bulk soil and isolated aggregates before and after activation with glucose, the actual and the potential enzyme activities were measured. Although C-org and C-mic as well as the activities of ß-glucosidase, phosphatase, and sulfatase were unaffected in bulk soil and in aggregate-size classes by elevated CO2, significant changes were observed in potential enzyme production after substrate amendment. After adding glucose, enzyme activities under elevated CO2 were 1.2-1.9-fold higher than under ambient CO2. In addition, µ values were significantly higher under elevated than ambient CO2 for bulk soil, small macroaggregates, and microaggregates. Based on changes in µ, GMB, and lag-period, we conclude that elevated atmospheric CO2 stimulated the r-selected microorganisms, especially in soil microaggregates. In contrast, significantly higher chitinase activity in bulk soil and in large macroaggregates under elevated CO2 revealed an increased contribution of fungi to turnover processes. We conclude that quantitative and

  4. ESTRADIOL-INDUCED ENHANCEMENT OF OBJECT MEMORY CONSOLIDATION INVOLVES HIPPOCAMPAL ERK ACTIVATION AND MEMBRANE-BOUND ESTROGEN RECEPTORS

    OpenAIRE

    Fernandez, Stephanie M.; Michael C. Lewis; Pechenino, Angela S.; Harburger, Lauren L.; Orr, Patrick T.; Gresack, Jodi E.; Schafe, Glenn E.; Frick, Karyn M.

    2008-01-01

    The extracellular signal-regulated kinase (ERK) pathway is critical for various forms of learning and memory, and is activated by the potent estrogen, 17β-estradiol (E2). Here, we asked whether E2 modulates memory via ERK activation and putative membrane-bound estrogen receptors (ERs). Using ovariectomized mice, we first demonstrate that intraperitoneal (i.p.) injection of 0.2 mg/kg E2 significantly increases dorsal hippocampal levels of phosphorylated ERK protein 1 hour after injection. Seco...

  5. Platelet-derived growth factor-D modulates extracellular matrix homeostasis and remodeling through TIMP-1 induction and attenuation of MMP-2 and MMP-9 gelatinase activities

    Energy Technology Data Exchange (ETDEWEB)

    Borkham-Kamphorst, Erawan, E-mail: ekamphorst@ukaachen.de; Alexi, Pascal; Tihaa, Lidia; Haas, Ute; Weiskirchen, Ralf, E-mail: rweiskirchen@ukaachen.de

    2015-02-13

    Platelet-derived growth factor-D (PDGF-D) is a more recent recognized growth factor involved in the regulation of several cellular processes, including cell proliferation, transformation, invasion, and angiogenesis by binding to and activating its cognate receptor PDGFR-β. After bile duct ligation or in the carbon tetrachloride-induced hepatic fibrosis model{sub ,} PDGF-D showed upregulation comparable to PDGF-B. Moreover, adenoviral PDGF-D gene transfer induced hepatic stellate cell proliferation and liver fibrosis. We here investigated the molecular mechanism of PDGF-D involvement in liver fibrogenesis. Therefore, the GRX mouse cell line was stimulated with PDGF-D and evaluated for fibrotic markers and PDGF-D signaling pathways in comparison to the other PDGF isoforms. We found that PDGF-D failed to enhance Col I and α-smooth muscle actin (α-SMA) production but has capacity to upregulate expression of the tissue inhibitor of metalloprotease 1 (TIMP-1) resulting in attenuation of MMP-2 and MMP-9 gelatinase activity as indicated by gelatinase zymography. This phenomenon was restored through application of a PDGF-D neutralizing antibody. Unexpectedly, PDGF-D incubation decreased both PDGFR-α and -β in mRNA and protein levels, and PDGF-D phosphorylated typrosines specific for PDGFR-α and -β. We conclude that PDGF-D intensifies fibrogenesis by interfering with the fibrolytic activity of the TIMP-1/MMP system and that PDGF-D signaling is mediated through both PDGF-α and -β receptors. - Highlights: • PDGF-D signals through PDGF receptor type α and β. • PDGF-D modulates extracellular matrix homeostasis and remodeling. • Like PDGF-B, PDGF-D triggers phosphorylation of PLC-γ, Akt/PKB, JNK, ERK1/2, and p38. • PDGF-D induces TIMP-1 expression through ERK and p38 MAPK. • PDGF-D attenuates MMP-2 and MMP-9 gelatinase activities.

  6. Slit/Robo1 signaling regulates neural tube development by balancing neuroepithelial cell proliferation and differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Guang; Li, Yan; Wang, Xiao-yu [Key Laboratory for Regenerative Medicine of The Ministry of Education, Department of Histology and Embryology, School of Medicine, Jinan University, Guangzhou 510632 (China); Han, Zhe [Institute of Vascular Biological Sciences, Guangdong Pharmaceutical University, Guangzhou 510224 (China); Chuai, Manli [College of Life Sciences Biocentre, University of Dundee, Dundee DD1 5EH (United Kingdom); Wang, Li-jing [Institute of Vascular Biological Sciences, Guangdong Pharmaceutical University, Guangzhou 510224 (China); Ho Lee, Kenneth Ka [Stem Cell and Regeneration Thematic Research Programme, School of Biomedical Sciences, Chinese University of Hong Kong, Shatin (Hong Kong); Geng, Jian-guo, E-mail: jgeng@umich.edu [Institute of Vascular Biological Sciences, Guangdong Pharmaceutical University, Guangzhou 510224 (China); Department of Biologic and Materials Sciences, University of Michigan School of Dentistry, Ann Arbor, MI 48109 (United States); Yang, Xuesong, E-mail: yang_xuesong@126.com [Key Laboratory for Regenerative Medicine of The Ministry of Education, Department of Histology and Embryology, School of Medicine, Jinan University, Guangzhou 510632 (China)

    2013-05-01

    development by tightly coordinating cell proliferation and differentiation during neurulation. - Highlights: ► The role of Slit/Robo1 signaling was investigated with chick and mouse models. ► Disturbance of Slit/Robo1 signaling resulted in neural tube defects. ► Slit/Robo1 signaling regulated the proliferation of neural tube cells. ► Slit/Robo1 signaling modulated the differentiation of neural tube cells. ► Slit/Robo1 signaling balanced the proliferation and differentiation of neural tube.

  7. Slit/Robo1 signaling regulates neural tube development by balancing neuroepithelial cell proliferation and differentiation

    International Nuclear Information System (INIS)

    coordinating cell proliferation and differentiation during neurulation. - Highlights: ► The role of Slit/Robo1 signaling was investigated with chick and mouse models. ► Disturbance of Slit/Robo1 signaling resulted in neural tube defects. ► Slit/Robo1 signaling regulated the proliferation of neural tube cells. ► Slit/Robo1 signaling modulated the differentiation of neural tube cells. ► Slit/Robo1 signaling balanced the proliferation and differentiation of neural tube

  8. Activation of MEK2 is sufficient to induce skin papilloma formation in transgenic zebrafish

    OpenAIRE

    Chou, Chih-Ming; Chen, Yi-Chung; SU, SAN; Chen, Gen-Der; Huang, Kai-Yun; Lien, Huang-Wei; Huang, Chang-Jen; Cheng, Chia-Hsiung

    2015-01-01

    Background Mutations in mitogen-activated protein kinase (MAPK) kinase 1 (MEK1) that occur during cell proliferation and tumor formation are well described. Information on the roles of MEK2 in these effects is still limited. We established a constitutive MEK2 transgenic zebrafish, Tg(krt14:MEK2S219D-GFP), to elucidate the role of MEK2 in skin tumor formation. Results We found that both constitutive MEK2 and MEK1 are able to phosphorylate the extracellular signal-regulated kinase 1 (ERK1) prot...

  9. Transcriptome of extracellular vesicles released by hepatocytes.

    Directory of Open Access Journals (Sweden)

    Felix Royo

    Full Text Available The discovery that the cells communicate through emission of vesicles has opened new opportunities for better understanding of physiological and pathological mechanisms. This discovery also provides a novel source for non-invasive disease biomarker research. Our group has previously reported that hepatocytes release extracellular vesicles with protein content reflecting the cell-type of origin. Here, we show that the extracellular vesicles released by hepatocytes also carry RNA. We report the messenger RNA composition of extracellular vesicles released in two non-tumoral hepatic models: primary culture of rat hepatocytes and a progenitor cell line obtained from a mouse foetal liver. We describe different subpopulations of extracellular vesicles with different densities and protein and RNA content. We also show that the RNA cargo of extracellular vesicles released by primary hepatocytes can be transferred to rat liver stellate-like cells and promote their activation. Finally, we provide in vitro and in vivo evidence that liver-damaging drugs galactosamine, acetaminophen, and diclofenac modify the RNA content of these vesicles. To summarize, we show that the extracellular vesicles secreted by hepatocytes contain various RNAs. These vesicles, likely to be involved in the activation of stellate cells, might become a new source for non-invasive identification of the liver toxicity markers.

  10. Contractions activate hormone-sensitive lipase in rat muscle by protein kinase C and mitogen-activated protein kinase

    DEFF Research Database (Denmark)

    Donsmark, Morten; Langfort, Jozef; Holm, Cecilia;

    2003-01-01

    Intramuscular triacylglycerol is an important energy store and is also related to insulin resistance. The mobilization of fatty acids from this pool is probably regulated by hormone-sensitive lipase (HSL), which has recently been shown to exist in muscle and to be activated by both adrenaline......-induced activation of HSL was abolished by the protein kinase C (PKC) inhibitors bisindolylmaleimide I and calphostin C and reduced 50% by the mitogen-activated protein kinase kinase (MEK) inhibitor U0126, which also completely blocked extracellular signal-regulated kinase (ERK) 1 and 2 phosphorylation. None...... of the inhibitors reduced adrenaline-induced HSL activation in soleus muscle. Both phorbol-12-myristate-13-acetate (PMA), which activates PKC and, in turn, ERK, and caffeine, which increases intracellular Ca2+ without eliciting contraction, increased HSL activity. Activated ERK increased HSL activity in supernatant...

  11. Enzymatically quiescent heparanase augments T cell interactions with VCAM-1 and extracellular matrix components under versatile dynamic contexts.

    Science.gov (United States)

    Sotnikov, Ilya; Hershkoviz, Rami; Grabovsky, Valentin; Ilan, Neta; Cahalon, Liora; Vlodavsky, Israel; Alon, Ronen; Lider, Ofer

    2004-05-01

    During their migration into inflammatory sites, immune cells, such as T cells, secrete extracellular matrix (ECM)-degrading enzymes, such as heparanase, which, under mildly acidic conditions, degrade heparan sulfate proteoglycans (HSPG). We have previously shown that at pH 7.2, human placental heparanase loses its enzymatic activity, while retaining its ability to bind HSPG and promote T cell adhesion to unfractionated ECM. We now demonstrate that the 65-kDa recombinant human heparanase, which is devoid of enzymatic activity, but can still bind HSPG, captures T cells under shear flow conditions and mediates their rolling and arrest, in the absence or presence of stromal cell-derived factor 1 alpha (SDF-1 alpha; CXCL12), in an alpha(4)beta(1)-VCAM-1-dependent manner. Furthermore, heparanase binds to and induces T cell adhesion to key ECM components, like fibronectin and hyaluronic acid, in beta(1) integrin- and CD44-specific manners, respectively, via the activation of the protein kinase C and phosphatidylinositol 3-kinase intracellular signaling machineries. Although the nature of the putative T cell heparanase-binding moiety is unknown, it appears that heparanase exerts its proadhesive activity by interacting with the T cells' surface HSPG, because pretreatment of the cells with heparinase abolished their subsequent response to heparanase. Also, heparanase augmented the SDF-1 alpha-triggered phosphorylation of Pyk-2 and extracellular signal-regulated kinase-2 implicated in integrin functioning. Moreover, heparanase, which had no chemotactic effect on T cells on its own, augmented the SDF-1 alpha-induced T cell chemotaxis across fibronectin. These findings add another dimension to the known versatility of heparanase as a key regulator of T cell activities during inflammation, both in the context of the vasculature and at extravascular sites. PMID:15100255

  12. Roles of Na+/H+ exchange in regulation of p38 mitogen-activated protein kinase activity and cell death after chemical anoxia in NIH3T3 fibroblasts

    DEFF Research Database (Denmark)

    Rentsch, Maria L; Ossum, Carlo G; Hoffmann, Else K;

    2007-01-01

    , p38 mitogen-activated protein kinase (MAPK), ERK1/2, p53, and Akt activity, and cell death, after chemical anoxia in NIH3T3 fibroblasts. The NHE1 inhibitor 5'-(N-ethyl-N-isopropyl) amiloride (EIPA) (5 muM), as well as removal of extracellular Na(+) [replaced by N-methyl-D: -glucamine (NMDG......) and extracellular signal-regulated kinase (ERK) (PD98059). In contrast, chemical anoxia activated p38 MAPK in an NHE-dependent manner, while ERK1/2 activity was unaffected. Anoxia-induced cell death was caspase-3-independent, mildly attenuated by EIPA, potently exacerbated by SB203580, and unaffected by PD98059...

  13. Tumor extracellular acidity-activated nanoparticles as drug delivery systems%肿瘤酸度活化的纳米药物载体

    Institute of Scientific and Technical Information of China (English)

    李洪军; 王均

    2014-01-01

    pH响应性的纳米粒(nanoparticles,NPs)作为药物输送载体用于肿瘤治疗倍受关注。实体瘤的弱酸性肿瘤微环境(pHe约为6.8)为抗肿瘤药物纳米载体的设计提供了新思路和发展契机。相对于靶向基团介导的肿瘤靶向策略,对pHe响应的纳米载体更具有普适性。本文概述了pHe活化纳米药物载体在肿瘤治疗中的设计和应用现状,重点介绍pHe触发表面电荷反转的纳米颗粒用于抗肿瘤药物和小干扰RNA(siRNA)输送的研究进展,并展望这种新型载体在增强肿瘤疗效方面的应用潜力。%pH-responsive nanoparticles (NPs) are currently under intense development as carriers of drug delivery systems for cancer therapy. Of these NPs, those that are designed to target the slightly acidic extracellular pH environment (pHe~6.8) of solid tu-mors offer a new paradigm of tumor-targeted drug delivery. Compared with conventional, specific, surface-targeting approaches, the pHe-targeting strategy is considered to be more general because of the common occurrence of an acidic microenvironment in solid tu-mors. This review mainly focuses on the design and applications of pHe-activated NPs, particularly on pHe-activated surface-charge re-versal NPs, for drug and siRNA delivery to tumors. The novel development of NPs described in this review has high potential for achieving stronger therapeutic effects in cancer treatment.

  14. Extracellular collagenases and the endocytic receptor, urokinase plasminogen activator receptor-associated protein/Endo180, cooperate in fibroblast-mediated collagen degradation

    DEFF Research Database (Denmark)

    Madsen, Daniel H; Engelholm, Lars H; Ingvarsen, Signe;

    2007-01-01

    The collagens of the extracellular matrix are the most abundant structural proteins in the mammalian body. In tissue remodeling and in the invasive growth of malignant tumors, collagens constitute an important barrier, and consequently, the turnover of collagen is a rate-limiting process in these...

  15. Extracellular S100A4(mts1) stimulates invasive growth of mouse endothelial cells and modulates MMP-13 matrix metalloproteinase activity

    DEFF Research Database (Denmark)

    Schmidt-Hansen, Birgitte; Ornås, Dorte; Grigorian, Mariam;

    2004-01-01

    S100A4(mts1) protein expression has been strongly associated with metastatic tumor progression. It has been suggested as a prognostic marker for a number of human cancers. It is proposed that extracellular S100A4 accelerates cancer progression by stimulating the motility of endothelial cells...

  16. The Na+/H+ exchanger, NHE1, differentially regulates mitogen-activated protein kinase subfamilies after osmotic shrinkage in Ehrlich Lettre Ascites cells

    DEFF Research Database (Denmark)

    Petersen, Stine Helene Falsig; Rasmussen, Maria; Darborg, Barbara Vasek;

    2007-01-01

    Osmotic stress modulates mitogen activated protein kinase (MAPK) activities, leading to altered gene transcription and cell death/survival balance, however, the mechanisms involved are incompletely elucidated. Here, we show, using a combination of biochemical and molecular biology approaches......, that three MAPKs exhibit unique interrelationships with the Na(+)/H(+) exchanger, NHE1, after osmotic cell shrinkage: Extracellular Signal Regulated Kinase (ERK1/2) is inhibited in an NHE1-dependent, pH(i)-independent manner, c-Jun N-terminal kinase (JNK1/2) is stimulated, in part through NHE1-mediated...

  17. Extracellular Gd-CA

    DEFF Research Database (Denmark)

    Thomsen, Henrik S; Marckmann, Peter

    2008-01-01

    Until recently it was believed that extracellular gadolinium-based contrast agents were safe for both the kidneys and all other organs within the dose range up to 0.3 mmol/kg body weight. However, in 2006, it was demonstrated that some gadolinium-based contrast agents may trig the development of ...

  18. Extracellular Vesicle (EV) Array

    DEFF Research Database (Denmark)

    Jørgensen, Malene; Bæk, Rikke; Pedersen, Shona;

    2013-01-01

    Exosomes are one of the several types of cell-derived vesicles with a diameter of 30-100 nm. These extracellular vesicles are recognized as potential markers of human diseases such as cancer. However, their use in diagnostic tests requires an objective and high-throughput method to define...

  19. Bacterial extracellular lignin peroxidase

    Science.gov (United States)

    Crawford, Donald L.; Ramachandra, Muralidhara

    1993-01-01

    A newly discovered lignin peroxidase enzyme is provided. The enzyme is obtained from a bacterial source and is capable of degrading the lignin portion of lignocellulose in the presence of hydrogen peroxide. The enzyme is extracellular, oxidative, inducible by lignin, larch wood xylan, or related substrates and capable of attacking certain lignin substructure chemical bonds that are not degradable by fungal lignin peroxidases.

  20. Extracellular granzymes in inflammation

    NARCIS (Netherlands)

    Wensink, A.C.

    2014-01-01

    It has been well established that granzymes released by cytotoxic lymphocytes induce cell death in virus-infected cells and tumor cells. Next to this intracellular role of granzymes in triggering apoptosis, granzymes also exist extracellularly in the circulation of patients with autoimmune diseases

  1. Activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates breast cancer cell metastatic behaviors through inhibition of plasminogen activation and extracellular proteolysis

    OpenAIRE

    Bazzi, Zainab A.; Lanoue, Danielle; El-Youssef, Mouhanned; Romagnuolo, Rocco; Tubman, Janice; Cavallo-Medved, Dora; Porter, Lisa A.; Boffa, Michael B.

    2016-01-01

    Background Thrombin activatable fibrinolysis inhibitor (TAFI) is a plasma zymogen, which can be converted to activated TAFI (TAFIa) through proteolytic cleavage by thrombin, plasmin, and most effectively thrombin in complex with the endothelial cofactor thrombomodulin (TM). TAFIa is a carboxypeptidase that cleaves carboxyl terminal lysine and arginine residues from protein and peptide substrates, including plasminogen-binding sites on cell surface receptors. Carboxyl terminal lysine residues ...

  2. Role of extracellular signal—regulated kinase in free radical—induced injury in kidney of rats treated with cephaloridine

    Institute of Scientific and Technical Information of China (English)

    GembM; HiraJ

    2002-01-01

    We examined the role of a down stream of intracellular signaling pathway,extracellular signal-regulated kinase(ERK),in cephaloridine (CER)-induced nephrotoxicity in rats.The increase in phosphorylated ERK(pERK,activated ERK) was detected in nucleus fraction prepared from rat kidney cortex 24h after injections of antibiotic CER with the increase in BUN level.The slices prepared from rat kidney cortex were incubated in the medium containing PD980-59,a MEK1/2 inhibitor,for the measurement of free radical production and cell injure(LDH leakage).CER caused not only the increases in lipid peroxidation as an index of free radical production and LDH leakage,but also ERK activation in nucleus fraction.MEK1/2 inhibitor ameliorated CER-induced injury and suppressed ERK activation in the slices.These results suggest a possible role of MEK/ERK signaling pathway in free radical-induced CER nephrotoxicity.

  3. Effect of lead on ERK activity and the protective function of bFGF in rat primary culture astroglia*

    OpenAIRE

    Zhang, Ying; Ye, Li-ping; Wang, Biao; Cao, Shi-cheng; Sun, Li-Guang

    2007-01-01

    Objective: To observe the effects of lead on levels of phosphorylated extracellular signal regulated kinase (p-ERK) in the cytoplasm of primary cultures of rat astroglial cells and the possible protective effect of basic fibroblast growth factor (bFGF) on lead-induced effects. Methods: The primary astroglia cells from 1~6 d old Wistar rats were cultured. The cells pretreated with the MEK1 (mitogen-activated protein kinase kinase 1) inhibitor PD98059 and bFGF, respectively, were exposed to Pb ...

  4. Complex coordinated extracellular metabolism: Acid phosphatases activate diluted human leukocyte proteins to generate energy flow as NADPH from purine nucleotide ribose.

    Science.gov (United States)

    Hibbs, John B; Vavrin, Zdenek; Cox, James E

    2016-08-01

    Complex metabolism is thought to occur exclusively in the crowded intracellular environment. Here we report that diluted enzymes from lysed human leukocytes produce extracellular energy. Our findings involve two pathways: the purine nucleotide catabolic pathway and the pentose phosphate pathway, which function together to generate energy as NADPH. Glucose6P fuel for NADPH production is generated from structural ribose of purine ribonucleoside monophosphates, ADP, and ADP-ribose. NADPH drives glutathione reductase to reduce an oxidized glutathione disulfide-glutathione redox couple. Acid phosphatases initiate ribose5P salvage from purine ribonucleoside monophosphates, and transaldolase controls the direction of carbon chain flow through the nonoxidative branch of the pentose phosphate pathway. These metabolic control points are regulated by pH. Biologically, this energy conserving metabolism could function in perturbed extracellular spaces. PMID:26895212

  5. Complex coordinated extracellular metabolism: Acid phosphatases activate diluted human leukocyte proteins to generate energy flow as NADPH from purine nucleotide ribose.

    Science.gov (United States)

    Hibbs, John B; Vavrin, Zdenek; Cox, James E

    2016-08-01

    Complex metabolism is thought to occur exclusively in the crowded intracellular environment. Here we report that diluted enzymes from lysed human leukocytes produce extracellular energy. Our findings involve two pathways: the purine nucleotide catabolic pathway and the pentose phosphate pathway, which function together to generate energy as NADPH. Glucose6P fuel for NADPH production is generated from structural ribose of purine ribonucleoside monophosphates, ADP, and ADP-ribose. NADPH drives glutathione reductase to reduce an oxidized glutathione disulfide-glutathione redox couple. Acid phosphatases initiate ribose5P salvage from purine ribonucleoside monophosphates, and transaldolase controls the direction of carbon chain flow through the nonoxidative branch of the pentose phosphate pathway. These metabolic control points are regulated by pH. Biologically, this energy conserving metabolism could function in perturbed extracellular spaces.

  6. Complex coordinated extracellular metabolism: Acid phosphatases activate diluted human leukocyte proteins to generate energy flow as NADPH from purine nucleotide ribose

    Directory of Open Access Journals (Sweden)

    John B. Hibbs Jr.

    2016-08-01

    Full Text Available Complex metabolism is thought to occur exclusively in the crowded intracellular environment. Here we report that diluted enzymes from lysed human leukocytes produce extracellular energy. Our findings involve two pathways: the purine nucleotide catabolic pathway and the pentose phosphate pathway, which function together to generate energy as NADPH. Glucose6P fuel for NADPH production is generated from structural ribose of purine ribonucleoside monophosphates, ADP, and ADP-ribose. NADPH drives glutathione reductase to reduce an oxidized glutathione disulfide-glutathione redox couple. Acid phosphatases initiate ribose5P salvage from purine ribonucleoside monophosphates, and transaldolase controls the direction of carbon chain flow through the nonoxidative branch of the pentose phosphate pathway. These metabolic control points are regulated by pH. Biologically, this energy conserving metabolism could function in perturbed extracellular spaces.

  7. Extracellular polysaccharide production by a strain of Pleurotus djamor isolated in the south of Brazil and antitumor activity on Sarcoma 180

    Directory of Open Access Journals (Sweden)

    Gisele Martini Borges

    2013-12-01

    Full Text Available Polysaccharides with medicinal properties can be obtained from fruiting bodies, mycelium and culture broth of several fungus species. This work was carried out in batch culture using a stirred tank reactor with two different initial glucose concentrations (40-50 g/L and pH values (3.0-4.0 to enhance extracellular polysaccharides production by Pleurotus djamor UNIVILLE 001 and evaluate antitumor effect of intraperitonial administration of Pleurotus djamor extract on sarcoma 180 animal model. According to factorial design, the low pH value (pH 3.0 led to a gain of 1.6 g/L on the extracellular polysaccharide concentration, while glucose concentration in the tested range had no significant effect on the concentration of polysaccharide. With 40 g/L initial glucose concentration and pH 3.0, it was observed that yield factor of extracellular polysaccharide on substrate (Y P/S = 0.072 and maximum extracellular polysaccharide productivity (Q Pmax = 11.26 mg/L.h were about 188% and 321% respectively higher than those obtained in the experiment performed at pH 4.0. Under these conditions, the highest values of the yield factor of biomass on substrate (Y X/S = 0.24 and maximal biomass productivity (Q Xmax = 32.2 mg/L.h were also reached. In tumor response study, mean tumor volume on the 21th day was 35.3 cm³ in untreated group and 1.6 cm³ in treated group (p = 0.05 with a tumor inhibition rate of 94%. These impressive results suggests an inhibitory effect of P.djamor extract on cancer cells.

  8. Bone marrow stromal/stem cell-derived extracellular vesicles regulate osteoblast activity and differentiation in vitro and promote bone regeneration in vivo

    OpenAIRE

    Yunhao Qin; Lian Wang; Zhengliang Gao; Genyin Chen; Changqing Zhang

    2016-01-01

    Emerging evidence suggests that extracellular vesicles (EVs) are secreted by diverse tissues and play important roles in cell-cell communication, organ interactions and tissue homeostasis. Studies have reported the use of EVs to stimulate tissue regeneration, such as hepatic cell regeneration, and to treat diseases, such as pulmonary hypertension. However, little is known about the osteogenic effect of EVs. In this study, we explore the role of bone marrow stromal cell-derived EVs in the regu...

  9. Tumor Necrosis Factor-α and Apoptosis Signal-Regulating Kinase 1 Control Reactive Oxygen Species Release, Mitochondrial Autophagy and C-Jun N-Terminal Kinase/P38 Phosphorylation During Necrotizing Enterocolitis

    Directory of Open Access Journals (Sweden)

    Naira Baregamian

    2009-01-01

    Full Text Available Background: Oxidative stress and inflammation may contribute to the disruption of the protective gut barrier through various mechanisms; mitochondrial dysfunction resulting from inflammatory and oxidative injury may potentially be a significant source of apoptosis during necrotizing enterocolitis (NEC. Tumor necrosis factor (TNFα is thought to generate reactive oxygen species (ROS and activate the apoptosis signal-regulating kinase 1 (ASK1-c-Jun N-terminal kinase (JNK/p38 pathway. Hence, the focus of our study was to examine the effects of TNFα/ROs on mitochondrial function, ASK1-JNK/p38 cascade activation in intestinal epithelial cells during NEC.

  10. Activation of the transcription factor FosB/activating protein-1 (AP-1) is a prominent downstream signal of the extracellular nucleotide receptor P2RX7 in monocytic and osteoblastic cells.

    Science.gov (United States)

    Gavala, Monica L; Hill, Lindsay M; Lenertz, Lisa Y; Karta, Maya R; Bertics, Paul J

    2010-10-29

    Activation of the ionotropic P2RX7 nucleotide receptor by extracellular ATP has been implicated in modulating inflammatory disease progression. Continuous exposure of P2RX7 to ligand can result in apoptosis in many cell types, including monocytic cells, whereas transient activation of P2RX7 is linked to inflammatory mediator production and the promotion of cell growth. Given the rapid hydrolysis of ATP in the circulation and interstitial space, transient activation of P2RX7 appears critically important for its action, yet its effects on gene expression are unclear. The present study demonstrates that short-term stimulation of human and mouse monocytic cells as well as mouse osteoblasts with P2RX7 agonists substantially induces the expression of several activating protein-1 (AP-1) members, particularly FosB. The potent activation of FosB after P2RX7 stimulation is especially noteworthy considering that little is known concerning the role of FosB in immunological regulation. Interestingly, the magnitude of FosB activation induced by P2RX7 stimulation appears greater than that observed with other known inducers of FosB expression. In addition, we have identified a previously unrecognized role for FosB in osteoblasts with respect to nucleotide-induced expression of cyclooxygenase-2 (COX-2), which is the rate-limiting enzyme in prostaglandin biosynthesis from arachidonic acid and is critical for osteoblastic differentiation and immune behavior. The present studies are the first to link P2RX7 action to FosB/AP-1 regulation in multiple cell types, including a role in nucleotide-induced COX-2 expression, and support a role for FosB in the control of immune and osteogenic function by P2RX7. PMID:20813842

  11. Determination of the Proportion of Total Soil Extracellular Acid Phosphomonoesterase (E.C. 3.1.3.2 Activity Represented by Roots in the Soil of Different Forest Ecosystems

    Directory of Open Access Journals (Sweden)

    Klement Rejsek

    2012-01-01

    Full Text Available The aim of this study is to present a new method for determining the root-derived extracellular acid phosphomonoesterase (EAPM activity fraction within the total EAPM activity of soil. EAPM activity was determined for roots, organic and mineral soil. Samples were collected using paired PVC cylinders, inserted to a depth of 15 cm, within seven selected forest stands. Root-derived EAPM formed between 4 and18% of the total EAPM activity of soil from forests of differing maturity. A new approach, presented in this work, enables separation of root-derived EAPM activity from total soil EAPM. Separation of root-derived EAPM from soil provides a better understanding of its role in P-cycling in terrestrial ecosystems. The method presented in this work is a first step towards the separation of root- and microbe-derived EAPM in soils, which are thought to possess different kinetic properties and different sensitivity to environmental change.

  12. Transcriptional Down-Regulation of Thromboxane A(2) Receptor Expression via Activation of MAPK ERK1/2, p38/NF-kappaB Pathways

    DEFF Research Database (Denmark)

    Zhang, Wei; Zhang, Yaping; Edvinsson, Lars;

    2008-01-01

    . Phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2), p38 and nuclear factor-kappaB (NF-kappaB) was seen by Western blot within 1-3 h after organ culture. Inhibition of ERK1/2, p38 or NF-kappaB reversed depressed contraction as well as decreased receptor mRNA expression. Actinomycin D...... arteries down-regulates VSMC TP receptor expression through activation of ERK1/2 and p38/NF-kappaB signal pathways....

  13. Transcriptional down-regulation of thromboxane A(2) receptor expression via activation of MAPK ERK1/2, p38/NF-kappaB pathways

    DEFF Research Database (Denmark)

    Zhang, Wei; Zhang, Yaping; Edvinsson, Lars;

    2009-01-01

    . Phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2), p38 and nuclear factor-kappaB (NF-kappaB) was seen by Western blot within 1-3 h after organ culture. Inhibition of ERK1/2, p38 or NF-kappaB reversed depressed contraction as well as decreased receptor mRNA expression. Actinomycin D...... arteries down-regulates VSMC TP receptor expression through activation of ERK1/2 and p38/NF-kappaB signal pathways....

  14. (Prorenin receptor triggers distinct angiotensin II-independent extracellular matrix remodeling and deterioration of cardiac function.

    Directory of Open Access Journals (Sweden)

    Anne-Mari Moilanen

    Full Text Available BACKGROUND: Activation of the renin-angiotensin-system (RAS plays a key pathophysiological role in heart failure in patients with hypertension and myocardial infarction. However, the function of (prorenin receptor ((PRR is not yet solved. We determined here the direct functional and structural effects of (PRR in the heart. METHODOLOGY/PRINCIPAL FINDINGS: (PRR was overexpressed by using adenovirus-mediated gene delivery in normal adult rat hearts up to 2 weeks. (PRR gene delivery into the anterior wall of the left ventricle decreased ejection fraction (P<0.01, fractional shortening (P<0.01, and intraventricular septum diastolic and systolic thickness, associated with approximately 2-fold increase in left ventricular (PRR protein levels at 2 weeks. To test whether the worsening of cardiac function and structure by (PRR gene overexpression was mediated by angiotensin II (Ang II, we infused an AT(1 receptor blocker losartan via osmotic minipumps. Remarkably, cardiac function deteriorated in losartan-treated (PRR overexpressing animals as well. Intramyocardial (PRR gene delivery also resulted in Ang II-independent activation of extracellular-signal-regulated kinase1/2 phosphorylation and myocardial fibrosis, and the expression of transforming growth factor-β1 and connective tissue growth factor genes. In contrast, activation of heat shock protein 27 phosphorylation and apoptotic cell death by (PRR gene delivery was Ang II-dependent. Finally, (PRR overexpression significantly increased direct protein-protein interaction between (PRR and promyelocytic zinc-finger protein. CONCLUSIONS/SIGNIFICANCE: These results indicate for the first time that (PRR triggers distinct Ang II-independent myocardial fibrosis and deterioration of cardiac function in normal adult heart and identify (PRR as a novel therapeutic target to optimize RAS blockade in failing hearts.

  15. Cypermethrin Induces Macrophages Death through Cell Cycle Arrest and Oxidative Stress-Mediated JNK/ERK Signaling Regulated Apoptosis

    Directory of Open Access Journals (Sweden)

    Fang Huang

    2016-06-01

    Full Text Available Cypermethrin is one of the most highly effective synthetic pyrethroid insecticides. The toxicity of cypermethrin to the reproductive and nervous systems has been well studied. However, little is known about the toxic effect of cypermethrin on immune cells such as macrophages. Here, we investigated the cytotoxicity of cypermethrin on macrophages and the underlying molecular mechanisms. We found that cypermethrin reduced cell viability and induced apoptosis in RAW 264.7 cells. Cypermethrin also increased reactive oxygen species (ROS production and DNA damage in a dose-dependent manner. Moreover, cypermethrin-induced G1 cell cycle arrest was associated with an enhanced expression of p21, wild-type p53, and down-regulation of cyclin D1, cyclin E and CDK4. In addition, cypermethrin treatment activated MAPK signal pathways by inducing c-Jun N-terminal kinase (JNK and extracellular regulated protein kinases 1/2 ERK1/2 phosphorylation, and increased the cleaved poly ADP-ribose polymerase (PARP. Further, pretreatment with antioxidant N-acetylcysteine (NAC effectively abrogated cypermethrin-induced cell cytotoxicity, G1 cell cycle arrest, DNA damage, PARP activity, and JNK and ERK1/2 activation. The specific JNK inhibitor (SP600125 and ERK1/2 inhibitor (PD98059 effectively reversed the phosphorylation level of JNK and ERK1/2, and attenuated the apoptosis. Taken together, these data suggested that cypermethrin caused immune cell death via inducing cell cycle arrest and apoptosis regulated by ROS-mediated JNK/ERK pathway.

  16. Extracellular enzyme kinetics scale with resource availability

    Science.gov (United States)

    Sinsabaugh, Robert L.; Belnap, Jayne; Findlay, Stuart G.; Follstad Shah, Jennifer J.; Hill, Brian H.; Kuehn, Kevin A.; Kuske, Cheryl; Litvak, Marcy E.; Martinez, Noelle G.; Moorhead, Daryl L.; Warnock, Daniel D.

    2014-01-01

    Microbial community metabolism relies on external digestion, mediated by extracellular enzymes that break down complex organic matter into molecules small enough for cells to assimilate. We analyzed the kinetics of 40 extracellular enzymes that mediate the degradation and assimilation of carbon, nitrogen and phosphorus by diverse aquatic and terrestrial microbial communities (1160 cases). Regression analyses were conducted by habitat (aquatic and terrestrial), enzyme class (hydrolases and oxidoreductases) and assay methodology (low affinity and high affinity substrates) to relate potential reaction rates to substrate availability. Across enzyme classes and habitats, the scaling relationships between apparent Vmax and apparent Km followed similar power laws with exponents of 0.44 to 0.67. These exponents, called elasticities, were not statistically distinct from a central value of 0.50, which occurs when the Km of an enzyme equals substrate concentration, a condition optimal for maintenance of steady state. We also conducted an ecosystem scale analysis of ten extracellular hydrolase activities in relation to soil and sediment organic carbon (2,000–5,000 cases/enzyme) that yielded elasticities near 1.0 (0.9 ± 0.2, n = 36). At the metabolomic scale, the elasticity of extracellular enzymatic reactions is the proportionality constant that connects the C:N:P stoichiometries of organic matter and ecoenzymatic activities. At the ecosystem scale, the elasticity of extracellular enzymatic reactions shows that organic matter ultimately limits effective enzyme binding sites. Our findings suggest that one mechanism by which microbial communities maintain homeostasis is regulating extracellular enzyme expression to optimize the short-term responsiveness of substrate acquisition. The analyses also show that, like elemental stoichiometry, the fundamental attributes of enzymatic reactions can be extrapolated from biochemical to community and ecosystem scales.

  17. Immunotherapeutic potential of extracellular vesicles

    Directory of Open Access Journals (Sweden)

    Bin eZhang

    2014-10-01

    Full Text Available Extracellular vesicles or EVs is a term that encompasses all classes of secreted lipid membrane vesicles. Despite being scientific novelties, EVs are gaining importance as a mediator of important physiological and pathological intercellular activities possibly through the transfer of their cargo of protein and RNA between cells. In particular, exosomes the currently best characterized EVs have been notable for their in vitro and in vivo immunomodulatory activities. Exosomes are nanometer-sized endosome-derived vesicles secreted by many cell types and their immunomodulatory potential is independent of their cell source. Besides immune cells such as dendritic cells, macrophages and T cells, cancer and stem cells also secrete immunologically active exosomes that could influence both physiological and pathological processes. The immunological activities of exosomes affect both innate and adaptive immunity and include antigen presentation, T cell activation, T cell polarisation to Tregs, immune suppression and anti-inflammation. As such, exosomes carry much immunotherapeutic potential as a therapeutic agent and a therapeutic target.

  18. Influence of Verticillium dahliae Mycelium on Extracellular Proteases and Antifungal Activity of Streptomyces%大丽轮枝菌菌体对链霉菌胞外蛋白酶活性及抑菌效果的影响

    Institute of Scientific and Technical Information of China (English)

    薛磊; 薛泉宏; 赵娟; 申光辉; 唐明; 卢建军

    2012-01-01

    We investigated the antifungal mechanism of antagonistic Streptomyces on Verticillium dahliae Kleb, using pathogenic mycelium as the sole carbon source. The inducing effects of V. Dahliae on extracellular proteases and antifungal active substance synthesis were examined in four tested Streptomyces isolates in liquid cultures. V. Dahliae induced Streptomyces synthesis of extracellular protease and active antifungal substances, whereas an associated culture filtrate induced disintegration of V. Dahliae mycelium. Optimal fermentation conditions for extracellular proteases synthesized by isolate Z13 involved addition of 10 g·L-1 of fungal mycelium at 28 ℃ for 7 d. The highest extracellular protease activity of this isolate was 4.24 U. With the addition of 20 g·L-1 of fungal mycelium, V. Dahliae was 95.7% inhibited by a 5-fold dilution of a culture filtrate of isolate B49.%为研究拮抗链霉菌对棉花黄萎病原大丽轮枝菌(Verticillium dahliae Kleb.)的抑菌机理,以大丽轮枝菌菌体为唯一碳氮能源,采用液体培养法及生长速率法研究供试病原菌菌体对4株拮抗链霉菌胞外蛋白酶合成的诱导作用及抑菌活性物质产生的影响.结果表明:大丽轮枝菌菌体可以诱导供试链霉菌合成蛋白酶;诱导粗酶液对病原真菌菌丝有溶解作用;当菌体添加量为10 g·L-1,28℃培养7d时菌株Z13蛋白酶活性高达4.24 U;以大丽轮枝菌菌体为碳氮能源时供试链霉菌能产生活性较强的抑菌物质;当菌体添加量为20 g· L-1,发酵液稀释5倍时菌株B49所产抑菌活性物质的最大抑菌率达95.7%.

  19. AMPK/Snf1 signaling regulates histone acetylation: Impact on gene expression and epigenetic functions.

    Science.gov (United States)

    Salminen, Antero; Kauppinen, Anu; Kaarniranta, Kai

    2016-08-01

    AMP-activated protein kinase (AMPK) and its yeast homolog, Snf1, are critical regulators in the maintenance of energy metabolic balance not only stimulating energy production but also inhibiting energy-consuming processes. The AMPK/Snf1 signaling controls energy metabolism by specific phosphorylation of many metabolic enzymes and transcription factors, enhancing or suppressing their functions. The AMPK/Snf1 complexes can be translocated from cytoplasm into nuclei where they are involved in the regulation of transcription. Recent studies have indicated that AMPK/Snf1 activation can control histone acetylation through different mechanisms affecting not only gene transcription but also many other epigenetic functions. For instance, AMPK/Snf1 enzymes can phosphorylate the histone H3S10 (yeast) and H2BS36 (mammalian) sites which activate specific histone acetyltransferases (HAT), consequently enhancing histone acetylation. Moreover, nuclear AMPK can phosphorylate type 2A histone deacetylases (HDAC), e.g. HDAC4 and HDAC5, triggering their export from nuclei thus promoting histone acetylation reactions. AMPK activation can also increase the level of acetyl CoA, e.g. by inhibiting fatty acid and cholesterol syntheses. Acetyl CoA is a substrate for HATs, thus increasing their capacity for histone acetylation. On the other hand, AMPK can stimulate the activity of nicotinamide phosphoribosyltransferase (NAMPT) which increases the level of NAD(+). NAD(+) is a substrate for nuclear sirtuins, especially for SIRT1 and SIRT6, which deacetylate histones and transcription factors, e.g. those regulating ribosome synthesis and circadian clocks. Histone acetylation is an important epigenetic modification which subsequently can affect chromatin remodeling, e.g. via bromodomain proteins. We will review the signaling mechanisms of AMPK/Snf1 in the control of histone acetylation and subsequently clarify their role in the epigenetic regulation of ribosome synthesis and circadian clocks

  20. Angiotensin II type 1 receptor signalling regulates microRNA differentially in cardiac fibroblasts and myocytes

    DEFF Research Database (Denmark)

    Jeppesen, Pia Lindgren; Christensen, Gitte Lund; Schneider, Mikael;

    2011-01-01

    analogue, [Sar1, Ile4, Ile8] Ang II (SII Ang II), which selectively activates Gaq/11-independent signalling, failed to regulate miRNAs in HEK293N cells Furthermore, Ang II induced miRNA regulation was blocked following Gaq/11 and Mek1 inhibition. The observed Ang II regulation of miRNA was confirmed...... in primary cultures of adult cardiac fibroblasts. Interestingly, Ang II did not regulate miRNA expression in cardiac myocytes, but SII Ang II significantly downregulated miR-129-3p. Conclusions and implications: Five miRNAs were regulated by Ang II through mechanisms depending on Gaq/11 and Erk1/2 activation...