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Sample records for activate intracellular signalling

  1. Steroid signaling activation and intracellular localization of sex steroid receptors.

    PubMed

    Giraldi, Tiziana; Giovannelli, Pia; Di Donato, Marzia; Castoria, Gabriella; Migliaccio, Antimo; Auricchio, Ferdinando

    2010-12-01

    In addition to stimulating gene transcription, sex steroids trigger rapid, non-genomic responses in the extra-nuclear compartment of target cells. These events take place within seconds or minutes after hormone administration and do not require transcriptional activity of sex steroid receptors. Depending on cell systems, activation of extra-nuclear signaling pathways by sex steroids fosters cell cycle progression, prevents apoptosis, leads to epigenetic modifications and increases cell migration through cytoskeleton changes. These findings have raised the question of intracellular localization of sex steroid receptors mediating these responses. During the past years, increasing evidence has shown that classical sex steroid receptors localized in the extra-nuclear compartment or close to membranes of target cells induce these events. The emerging picture is that a process of bidirectional control between signaling activation and sex steroid receptor localization regulates the outcome of hormonal responses in target cells. This mechanism ensures cell cycle progression in estradiol-treated breast cancer cells, and its derangement might occur in progression of human proliferative diseases. These findings will be reviewed here together with unexpected examples of the relationship between sex steroid receptor localization, signaling activation and biological responses in target cells. We apologize to scientists whose reports are not mentioned or extensively discussed owing to space limitations.

  2. Intracellular Signaling by Hydrolysis of Phospholipids and Activation of Protein Kinase C

    NASA Astrophysics Data System (ADS)

    Nishizuka, Yasutomi

    1992-10-01

    Hydrolysis of inositol phospholipids by phospholipase C is initiated by either receptor stimulation or opening of Ca2+ channels. This was once thought to be the sole mechanism to produce the diacylglycerol that links extracellular signals to intracellular events through activation of protein kinase C. It is becoming clear that agonist-induced hydrolysis of other membrane phospholipids, particularly choline phospholipids, by phospholipase D and phospholipase A_2 may also take part in cell signaling. The products of hydrolysis of these phospholipids may enhance and prolong the activation of protein kinase C. Such prolonged activation of protein kinase C is essential for long-term cellular responses such as cell proliferation and differentiation.

  3. Key mediators of intracellular amino acids signaling to mTORC1 activation.

    PubMed

    Duan, Yehui; Li, Fengna; Tan, Kunrong; Liu, Hongnan; Li, Yinghui; Liu, Yingying; Kong, Xiangfeng; Tang, Yulong; Wu, Guoyao; Yin, Yulong

    2015-05-01

    Mammalian target of rapamycin complex 1 (mTORC1) is activated by amino acids to promote cell growth via protein synthesis. Specifically, Ras-related guanosine triphosphatases (Rag GTPases) are activated by amino acids, and then translocate mTORC1 to the surface of late endosomes and lysosomes. Ras homolog enriched in brain (Rheb) resides on this surface and directly activates mTORC1. Apart from the presence of intracellular amino acids, Rag GTPases and Rheb, other mediators involved in intracellular amino acid signaling to mTORC1 activation include human vacuolar sorting protein-34 (hVps34) and mitogen-activating protein kinase kinase kinase kinase-3 (MAP4K3). Those molecular links between mTORC1 and its mediators form a complicate signaling network that controls cellular growth, proliferation, and metabolism. Moreover, it is speculated that amino acid signaling to mTORC1 may start from the lysosomal lumen. In this review, we discussed the function of these mediators in mTORC1 pathway and how these mediators are regulated by amino acids in details.

  4. Pharmacology of intracellular signalling pathways

    PubMed Central

    Nahorski, Stefan R

    2006-01-01

    This article provides a brief and somewhat personalized review of the dramatic developments that have occurred over the last 45 years in our understanding of intracellular signalling pathways associated with G-protein-coupled receptor activation. Signalling via cyclic AMP, the phosphoinositides and Ca2+ is emphasized and these systems have already been revealed as new pharmacological targets. The therapeutic benefits of most of such targets are, however, yet to be realized, but it is certain that the discipline of pharmacology needs to widen its boundaries to meet these challenges in the future. PMID:16402119

  5. Novel robust biomarkers for human bladder cancer based on activation of intracellular signaling pathways.

    PubMed

    Lezhnina, Ksenia; Kovalchuk, Olga; Zhavoronkov, Alexander A; Korzinkin, Mikhail B; Zabolotneva, Anastasia A; Shegay, Peter V; Sokov, Dmitry G; Gaifullin, Nurshat M; Rusakov, Igor G; Aliper, Alexander M; Roumiantsev, Sergey A; Alekseev, Boris Y; Borisov, Nikolay M; Buzdin, Anton A

    2014-10-15

    We recently proposed a new bioinformatic algorithm called OncoFinder for quantifying the activation of intracellular signaling pathways. It was proved advantageous for minimizing errors of high-throughput gene expression analyses and showed strong potential for identifying new biomarkers. Here, for the first time, we applied OncoFinder for normal and cancerous tissues of the human bladder to identify biomarkers of bladder cancer. Using Illumina HT12v4 microarrays, we profiled gene expression in 17 cancer and seven non-cancerous bladder tissue samples. These experiments were done in two independent laboratories located in Russia and Canada. We calculated pathway activation strength values for the investigated transcriptomes and identified signaling pathways that were regulated differently in bladder cancer (BC) tissues compared with normal controls. We found, for both experimental datasets, 44 signaling pathways that serve as excellent new biomarkers of BC, supported by high area under the curve (AUC) values. We conclude that the OncoFinder approach is highly efficient in finding new biomarkers for cancer. These markers are mathematical functions involving multiple gene products, which distinguishes them from "traditional" expression biomarkers that only assess concentrations of single genes. PMID:25296972

  6. INTRACELLULAR SIGNALING AND DEVELOPMENTAL NEUROTOXICITY.

    EPA Science Inventory

    A book chapter in ?Molecular Toxicology: Transcriptional Targets? reviewed the role of intracellular signaling in the developmental neurotoxicity of environmental chemicals. This chapter covered a number of aspects including the development of the nervous system, role of intrace...

  7. G protein coupled receptor signaled apoptosis is associated with activation of a cation insensitive acidic endonuclease and intracellular acidification.

    PubMed

    Sharma, K; Srikant, C B

    1998-01-01

    Apoptosis associated oligonucleosomal fragmentation of DNA can result from the activation of endonucleases that exhibit different pH optima and are either sensitive or insensitive to divalent cations. DNA fragmentation due to activation of cation sensitive endonucleases occurs in the absence of a change in intracellular pH whereas intracellular acidification is a feature of apoptosis characterized by activation of cation insensitive acidic endonuclease. We have reported earlier that somatostatin (SST) induced DNA fragmentation and apoptosis is signaled in a receptor subtype selective manner uniquely via human somatostatin receptor subtype 3 (hSSTR3). In the present study we investigated the pH dependence and cation sensitivity of endonuclease induced in hSSTR3 expressing CHO-K1 cells by the SST agonist octreotide (OCT) and its effect on intracellular pH. We show that OCT induced apoptosis is associated with selective stimulation of a divalent cation insensitive acidic endonuclease. The intracellular pH of of cells undergoing OCT induced apoptosis was 0.9 pH units lower than that of control cells. The effect of OCT on endonuclease and pH was inhibited by orthovanadate as well as by pretreatment with pertussis toxin, suggesting that hSSTR3 initiated cytotoxic signaling is protein tyrosine phosphatase mediated and is G protein dependent. These findings suggest that intracellular acidification and activation of acidic endonuclease mediate wild type p53 associated apoptosis signaled by hormones acting via G protein coupled receptors.

  8. [A role of some intracellular signaling cascades in planarian regeneration activated under irradiation with low-temperature argon plasma].

    PubMed

    Ermakov, A M; Ermakova, O N; Maevskiĭ, E I

    2014-01-01

    Using inhibitory analysis the role of some intracellular signaling pathways in activation of planarian regeneration under the influence of low-temperature argon plasma (LTAP) has been investigated. Inactivation of specific inhibitors of intracellular signaling enzymes such as the receptor tyrosine kinase (EGFR), TGF β receptor, calmodulin, adenylate cyclase, phospholipase A2, phospholipase C, cyclin-dependent protein kinase, JAK2-protein kinase, JNK-protein kinase MEK-protein kinase led to inhibition of the head growth during its regeneration in planarians. Pretreatment with LTAP irradiation provided no inhibitory action of some cascades regulating proliferation. However, the inhibitors of the key regulators of regeneration: TGF β receptor, calmodulin and MEK-protein kinase completely suppressed the activating effect of plasma. Thus, by the example of regenerating planarians it is shown, that biological activity of low-temperature argon plasma LTAP is caused by modulation of a plurality of cellular signaling systems.

  9. Ehrlichia chaffeensis TRP120 Activates Canonical Notch Signaling To Downregulate TLR2/4 Expression and Promote Intracellular Survival

    PubMed Central

    Lina, Taslima T.; Dunphy, Paige S.; Luo, Tian

    2016-01-01

    ABSTRACT Ehrlichia chaffeensis preferentially targets mononuclear phagocytes and survives through a strategy of subverting innate immune defenses, but the mechanisms are unknown. We have shown E. chaffeensis type 1 secreted tandem repeat protein (TRP) effectors are involved in diverse molecular pathogen-host interactions, such as the TRP120 interaction with the Notch receptor-cleaving metalloprotease ADAM17. In the present study, we demonstrate E. chaffeensis, via the TRP120 effector, activates the canonical Notch signaling pathway to promote intracellular survival. We found that nuclear translocation of the transcriptionally active Notch intracellular domain (NICD) occurs in response to E. chaffeensis or recombinant TRP120, resulting in upregulation of Notch signaling pathway components and target genes notch1, adam17, hes, and hey. Significant differences in canonical Notch signaling gene expression levels (>40%) were observed during early and late stages of infection, indicating activation of the Notch pathway. We linked Notch pathway activation specifically to the TRP120 effector, which directly interacts with the Notch metalloprotease ADAM17. Using pharmacological inhibitors and small interfering RNAs (siRNAs) against γ-secretase enzyme, Notch transcription factor complex, Notch1, and ADAM17, we demonstrated that Notch signaling is required for ehrlichial survival. We studied the downstream effects and found that E. chaffeensis TRP120-mediated activation of the Notch pathway causes inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways required for PU.1 and subsequent Toll-like receptor 2/4 (TLR2/4) expression. This investigation reveals a novel mechanism whereby E. chaffeensis exploits the Notch pathway to evade the host innate immune response for intracellular survival. PMID:27381289

  10. Synaptic generation of an intracellular retrograde signal requires activation of the tyrosine kinase and mitogen-activated protein kinase signaling cascades in Aplysia.

    PubMed

    Stough, Shara; Kopec, Ashley M; Carew, Thomas J

    2015-11-01

    Cellular changes underlying memory formation can be generated in an activity-dependent manner at specific synapses. Thus an important question concerns the mechanisms by which synaptic signals communicate with the cell body to mediate these cellular changes. A monosynaptic circuit that is enhanced by sensitization in Aplysia is well-suited to study this question because three different subcellular compartments: (i) the sensorimotor SN-MN synapses, (ii) the SN projections to MNs via axonal connections, (iii) the SN cell bodies, can all be manipulated and studied independently. Here, we report that activity-dependent (AD) training in either the entire SN-MN circuit or in only the synaptic compartment, activates MAPK in a temporally and spatially specific pattern. Specifically, we find (i) MAPK activation is first transiently generated at SN-MN synapses during training, (ii) immediately after training MAPK is transiently activated in SN-MN axonal connections and persistently activated in SN cell bodies, and finally, (iii) MAPK is activated in SN cell bodies and SN-MN synapses 1h after training. These data suggest that there is an intracellularly transported retrograde signal generated at the synapse which is later responsible for delayed MAPK activation at SN somata. Finally, we find that this retrograde signal requires activation of tyrosine kinase (TK) and MEK signaling cascades at the synapses.

  11. Miro1 Regulates Activity-Driven Positioning of Mitochondria within Astrocytic Processes Apposed to Synapses to Regulate Intracellular Calcium Signaling

    PubMed Central

    Stephen, Terri-Leigh; Higgs, Nathalie F.; Sheehan, David F.; Al Awabdh, Sana; López-Doménech, Guillermo; Arancibia-Carcamo, I. Lorena

    2015-01-01

    It is fast emerging that maintaining mitochondrial function is important for regulating astrocyte function, although the specific mechanisms that govern astrocyte mitochondrial trafficking and positioning remain poorly understood. The mitochondrial Rho-GTPase 1 protein (Miro1) regulates mitochondrial trafficking and detachment from the microtubule transport network to control activity-dependent mitochondrial positioning in neurons. However, whether Miro proteins are important for regulating signaling-dependent mitochondrial dynamics in astrocytic processes remains unclear. Using live-cell confocal microscopy of rat organotypic hippocampal slices, we find that enhancing neuronal activity induces transient mitochondrial remodeling in astrocytes, with a concomitant, transient reduction in mitochondrial trafficking, mediated by elevations in intracellular Ca2+. Stimulating neuronal activity also induced mitochondrial confinement within astrocytic processes in close proximity to synapses. Furthermore, we show that the Ca2+-sensing EF-hand domains of Miro1 are important for regulating mitochondrial trafficking in astrocytes and required for activity-driven mitochondrial confinement near synapses. Additionally, activity-dependent mitochondrial positioning by Miro1 reciprocally regulates the levels of intracellular Ca2+ in astrocytic processes. Thus, the regulation of intracellular Ca2+ signaling, dependent on Miro1-mediated mitochondrial positioning, could have important consequences for astrocyte Ca2+ wave propagation, gliotransmission, and ultimately neuronal function. SIGNIFICANCE STATEMENT Mitochondria are key cellular organelles that play important roles in providing cellular energy and buffering intracellular calcium ions. The mechanisms that control mitochondrial distribution within the processes of glial cells called astrocytes and the impact this may have on calcium signaling remains unclear. We show that activation of glutamate receptors or increased neuronal

  12. Miro1 Regulates Activity-Driven Positioning of Mitochondria within Astrocytic Processes Apposed to Synapses to Regulate Intracellular Calcium Signaling.

    PubMed

    Stephen, Terri-Leigh; Higgs, Nathalie F; Sheehan, David F; Al Awabdh, Sana; López-Doménech, Guillermo; Arancibia-Carcamo, I Lorena; Kittler, Josef T

    2015-12-01

    It is fast emerging that maintaining mitochondrial function is important for regulating astrocyte function, although the specific mechanisms that govern astrocyte mitochondrial trafficking and positioning remain poorly understood. The mitochondrial Rho-GTPase 1 protein (Miro1) regulates mitochondrial trafficking and detachment from the microtubule transport network to control activity-dependent mitochondrial positioning in neurons. However, whether Miro proteins are important for regulating signaling-dependent mitochondrial dynamics in astrocytic processes remains unclear. Using live-cell confocal microscopy of rat organotypic hippocampal slices, we find that enhancing neuronal activity induces transient mitochondrial remodeling in astrocytes, with a concomitant, transient reduction in mitochondrial trafficking, mediated by elevations in intracellular Ca(2+). Stimulating neuronal activity also induced mitochondrial confinement within astrocytic processes in close proximity to synapses. Furthermore, we show that the Ca(2+)-sensing EF-hand domains of Miro1 are important for regulating mitochondrial trafficking in astrocytes and required for activity-driven mitochondrial confinement near synapses. Additionally, activity-dependent mitochondrial positioning by Miro1 reciprocally regulates the levels of intracellular Ca(2+) in astrocytic processes. Thus, the regulation of intracellular Ca(2+) signaling, dependent on Miro1-mediated mitochondrial positioning, could have important consequences for astrocyte Ca(2+) wave propagation, gliotransmission, and ultimately neuronal function. PMID:26631479

  13. Simultaneous intracellular β-D-glucosidase and phosphodiesterase I activities measurements based on a triple-signaling fluorescent probe.

    PubMed

    Li, Yinhui; Wang, Hao; Li, Jishan; Zheng, Jing; Xu, Xinhua; Yang, Ronghua

    2011-02-15

    Despite that considerable efforts have been devoted to the design of various fluorogenic enzyme substrates, they are single-enzyme assay approaches that cannot afford detection of multienzyme activity. In this study, we set out our first attempt to design a new probe that could measure intracellular β-D-glucosidase and phosphodiesterase I activities. Unlike the commonly used fluorogenic enzyme substrates that contain one recognition site and signaling reporter, the new probe molecule possesses two cleavage sites, specificly corresponding to β-D-glucosidase and phosphodiesterase I, and three fluorescent reporters, 7-β-D-glucopyranosyloxycoumarin, 7-hydroxycoumarin, and meso-tetraphenylporphyrin. On the basis of intramolecular photoinduced electron transfer and fluorescence resonance energy transfer mechanisms, interaction of the probe with the two enzymes, whether only one or both, produces different signal readouts with high sensitivity. Remarkably, the probe is chemically stable in complex biological fluids. Fluorescence outputs are not significantly affected by biologically related metal ions, anions, amino acids, and proteins. Furthermore, fluorescence microscopy confirmed that the probe is an excellent candidate for intracellular delivery and can be accumulated intensively in cells. We demonstrated the applicability for the simultaneous images of intracellular β-D-glucosidase and phosphodiesterase I activities using the different optical imaging modes.

  14. Phospholipase C-η1 is activated by intracellular Ca(2+) mobilization and enhances GPCRs/PLC/Ca(2+) signaling.

    PubMed

    Kim, Jung Kuk; Choi, Jung Woong; Lim, Seyoung; Kwon, Ohman; Seo, Jeong Kon; Ryu, Sung Ho; Suh, Pann-Ghill

    2011-06-01

    Phospholipase C-η1 (PLC-η1) is the most recently identified PLC isotype and is primarily expressed in nerve tissue. However, its functional role is unclear. In the present study, we report for the first time that PLC-η1 acts as a signal amplifier in G protein-coupled receptor (GPCR)-mediated PLC and Ca(2+) signaling. Short-hairpin RNA (shRNA)-mediated knockdown of endogenous PLC-η1 reduced lysophosphatidic acid (LPA)-, bradykinin (BK)-, and PACAP-induced PLC activity in mouse neuroblastoma Neuro2A (N2A) cells, indicating that PLC-η1 participates in GPCR-mediated PLC activation. Interestingly, ionomycin-induced PLC activity was significantly decreased by PLC-η1, but not PLC-η2, knockdown. In addition, we found that intracellular Ca(2+) source is enough for PLC-η1 activation. Furthermore, the IP(3) receptor inhibitor, 2-APB, inhibited LPA-induced PLC activity in control N2A cells, whereas this effect was not observed in PLC-η1 knockdown N2A cells, suggesting a pivotal role of intracellular Ca(2+) mobilization in PLC-η1 activation. Finally, we found that LPA-induced ERK1/2 phosphorylation and expression of the downstream target gene, krox-24, were significantly decreased by PLC-η1 knockdown, and these knockdown effects were abolished by 2-APB. Taken together, our results strongly suggest that PLC-η1 is activated via intracellular Ca(2+) mobilization from the ER, and therefore amplifies GPCR-mediated signaling.

  15. Modeling and analysis of early events in T-lymphocyte antigen-activated intracellular-signaling pathways

    NASA Astrophysics Data System (ADS)

    Zheng, Yanan; Balakrishnan, Venkataramanan; Buzzard, Greg; Geahlen, Robert; Harrison, Marietta; Rundell, Ann

    2005-12-01

    The T-cell antigen-activated signaling pathway is a highly regulated intracellular biochemical system that is crucial for initiating an appropriate adaptive immune response. To improve the understanding of the complex regulatory mechanisms controlling the early events in T-cell signaling, a detailed mathematical model was developed that utilizes ordinary differential equations to describe chemical reactions of the signaling pathway. The model parameter values were constrained by experimental data on the activation of a specific signaling intermediate and indicated an initial rapid cascade of phosphorylation events followed by a comparatively slow signal downregulation. Nonlinear analysis of the model suggested that thresholding and bistability occur as a result of the embedded positive and negative feedback loops within the model. These nonlinear system properties may enhance the T-cell receptor specificity and provide sub-threshold noise filtering with switch-like behavior to ensure proper cell response. Additional analysis using a reduced second-order model led to further understanding of the observed system behavior. Moreover, the interactions between the positive and negative feedback loops enabled the model to exhibit, among a variety of other feasible dynamics, a sustained oscillation that corresponds to a stable limit cycle in the two-dimensional phase plane. Quantitative analysis in this paper has helped identify potential regulatory mechanisms in the early T-cell signaling events. This integrated approach provides a framework to quantify and discover the ensemble of interconnected T-cell antigen-activated signaling pathways from limited experimental data.

  16. Intracellular Ca2+ signaling and preimplantation development.

    PubMed

    Armant, D Randall

    2015-01-01

    The key, versatile role of intracellular Ca2+ signaling during egg activation after fertilization has been appreciated for several decades. More recently, evidence has accumulated supporting the concept that cytoplasmic Ca2+ is also a major signaling nexus during subsequent development of the fertilized ovum. This chapter will review the molecular reactions that regulate intracellular Ca2+ levels and cell function, the role of Ca2+ signaling during egg activation and specific examples of repetitive Ca2+ signaling found throughout pre- and peri-implantation development. Many of the upstream and downstream pathways utilized during egg activation are also critical for specific processes that take place during embryonic development. Much remains to be done to elucidate the full complexity of Ca2+ signaling mechanisms in preimplantation embryos to the level of detail accomplished for egg activation. However, an emerging concept is that because this second messenger can be modulated downstream of numerous receptors and is able to bind and activate multiple cytoplasmic signaling proteins, it can help the coordination of development through up- and downstream pathways that change with each embryonic stage.

  17. An inside job: hacking into Janus kinase/signal transducer and activator of transcription signaling cascades by the intracellular protozoan Toxoplasma gondii.

    PubMed

    Denkers, Eric Y; Bzik, David J; Fox, Barbara A; Butcher, Barbara A

    2012-02-01

    The intracellular protozoan Toxoplasma gondii is well known for its skill at invading and living within host cells. New discoveries are now also revealing the astounding ability of the parasite to inject effector proteins into the cytoplasm to seize control of the host cell. This review summarizes recent advances in our understanding of one such secretory protein called ROP16. This molecule is released from rhoptries into the host cell during invasion. The ROP16 molecule acts as a kinase, directly activating both signal transducer and activator of transcription 3 (STAT3) and STAT6 signaling pathways. In macrophages, an important and preferential target cell of parasite infection, the injection of ROP16 has multiple consequences, including downregulation of proinflammatory cytokine signaling and macrophage deviation to an alternatively activated phenotype. PMID:22104110

  18. An inside job: hacking into Janus kinase/signal transducer and activator of transcription signaling cascades by the intracellular protozoan Toxoplasma gondii.

    PubMed

    Denkers, Eric Y; Bzik, David J; Fox, Barbara A; Butcher, Barbara A

    2012-02-01

    The intracellular protozoan Toxoplasma gondii is well known for its skill at invading and living within host cells. New discoveries are now also revealing the astounding ability of the parasite to inject effector proteins into the cytoplasm to seize control of the host cell. This review summarizes recent advances in our understanding of one such secretory protein called ROP16. This molecule is released from rhoptries into the host cell during invasion. The ROP16 molecule acts as a kinase, directly activating both signal transducer and activator of transcription 3 (STAT3) and STAT6 signaling pathways. In macrophages, an important and preferential target cell of parasite infection, the injection of ROP16 has multiple consequences, including downregulation of proinflammatory cytokine signaling and macrophage deviation to an alternatively activated phenotype.

  19. An Inside Job: Hacking into Janus Kinase/Signal Transducer and Activator of Transcription Signaling Cascades by the Intracellular Protozoan Toxoplasma gondii

    PubMed Central

    Bzik, David J.; Fox, Barbara A.; Butcher, Barbara A.

    2012-01-01

    The intracellular protozoan Toxoplasma gondii is well known for its skill at invading and living within host cells. New discoveries are now also revealing the astounding ability of the parasite to inject effector proteins into the cytoplasm to seize control of the host cell. This review summarizes recent advances in our understanding of one such secretory protein called ROP16. This molecule is released from rhoptries into the host cell during invasion. The ROP16 molecule acts as a kinase, directly activating both signal transducer and activator of transcription 3 (STAT3) and STAT6 signaling pathways. In macrophages, an important and preferential target cell of parasite infection, the injection of ROP16 has multiple consequences, including downregulation of proinflammatory cytokine signaling and macrophage deviation to an alternatively activated phenotype. PMID:22104110

  20. Intracellular Signal Modulation by Nanomaterials

    PubMed Central

    Hussain, Salik; Garantziotis, Stavros; Rodrigues-Lima, Fernando; Dupret, Jean-Marie; Baeza-Squiban, Armelle; Boland, Sonja

    2016-01-01

    A thorough understanding of the interactions of nanomaterials with biological systems and the resulting activation of signal transduction pathways is essential for the development of safe and consumer friendly nanotechnology. Here we present an overview of signaling pathways induced by nanomaterial exposures and describe the possible correlation of their physicochemical characteristics with biological outcomes. In addition to the hierarchical oxidative stress model and a review of the intrinsic and cell-mediated mechanisms of reactive Oxygen species (ROS) generating capacities of nanomaterials, we also discuss other oxidative stress dependent and independent cellular signaling pathways. Induction of the inflammasome, calcium signaling, and endoplasmic reticulum stress are reviewed. Furthermore, the uptake mechanisms can crucially affect the cytotoxicity of nanomaterials and membrane-dependent signaling pathways can be responsible for cellular effects of nanomaterials. Epigenetic regulation by nanomaterials effects of nanoparticle-protein interactions on cell signaling pathways, and the induction of various cell death modalities by nanomaterials are described. We describe the common trigger mechanisms shared by various nanomaterials to induce cell death pathways and describe the interplay of different modalities in orchestrating the final outcome after nanomaterial exposures. A better understanding of signal modulations induced by nanomaterials is not only essential for the synthesis and design of safer nanomaterials but will also help to discover potential nanomedical applications of these materials. Several biomedical applications based on the different signaling pathways induced by nanomaterials are already proposed and will certainly gain a great deal of attraction in the near future. PMID:24683030

  1. Juglanthraquinone C Induces Intracellular ROS Increase and Apoptosis by Activating the Akt/Foxo Signal Pathway in HCC Cells

    PubMed Central

    2016-01-01

    Juglanthraquinone C (JC), a naturally occurring anthraquinone extracted from Juglans mandshurica, could induce apoptosis of cancer cells. This study aims to investigate the detailed cytotoxicity mechanism of JC in HepG2 and BEL-7402 cells. The Affymetrix HG-U133 Plus 2.0 arrays were first used to analyze the mRNA expression exposed to JC or DMSO in HepG2 cells. Consistent with the previous results, the data indicated that JC could induce apoptosis and hyperactivated Akt. The Western blot analysis further revealed that Akt, a well-known survival protein, was strongly activated in HepG2 and BEL-7402 cells. Furthermore, an obvious inhibitory effect on JC-induced apoptosis was observed when the Akt levels were decreased, while the overexpression of constitutively active mutant Akt greatly accelerated JC-induced apoptosis. The subsequent results suggested that JC treatment suppressed nuclear localization and increased phosphorylated levels of Foxo3a, and the overexpression of Foxo3a abrogated JC-induced apoptosis. Most importantly, the inactivation of Foxo3a induced by JC further led to an increase of intracellular ROS levels by suppressing ROS scavenging enzymes, and the antioxidant N-acetyl-L-cysteine and catalase successfully decreased JC-induced apoptosis. Collectively, this study demonstrated that JC induced the apoptosis of hepatocellular carcinoma (HCC) cells by activating Akt/Foxo signaling pathway and increasing intracellular ROS levels. PMID:26682007

  2. Intracellular signalling by C-peptide.

    PubMed

    Hills, Claire E; Brunskill, Nigel J

    2008-01-01

    C-peptide, a cleavage product of the proinsulin molecule, has long been regarded as biologically inert, serving merely as a surrogate marker for insulin release. Recent findings demonstrate both a physiological and protective role of C-peptide when administered to individuals with type I diabetes. Data indicate that C-peptide appears to bind in nanomolar concentrations to a cell surface receptor which is most likely to be G-protein coupled. Binding of C-peptide initiates multiple cellular effects, evoking a rise in intracellular calcium, increased PI-3-kinase activity, stimulation of the Na(+)/K(+) ATPase, increased eNOS transcription, and activation of the MAPK signalling pathway. These cell signalling effects have been studied in multiple cell types from multiple tissues. Overall these observations raise the possibility that C-peptide may serve as a potential therapeutic agent for the treatment or prevention of long-term complications associated with diabetes. PMID:18382618

  3. Intracellular signaling and hepatocellular carcinoma.

    PubMed

    Iakova, Polina; Timchenko, Lubov; Timchenko, Nikolai A

    2011-02-01

    Liver cancer is the fifth most common cancer and the third most common cause of cancer related death in the world. The recent development of new techniques for the investigations of global change in the gene expression, signaling pathways and wide genome binding has provided novel information for the mechanisms underlying liver cancer progression. Although these studies identified gene expression signatures in hepatocellular carcinoma, the early steps of the development of hepatocellular carcinomas (HCC) are not well understood. The development of HCC is a multistep process which includes the progressive alterations of gene expression leading to the increased proliferation and to liver cancer. This review summarizes recent progress in the identification of the key steps of the development of HCC with the focus on early events of carcinogenesis and on the role of translational and epigenetic alterations in the development of HCC. Quiescent stage of the liver is supported by several tumor suppressor proteins including p53, Rb and C/EBPα. Studies with chemical models of liver carcinogenesis and with human HCC have shown that the elevation of gankyrin is responsible for the elimination of these three proteins at early steps of carcinogenesis. Later stages of progression of the liver cancer are associated with alterations in many signaling pathways including translation which leads to epigenetic silencing/activation of many genes. Particularly, recent reports suggest a critical role of histone deacetylase 1, HDAC1, in the development of HCC through the interactions with transcription factors such as C/EBP family proteins. PMID:20850540

  4. Coxiella subversion of intracellular host signaling.

    PubMed

    Hussain, S Kauser; Voth, Daniel E

    2012-01-01

    Coxiella burnetii is a highly infectious bacterial pathogen that replicates in a specialized vacuole inside eukaryotic cells. Due to a prolonged growth cycle, Coxiella continuously manipulates cellular processes to generate this parasitophorous vacuole (PV) and promote host cell viability. Here, we discuss recent findings that indicate Coxiella modulates several host signaling pathways to influence survival and ensure intracellular replication. The pathogen actively inhibits apoptotic cell death and activates the pro-survival kinases Akt and Erk1/2 to promote host viability. Coxiella's anti-apoptotic activity also involves the interface between autophagy and apoptosis, which is regulated by the interaction of autophagy-related Beclin-1 and anti-apoptotic Bcl-2. Additionally, Coxiella requires host kinase activity for PV biogenesis and maintenance. Thus, signaling modulation by Coxiella is critical for multiple aspects of host cell parasitism. Collectively, recent signaling studies have enhanced our understanding of the unique Coxiella-host cell interaction. Identification of bacterial factors that regulate signaling events will further our ability to model this intriguing infectious process.

  5. Control of Intracellular Calcium Signaling as a Neuroprotective Strategy

    PubMed Central

    Duncan, R. Scott; Goad, Daryl L.; Grillo, Michael A.; Kaja, Simon; Payne, Andrew J.; Koulen, Peter

    2010-01-01

    Both acute and chronic degenerative diseases of the nervous system reduce the viability and function of neurons through changes in intracellular calcium signaling. In particular, pathological increases in the intracellular calcium concentration promote such pathogenesis. Disease involvement of numerous regulators of intracellular calcium signaling located on the plasma membrane and intracellular organelles has been documented. Diverse groups of chemical compounds targeting ion channels, G-protein coupled receptors, pumps and enzymes have been identified as potential neuroprotectants. The present review summarizes the discovery, mechanisms and biological activity of neuroprotective molecules targeting proteins that control intracellular calcium signaling to preserve or restore structure and function of the nervous system. Disease relevance, clinical applications and new technologies for the identification of such molecules are being discussed. PMID:20335972

  6. Intracellular labile iron determines H2O2-induced apoptotic signaling via sustained activation of ASK1/JNK-p38 axis.

    PubMed

    Mantzaris, M D; Bellou, S; Skiada, V; Kitsati, N; Fotsis, T; Galaris, D

    2016-08-01

    Hydrogen peroxide (H2O2) acts as a second messenger in signal transduction participating in several redox regulated pathways, including cytokine and growth factor stimulated signals. However, the exact molecular mechanisms underlying these processes remain poorly understood and require further investigation. In this work, using Jurkat T lymphoma cells and primary human umbilical vein endothelial cells, it was observed that changes in intracellular "labile iron" were able to modulate signal transduction in H2O2-induced apoptosis. Chelation of intracellular labile iron by desferrioxamine rendered cells resistant to H2O2-induced apoptosis. In order to identify the exact points of iron action, we investigated selected steps in H2O2-mediated apoptotic pathway, focusing on mitogen activated protein kinases (MAPKs) JNK, p38 and ERK. It was observed that spatiotemporal changes in intracellular labile iron, induced by H2O2, influenced the oxidation pattern of the upstream MAP3K ASK1 and promoted the sustained activation of JNK-p38 axis in a defined time-dependent context. Moreover, we indicate that H2O2 induced spatiotemporal changes in intracellular labile iron, at least in part, by triggering the destabilization of lysosomal compartments, promoting a concomitant early response in proteins of iron homeostasis. These results raise the possibility that iron-mediated oxidation of distinct proteins may be implicated in redox signaling processes. Since labile iron can be pharmacologically modified in vivo, it may represent a promising target for therapeutic interventions in related pathological conditions.

  7. Optogenetic activation of intracellular adenosine A2A receptor signaling in the hippocampus is sufficient to trigger CREB phosphorylation and impair memory.

    PubMed

    Li, P; Rial, D; Canas, P M; Yoo, J-H; Li, W; Zhou, X; Wang, Y; van Westen, G J P; Payen, M-P; Augusto, E; Gonçalves, N; Tomé, A R; Li, Z; Wu, Z; Hou, X; Zhou, Y; IJzerman, A P; PIJzerman, Ad; Boyden, E S; Cunha, R A; Qu, J; Chen, J-F

    2015-11-01

    Human and animal studies have converged to suggest that caffeine consumption prevents memory deficits in aging and Alzheimer's disease through the antagonism of adenosine A2A receptors (A2ARs). To test if A2AR activation in the hippocampus is actually sufficient to impair memory function and to begin elucidating the intracellular pathways operated by A2AR, we have developed a chimeric rhodopsin-A2AR protein (optoA2AR), which retains the extracellular and transmembrane domains of rhodopsin (conferring light responsiveness and eliminating adenosine-binding pockets) fused to the intracellular loop of A2AR to confer specific A2AR signaling. The specificity of the optoA2AR signaling was confirmed by light-induced selective enhancement of cAMP and phospho-mitogen-activated protein kinase (p-MAPK) (but not cGMP) levels in human embryonic kidney 293 (HEK293) cells, which was abolished by a point mutation at the C terminal of A2AR. Supporting its physiological relevance, optoA2AR activation and the A2AR agonist CGS21680 produced similar activation of cAMP and p-MAPK signaling in HEK293 cells, of p-MAPK in the nucleus accumbens and of c-Fos/phosphorylated-CREB (p-CREB) in the hippocampus, and similarly enhanced long-term potentiation in the hippocampus. Remarkably, optoA2AR activation triggered a preferential p-CREB signaling in the hippocampus and impaired spatial memory performance, while optoA2AR activation in the nucleus accumbens triggered MAPK signaling and modulated locomotor activity. This shows that the recruitment of intracellular A2AR signaling in the hippocampus is sufficient to trigger memory dysfunction. Furthermore, the demonstration that the biased A2AR signaling and functions depend on intracellular A2AR loops prompts the possibility of targeting the intracellular A2AR-interacting partners to selectively control different neuropsychiatric behaviors. PMID:25687775

  8. Optogenetic control of intracellular signaling pathways.

    PubMed

    Zhang, Kai; Cui, Bianxiao

    2015-02-01

    Cells employ a plethora of signaling pathways to make their life-and-death decisions. Extensive genetic, biochemical, and physiological studies have led to the accumulation of knowledge about signaling components and their interactions within signaling networks. These conventional approaches, although useful, lack the ability to control the spatial and temporal aspects of signaling processes. The recently emerged optogenetic tools open exciting opportunities by enabling signaling regulation with superior temporal and spatial resolution, easy delivery, rapid reversibility, fewer off-target side effects, and the ability to dissect complex signaling networks. Here we review recent achievements in using light to control intracellular signaling pathways and discuss future prospects for the field, including integration of new genetic approaches into optogenetics.

  9. Optogenetic control of intracellular signaling pathways

    PubMed Central

    Zhang, Kai; Cui, Bianxiao

    2014-01-01

    Cells employ a plethora of signaling pathways to make their life-and-death decisions. Extensive genetic, biochemical, and physiological studies have led to the accumulation of knowledge about signaling components and their interactions within signaling networks. These conventional approaches, though useful, lack the ability to control the spatial and temporal aspects of signaling processes. The recently emerged optogenetic tools open up exciting opportunities by enabling signaling regulation with superior temporal and spatial resolution, easy delivery, rapid reversibility, fewer off-target side effects, and the ability to dissect complex signaling networks. Here we review recent achievements in using light to control intracellular signaling pathways, and discuss future prospects for the field, including integration of new genetic approaches into optogenetics. PMID:25529484

  10. Alan [corrected] N. Epstein award: Intracellular signaling and ingestive behaviors.

    PubMed

    Daniels, Derek

    2010-07-14

    Understanding the role of intracellular signaling pathways in ingestive behavior is a challenging problem in behavioral neuroscience. This review summarizes work conducted on two systems with the aim of identifying intracellular events that relate to food and fluid intake. The first set of experiments focused on melanocortin receptors and their ability to signal through members of the mitogen-activated protein (MAP) kinase family. The second set of experiments focused on the role of intracellular signaling pathways in water and saline intakes that are stimulated by angiotensin II (AngII). The initial findings in each line of research have been extended by subsequent research that is discussed in turn. The paper represents an invited review by a symposium, award winner or keynote speaker at the Society for the Study of Ingestive Behavior [SSIB] Annual Meeting in Portland, July 2009.

  11. Stochastic models of intracellular calcium signals

    NASA Astrophysics Data System (ADS)

    Rüdiger, Sten

    2014-01-01

    Cellular signaling operates in a noisy environment shaped by low molecular concentrations and cellular heterogeneity. For calcium release through intracellular channels-one of the most important cellular signaling mechanisms-feedback by liberated calcium endows fluctuations with critical functions in signal generation and formation. In this review it is first described, under which general conditions the environment makes stochasticity relevant, and which conditions allow approximating or deterministic equations. This analysis provides a framework, in which one can deduce an efficient hybrid description combining stochastic and deterministic evolution laws. Within the hybrid approach, Markov chains model gating of channels, while the concentrations of calcium and calcium binding molecules (buffers) are described by reaction-diffusion equations. The article further focuses on the spatial representation of subcellular calcium domains related to intracellular calcium channels. It presents analysis for single channels and clusters of channels and reviews the effects of buffers on the calcium release. For clustered channels, we discuss the application and validity of coarse-graining as well as approaches based on continuous gating variables (Fokker-Planck and chemical Langevin equations). Comparison with recent experiments substantiates the stochastic and spatial approach, identifies minimal requirements for a realistic modeling, and facilitates an understanding of collective channel behavior. At the end of the review, implications of stochastic and local modeling for the generation and properties of cell-wide release and the integration of calcium dynamics into cellular signaling models are discussed.

  12. 4-Hydroxy-2-nonenal induces apoptosis by activating ERK1/2 signaling and depleting intracellular glutathione in intestinal epithelial cells

    PubMed Central

    Ji, Yun; Dai, Zhaolai; Wu, Guoyao; Wu, Zhenlong

    2016-01-01

    Excessive reactive oxygen species (ROS) induces oxidative damage to cellular constituents, ultimately leading to induction of apoptotic cell death and the pathogenesis of various diseases. The molecular mechanisms for the action of ROS in intestinal diseases remain poorly defined. Here, we reported that 4-hydroxy-2-nonenal (4-HNE) treatment led to capses-3-dependent apoptosis accompanied by increased intracellular ROS level and reduced glutathione concentration in intestinal epithelial cells. These effects of 4-HNE were markedly abolished by the antioxidant L-cysteine derivative N-acetylcysteine (NAC). Further studies demonstrated that the protective effect of NAC was associated with restoration of intracellular redox state by Nrf2-related regulation of expression of genes involved in intracellular glutathione (GSH) biosynthesis and inactivation of 4-HNE-induced phosphorylation of extracellular signal-regulated protein kinases (ERK1/2). The 4-HNE-induced ERK1/2 activation was mediated by repressing mitogen-activated protein kinase phosphatase-1 (MKP-1), a negative regulator of ERK1/2, through a proteasome-dependent degradation mechanism. Importantly, either overexpression of MKP-1 or NAC treatment blocked 4-HNE-induced MKP-1 degradation, thereby protecting cell from apoptosis. These novel findings provide new insights into a functional role of MKP-1 in oxidative stress-induced cell death by regulating ERK1/2 MAP kinase in intestinal epithelial cells. PMID:27620528

  13. 4-Hydroxy-2-nonenal induces apoptosis by activating ERK1/2 signaling and depleting intracellular glutathione in intestinal epithelial cells.

    PubMed

    Ji, Yun; Dai, Zhaolai; Wu, Guoyao; Wu, Zhenlong

    2016-01-01

    Excessive reactive oxygen species (ROS) induces oxidative damage to cellular constituents, ultimately leading to induction of apoptotic cell death and the pathogenesis of various diseases. The molecular mechanisms for the action of ROS in intestinal diseases remain poorly defined. Here, we reported that 4-hydroxy-2-nonenal (4-HNE) treatment led to capses-3-dependent apoptosis accompanied by increased intracellular ROS level and reduced glutathione concentration in intestinal epithelial cells. These effects of 4-HNE were markedly abolished by the antioxidant L-cysteine derivative N-acetylcysteine (NAC). Further studies demonstrated that the protective effect of NAC was associated with restoration of intracellular redox state by Nrf2-related regulation of expression of genes involved in intracellular glutathione (GSH) biosynthesis and inactivation of 4-HNE-induced phosphorylation of extracellular signal-regulated protein kinases (ERK1/2). The 4-HNE-induced ERK1/2 activation was mediated by repressing mitogen-activated protein kinase phosphatase-1 (MKP-1), a negative regulator of ERK1/2, through a proteasome-dependent degradation mechanism. Importantly, either overexpression of MKP-1 or NAC treatment blocked 4-HNE-induced MKP-1 degradation, thereby protecting cell from apoptosis. These novel findings provide new insights into a functional role of MKP-1 in oxidative stress-induced cell death by regulating ERK1/2 MAP kinase in intestinal epithelial cells. PMID:27620528

  14. Estrogenic xenobiotics affect the intracellular activation signal in mitogen-induced human peripheral blood lymphocytes: immunotoxicological impact.

    PubMed

    Sakabe, K; Okuma, M; Kazuno, M; Yamaguchi, T; Yoshida, T; Furuya, H; Kayama, F; Suwa, Y; Fujii, W; Fresa, K L

    1998-01-01

    The present study was an attempt to elucidate the effect of estrogenic xenobiotics on the proliferation of mitogen-stimulated human peripheral blood lymphocyte (PBL). Our findings follow: (a) the proliferation of PBL in response to phytohemagglutinin (PHA) was mediated by protein kinase C activity, but estrogenic xenobiotics had a strong inhibitory effect on protein kinase C activity of PHA-stimulated PBL; (b) cytoplasmic extracts from PHA-stimulated PBL greatly activated DNA replication, but estrogenic xenobiotics had a strong inhibitory effect on these activities. The results suggest that the cytoplasmic signal-generating system in mitogen-treated PBL is inhibited by estrogenic xenobiotics, and that the defect occurs at all stages in the sequence of events leading to DNA synthesis and cell proliferation. PMID:9730256

  15. Evaluation of Intracellular Signaling Downstream Chimeric Antigen Receptors

    PubMed Central

    Karlsson, Hannah; Svensson, Emma; Gigg, Camilla; Jarvius, Malin; Olsson-Strömberg, Ulla; Savoldo, Barbara; Dotti, Gianpietro; Loskog, Angelica

    2015-01-01

    CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G) CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G) CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs. PMID:26700307

  16. Spatio-temporal modelling of the NF-κB intracellular signalling pathway: the roles of diffusion, active transport, and cell geometry.

    PubMed

    Terry, Alan J; Chaplain, Mark A J

    2011-12-01

    The nuclear factor kappa B (NF-κB) intracellular signalling pathway is central to many stressful, inflammatory, and innate immune responses. NF-κB proteins themselves are transcription factors for hundreds of genes. Experiments have shown that the NF-κB pathway can exhibit oscillatory dynamics-a negative feedback loop causes oscillatory nuclear-cytoplasmic translocation of NF-κB. Given that cell size and shape are known to influence intracellular signal transduction, we consider a spatio-temporal model of partial differential equations for the NF-κB pathway, where we model molecular movement by diffusion and, for several key species including NF-κB, by active transport as well. Through numerical simulations we find values for model parameters such that sustained oscillatory dynamics occur. Our spatial profiles and animations bear a striking resemblance to experimental images and movie clips employing fluorescent fusion proteins. We discover that oscillations in nuclear NF-κB may occur when active transport is across the nuclear membrane only, or when no species are subject to active transport. However, when active transport is across the nuclear membrane and NF-κB is additionally actively transported through the cytoplasm, oscillations are lost. Hence transport mechanisms in a cell will influence its response to activation of its NF-κB pathway. We also demonstrate that sustained oscillations in nuclear NF-κB are somewhat robust to changes in the shape of the cell, or the shape, location, and size of its nucleus, or the location of ribosomes. Yet if the cell is particularly flat or the nucleus sufficiently small, then oscillations are lost. Thus the geometry of a cell may partly determine its response to NF-κB activation. The NF-κB pathway is known to be constitutively active in several human cancers. Our spatially explicit modelling approach will allow us, in future work, to investigate targeted drug therapy of tumours.

  17. Spatio-temporal modelling of the NF-κB intracellular signalling pathway: the roles of diffusion, active transport, and cell geometry.

    PubMed

    Terry, Alan J; Chaplain, Mark A J

    2011-12-01

    The nuclear factor kappa B (NF-κB) intracellular signalling pathway is central to many stressful, inflammatory, and innate immune responses. NF-κB proteins themselves are transcription factors for hundreds of genes. Experiments have shown that the NF-κB pathway can exhibit oscillatory dynamics-a negative feedback loop causes oscillatory nuclear-cytoplasmic translocation of NF-κB. Given that cell size and shape are known to influence intracellular signal transduction, we consider a spatio-temporal model of partial differential equations for the NF-κB pathway, where we model molecular movement by diffusion and, for several key species including NF-κB, by active transport as well. Through numerical simulations we find values for model parameters such that sustained oscillatory dynamics occur. Our spatial profiles and animations bear a striking resemblance to experimental images and movie clips employing fluorescent fusion proteins. We discover that oscillations in nuclear NF-κB may occur when active transport is across the nuclear membrane only, or when no species are subject to active transport. However, when active transport is across the nuclear membrane and NF-κB is additionally actively transported through the cytoplasm, oscillations are lost. Hence transport mechanisms in a cell will influence its response to activation of its NF-κB pathway. We also demonstrate that sustained oscillations in nuclear NF-κB are somewhat robust to changes in the shape of the cell, or the shape, location, and size of its nucleus, or the location of ribosomes. Yet if the cell is particularly flat or the nucleus sufficiently small, then oscillations are lost. Thus the geometry of a cell may partly determine its response to NF-κB activation. The NF-κB pathway is known to be constitutively active in several human cancers. Our spatially explicit modelling approach will allow us, in future work, to investigate targeted drug therapy of tumours. PMID:21907212

  18. Pharmacological analysis of intracellular Ca2+ signalling: problems and pitfalls.

    PubMed

    Taylor, C W; Broad, L M

    1998-09-01

    The complex changes in intracellular Ca2+ concentration that follow cell stimulation reflect the concerted activities of Ca2+ channels in the plasma membrane and in the membranes of intracellular stores, and the opposing actions of the mechanisms that extrude Ca2+ from the cytosol. Disentangling the roles of each of these processes is hampered by the lack of adequately selective pharmacological tools. In this review, Colin Taylor and Lisa Broad summarize the more serious problems associated with some of the commonly used drugs, and describe specific situations in which the multiple effects of drugs on Ca2(+)-signalling pathways have confused analysis of these pathways.

  19. Domain-Specific Activation of Death-Associated Intracellular Signalling Cascades by the Cellular Prion Protein in Neuroblastoma Cells.

    PubMed

    Vilches, Silvia; Vergara, Cristina; Nicolás, Oriol; Mata, Ágata; Del Río, José A; Gavín, Rosalina

    2016-09-01

    The biological functions of the cellular prion protein remain poorly understood. In fact, numerous studies have aimed to determine specific functions for the different protein domains. Studies of cellular prion protein (PrP(C)) domains through in vivo expression of molecules carrying internal deletions in a mouse Prnp null background have provided helpful data on the implication of the protein in signalling cascades in affected neurons. Nevertheless, understanding of the mechanisms underlying the neurotoxicity induced by these PrP(C) deleted forms is far from complete. To better define the neurotoxic or neuroprotective potential of PrP(C) N-terminal domains, and to overcome the heterogeneity of results due to the lack of a standardized model, we used neuroblastoma cells to analyse the effects of overexpressing PrP(C) deleted forms. Results indicate that PrP(C) N-terminal deleted forms were properly processed through the secretory pathway. However, PrPΔF35 and PrPΔCD mutants led to death by different mechanisms sharing loss of alpha-cleavage and activation of caspase-3. Our data suggest that both gain-of-function and loss-of-function pathogenic mechanisms may be associated with N-terminal domains and may therefore contribute to neurotoxicity in prion disease. Dissecting the molecular response induced by PrPΔF35 may be the key to unravelling the physiological and pathological functions of the prion protein. PMID:26250617

  20. Domain-Specific Activation of Death-Associated Intracellular Signalling Cascades by the Cellular Prion Protein in Neuroblastoma Cells.

    PubMed

    Vilches, Silvia; Vergara, Cristina; Nicolás, Oriol; Mata, Ágata; Del Río, José A; Gavín, Rosalina

    2016-09-01

    The biological functions of the cellular prion protein remain poorly understood. In fact, numerous studies have aimed to determine specific functions for the different protein domains. Studies of cellular prion protein (PrP(C)) domains through in vivo expression of molecules carrying internal deletions in a mouse Prnp null background have provided helpful data on the implication of the protein in signalling cascades in affected neurons. Nevertheless, understanding of the mechanisms underlying the neurotoxicity induced by these PrP(C) deleted forms is far from complete. To better define the neurotoxic or neuroprotective potential of PrP(C) N-terminal domains, and to overcome the heterogeneity of results due to the lack of a standardized model, we used neuroblastoma cells to analyse the effects of overexpressing PrP(C) deleted forms. Results indicate that PrP(C) N-terminal deleted forms were properly processed through the secretory pathway. However, PrPΔF35 and PrPΔCD mutants led to death by different mechanisms sharing loss of alpha-cleavage and activation of caspase-3. Our data suggest that both gain-of-function and loss-of-function pathogenic mechanisms may be associated with N-terminal domains and may therefore contribute to neurotoxicity in prion disease. Dissecting the molecular response induced by PrPΔF35 may be the key to unravelling the physiological and pathological functions of the prion protein.

  1. Heparin-disaccharide affects T cells: inhibition of NF-kappaB activation, cell migration, and modulation of intracellular signaling.

    PubMed

    Hecht, Iris; Hershkoviz, Rami; Shivtiel, Shoham; Lapidot, Tzvi; Cohen, Irun R; Lider, Ofer; Cahalon, Liora

    2004-06-01

    We previously reported that disaccharides (DS), generated by enzymatic degradation of heparin or heparan sulfate, inhibit T cell-mediated immune reactions in rodents and regulate cytokine [tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-8, and IL-1beta] secretion by T cells, macrophages, or intestinal epithelial cells. Here, we investigated the effects of a trisulfated heparin DS (3S-DS) on two aspects of T cell function: secretion of proinflammatory cytokines and migration to an inflamed site. 3S-DS down-regulated nuclear factor-kappaB activity and reduced the secretion of TNF-alpha and interferon-gamma (IFN-gamma) by anti-CD3-activated T cells. In addition, 3S-DS inhibited CXC chemokine ligand 12 (CXCL12; stromal cell-derived factor-1alpha)-dependent migration in vitro and in vivo and decreased CXCL12-induced T cell adhesion to the extracellular matrix glycoprotein, fibronectin (FN). This inhibition was accompanied by attenuation of CXCL12-induced Pyk2 phosphorylation but did not involve internalization of the CXCL12 receptor, CXCR4, or phosphorylation of extracellular-regulated kinase. Despite inhibiting CXCL12-induced adhesion, 3S-DS, on its own, induced T cell adhesion to FN, which was accompanied by phosphorylation of Pyk2. A monosulfated DS showed no effect. Taken together, these data provide evidence that 3S-DS can regulate inflammation by inducing and modulating T cell-signaling events, desensitizing CXCR4, and modulating T cell receptor-induced responses. PMID:15020655

  2. Inhibitor of apoptosis proteins as intracellular signaling intermediates.

    PubMed

    Kocab, Andrew J; Duckett, Colin S

    2016-01-01

    Inhibitor of apoptosis (IAP) proteins have often been considered inhibitors of cell death due to early reports that described their ability to directly bind and inhibit caspases, the primary factors that implement apoptosis. However, a greater understanding is evolving regarding the vital roles played by IAPs as transduction intermediates in a diverse set of signaling cascades associated with functions ranging from the innate immune response to cell migration to cell-cycle regulation. In this review, we discuss the functions of IAPs in signaling, focusing primarily on the cellular IAP (c-IAP) proteins. The c-IAPs are important components in tumor necrosis factor receptor superfamily signaling cascades, which include activation of the NF-κB transcription factor family. As these receptors modulate cell proliferation and cell death, the involvement of the c-IAPs in these pathways provides an additional means of controlling cellular fate beyond simply inhibiting caspase activity. Additionally, IAP-binding proteins, such as Smac and caspases, which have been described as having cell death-independent roles, may affect c-IAP activity in intracellular signaling. Collectively, the multi-faceted functions and complex regulation of the c-IAPs illustrate their importance as intracellular signaling intermediates.

  3. Microdamage induced calcium efflux from bone matrix activates intracellular calcium signaling in osteoblasts via L-type and T-type voltage-gated calcium channels.

    PubMed

    Jung, Hyungjin; Best, Makenzie; Akkus, Ozan

    2015-07-01

    Mechanisms by which bone microdamage triggers repair response are not completely understood. It has been shown that calcium efflux ([Ca(2+)]E) occurs from regions of bone undergoing microdamage. Such efflux has also been shown to trigger intracellular calcium signaling ([Ca(2+)]I) in MC3T3-E1 cells local to damaged regions. Voltage-gated calcium channels (VGCCs) are implicated in the entry of [Ca(2+)]E to the cytoplasm. We investigated the involvement of VGCC in the extracellular calcium induced intracellular calcium response (ECIICR). MC3T3-E1 cells were subjected to one dimensional calcium efflux from their basal aspect which results in an increase in [Ca(2+)]I. This increase was concomitant with membrane depolarization and it was significantly reduced in the presence of Bepridil, a non-selective VGCC inhibitor. To identify specific type(s) of VGCC in ECIICR, the cells were treated with selective inhibitors for different types of VGCC. Significant changes in the peak intensity and the number of [Ca(2+)]I oscillations were observed when L-type and T-type specific VGCC inhibitors (Verapamil and NNC55-0396, respectively) were used. So as to confirm the involvement of L- and T-type VGCC in the context of microdamage, cells were seeded on devitalized notched bone specimen, which were loaded to induce microdamage in the presence and absence of Verapamil and NNC55-0396. The results showed significant decrease in [Ca(2+)]I activity of cells in the microdamaged regions of bone when L- and T-type blockers were applied. This study demonstrated that extracellular calcium increase in association with damage depolarizes the cell membrane and the calcium ions enter the cell cytoplasm by L- and T-type VGCCs.

  4. Calcium, channels, intracellular signaling and autoimmunity.

    PubMed

    Izquierdo, Jorge-Hernán; Bonilla-Abadía, Fabio; Cañas, Carlos A; Tobón, Gabriel J

    2014-01-01

    Calcium (Ca²⁺) is an important cation able to function as a second messenger in different cells of the immune system, particularly in B and T lymphocytes, macrophages and mastocytes, among others. Recent discoveries related to the entry of Ca²⁺ through the store-operated calcium entry (SOCE) has opened a new investigation area about the cell destiny regulated by Ca²⁺ especially in B and T lymphocytes. SOCE acts through calcium-release-activated calcium (CRAC) channels. The function of CRAC depends of two recently discovered regulators: the Ca²⁺ sensor in the endoplasmic reticulum or stromal interaction molecule (STIM-1) and one subunit of CRAC channels called Orai1. This review focuses on the role of Ca²⁺ signals in B and T lymphocytes functions, the signalling pathways leading to Ca²⁺ influx, and the relationship between Ca²⁺ signals and autoimmune diseases.

  5. C-Peptide and its intracellular signaling.

    PubMed

    Hills, Claire E; Brunskill, Nigel J

    2009-01-01

    Although long believed to be inert, C-peptide has now been shown to have definite biological effects both in vitro and in vivo in diabetic animals and in patients with type 1 diabetes. These effects point to a protective action of C-peptide against the development of diabetic microvascular complications. Underpinning these observations is undisputed evidence of C-peptide binding to a variety of cell types at physiologically relevant concentrations, and the downstream stimulation of multiple cell signaling pathways and gene transcription via the activation of numerous transcription factors. These pathways affect such fundamental cellular processes as re-absorptive and/or secretory phenotype, migration, growth, and survival. Whilst the receptor remains to be identified, experimental data points strongly to the existence of a specific G-protein-coupled receptor for C-peptide. Of the cell types studied so far, kidney tubular cells express the highest number of C-peptide binding sites. Accordingly, C-peptide exerts major effects on the function of these cells, and in the context of diabetic nephropathy appears to antagonise the pathophysiological effects of major disease mediators such as TGFbeta1 and TNFalpha. Therefore, based on its cellular activity profile C-peptide appears well positioned for development as a therapeutic tool to treat microvascular complications in type 1 diabetes. PMID:20039003

  6. Visualization of Intracellular Tyrosinase Activity in vitro

    PubMed Central

    Setty, Subba Rao Gangi

    2016-01-01

    Melanocytes produce the melanin pigments in melanosomes and these organelles protect the skin against harmful ultraviolet rays. Tyrosinase is the key cuproenzyme which initiates the pigment synthesis using its substrate amino acid tyrosine or L-DOPA (L-3, 4-dihydroxyphenylalanine). Moreover, the activity of tyrosinase directly correlates to the cellular pigmentation. Defects in tyrosinase transport to melanosomes or mutations in the enzyme or reduced intracellular copper levels results in loss of tyrosinase activity in melanosomes, commonly observed in albinism. Here, we described a method to detect the intracellular activity of tyrosinase in mouse melanocytes. This protocol will visualize the active tyrosinase present in the intracellular vesicles or organelles including melanosomes. PMID:27231711

  7. [Neurotensin: reception and intracellular mechanisms of signaling].

    PubMed

    Osadchiĭ, O E

    2006-01-01

    The review coveres the features of neurotensin receptor, functional role ot its structural elements, nature of conjugation with effectoral cell systems, and mechanisms of receptor decensitization developing as results of prolonged effect of agonist. The author provides pharmacological description of neurotensin antagonists and special features of three subtypes of its receptors. The author reviews the research results establishing a correlation between structural modification of various section of neurotensin molecula and manifestations of its physiological activity. Special focus is mage on discussion of neurotensin's physiological effects developing as results of its modulating impact on discharge of other biologically active substances.

  8. How complex are intracellular immune receptor signaling complexes?

    PubMed

    Bonardi, Vera; Dangl, Jeffery L

    2012-01-01

    Nucleotide binding leucine-rich repeat proteins (NLRs) are the major class of intracellular immune receptors in plants. NLRs typically function to specifically recognize pathogen effectors and to initiate and control defense responses that severely limit pathogen growth in plants (termed effector-triggered immunity, or ETI). Despite numerous reports supporting a central role in innate immunity, the molecular mechanisms driving NLR activation and downstream signaling remain largely elusive. Recent reports shed light on the pre- and post-activation dynamics of a few NLR-containing protein complexes. Recent technological advances in the use of proteomics may enable high-resolution definition of immune protein complexes and possible activation-relevant post-translational modifications of the components in these complexes. In this review, we focus on research aimed at characterizing pre- and post-activation NLR protein complexes and the molecular events that follow activation. We discuss the use of new or improved technologies as tools to unveil the molecular mechanisms that define NLR-mediated pathogen recognition.

  9. Intracellular Signaling by Diffusion: Can Waves of Hydrogen Peroxide Transmit Intracellular Information in Plant Cells?

    PubMed Central

    Vestergaard, Christian Lyngby; Flyvbjerg, Henrik; Møller, Ian Max

    2012-01-01

    Amplitude- and frequency-modulated waves of Ca2+ ions transmit information inside cells. Reactive Oxygen Species (ROS), specifically hydrogen peroxide, have been proposed to have a similar role in plant cells. We consider the feasibility of such an intracellular communication system in view of the physical and biochemical conditions in plant cells. As model system, we use a H2O2 signal originating at the plasma membrane (PM) and spreading through the cytosol. We consider two maximally simple types of signals, isolated pulses and harmonic oscillations. First we consider the basic limits on such signals as regards signal origin, frequency, amplitude, and distance. Then we establish the impact of ROS-removing enzymes on the ability of H2O2 to transmit signals. Finally, we consider to what extent cytoplasmic streaming distorts signals. This modeling allows us to predict the conditions under which diffusion-mediated signaling is possible. We show that purely diffusive transmission of intracellular information by H2O2 over a distance of 1 μm (typical distance between organelles, which may function as relay stations) is possible at frequencies well above 1 Hz, which is the highest frequency observed experimentally. This allows both frequency and amplitude modulation of the signal. Signaling over a distance of 10 μm (typical distance between the PM and the nucleus) may be possible, but requires high signal amplitudes or, equivalently, a very low detection threshold. Furthermore, at this longer distance a high rate of enzymatic degradation is required to make signaling at frequencies above 0.1 Hz possible. In either case, cytoplasmic streaming does not seriously disturb signals. We conclude that although purely diffusion-mediated signaling without relaying stations is theoretically possible, it is unlikely to work in practice, since it requires a much faster enzymatic degradation and a much lower cellular background concentration of H2O2 than observed

  10. Intracellular delivery of a cell-penetrating SOCS1 that targets IFN-gamma signaling.

    PubMed

    DiGiandomenico, Antonio; Wylezinski, Lukasz S; Hawiger, Jacek

    2009-07-21

    Suppressor of cytokine signaling-1 (SOCS1) is an intracellular inhibitor of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway that couples interferon-gamma (IFN-gamma) signaling to the nucleus. Because several inflammatory diseases are associated with uncontrolled IFN-gamma signaling, we engineered a recombinant cell-penetrating SOCS1 (CP-SOCS1) to target this pathway. Here, we show that CP-SOCS1, analogous to endogenous SOCS1, interacted with components of the IFN-gamma signaling complex and functionally attenuated the phosphorylation of STAT1, which resulted in the subsequent inhibition of the production of proinflammatory chemokines and cytokines. Thus, controlled, intracellular delivery of recombinant CP-SOCS1 boosted the anti-inflammatory potential of the cell by restoring the homeostatic balance between pro- and anti-inflammatory signaling. This approach to controlling signal transduction has potential use for therapeutic targeting of signaling pathways associated with inflammatory diseases.

  11. Evolution of the Calcium-Based Intracellular Signaling System

    PubMed Central

    Marchadier, Elodie; Oates, Matt E.; Fang, Hai; Donoghue, Philip C.J.; Hetherington, Alistair M.; Gough, Julian

    2016-01-01

    To progress our understanding of molecular evolution from a collection of well-studied genes toward the level of the cell, we must consider whole systems. Here, we reveal the evolution of an important intracellular signaling system. The calcium-signaling toolkit is made up of different multidomain proteins that have undergone duplication, recombination, sequence divergence, and selection. The picture of evolution, considering the repertoire of proteins in the toolkit of both extant organisms and ancestors, is radically different from that of other systems. In eukaryotes, the repertoire increased in both abundance and diversity at a far greater rate than general genomic expansion. We describe how calcium-based intracellular signaling evolution differs not only in rate but in nature, and how this correlates with the disparity of plants and animals. PMID:27358427

  12. Intracellular angiotensin II activates rat myometrium.

    PubMed

    Deliu, Elena; Tica, Andrei A; Motoc, Dana; Brailoiu, G Cristina; Brailoiu, Eugen

    2011-09-01

    Angiotensin II is a modulator of myometrial activity; both AT(1) and AT(2) receptors are expressed in myometrium. Since in other tissues angiotensin II has been reported to activate intracellular receptors, we assessed the effects of intracellular administration of angiotensin II via microinjection on myometrium, using calcium imaging. Intracellular injection of angiotensin II increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) in myometrial cells in a dose-dependent manner. The effect was abolished by the AT(1) receptor antagonist losartan but not by the AT(2) receptor antagonist PD-123319. Disruption of the endo-lysosomal system, but not that of Golgi apparatus, prevented the angiotensin II-induced increase in [Ca(2+)](i). Blockade of AT(1) receptor internalization had no effect, whereas blockade of microautophagy abolished the increase in [Ca(2+)](i) produced by intracellular injection of angiotensin II; this indicates that microautophagy is a critical step in transporting the peptide into the endo-lysosomes lumenum. The response to angiotensin II was slightly reduced in Ca(2+)-free saline, indicating a major involvement of Ca(2+) release from internal stores. Blockade of inositol 1,4,5-trisphosphate (IP(3)) receptors with heparin and xestospongin C or inhibition of phospholipase C (PLC) with U-73122 abolished the response to angiotensin II, supporting the involvement of PLC-IP(3) pathway. Angiotensin II-induced increase in [Ca(2+)](i) was slightly reduced by antagonism of ryanodine receptors. Taken together, our results indicate for the first time that in myometrial cells, intracellular angiotensin II activates AT(1)-like receptors on lysosomes and activates PLC-IP(3)-dependent Ca(2+) release from endoplasmic reticulum; the response is further augmented by a Ca(2+)-induced Ca(2+) release mechanism via ryanodine receptors activation.

  13. Activation of ATP-sensitive potassium channels in rat pancreatic beta-cells by linoleic acid through both intracellular metabolites and membrane receptor signalling pathway.

    PubMed

    Zhao, Yu-Feng; Pei, Jianming; Chen, Chen

    2008-09-01

    ATP-sensitive potassium channels (K(ATP) channels) determine the excitability of pancreatic beta-cells and importantly regulate glucose-stimulated insulin secretion (GSIS). Long-chain free fatty acids (FFAs) decrease GSIS after long-term exposure to beta-cells, but the effects of exogenous FFAs on K(ATP) channels are not yet well clarified. In this study, the effects of linoleic acid (LA) on membrane potential (MP) and K(ATP) channels were observed in primary cultured rat pancreatic beta-cells. LA (20 microM) induced hyperpolarization of MP and opening of K(ATP) channels, which was totally reversed and inhibited by tolbutamide, a K(ATP) channel blocker. Inhibition of LA metabolism by acyl-CoA synthetase inhibitor, triacsin C (10 microM), partially inhibited LA-induced opening of K(ATP) channels by 64%. The non-FFA G protein-coupled receptor (GPR) 40 agonist, GW9508 (40 microM), induced an opening of K(ATP) channels, which was similar to that induced by LA under triacsin C treatment. Blockade of protein kinases A and C did not influence the opening of K(ATP) channels induced by LA and GW9508, indicating that these two protein kinase pathways are not involved in the action of LA on K(ATP) channels. The present study demonstrates that LA induces hyperpolarization of MP by activating K(ATP) channels via both intracellular metabolites and activation of GPR40. It indicates that not only intracellular metabolites of FFAs but also GPR40-mediated pathways take part in the inhibition of GSIS and beta-cell dysfunction induced by FFAs.

  14. Analysis of Intracellular Calcium Signaling in Human Embryonic Stem Cells.

    PubMed

    Péntek, Adrienn; Pászty, Katalin; Apáti, Ágota

    2016-01-01

    Measurement of changes in intracellular calcium concentration is one of the most common and useful tools for studying signal transduction pathways or cellular responses in basic research and drug screening purposes as well. Increasing number of such applications using human pluripotent stem cells and their derivatives requires development of calcium signal measurements for this special cell type. Here we describe a modified protocol for analysis of calcium signaling events in human embryonic stem cells, which can be used for other pluripotent cell types (such as iPSC) or their differentiated offspring as well.

  15. Application of atomic force microscopy for studying intracellular signalization in neurons

    NASA Astrophysics Data System (ADS)

    Ankudinov, A. V.; Khalisov, M. M.; Penniyainen, V. A.; Podzorova, S. A.; Krylov, B. V.

    2015-10-01

    The first attempt is made at applying the method of atomic force microscopy (AFM) for determining the molecular mechanisms of intracellular signalization with the participation of Na+, K+-ATPhase playing an important role of a signal transductor (amplifier). The AFM method combined with the organotypic cultivation makes it possible to obtain quantitative information on the Young moduli of living neurons and cells subjected to the action of very low concentrations of ouabain. This substance is known to trigger in this case the intracellular signalization processes by transferring a molecular signal to the genome of a cell. The cell response is manifested in a sharp intensification of protein synthesis accompanied by a rearrangement of the cytoskeleton and activation of enzyme signal pathways in a cytosol. AFM measurements of the images of the cell surface relief are performed using the PeakForce quantitative nanomechanical properties mapping PeakForce QNM mode. The Young moduli of control neurons and of sensory neurons under the action of ouabain are measured simultaneously. It is found that the activation of the signal-transducing function of Na+, K+-ATPhase triggers intracellular signal cascades, which increase the cell stiffness. The application of the AFM method in further studies of the mechanisms of intracellular molecular processes appears as promising. Its combination with inhibitory analysis will clarify the role of individual molecules (e.g., a number of ferments) in regulation of growth and development of living organisms.

  16. H⁺-activated Na⁺ influx in the ventricular myocyte couples Ca²⁺-signalling to intracellular pH.

    PubMed

    Garciarena, Carolina D; Youm, Jae Boum; Swietach, Pawel; Vaughan-Jones, Richard D

    2013-08-01

    Acid extrusion on Na(+)-coupled pH-regulatory proteins (pH-transporters), Na(+)/H(+) exchange (NHE1) and Na(+)-HCO3(-) co-transport (NBC), drives Na(+) influx into the ventricular myocyte. This H(+)-activated Na(+)-influx is acutely up-regulated at pHi<7.2, greatly exceeding Na(+)-efflux on the Na(+)/K(+) ATPase. It is spatially heterogeneous, due to the co-localisation of NHE1 protein (the dominant pH-transporter) with gap-junctions at intercalated discs. Overall Na(+)-influx via NBC is considerably lower, but much is co-localised with L-type Ca(2+)-channels in transverse-tubules. Through a functional coupling with Na(+)/Ca(2+) exchange (NCX), H(+)-activated Na(+)-influx increases sarcoplasmic-reticular Ca(2+)-loading and release during intracellular acidosis. This raises Ca(2+)-transient amplitude, rescuing it from direct H(+)-inhibition. Functional coupling is biochemically regulated and linked to membrane receptors, through effects on NHE1 and NBC. It requires adequate cytoplasmic Na(+)-mobility, as NHE1 and NCX are spatially separated (up to 60μm). The relevant functional NCX activity must be close to dyads, as it exerts no effect on bulk diastolic Ca(2+). H(+)-activated Na(+)-influx is up-regulated during ischaemia-reperfusion and some forms of maladaptive hypertrophy and heart failure. It is thus an attractive system for therapeutic manipulation. This article is part of a Special Issue entitled "Na(+) Regulation in Cardiac Myocytes".

  17. Signal Transduction and Intracellular Trafficking by the Interleukin 36 Receptor*

    PubMed Central

    Saha, Siddhartha S.; Singh, Divyendu; Raymond, Ernest L.; Ganesan, Rajkumar; Caviness, Gary; Grimaldi, Christine; Woska, Joseph R.; Mennerich, Detlev; Brown, Su-Ellen; Mbow, M. Lamine; Kao, C. Cheng

    2015-01-01

    Improper signaling of the IL-36 receptor (IL-36R), a member of the IL-1 receptor family, has been associated with various inflammation-associated diseases. However, the requirements for IL-36R signal transduction remain poorly characterized. This work seeks to define the requirements for IL-36R signaling and intracellular trafficking. In the absence of cognate agonists, IL-36R was endocytosed and recycled to the plasma membrane. In the presence of IL-36, IL-36R increased accumulation in LAMP1+ lysosomes. Endocytosis predominantly used a clathrin-mediated pathway, and the accumulation of the IL-36R in lysosomes did not result in increased receptor turnover. The ubiquitin-binding Tollip protein contributed to IL-36R signaling and increased the accumulation of both subunits of the IL-36R. PMID:26269592

  18. Temporally precise in vivo control of intracellular signalling.

    PubMed

    Airan, Raag D; Thompson, Kimberly R; Fenno, Lief E; Bernstein, Hannah; Deisseroth, Karl

    2009-04-23

    In the study of complex mammalian behaviours, technological limitations have prevented spatiotemporally precise control over intracellular signalling processes. Here we report the development of a versatile family of genetically encoded optical tools ('optoXRs') that leverage common structure-function relationships among G-protein-coupled receptors (GPCRs) to recruit and control, with high spatiotemporal precision, receptor-initiated biochemical signalling pathways. In particular, we have developed and characterized two optoXRs that selectively recruit distinct, targeted signalling pathways in response to light. The two optoXRs exerted opposing effects on spike firing in nucleus accumbens in vivo, and precisely timed optoXR photostimulation in nucleus accumbens by itself sufficed to drive conditioned place preference in freely moving mice. The optoXR approach allows testing of hypotheses regarding the causal impact of biochemical signalling in behaving mammals, in a targetable and temporally precise manner.

  19. Intracellular autocrine VEGF signaling promotes EBDC cell proliferation, which can be inhibited by Apatinib.

    PubMed

    Peng, Sui; Zhang, Yanyan; Peng, Hong; Ke, Zunfu; Xu, Lixia; Su, Tianhong; Tsung, Allan; Tohme, Samer; Huang, Hai; Zhang, Qiuyang; Lencioni, Riccardo; Zeng, Zhirong; Peng, Baogang; Chen, Minhu; Kuang, Ming

    2016-04-10

    Tumor cells produce vascular endothelial growth factor (VEGF) which can interact with membrane or cytoplasmic VEGF receptors (VEGFRs) to promote cell growth. We aimed to investigate the role of extracellular/intracellular autocrine VEGF signaling and Apatinib, a highly selective VEGFR2 inhibitor, in extrahepatic bile duct cancer (EBDC). We found conditioned medium or recombinant human VEGF treatment promoted EBDC cell proliferation through a phospholipase C-γ1-dependent pathway. This pro-proliferative effect was diminished by VEGF, VEGFR1 or VEGFR2 neutralizing antibodies, but more significantly suppressed by intracellular VEGFR inhibitor. The rhVEGF induced intracellular VEGF signaling by promoting nuclear accumulation of pVEGFR1/2 and enhancing VEGF promoter activity, mRNA and protein expression. Internal VEGFR2 inhibitor Apatinib significantly inhibited intracellular VEGF signaling, suppressed cell proliferation in vitro and delayed xenograft tumor growth in vivo, while anti-VEGF antibody Bevacizumab showed no effect. Clinically, overexpression of pVEGFR1 and pVEGFR2 was significantly correlated with poorer overall survival (P = .007 and P = .020, respectively). In conclusion, the intracellular autocrine VEGF loop plays a predominant role in VEGF-induced cell proliferation. Apatinib is an effective intracellular VEGF pathway blocker that presents a great therapeutic potential in EBDC. PMID:26805764

  20. Phytanic acid and pristanic acid, branched-chain fatty acids associated with Refsum disease and other inherited peroxisomal disorders, mediate intracellular Ca2+ signaling through activation of free fatty acid receptor GPR40.

    PubMed

    Kruska, Nicol; Reiser, Georg

    2011-08-01

    The accumulation of the two branched-chain fatty acids phytanic acid and pristanic acid is known to play an important role in several diseases with peroxisomal impairment, like Refsum disease, Zellweger syndrome and α-methylacyl-CoA racemase deficiency. Recent studies elucidated that the toxic activity of phytanic acid and pristanic acid is mediated by multiple mitochondrial dysfunctions, generation of reactive oxygen species and Ca2+ deregulation via the InsP3-Ca2+ signaling pathway in glial cells. However, the exact signaling mechanism through which both fatty acids mediate toxicity is still under debate. Here, we studied the ability of phytanic acid and pristanic acid to activate the free fatty acid receptor GPR40, a G-protein-coupled receptor, which was described to be involved in the Ca2+ signaling of fatty acids. We treated HEK 293 cells expressing the GPR40 receptor with phytanic acid or pristanic acid. This resulted in a significant increase in the intracellular Ca2+ level, similar to the effect seen after treatment with the synthetic GPR40 agonist GW9508. Furthermore, we demonstrate that the GPR40 activation might be due to an interaction of the carboxylate moiety of fatty acids with the receptor. Our findings indicate that the phytanic acid- and pristanic acid-mediated Ca2+ deregulation can involve the activation of GPR40. Therefore, we suppose that activation of GPR40 might be part of the signaling cascade of the toxicity of phytanic and pristanic acids.

  1. Detection and Quantification of Intracellular Signaling Using FRET-Based Biosensors and High Content Imaging.

    PubMed

    Halls, Michelle L; Poole, Daniel P; Ellisdon, Andrew M; Nowell, Cameron J; Canals, Meritxell

    2015-01-01

    Förster resonance energy transfer (FRET) biosensors represent invaluable tools to detect the spatiotemporal context of second messenger production and intracellular signaling that cannot be attained using traditional methods. Here, we describe a detailed protocol for the use of high content imaging in combination with FRET biosensors to assess second messenger production and intracellular signaling in a time-effective manner. We use four different FRET biosensors to measure cAMP levels, kinase (ERK and PKC), and GTPase activity. Importantly, we provide the protocols to express and measure these sensors in a variety of model cell lines and primary dorsal root ganglia neurons.

  2. Detection and Quantification of Intracellular Signaling Using FRET-Based Biosensors and High Content Imaging.

    PubMed

    Halls, Michelle L; Poole, Daniel P; Ellisdon, Andrew M; Nowell, Cameron J; Canals, Meritxell

    2015-01-01

    Förster resonance energy transfer (FRET) biosensors represent invaluable tools to detect the spatiotemporal context of second messenger production and intracellular signaling that cannot be attained using traditional methods. Here, we describe a detailed protocol for the use of high content imaging in combination with FRET biosensors to assess second messenger production and intracellular signaling in a time-effective manner. We use four different FRET biosensors to measure cAMP levels, kinase (ERK and PKC), and GTPase activity. Importantly, we provide the protocols to express and measure these sensors in a variety of model cell lines and primary dorsal root ganglia neurons. PMID:26260599

  3. Fatty Acid Signaling: The New Function of Intracellular Lipases

    PubMed Central

    Papackova, Zuzana; Cahova, Monika

    2015-01-01

    Until recently, intracellular triacylglycerols (TAG) stored in the form of cytoplasmic lipid droplets have been considered to be only passive “energy conserves”. Nevertheless, degradation of TAG gives rise to a pleiotropic spectrum of bioactive intermediates, which may function as potent co-factors of transcription factors or enzymes and contribute to the regulation of numerous cellular processes. From this point of view, the process of lipolysis not only provides energy-rich equivalents but also acquires a new regulatory function. In this review, we will concentrate on the role that fatty acids liberated from intracellular TAG stores play as signaling molecules. The first part provides an overview of the transcription factors, which are regulated by fatty acids derived from intracellular stores. The second part is devoted to the role of fatty acid signaling in different organs/tissues. The specific contribution of free fatty acids released by particular lipases, hormone-sensitive lipase, adipose triacylglycerol lipase and lysosomal lipase will also be discussed. PMID:25674855

  4. Intracellular mechanisms of lymphoid cell activation.

    PubMed

    Fresa, K; Hameed, M; Cohen, S

    1989-01-01

    Activation of lymphocytes for proliferation is associated with the appearance of an intracellular factor (ADR) that can induce DNA synthesis in isolated quiescent nuclei. ADR plays a role in the sequence of intracellular events leading to activation for IL-2-mediated proliferation. Because of the nature of the defining assay, the locus of ADR action appears to be near the terminal end of the transduction pathway. Interestingly, although lymphocytes from aged individuals respond poorly to proliferative stimuli, they appear to produce normal to above-normal levels of ADR. In contrast, their nuclei are only poorly responsive to stimulation by ADR. Preparations rich in ADR activity have proteolytic activity as well. In addition, aprotinin, as well as a variety of other protease inhibitors, suppresses ADR-induced DNA synthesis in a dose-dependent manner. ADR activity can be removed from active extracts by absorption with aprotinin-conjugated agarose beads, and can be removed from the beads by elution at pH 5.0. This latter suggests that ADR itself is a protease. However, its endogenous substrate is not yet known. We have also detected an inhibitor of ADR activity in the cytoplasm of resting lymphocytes. This is a heat-stable protein of approximately 60,000 Da. In addition to suppressing the interaction of ADR with quiescent nuclei, the inhibitor can suppress DNA synthetic activity of replicative nuclei isolated from mitogen-activated lymphocytes. Interestingly, these preparations had little or no activity on replicative nuclei derived from several neoplastic cell lines. The resistance of tumor cell nuclei to spontaneously occurring cytoplasmic inhibitory factors such as the one described here may provide one explanation for the loss of growth control in neoplastic cells. PMID:2642767

  5. Modeling intracellular signaling underlying striatal function in health and disease.

    PubMed

    Nair, Anu G; Gutierrez-Arenas, Omar; Eriksson, Olivia; Jauhiainen, Alexandra; Blackwell, Kim T; Kotaleski, Jeanette H

    2014-01-01

    Striatum, which is the input nucleus of the basal ganglia, integrates cortical and thalamic glutamatergic inputs with dopaminergic afferents from the substantia nigra pars compacta. The combination of dopamine and glutamate strongly modulates molecular and cellular properties of striatal neurons and the strength of corticostriatal synapses. These actions are performed via intracellular signaling networks, containing several intertwined feedback loops. Understanding the role of dopamine and other neuromodulators requires the development of quantitative dynamical models for describing the intracellular signaling, in order to provide precise unambiguous descriptions and quantitative predictions. Building such models requires integration of data from multiple data sources containing information regarding the molecular interactions, the strength of these interactions, and the subcellular localization of the molecules. Due to the uncertainty, variability, and sparseness of these data, parameter estimation techniques are critical for inferring or constraining the unknown parameters, and sensitivity analysis evaluates which parameters are most critical for a given observed macroscopic behavior. Here, we briefly review the modeling approaches and tools that have been used to investigate biochemical signaling in the striatum, along with some of the models built around striatum. We also suggest a future direction for the development of such models from the, now becoming abundant, high-throughput data.

  6. Modeling intracellular signaling underlying striatal function in health and disease.

    PubMed

    Nair, Anu G; Gutierrez-Arenas, Omar; Eriksson, Olivia; Jauhiainen, Alexandra; Blackwell, Kim T; Kotaleski, Jeanette H

    2014-01-01

    Striatum, which is the input nucleus of the basal ganglia, integrates cortical and thalamic glutamatergic inputs with dopaminergic afferents from the substantia nigra pars compacta. The combination of dopamine and glutamate strongly modulates molecular and cellular properties of striatal neurons and the strength of corticostriatal synapses. These actions are performed via intracellular signaling networks, containing several intertwined feedback loops. Understanding the role of dopamine and other neuromodulators requires the development of quantitative dynamical models for describing the intracellular signaling, in order to provide precise unambiguous descriptions and quantitative predictions. Building such models requires integration of data from multiple data sources containing information regarding the molecular interactions, the strength of these interactions, and the subcellular localization of the molecules. Due to the uncertainty, variability, and sparseness of these data, parameter estimation techniques are critical for inferring or constraining the unknown parameters, and sensitivity analysis evaluates which parameters are most critical for a given observed macroscopic behavior. Here, we briefly review the modeling approaches and tools that have been used to investigate biochemical signaling in the striatum, along with some of the models built around striatum. We also suggest a future direction for the development of such models from the, now becoming abundant, high-throughput data. PMID:24560149

  7. Kinetic insulation as an effective mechanism for achieving pathway specificity in intracellular signaling networks

    PubMed Central

    Behar, Marcelo; Dohlman, Henrik G.; Elston, Timothy C.

    2007-01-01

    Intracellular signaling pathways that share common components often elicit distinct physiological responses. In most cases, the biochemical mechanisms responsible for this signal specificity remain poorly understood. Protein scaffolds and cross-inhibition have been proposed as strategies to prevent unwanted cross-talk. Here, we report a mechanism for signal specificity termed “kinetic insulation.” In this approach signals are selectively transmitted through the appropriate pathway based on their temporal profile. In particular, we demonstrate how pathway architectures downstream of a common component can be designed to efficiently separate transient signals from signals that increase slowly over time. Furthermore, we demonstrate that upstream signaling proteins can generate the appropriate input to the common pathway component regardless of the temporal profile of the external stimulus. Our results suggest that multilevel signaling cascades may have evolved to modulate the temporal profile of pathway activity so that stimulus information can be efficiently encoded and transmitted while ensuring signal specificity. PMID:17913886

  8. Heat shock protein 70 (Hsp70) interacts with the Notch1 intracellular domain and contributes to the activity of Notch signaling in myelin-reactive CD4 T cells.

    PubMed

    Juryńczyk, Maciej; Lewkowicz, Przemysław; Domowicz, Małgorzata; Mycko, Marcin P; Selmaj, Krzysztof W

    2015-10-15

    Notch receptors (Notch1-4) are involved in the differentiation of CD4 T cells and the development of autoimmunity. Mechanisms regulating Notch signaling in CD4 T cells are not fully elucidated. In this study we investigated potential crosstalk between Notch pathway molecules and heat shock protein 70 (Hsp70), the major intracellular chaperone involved in the protein transport during immune responses and other stress conditions. Using Hsp70(-/-) mice we found that Hsp70 is critical for up-regulation of NICD1 and induction of Notch target genes in Jagged1- and Delta-like1-stimulated CD4 T cells. Co-immunoprecipitation analysis of wild-type CD4 T cells stimulated with either Jagged1 or Delta-like1 showed a direct interaction between NICD1 and Hsp70. Both molecules co-localized within the nucleus of CD4 T cells stimulated with Notch ligands. Molecular interaction and nuclear colocalization of NICD1 and Hsp70 were also detected in CD4 T cells reactive against myelin oligodendrocyte glycoprotein (MOG)35-55, which showed Hsp70-dependent up-regulation of both NICD1 and Notch target genes. In conclusion, we demonstrate for the first time that Hsp70 interacts with NICD1 and contributes to the activity of Notch signaling in CD4 T cells. Interaction between Hsp70 and NICD1 may represent a novel mechanism regulating Notch signaling in activated CD4 T cells.

  9. Acoustic tweezers for studying intracellular calcium signaling in SKBR-3 human breast cancer cells.

    PubMed

    Hwang, Jae Youn; Yoon, Chi Woo; Lim, Hae Gyun; Park, Jin Man; Yoon, Sangpil; Lee, Jungwoo; Shung, K Kirk

    2015-12-01

    Extracellular matrix proteins such as fibronectin (FNT) play crucial roles in cell proliferation, adhesion, and migration. For better understanding of these associated cellular activities, various microscopic manipulation tools have been used to study their intracellular signaling pathways. Recently, it has appeared that acoustic tweezers may possess similar capabilities in the study. Therefore, we here demonstrate that our newly developed acoustic tweezers with a high-frequency lithium niobate ultrasonic transducer have potentials to study intracellular calcium signaling by FNT-binding to human breast cancer cells (SKBR-3). It is found that intracellular calcium elevations in SKBR-3 cells, initially occurring on the microbead-contacted spot and then eventually spreading over the entire cell, are elicited by attaching an acoustically trapped FNT-coated microbead. Interestingly, they are suppressed by either extracellular calcium elimination or phospholipase C (PLC) inhibition. Hence, this suggests that our acoustic tweezers may serve as an alternative tool in the study of intracellular signaling by FNT-binding activities.

  10. 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.

    PubMed

    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.

  11. Capsaicin mimics mechanical load-induced intracellular signaling events: involvement of TRPV1-mediated calcium signaling in induction of skeletal muscle hypertrophy.

    PubMed

    Ito, Naoki; Ruegg, Urs T; Kudo, Akira; Miyagoe-Suzuki, Yuko; Takeda, Shin'ichi

    2013-01-01

    Mechanical load-induced intracellular signaling events are important for subsequent skeletal muscle hypertrophy. We previously showed that load-induced activation of the cation channel TRPV1 caused an increase in intracellular calcium concentrations ([Ca ( 2+) ]i) and that this activated mammalian target of rapamycin (mTOR) and promoted muscle hypertrophy. However, the link between mechanical load-induced intracellular signaling events, and the TRPV1-mediated increases in [Ca ( 2+) ]i are not fully understood. Here we show that administration of the TRPV1 agonist, capsaicin, induces phosphorylation of mTOR, p70S6K, S6, Erk1/2 and p38 MAPK, but not Akt, AMPK or GSK3β. Furthermore, the TRPV1-induced phosphorylation patterns resembled those induced by mechanical load. Our results continue to highlight the importance of TRPV1-mediated calcium signaling in load-induced intracellular signaling pathways.

  12. Simplest relationship between local field potential and intracellular signals in layered neural tissue

    NASA Astrophysics Data System (ADS)

    Chizhov, Anton V.; Sanchez-Aguilera, Alberto; Rodrigues, Serafim; de la Prida, Liset Menendez

    2015-12-01

    The relationship between the extracellularly measured electric field potential resulting from synaptic activity in an ensemble of neurons and intracellular signals in these neurons is an important but still open question. Based on a model neuron with a cylindrical dendrite and lumped soma, we derive a formula that substantiates a proportionality between the local field potential and the total somatic transmembrane current that emerges from the difference between the somatic and dendritic membrane potentials. The formula is tested by intra- and extracellular recordings of evoked synaptic responses in hippocampal slices. Additionally, the contribution of different membrane currents to the field potential is demonstrated in a two-population mean-field model. Our formalism, which allows for a simple estimation of unknown dendritic currents directly from somatic measurements, provides an interpretation of the local field potential in terms of intracellularly measurable synaptic signals. It is also applicable to the study of cortical activity using two-compartment neuronal population models.

  13. Simplest relationship between local field potential and intracellular signals in layered neural tissue.

    PubMed

    Chizhov, Anton V; Sanchez-Aguilera, Alberto; Rodrigues, Serafim; de la Prida, Liset Menendez

    2015-12-01

    The relationship between the extracellularly measured electric field potential resulting from synaptic activity in an ensemble of neurons and intracellular signals in these neurons is an important but still open question. Based on a model neuron with a cylindrical dendrite and lumped soma, we derive a formula that substantiates a proportionality between the local field potential and the total somatic transmembrane current that emerges from the difference between the somatic and dendritic membrane potentials. The formula is tested by intra- and extracellular recordings of evoked synaptic responses in hippocampal slices. Additionally, the contribution of different membrane currents to the field potential is demonstrated in a two-population mean-field model. Our formalism, which allows for a simple estimation of unknown dendritic currents directly from somatic measurements, provides an interpretation of the local field potential in terms of intracellularly measurable synaptic signals. It is also applicable to the study of cortical activity using two-compartment neuronal population models. PMID:26764724

  14. Intracellular sensing of complement C3 activates cell autonomous immunity

    PubMed Central

    Tam, Jerry C.H.; Bidgood, Susanna R.; McEwan, William A.; James, Leo C.

    2014-01-01

    Pathogens traverse multiple barriers during infection including cell membranes. Here we show that during this transition pathogens carry covalently attached complement C3 into the cell, triggering immediate signalling and effector responses. Sensing of C3 in the cytosol activates MAVS-dependent signalling cascades and induces proinflammatory cytokine secretion. C3 also flags viruses for rapid proteasomal degradation, thereby preventing their replication. This system can detect both viral and bacterial pathogens but is antagonized by enteroviruses, such as rhinovirus and poliovirus, which cleave C3 using their 3C protease. The antiviral Rupintrivir inhibits 3C protease and prevents C3 cleavage, rendering enteroviruses susceptible to intracellular complement sensing. Thus, complement C3 allows cells to detect and disable pathogens that have invaded the cytosol. PMID:25190799

  15. Ultraviolet B radiation-mediated inhibition of interferon-gamma-induced keratinocyte activation is independent of interleukin-10 and other soluble mediators but associated with enhanced intracellular suppressors of cytokine-signaling expression.

    PubMed

    Friedrich, Markus; Holzmann, Ruth; Sterry, Wolfram; Wolk, Kerstin; Truppel, Andreas; Piazena, Helmut; Schonbein, Christiane; Sabat, Robert; Asadullah, Khusru

    2003-10-01

    Ultraviolet irradiation represents a well-established treatment modality for inflammatory skin diseases. The aim of this study was to investigate the mechanisms of ultraviolet B radiation-induced keratinocyte insensitivity towards interferon-gamma. Flow cytometric analyses indicated that ultraviolet B radiation temporarily inhibits the interferon-gamma-induced activation of primary keratinocyte and HaCaT cells as measured by reduced intercellular adhesion molecule-1 (CD54) and HLA-DR upregulation. Western blot experiments have suggested that this is mediated by the ultraviolet B radiation-induced inhibition of signal transduction and transcription factor-1 phosphorylation. Neither interleukin-10 neutralization nor interleukin-10 addition had any effect on the ultraviolet B radiation-induced inhibition of interferon-gamma induced intercellular adhesion molecule-1 expression. Furthermore, the supernatant from ultraviolet B-irradiated cells failed to inhibit the interferon-gamma-induced CD54 and HLA-DR upregulation in nonradiated HaCaT cells. Moreover, irradiated cells from whom the supernatant was withdrawn 4 h after irradiation still showed a diminished interferon-gamma-induced response after 24 h. Thus, not soluble but intracellular factors might be involved in the ultraviolet B radiation-induced inhibition of interferon-gamma-induced keratinocyte activation. Therefore, we analyzed the expression of members of suppressors of cytokine-signaling (SOCS) molecules using real-time polymerase chain reaction. We found a fast and strong upregulation of SOCS1 and SOCS3 but not of SOCS2 after ultraviolet B radiation. Similarly, ultraviolet B radiation induced the expression of these particular SOCS molecules in lesional psoriatic skin. As SOCS molecules are known inhibitors of signal transduction and transcription factor phosphorylation, which is essential for interferon-gamma-induced intercellular adhesion molecule-1 and HLA-DR upregulation, this may explain the interferon

  16. Noise decomposition of intracellular biochemical signaling networks using nonequivalent reporters.

    PubMed

    Rhee, Alex; Cheong, Raymond; Levchenko, Andre

    2014-12-01

    Experimental measurements of biochemical noise have primarily focused on sources of noise at the gene expression level due to limitations of existing noise decomposition techniques. Here, we introduce a mathematical framework that extends classical extrinsic-intrinsic noise analysis and enables mapping of noise within upstream signaling networks free of such restrictions. The framework applies to systems for which the responses of interest are linearly correlated on average, although the framework can be easily generalized to the nonlinear case. Interestingly, despite the high degree of complexity and nonlinearity of most mammalian signaling networks, three distinct tumor necrosis factor (TNF) signaling network branches displayed linearly correlated responses, in both wild-type and perturbed versions of the network, across multiple orders of magnitude of ligand concentration. Using the noise mapping analysis, we find that the c-Jun N-terminal kinase (JNK) pathway generates higher noise than the NF-κB pathway, whereas the activation of c-Jun adds a greater amount of noise than the activation of ATF-2. In addition, we find that the A20 protein can suppress noise in the activation of ATF-2 by separately inhibiting the TNF receptor complex and JNK pathway through a negative feedback mechanism. These results, easily scalable to larger and more complex networks, pave the way toward assessing how noise propagates through cellular signaling pathways and create a foundation on which we can further investigate the relationship between signaling system architecture and biological noise.

  17. Noise decomposition of intracellular biochemical signaling networks using nonequivalent reporters

    PubMed Central

    Rhee, Alex; Cheong, Raymond; Levchenko, Andre

    2014-01-01

    Experimental measurements of biochemical noise have primarily focused on sources of noise at the gene expression level due to limitations of existing noise decomposition techniques. Here, we introduce a mathematical framework that extends classical extrinsic–intrinsic noise analysis and enables mapping of noise within upstream signaling networks free of such restrictions. The framework applies to systems for which the responses of interest are linearly correlated on average, although the framework can be easily generalized to the nonlinear case. Interestingly, despite the high degree of complexity and nonlinearity of most mammalian signaling networks, three distinct tumor necrosis factor (TNF) signaling network branches displayed linearly correlated responses, in both wild-type and perturbed versions of the network, across multiple orders of magnitude of ligand concentration. Using the noise mapping analysis, we find that the c-Jun N-terminal kinase (JNK) pathway generates higher noise than the NF-κB pathway, whereas the activation of c-Jun adds a greater amount of noise than the activation of ATF-2. In addition, we find that the A20 protein can suppress noise in the activation of ATF-2 by separately inhibiting the TNF receptor complex and JNK pathway through a negative feedback mechanism. These results, easily scalable to larger and more complex networks, pave the way toward assessing how noise propagates through cellular signaling pathways and create a foundation on which we can further investigate the relationship between signaling system architecture and biological noise. PMID:25404303

  18. The emerging role of phosphoinositide clustering in intracellular trafficking and signal transduction

    PubMed Central

    Picas, Laura; Gaits-Iacovoni, Frederique; Goud, Bruno

    2016-01-01

    Phosphoinositides are master regulators of multiple cellular processes: from vesicular trafficking to signaling, cytoskeleton dynamics, and cell growth. They are synthesized by the spatiotemporal regulated activity of phosphoinositide-metabolizing enzymes. The recent observation that some protein modules are able to cluster phosphoinositides suggests that alternative or complementary mechanisms might operate to stabilize the different phosphoinositide pools within cellular compartments. Herein, we discuss the different known and potential molecular players that are prone to engage phosphoinositide clustering and elaborate on how such a mechanism might take part in the regulation of intracellular trafficking and signal transduction. PMID:27092250

  19. An adipocentric view of signaling and intracellular trafficking.

    PubMed

    Mora, Silvia; Pessin, Jeffrey E

    2002-01-01

    Adipocytes have traditionally been considered to be the primary site for whole body energy storage mainly in the form of triglycerides and fatty acids. This occurs through the ability of insulin to markedly stimulate both glucose uptake and lipogenesis. Conventional wisdom held that defects in fuel partitioning into adipocytes either because of increased adipose tissue mass and/or increased lipolysis and circulating free fatty acids resulted in dyslipidemia, obesity, insulin resistance and perhaps diabetes. However, it has become increasingly apparent that loss of adipose tissue (lipodystrophies) in both animal models and humans also leads to metabolic disorders that result in severe states of insulin resistance and potential diabetes. These apparently opposite functions can be resolved by the establishment of adipocytes not only as a fuel storage depot but also as a critical endocrine organ that secretes a variety of signaling molecules into the circulation. Although the molecular function of these adipocyte-derived signals are poorly understood, they play a central role in the maintenance of energy homeostasis by regulating insulin secretion, insulin action, glucose and lipid metabolism, energy balance, host defense and reproduction. The diversity of these secretory factors include enzymes (lipoprotein lipase (LPL) and adipsin), growth factors [vascular endothelial growth factor (VEGF)], cytokines (tumor necrosis factor-alpha, interleukin 6) and several other hormones involved in fatty acid and glucose metabolism (leptin, Acrp30, resistin and acylation stimulation protein). Despite the large number of molecules secreted by adipocytes, our understanding of the pathways and mechanisms controlling intracellular trafficking and exocytosis in adipocytes is poorly understood. In this article, we will review the current knowledge of the trafficking and secretion processes that take place in adipocytes, focusing our attention on two of the best characterized adipokine

  20. Mechanisms by which the inhibition of specific intracellular signaling pathways increase osteoblast proliferation on apatite surfaces.

    PubMed

    Yang, Seungwon; Tian, Yu-Shun; Lee, Yun-Jung; Yu, Frank H; Kim, Hyun-Man

    2011-04-01

    Osteoblasts proliferate slowly on the surface of calcium phosphate apatite which is widely used as a substrate biomaterial in bone regeneration. Owing to poor adhesion signaling in the cells grown on the calcium phosphate surface, inadequate growth factor signaling is generated to trigger cell cycle progression. The present study investigated an intracellular signal transduction pathway involved in the slow cell proliferation in osteoblasts grown on the calcium phosphate surface. Small GTPase RhoA and phosphatase and tensin homolog (PTEN) were more activated in cells grown on the surface of calcium phosphate apatite than on tissue culture plate. Specific inhibition of RhoA and PTEN induced the cells on calcium phosphate apatite surface to proliferate at a similar rate as cells on tissue culture plate surface. Specific inhibition of ROCK, which is a downstream effector of RhoA and an upstream activator of PTEN also increased proliferation of these osteoblasts. Present results indicate that physical property of calcium phosphate crystals that impede cell proliferation may be surmounted by the inhibition of the RhoA/ROCK/PTEN pathway to rescue delayed proliferation of osteoblasts on the calcium phosphate apatite surface. In addition, specific inhibition of ROCK promoted cell migration and osteoblast differentiation. Inhibition of the RhoA/ROCK/PTEN intracellular signaling pathway is expected to enhance cell activity to promote and accelerate bone regeneration on the calcium phosphate apatite surface.

  1. Mitochondrial transporters as novel targets for intracellular calcium signaling.

    PubMed

    Satrústegui, Jorgina; Pardo, Beatriz; Del Arco, Araceli

    2007-01-01

    Ca(2+) signaling in mitochondria is important to tune mitochondrial function to a variety of extracellular stimuli. The main mechanism is Ca(2+) entry in mitochondria via the Ca(2+) uniporter followed by Ca(2+) activation of three dehydrogenases in the mitochondrial matrix. This results in increases in mitochondrial NADH/NAD ratios and ATP levels and increased substrate uptake by mitochondria. We review evidence gathered more than 20 years ago and recent work indicating that substrate uptake, mitochondrial NADH/NAD ratios, and ATP levels may be also activated in response to cytosolic Ca(2+) signals via a mechanism that does not require the entry of Ca(2+) in mitochondria, a mechanism depending on the activity of Ca(2+)-dependent mitochondrial carriers (CaMC). CaMCs fall into two groups, the aspartate-glutamate carriers (AGC) and the ATP-Mg/P(i) carriers, also named SCaMC (for short CaMC). The two mammalian AGCs, aralar and citrin, are members of the malate-aspartate NADH shuttle, and citrin, the liver AGC, is also a member of the urea cycle. Both types of CaMCs are activated by Ca(2+) in the intermembrane space and function together with the Ca(2+) uniporter in decoding the Ca(2+) signal into a mitochondrial response.

  2. Nanosecond pulsed electric fields modulate cell function through intracellular signal transduction mechanisms.

    PubMed

    Beebe, Stephen J; Blackmore, Peter F; White, Jody; Joshi, Ravindra P; Schoenbach, Karl H

    2004-08-01

    These studies describe the effects of nanosecond (10-300 ns) pulsed electric fields (nsPEF) on mammalian cell structure and function. As the pulse durations decrease, effects on the plasma membrane (PM) decrease and effects on intracellular signal transduction mechanisms increase. When nsPEF-induced PM electroporation effects occur, they are distinct from classical PM electroporation effects, suggesting unique, nsPEF-induced PM modulations. In HL-60 cells, nsPEF that are well below the threshold for PM electroporation and apoptosis induction induce effects that are similar to purinergic agonistmediated calcium release from intracellular stores, which secondarily initiate capacitive calcium influx through store-operated calcium channels in the PM. NsPEF with durations and electric field intensities that do or do not cause PM electroporation, induce apoptosis in mammalian cells with a well-characterized phenotype typified by externalization of phosphatidylserine on the outer PM and activation of caspase proteases. Treatment of mouse fibrosarcoma tumors with nsPEF also results in apoptosis induction. When Jurkat cells were transfected by electroporation and then treated with nsPEF, green fluorescent protein expression was enhanced compared to electroporation alone. The results indicate that nsPEF activate intracellular mechanisms that can determine cell function and fate, providing an important new tool for probing signal transduction mechanisms that modulate cell structure and function and for potential therapeutic applications for cancer and gene therapy.

  3. Host Intracellular Signaling Events and Pro-inflammatory Cytokine Production in African Trypanosomiasis.

    PubMed

    Kuriakose, Shiby M; Singh, Rani; Uzonna, Jude E

    2016-01-01

    Pathogens, such as bacteria, viruses, and parasites, possess specific molecules or proteins that are recognized by several host innate immune receptors, leading to the activation of several intracellular signaling molecules and pathways. The magnitude and quality of these events significantly affect the outcome of infection. African trypanosomes, including Trypanosoma congolense, are capable of manipulating the host immune response, including the activity of macrophages, which are the key immune cells that contribute to the immunopathogenesis of African trypanosomiasis. Although it is known that immune hyperactivation and excessive pro-inflammatory cytokine production are the hallmarks of African trypanosomiasis, the mechanisms through which these events are triggered are poorly defined. However, it is known that macrophages may play a significant role in these processes, because phagocytosis of trypanosomes by macrophages initiates intracellular signal transduction cascades that lead to the release of pro-inflammatory cytokines and alteration in cell function. This review highlights recent progress in our understanding of the innate immune receptors, signaling pathways, and transcription factors involved in T. congolense-induced pro-inflammatory cytokine production in macrophages. It will reveal the existence of complex signaling events through which the parasite modulates the host immune response, thus identifying novel targets that could aid in designing strategies to effectively control the disease. PMID:27242788

  4. Host Intracellular Signaling Events and Pro-inflammatory Cytokine Production in African Trypanosomiasis

    PubMed Central

    Kuriakose, Shiby M.; Singh, Rani; Uzonna, Jude E.

    2016-01-01

    Pathogens, such as bacteria, viruses, and parasites, possess specific molecules or proteins that are recognized by several host innate immune receptors, leading to the activation of several intracellular signaling molecules and pathways. The magnitude and quality of these events significantly affect the outcome of infection. African trypanosomes, including Trypanosoma congolense, are capable of manipulating the host immune response, including the activity of macrophages, which are the key immune cells that contribute to the immunopathogenesis of African trypanosomiasis. Although it is known that immune hyperactivation and excessive pro-inflammatory cytokine production are the hallmarks of African trypanosomiasis, the mechanisms through which these events are triggered are poorly defined. However, it is known that macrophages may play a significant role in these processes, because phagocytosis of trypanosomes by macrophages initiates intracellular signal transduction cascades that lead to the release of pro-inflammatory cytokines and alteration in cell function. This review highlights recent progress in our understanding of the innate immune receptors, signaling pathways, and transcription factors involved in T. congolense-induced pro-inflammatory cytokine production in macrophages. It will reveal the existence of complex signaling events through which the parasite modulates the host immune response, thus identifying novel targets that could aid in designing strategies to effectively control the disease. PMID:27242788

  5. Host Intracellular Signaling Events and Pro-inflammatory Cytokine Production in African Trypanosomiasis.

    PubMed

    Kuriakose, Shiby M; Singh, Rani; Uzonna, Jude E

    2016-01-01

    Pathogens, such as bacteria, viruses, and parasites, possess specific molecules or proteins that are recognized by several host innate immune receptors, leading to the activation of several intracellular signaling molecules and pathways. The magnitude and quality of these events significantly affect the outcome of infection. African trypanosomes, including Trypanosoma congolense, are capable of manipulating the host immune response, including the activity of macrophages, which are the key immune cells that contribute to the immunopathogenesis of African trypanosomiasis. Although it is known that immune hyperactivation and excessive pro-inflammatory cytokine production are the hallmarks of African trypanosomiasis, the mechanisms through which these events are triggered are poorly defined. However, it is known that macrophages may play a significant role in these processes, because phagocytosis of trypanosomes by macrophages initiates intracellular signal transduction cascades that lead to the release of pro-inflammatory cytokines and alteration in cell function. This review highlights recent progress in our understanding of the innate immune receptors, signaling pathways, and transcription factors involved in T. congolense-induced pro-inflammatory cytokine production in macrophages. It will reveal the existence of complex signaling events through which the parasite modulates the host immune response, thus identifying novel targets that could aid in designing strategies to effectively control the disease.

  6. Anti-cancer drugs interfere with intracellular calcium signaling.

    PubMed

    Florea, Ana-Maria; Büsselberg, Dietrich

    2009-09-01

    (Neuro-)toxicity of metal and metal compounds is frequently highlighted. While specific metals or metal compounds are essential for cellular function, other metals are toxic and/or carcinogens. Metals can trigger accidental cell death in the form of necrosis, or activate programmed cell death in the form of apoptosis. The aim of anti-cancer therapy is induction of apoptosis in tumor cells. Therefore, there is an interesting twist in the toxicity of metals and metal compounds (e.g., arsenic trioxide, cisplatin); since they have a higher specificity to induce apoptosis in cancer cells (possibly due to the high turnover in these cells) they are used to cure some forms of cancer. A body of evidence suggests that second messengers, such as modulations in the intracellular calcium concentration, could be involved in metals induced toxicity as well as in the beneficial effects shown by anti-cancer drugs. Here we review the influence on calcium homeostasis induced by some metallic compounds: cisplatin, arsenic trioxide and trimethyltin chloride.

  7. Study of neurotoxic intracellular calcium signalling triggered by amyloids.

    PubMed

    Villalobos, Carlos; Caballero, Erica; Sanz-Blasco, Sara; Núñez, Lucía

    2012-01-01

    Neurotoxicity in Alzheimer's disease (AD) is associated to dishomeostasis of intracellular Ca(2+) induced by amyloid β peptide (Aβ) species. Understanding of the effects of Aβ on intracellular Ca(2+) homeostasis requires preparation of the different Aβ assemblies including oligomers and fibrils and the testing of their effects on cytosolic and mitochondrial Ca(2+) in neurons. Procedures for cerebellar granule cell culture, preparation of Aβ species as well as fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca(2+) in neurons are described.

  8. Role of conserved intracellular motifs in Serrate signalling, cis-inhibition and endocytosis

    PubMed Central

    Glittenberg, Marcus; Pitsouli, Chrysoula; Garvey, Clare; Delidakis, Christos; Bray, Sarah

    2006-01-01

    Notch is the receptor in a signalling pathway that operates in a diverse spectrum of developmental processes. Its ligands (e.g. Serrate) are transmembrane proteins whose signalling competence is regulated by the endocytosis-promoting E3 ubiquitin ligases, Mindbomb1 and Neuralized. The ligands also inhibit Notch present in the same cell (cis-inhibition). Here, we identify two conserved motifs in the intracellular domain of Serrate that are required for efficient endocytosis. The first, a dileucine motif, is dispensable for trans-activation and cis-inhibition despite the endocytic defect, demonstrating that signalling can be separated from bulk endocytosis. The second, a novel motif, is necessary for interactions with Mindbomb1/Neuralized and is strictly required for Serrate to trans-activate and internalise efficiently but not for it to inhibit Notch signalling. Cis-inhibition is compromised when an ER retention signal is added to Serrate, or when the levels of Neuralized are increased, and together these data indicate that cis-inhibitory interactions occur at the cell surface. The balance of ubiquitinated/unubiquitinated ligand will thus affect the signalling capacity of the cell at several levels. PMID:17006545

  9. Intracellular observation of nanocarriers modified with a mitochondrial targeting signal peptide.

    PubMed

    Kawamura, Eriko; Yamada, Yuma; Yasuzaki, Yukari; Hyodo, Mamoru; Harashima, Hideyoshi

    2013-11-01

    This study focused on the intracellular observation of nanocarriers modified with a mitochondrial targeting signal peptide (MTS). The nanocarriers showed an efficient cellular uptake, and the MTS had a positive effect on their mitochondrial targeting. This is the first report of an intracellular observation of nanocarriers modified with MTS.

  10. Intracellular Signaling Pathways Involved in Childhood Acute Lymphoblastic Leukemia; Molecular Targets.

    PubMed

    Layton Tovar, Cristian Fabián; Mendieta Zerón, Hugo

    2016-06-01

    Acute lymphoblastic leukemia (ALL) is a malignant disease characterized by an uncontrolled proliferation of immature lymphoid cells. ALL is the most common hematologic malignancy in early childhood, and it reaches peak incidence between the ages of 2 and 3 years. The prognosis of ALL is associated with aberrant gene expression, in addition to the presence of numerical or structural chromosomal alterations, age, race, and immunophenotype. The Relapse rate with regard to pharmacological treatment rises in childhood; thus, the expression of biomarkers associated with the activation of cell signaling pathways is crucial to establish the disease prognosis. Intracellular pathways involved in ALL are diverse, including Janus kinase/Signal transducers and transcription activators (JAK-STAT), Phosphoinositide-3-kinase-protein kinase B (PI3K-AKT), Ras mitogen-activated protein kinase (Ras-MAPK), Glycogen synthase kinase-3β (GSK-3β), Nuclear factor-kappa beta (NF-κB), and Hypoxia-inducible transcription factor 1α (HIF-1α), among others. In this review, we present several therapeutic targets, intracellular pathways, and molecular markers that are being studied extensively at present.

  11. Minireview: Role of intracellular scaffolding proteins in the regulation of endocrine G protein-coupled receptor signaling.

    PubMed

    Walther, Cornelia; Ferguson, Stephen S G

    2015-06-01

    The majority of hormones stimulates and mediates their signal transduction via G protein-coupled receptors (GPCRs). The signal is transmitted into the cell due to the association of the GPCRs with heterotrimeric G proteins, which in turn activates an extensive array of signaling pathways to regulate cell physiology. However, GPCRs also function as scaffolds for the recruitment of a variety of cytoplasmic protein-interacting proteins that bind to both the intracellular face and protein interaction motifs encoded by GPCRs. The structural scaffolding of these proteins allows GPCRs to recruit large functional complexes that serve to modulate both G protein-dependent and -independent cellular signaling pathways and modulate GPCR intracellular trafficking. This review focuses on GPCR interacting PSD95-disc large-zona occludens domain containing scaffolds in the regulation of endocrine receptor signaling as well as their potential role as therapeutic targets for the treatment of endocrinopathies.

  12. Feedback Regulation of Cell-Substratum Adhesion by Integrin-Mediated Intracellular Ca2+ Signaling

    NASA Astrophysics Data System (ADS)

    Sjaastad, Michael D.; Angres, Brigitte; Lewis, Richard S.; Nelson, W. James

    1994-08-01

    Integrin binding to extracellular matrix (ECM) regulates cell migration and gene expression in embryogenesis, metastasis, wound healing, and the inflammatory response. In many cases, binding of integrins to ECM triggers intracellular signaling pathways. The regulatory roles of intracellular signaling mechanisms in these events are poorly understood. Using single-cell analysis, we demonstrate that beads coated with peptide containing Arg-Gly-Asp (RGD), an integrin recognition motif found in many ECM proteins, elicit a rapid transient increase in intracellular calcium in Madin-Darby canine kidney (MDCK) epithelial cells. Also, significantly more beads bind to responding cells than to nonresponders. Several independent methods that inhibit RGD-induced Ca2+ signaling decrease both the number of beads bound and the strength of adhesion to an RGD-coated substratum. These results indicate that intracellular Ca2+ signaling participates in a positive feedback loop that enhances integrin-mediated cell adhesion

  13. Intracellular signaling via ERK/MAPK completes the pathway for tubulogenic fibronectin in MDCK cells.

    PubMed

    Liu, Zhao; Greco, Andres J; Hellman, Nathan E; Spector, June; Robinson, Jonathan; Tang, Oliver T; Lipschutz, Joshua H

    2007-02-16

    A classic in vitro model of branching morphogenesis utilizes the Madin-Darby canine kidney (MDCK) cell line. MDCK Strain II cells form hollow monoclonal cysts in a three-dimensional collagen matrix over the course of 10 days and tubulate in response to hepatocyte growth factor (HGF). We and our colleagues previously showed that activation of the extracellular-signal regulated kinase (ERK, aka MAPK) pathway is necessary and sufficient to induce tubulogenesis in MDCK cells. We also showed in a microarray study that one of the genes upregulated by HGF was the known tubulogene fibronectin. Given that HGF activates a multitude of signaling pathways, including ERK/MAPK, to test the intracellular regulatory pathway, we used two distinct inhibitors of ERK activation (U0126 and PD098059). Following induction of MDCK Type II cells with HGF, tubulogenic fibronectin mRNA was upregulated fourfold by real-time PCR, and minimal or no change in fibronectin expression was seen when HGF was added with either U0126 or PD098059. We confirmed these results using an MDCK cell line inducible for Raf, which is upstream of ERK. Following activation of Raf, fibronectin mRNA and protein expression were increased to a similar degree as was seen following HGF induction. Furthermore, MDCK Strain I cells, which originate from collecting ducts and have constitutively active ERK, spontaneously initiate tubulogenesis. We show here that MDCK Strain I cells have high levels of fibronectin mRNA and protein compared to MDCK Strain II cells. When U0126 and PD098059 were added to MDCK Strain I cells, fibronectin mRNA, and protein levels were decreased to levels seen in MDCK Strain II cells. These data allow us to complete what we believe is the first description of a tubulogenic pathway from receptor/ligand (HGF/CMET), through an intracellular signaling pathway (ERK/MAPK), to transcription and, finally, secretion of a critical tubuloprotein (fibronectin).

  14. Intracellular Redox Compartmentation and ROS-Related Communication in Regulation and Signaling.

    PubMed

    Noctor, Graham; Foyer, Christine H

    2016-07-01

    Recent years have witnessed enormous progress in understanding redox signaling related to reactive oxygen species (ROS) in plants. The consensus view is that such signaling is intrinsic to many developmental processes and responses to the environment. ROS-related redox signaling is tightly wedded to compartmentation. Because membranes function as barriers, highly redox-active powerhouses such as chloroplasts, peroxisomes, and mitochondria may elicit specific signaling responses. However, transporter functions allow membranes also to act as bridges between compartments, and so regulated capacity to transmit redox changes across membranes influences the outcome of triggers produced at different locations. As well as ROS and other oxidizing species, antioxidants are key players that determine the extent of ROS accumulation at different sites and that may themselves act as signal transmitters. Like ROS, antioxidants can be transported across membranes. In addition, the intracellular distribution of antioxidative enzymes may be modulated to regulate or facilitate redox signaling appropriate to the conditions. Finally, there is substantial plasticity in organellar shape, with extensions such as stromules, peroxules, and matrixules playing potentially crucial roles in organelle-organelle communication. We provide an overview of the advances in subcellular compartmentation, identifying the gaps in our knowledge and discussing future developments in the area. PMID:27208308

  15. Intracellular Redox Compartmentation and ROS-Related Communication in Regulation and Signaling1[OPEN

    PubMed Central

    2016-01-01

    Recent years have witnessed enormous progress in understanding redox signaling related to reactive oxygen species (ROS) in plants. The consensus view is that such signaling is intrinsic to many developmental processes and responses to the environment. ROS-related redox signaling is tightly wedded to compartmentation. Because membranes function as barriers, highly redox-active powerhouses such as chloroplasts, peroxisomes, and mitochondria may elicit specific signaling responses. However, transporter functions allow membranes also to act as bridges between compartments, and so regulated capacity to transmit redox changes across membranes influences the outcome of triggers produced at different locations. As well as ROS and other oxidizing species, antioxidants are key players that determine the extent of ROS accumulation at different sites and that may themselves act as signal transmitters. Like ROS, antioxidants can be transported across membranes. In addition, the intracellular distribution of antioxidative enzymes may be modulated to regulate or facilitate redox signaling appropriate to the conditions. Finally, there is substantial plasticity in organellar shape, with extensions such as stromules, peroxules, and matrixules playing potentially crucial roles in organelle-organelle communication. We provide an overview of the advances in subcellular compartmentation, identifying the gaps in our knowledge and discussing future developments in the area. PMID:27208308

  16. Fluoxetine suppresses calcium signaling in human T lymphocytes through depletion of intracellular calcium stores.

    PubMed

    Gobin, V; De Bock, M; Broeckx, B J G; Kiselinova, M; De Spiegelaere, W; Vandekerckhove, L; Van Steendam, K; Leybaert, L; Deforce, D

    2015-09-01

    Selective serotonin reuptake inhibitors, such as fluoxetine, have recently been shown to exert anti-inflammatory and immunosuppressive effects. Although the effects on cytokine secretion, proliferation and viability of T lymphocytes have been extensively characterized, little is known about the mechanism behind these effects. It is well known that Ca(2+) signaling is an important step in the signaling transduction pathway following T cell receptor activation. Therefore, we investigated if fluoxetine interferes with Ca(2+) signaling in Jurkat T lymphocytes. Fluoxetine was found to suppress Ca(2+) signaling in response to T cell receptor activation. Moreover, fluoxetine was found to deplete intracellular Ca(2+) stores, thereby leaving less Ca(2+) available for release upon IP3- and ryanodine-receptor activation. The Ca(2+)-modifying effects of fluoxetine are not related to its capability to block the serotonin transporter, as even a large excess of 5HT did not abolish the effects. In conclusion, these data show that fluoxetine decreases IP3- and ryanodine-receptor mediated Ca(2+) release in Jurkat T lymphocytes, an effect likely to be at the basis of the observed immunosuppression.

  17. Regulation of Epidermal Growth Factor Receptor Signaling by Endocytosis and Intracellular Trafficking

    SciTech Connect

    Burke, Patrick; Schooler, Kevin; Wiley, H S.

    2001-06-01

    Ligand activation of the epidermal growth factor receptor (EGFR) leads to its rapid internalization and eventual delivery to lysosomes. This process is thought to be a mechanism to attenuate signaling, but signals could potentially be generated following endocytosis. To directly evaluate EGFR signaling during receptor trafficking, we developed a technique to rapidly and selectively isolate internalized EGFR and associated molecules using reversibly-biotinylated anti-EGFR antibodies. In addition, we developed antibodies specific to tyrosine-phosphorylated EGFR. Using a combination of fluorescence imaging and affinity precipitation approaches, we evaluated the state of EGFR activation and substrate association during trafficking in epithelial cells. We found that following internalization, EGFR remained active in the early endosomes. However, receptors were inactivated prior to degradation, apparently due to ligand removal from endosomes. Adapter molecules, such as Shc, were associated with EGFR both at the cell surface and within endosomes. Some molecules, such as Grb2, were primarily found associated with surface EGFR, while others, such as Eps8, were only found with intracellular receptors. During the inactivation phase, c-Cbl became EGFR-associated, consistent with its postulated role in receptor attenuation. We conclude that the association of the EGFR with different proteins is compartment-specific . In addition, ligand loss is the proximal cause of EGFR inactivation. Thus, regulated trafficking could potentially influence the pattern as well as the duration of signal transduction.

  18. Ancient origins and evolutionary conservation of intracellular and neural signaling pathways engaged by the leptin receptor.

    PubMed

    Cui, Melissa Y; Hu, Caroline K; Pelletier, Chris; Dziuba, Adam; Slupski, Rose H; Li, Choi; Denver, Robert J

    2014-11-01

    In mammals, leptin acts on leptin receptor (LepR) -expressing neurons in the brain to suppress food intake and stimulate whole-body metabolism. A similar action of leptin on food intake has been reported in the frog Xenopus laevis and in several bony fishes. However, the intracellular signaling and neural pathways by which leptin regulates energy balance have not been investigated outside of mammals. Using reporter assays and site-directed mutagenesis we show that the frog LepR signals via signal transducer and activator of transcription (STAT) 3 and STAT5 through evolutionarily conserved tyrosine residues in the LepR cytoplasmic domain. In situ hybridization histochemistry for LepR mRNA in brain and pituitary showed strong expression in the magno- and parvocellular divisions of the anterior preoptic area (homologous to the mammalian paraventricular nucleus), the suprachiasmatic nucleus, ventral hypothalamus, and pars intermedia and pars distalis of the anterior pituitary. Leptin injection increased phosphorylated STAT3 immunoreactivity in LepR mRNA-positive cells, and induced socs3 and pomc mRNAs. Microarray analysis of preoptic area/hypothalamus/pituitary 2 hours after leptin injection identified leptin-regulated genes that included c-fos, a known leptin-activated gene; pituitary follicle-stimulating hormone subunit β, suggesting an important role for leptin in the reproductive axis of frogs; and B-cell translocation factor 2, which has important functions in neurogenesis. Our findings support that the intracellular signaling pathways and neural substrates that mediate leptin actions on energy balance were present in the common ancestor of modern amphibians and amniotes and have been conserved over 350 million years of evolutionary time.

  19. Roles of Intracellular Cyclic AMP Signal Transduction in the Capacitation and Subsequent Hyperactivation of Mouse and Boar Spermatozoa

    PubMed Central

    HARAYAMA, Hiroshi

    2013-01-01

    It is not until accomplishment of a variety of molecular changes during the transit through the female reproductive tract that mammalian spermatozoa are capable of exhibiting highly activated motility with asymmetric whiplash beating of the flagella (hyperactivation) and undergoing acrosomal exocytosis in the head (acrosome reaction). These molecular changes of the spermatozoa are collectively termed capacitation and promoted by bicarbonate, calcium and cholesterol acceptors. Such capacitation-promoting factors can stimulate intracellular cyclic AMP (cAMP) signal transduction in the spermatozoa. Meanwhile, hyperactivation and the acrosome reaction are essential to sperm fertilization with oocytes and are apparently triggered by a sufficient increase of intracellular Ca2+ in the sperm flagellum and head, respectively. Thus, it is necessary to investigate the relationship between cAMP signal transduction and calcium signaling cascades in the spermatozoa for the purpose of understanding the molecular basis of capacitation. In this review, I cover updated insights regarding intracellular cAMP signal transduction, the acrosome reaction and flagellar motility in mammalian spermatozoa and then account for possible roles of intracellular cAMP signal transduction in the capacitation and subsequent hyperactivation of mouse and boar spermatozoa. PMID:24162806

  20. Inhibitors of intracellular signaling pathways that lead to stimulated epidermal pigmentation: perspective of anti-pigmenting agents.

    PubMed

    Imokawa, Genji; Ishida, Koichi

    2014-01-01

    Few anti-pigmenting agents have been designed and developed according to their known hyperpigmentation mechanisms and corresponding intracellular signaling cascades. Most anti-pigmenting agents developed so far are mechanistically involved in the interruption of constitutional melanogenic mechanisms by which skin color is maintained at a normal and unstimulated level. Thus, owing to the difficulty of confining topical application to a specific hyperpigmented skin area, potent anti-pigmenting agents capable of attenuating the natural unstimulated pigmentation process have the risk of leading to hypopigmentation. Since intracellular signaling pathways within melanocytes do not function substantially in maintaining normal skin color and are activated only by environmental stimuli such as UV radiation, specifically down-regulating the activation of melanogenesis to the constitutive level would be an appropriate strategy to develop new potent anti-pigmenting agents with a low risk of hypopigmentation. In this article, we review the hyperpigmentation mechanisms and intracellular signaling pathways that lead to the stimulation of melanogenesis. We also discuss a screening and evaluation system to select candidates for new anti-melanogenic substances by focusing on inhibitors of endothelin-1 or stem cell factor-triggered intracellular signaling cascades. From this viewpoint, we show that extracts of the herbs Withania somnifera and Melia toosendan and the natural chemicals Withaferin A and Astaxanthin are new candidates for potent anti-pigmenting substances that avoid the risk of hypopigmentation. PMID:24823877

  1. Intracellular calcium signals display an avalanche-like behavior over multiple lengthscales

    PubMed Central

    Lopez, Lucía; Piegari, Estefanía; Sigaut, Lorena; Ponce Dawson, Silvina

    2012-01-01

    Many natural phenomena display “self-organized criticality” (SOC), (Bak et al., 1987). This refers to spatially extended systems for which patterns of activity characterized by different lengthscales can occur with a probability density that follows a power law with pattern size. Differently from power laws at phase transitions, systems displaying SOC do not need the tuning of an external parameter. Here we analyze intracellular calcium (Ca2+) signals, a key component of the signaling toolkit of almost any cell type. Ca2+ signals can either be spatially restricted (local) or propagate throughout the cell (global). Different models have suggested that the transition from local to global signals is similar to that of directed percolation. Directed percolation has been associated, in turn, to the appearance of SOC. In this paper we discuss these issues within the framework of simple models of Ca2+ signal propagation. We also analyze the size distribution of local signals (“puffs”) observed in immature Xenopus Laevis oocytes. The puff amplitude distribution obtained from observed local signals is not Gaussian with a noticeable fraction of large size events. The experimental distribution of puff areas in the spatio-temporal record of the image has a long tail that is approximately log-normal. The distribution can also be fitted with a power law relationship albeit with a smaller goodness of fit. The power law behavior is encountered within a simple model that includes some coupling among individual signals for a wide range of parameter values. An analysis of the model shows that a global elevation of the Ca2+ concentration plays a major role in determining whether the puff size distribution is long-tailed or not. This suggests that Ca2+-clearing from the cytosol is key to determine whether IP3-mediated Ca2+ signals can display a SOC-like behavior or not. PMID:22969730

  2. ER functions of oncogenes and tumor suppressors: Modulators of intracellular Ca(2+) signaling.

    PubMed

    Bittremieux, Mart; Parys, Jan B; Pinton, Paolo; Bultynck, Geert

    2016-06-01

    Intracellular Ca(2+) signals that arise from the endoplasmic reticulum (ER), the major intracellular Ca(2+)-storage organelle, impact several mitochondrial functions and dictate cell survival and cell death processes. Furthermore, alterations in Ca(2+) signaling in cancer cells promote survival and establish a high tolerance towards cell stress and damage, so that the on-going oncogenic stress does not result in the activation of cell death. Over the last years, the mechanisms underlying these oncogenic alterations in Ca(2+) signaling have started to emerge. An important aspect of this is the identification of several major oncogenes, including Bcl-2, Bcl-XL, Mcl-1, PKB/Akt, and Ras, and tumor suppressors, such as p53, PTEN, PML, BRCA1, and Beclin 1, as direct and critical regulators of Ca(2+)-transport systems located at the ER membranes, including IP3 receptors and SERCA Ca(2+) pumps. In this way, these proteins execute part of their function by controlling the ER-mitochondrial Ca(2+) fluxes, favoring either survival (oncogenes) or cell death (tumor suppressors). Oncogenic mutations, gene deletions or amplifications alter the expression and/or function of these proteins, thereby changing the delicate balance between oncogenes and tumor suppressors, impacting oncogenesis and favoring malignant cell function and behavior. In this review, we provided an integrated overview of the impact of the major oncogenes and tumor suppressors, often altered in cancer cells, on Ca(2+) signaling from the ER Ca(2+) stores. This article is part of a Special Issue entitled: Calcium and Cell Fate. Guest Editors: Jacques Haiech, Claus Heizmann, Joachim Krebs, Thierry Capiod and Olivier Mignen.

  3. Multiple Model-Informed Open-Loop Control of Uncertain Intracellular Signaling Dynamics

    PubMed Central

    Perley, Jeffrey P.; Mikolajczak, Judith; Harrison, Marietta L.; Buzzard, Gregery T.; Rundell, Ann E.

    2014-01-01

    Computational approaches to tune the activation of intracellular signal transduction pathways both predictably and selectively will enable researchers to explore and interrogate cell biology with unprecedented precision. Techniques to control complex nonlinear systems typically involve the application of control theory to a descriptive mathematical model. For cellular processes, however, measurement assays tend to be too time consuming for real-time feedback control and models offer rough approximations of the biological reality, thus limiting their utility when considered in isolation. We overcome these problems by combining nonlinear model predictive control with a novel adaptive weighting algorithm that blends predictions from multiple models to derive a compromise open-loop control sequence. The proposed strategy uses weight maps to inform the controller of the tendency for models to differ in their ability to accurately reproduce the system dynamics under different experimental perturbations (i.e. control inputs). These maps, which characterize the changing model likelihoods over the admissible control input space, are constructed using preexisting experimental data and used to produce a model-based open-loop control framework. In effect, the proposed method designs a sequence of control inputs that force the signaling dynamics along a predefined temporal response without measurement feedback while mitigating the effects of model uncertainty. We demonstrate this technique on the well-known Erk/MAPK signaling pathway in T cells. In silico assessment demonstrates that this approach successfully reduces target tracking error by 52% or better when compared with single model-based controllers and non-adaptive multiple model-based controllers. In vitro implementation of the proposed approach in Jurkat cells confirms a 63% reduction in tracking error when compared with the best of the single-model controllers. This study provides an experimentally

  4. Multiple model-informed open-loop control of uncertain intracellular signaling dynamics.

    PubMed

    Perley, Jeffrey P; Mikolajczak, Judith; Harrison, Marietta L; Buzzard, Gregery T; Rundell, Ann E

    2014-04-01

    Computational approaches to tune the activation of intracellular signal transduction pathways both predictably and selectively will enable researchers to explore and interrogate cell biology with unprecedented precision. Techniques to control complex nonlinear systems typically involve the application of control theory to a descriptive mathematical model. For cellular processes, however, measurement assays tend to be too time consuming for real-time feedback control and models offer rough approximations of the biological reality, thus limiting their utility when considered in isolation. We overcome these problems by combining nonlinear model predictive control with a novel adaptive weighting algorithm that blends predictions from multiple models to derive a compromise open-loop control sequence. The proposed strategy uses weight maps to inform the controller of the tendency for models to differ in their ability to accurately reproduce the system dynamics under different experimental perturbations (i.e. control inputs). These maps, which characterize the changing model likelihoods over the admissible control input space, are constructed using preexisting experimental data and used to produce a model-based open-loop control framework. In effect, the proposed method designs a sequence of control inputs that force the signaling dynamics along a predefined temporal response without measurement feedback while mitigating the effects of model uncertainty. We demonstrate this technique on the well-known Erk/MAPK signaling pathway in T cells. In silico assessment demonstrates that this approach successfully reduces target tracking error by 52% or better when compared with single model-based controllers and non-adaptive multiple model-based controllers. In vitro implementation of the proposed approach in Jurkat cells confirms a 63% reduction in tracking error when compared with the best of the single-model controllers. This study provides an experimentally

  5. Regulation of Transgene Expression in Tumor Cells by Exploiting Endogenous Intracellular Signals

    NASA Astrophysics Data System (ADS)

    Asai, Daisuke; Kang, Jeong-Hun; Toita, Riki; Tsuchiya, Akira; Niidome, Takuro; Nakashima, Hideki; Katayama, Yoshiki

    2009-03-01

    Recently, we have proposed a novel strategy for a cell-specific gene therapy system based on responses to intracellular signals. In this system, an intracellular signal that is specifically and abnormally activated in the diseased cells is used for the activation of transgene expression. In this study, we used protein kinase C (PKC)α as a trigger to activate transgene expression. We prepared a PKCα-responsive polymer conjugate [PPC(S)] and a negative control conjugate [PPC(A)], in which the phosphorylation site serine (Ser) was replaced with alanine (Ala). The phosphorylation for polymer/DNA complexes was determined with a radiolabel assay using [γ-32P]ATP. PPC(S)/DNA complexes were phosphorylated by the addition of PKCα, but no phosphorylation of the PPC(A)/DNA complex was observed. Moreover, after microinjection of polymer/GFP-encoding DNA complexes into HepG2 cells at cation/anion (C/A) ratios of 0.5 to 2.0, significant expression of GFP was observed in all cases using PPC(S)/DNA complexes, but no GFP expression was observed in the negative control PPC(A)/DNA complex-microinjected cells at C/A ratios of 1.0 and 2.0. On the other hand, GFP expression from PPC(S)/DNA complexes was completely suppressed in cells pretreated with PKCα inhibitor (Ro31-7549). These results suggest that our gene regulation system can be used for tumor cell-specific expression of a transgene in response to PKCα activity.

  6. PRG-1 Regulates Synaptic Plasticity via Intracellular PP2A/β1-Integrin Signaling.

    PubMed

    Liu, Xingfeng; Huai, Jisen; Endle, Heiko; Schlüter, Leslie; Fan, Wei; Li, Yunbo; Richers, Sebastian; Yurugi, Hajime; Rajalingam, Krishnaraj; Ji, Haichao; Cheng, Hong; Rister, Benjamin; Horta, Guilherme; Baumgart, Jan; Berger, Hendrik; Laube, Gregor; Schmitt, Ulrich; Schmeisser, Michael J; Boeckers, Tobias M; Tenzer, Stefan; Vlachos, Andreas; Deller, Thomas; Nitsch, Robert; Vogt, Johannes

    2016-08-01

    Alterations in dendritic spine numbers are linked to deficits in learning and memory. While we previously revealed that postsynaptic plasticity-related gene 1 (PRG-1) controls lysophosphatidic acid (LPA) signaling at glutamatergic synapses via presynaptic LPA receptors, we now show that PRG-1 also affects spine density and synaptic plasticity in a cell-autonomous fashion via protein phosphatase 2A (PP2A)/β1-integrin activation. PRG-1 deficiency reduces spine numbers and β1-integrin activation, alters long-term potentiation (LTP), and impairs spatial memory. The intracellular PRG-1 C terminus interacts in an LPA-dependent fashion with PP2A, thus modulating its phosphatase activity at the postsynaptic density. This results in recruitment of adhesome components src, paxillin, and talin to lipid rafts and ultimately in activation of β1-integrins. Consistent with these findings, activation of PP2A with FTY720 rescues defects in spine density and LTP of PRG-1-deficient animals. These results disclose a mechanism by which bioactive lipid signaling via PRG-1 could affect synaptic plasticity and memory formation. PMID:27453502

  7. Subcompartmentalized Nanoreactors as Artificial Organelle with Intracellular Activity.

    PubMed

    Thingholm, Bo; Schattling, Philipp; Zhang, Yan; Städler, Brigitte

    2016-04-01

    Cell mimicry is an approach which aims at substituting missing or lost activity. In this context, the goal of artificial organelles is to provide intracellularly active nanoreactors to affect the cellular performance. So far, only a handful of reports discuss concepts addressing this challenge based on single-component reactors. Here, the assembly of nanoreactors equipped with glucose oxidase (GOx)-loaded liposomal subunits coated with a poly(dopamine) polymer layer and RGD targeting units is reported. When comparing different surface modifications, the uptake of the nanoreactors by endothelial cells and macrophages with applied shear stress is confirmed without inherent cytotoxicity. Furthermore, the encapsulation and preserved activity of GOx within the nanoreactors is shown. The intracellular activity is demonstrated by exposing macrophages with internalized nanoreactors to glucose and assessment of the cell viability after 6 and 24 h. The macrophage viability is found to be reduced due to the intracellularly produced hydrogen peroxide by GOx. This report on the first intracellular active subcompartmentalized nanoreactors is a considerable step in therapeutic cell mimicry.

  8. Gamma Band Activity in the RAS-intracellular mechanisms

    PubMed Central

    Garcia-Rill, E.; Kezunovic, N.; D’Onofrio, S.; Luster, B.; Hyde, J.; Bisagno, V.; Urbano, F.J.

    2014-01-01

    Gamma band activity participates in sensory perception, problem solving, and memory. This review considers recent evidence showing that cells in the reticular activating system (RAS) exhibit gamma band activity, and describes the intrinsic membrane properties behind such manifestation. Specifically, we discuss how cells in the mesopontine pedunculopontine nucleus (PPN), intralaminar parafascicular nucleus (Pf), and pontine Subcoeruleus nucleus dorsalis (SubCD) all fire in the gamma band range when maximally activated, but no higher. The mechanisms involve high threshold, voltage-dependent P/Q-type calcium channels or sodium-dependent subthreshold oscillations. Rather than participating in the temporal binding of sensory events as in the cortex, gamma band activity in the RAS may participate in the processes of preconscious awareness, and provide the essential stream of information for the formulation of many of our actions. We address three necessary next steps resulting from these discoveries, an intracellular mechanism responsible for maintaining gamma band activity based on persistent G-protein activation, separate intracellular pathways that differentiate between gamma band activity during waking vs during REM sleep, and an intracellular mechanism responsible for the dysregulation in gamma band activity in schizophrenia. These findings open several promising research avenues that have not been thoroughly explored. What are the effects of sleep or REM sleep deprivation on these RAS mechanisms? Are these mechanisms involved in memory processing during waking and/or during REM sleep? Does gamma band processing differ during waking vs REM sleep after sleep or REM sleep deprivation? PMID:24309750

  9. Identification of intracellular domains in the growth hormone receptor involved in signal transduction

    SciTech Connect

    Billestrup, N.; Allevato, G.; Moldrup, A.

    1994-12-31

    The growth hormone (GH) receptor belongs to the GH/prolactin/cytokine super-family of receptors. The signal transduction mechanism utilized by this class of receptors remains largely unknown. In order to identify functional domains in the intracellular region of the GH receptor we generated a number of GH receptor mutants and analyzed their function after transfection into various cell lines. A truncated GH receptor missing 184 amino acids at the C-terminus was unable to medite GH effects on transcription of the Spi 2.1 and insulin genes. However, this mutant was fully active in mediating GH-stimulated metabolic effects such as protein synthesis and lipolysis. Furthermore, this mutant GH receptor internalized rapidly following GH binding. Another truncated GH receptor lacking all but five amino acids of the cytoplasmic domain could not mediate any effects of GH nor did it internalize. Deletion of the proline-rich region or changing the four prolines to alanines also resulted in a GH receptor deficient in signaling. Mutation of phenylalanine 346 to alanine resulted in a GH receptor which did not internalize rapidly; however, this mutant GH receptor was capable of mediating GH-stimulated transcription as well as metabolic effects. These results indicate that the intracellular part of the GH receptor can be divided into at least three functional domains: (1) for transcriptional activity, two domains are involved, one located in the C-terminal 184 amino acids and the other in the proline-rich domain; (2) for metabolic effects, a domain located in or near the proline-rich region is of importance; and (3) for internalization, phenylalanine 346 is necessary. 28 refs., 1 fig.

  10. Xenopus tropicalis oocytes as an advantageous model system for the study of intracellular Ca2+ signalling

    PubMed Central

    Marchant, Jonathan S; Parker, Ian

    2001-01-01

    The purpose of this study was to compare oocytes from the pipid frogs Xenopus tropicalis and Xenopus laevis, with respect to their utility for studying Ca2+ signalling mechanisms and for expression of heterologous proteins. We show that X. tropicalis oocytes possess an intracellular Ca2+ store that is mobilized by inositol (1,4,5) trisphosphate (IP3). Ca2+ signalling is activated by endogenous lysophosphatidic acid receptors and cytosolic Ca2+ activates a plasma membrane chloride conductance. The spatiotemporal organization of cytosolic Ca2+ signals, from the microscopic architecture of elementary Ca2+ ‘puffs' to the macroscopic patterns of Ca2+ spiking are closely similar to the local and global patterns of Ca2+ release previously characterized in oocytes from X. laevis. By injecting X. tropicalis oocytes with cDNA encoding an ER-targeted fluorescent protein construct, we demonstrate the capacity of the X. tropicalis oocyte to readily express heterologous proteins. The organization of ER is polarized across the oocyte, with the IP3-releaseable store targeted within an ∼8 μm wide band that circumscribes the cell. We conclude that the X. tropicalis oocyte shares many of the characteristics that have made oocytes of X. laevis a favoured system for studying Ca2+ signalling mechanisms. Moreover, X. tropicalis oocytes display further practical advantages in terms of imaging depth, Ca2+ signal magnitude and electrical properties. These further enhance the appeal of X. tropicalis as an experimental system, in addition to its greater amenability to transgenic approaches. PMID:11264232

  11. Different intracellular signalling pathways triggered by an anti-prolactin receptor (PRLR) antibody: Implication for a signal-specific PRLR agonist.

    PubMed

    Kan, Quan-E; Su, Yong; Yang, Huihui; Man, Hua

    2016-01-01

    In this work, we prepared a panel of monoclonal antibodies directed against prolactin receptor (PRLR) using the hybridoma technique. Of these monoclonal antibodies (Mabs), the Mab designated B6 was chosen for further characterization based on its biological activity. We first demonstrated that B6 can specifically bind to the prolactin receptor (PRLR) expressed on target cells by immunoprecipitation and Western blotting analysis. Subsequently, epitope mapping studies using a competitive receptor-binding assay indicated that B6 epitopes partially overlapped with those of prolactin (PRL). We then examined the resulting signal transduction pathways activated by this antibody in T-47D and CHO-PRLR cells and found that B6 induced different intracellular signalling compared with prolactin, which activates serine-threonine kinase (AKT), extracellular signal-regulated kinase 1/2 (ERK1/2), signal transducer and activator of transcription1 (STAT1) and STAT3 but not STAT5. The present study suggests that: (a) B6 may be a signal-specific prolactin receptor (PRLR) agonist; (b) B6 may be a biological reagent that can be used to explore the mechanism of PRLR-mediated intracellular signalling. In addition, this work also implies a strategy for preparing signal-specific cytokine agonists.

  12. Hierarchic stochastic modelling applied to intracellular Ca(2+) signals.

    PubMed

    Moenke, Gregor; Falcke, Martin; Thurley, Keven

    2012-01-01

    Important biological processes like cell signalling and gene expression have noisy components and are very complex at the same time. Mathematical analysis of such systems has often been limited to the study of isolated subsystems, or approximations are used that are difficult to justify. Here we extend a recently published method (Thurley and Falcke, PNAS 2011) which is formulated in observable system configurations instead of molecular transitions. This reduces the number of system states by several orders of magnitude and avoids fitting of kinetic parameters. The method is applied to Ca(2+) signalling. Ca(2+) is a ubiquitous second messenger transmitting information by stochastic sequences of concentration spikes, which arise by coupling of subcellular Ca(2+) release events (puffs). We derive analytical expressions for a mechanistic Ca(2+) model, based on recent data from live cell imaging, and calculate Ca(2+) spike statistics in dependence on cellular parameters like stimulus strength or number of Ca(2+) channels. The new approach substantiates a generic Ca(2+) model, which is a very convenient way to simulate Ca(2+) spike sequences with correct spiking statistics.

  13. Irisin Controls Growth, Intracellular Ca2+ Signals, and Mitochondrial Thermogenesis in Cardiomyoblasts

    PubMed Central

    Xie, Chao; Zhang, Yuan; Tran, Tran D. N.; Wang, Hai; Li, Shiwu; George, Eva Vertes; Zhuang, Haoyang; Zhang, Peilan; Kandel, Avi; Lai, Yimu; Tang, Dongqi; Reeves, Westley H.; Cheng, Henrique; Ding, Yousong; Yang, Li-Jun

    2015-01-01

    Exercise offers short-term and long-term health benefits, including an increased metabolic rate and energy expenditure in myocardium. The newly-discovered exercise-induced myokine, irisin, stimulates conversion of white into brown adipocytes as well as increased mitochondrial biogenesis and energy expenditure. Remarkably, irisin is highly expressed in myocardium, but its physiological effects in the heart are unknown. The objective of this work is to investigate irisin’s potential multifaceted effects on cardiomyoblasts and myocardium. For this purpose, H9C2 cells were treated with recombinant irisin produced in yeast cells (r-irisin) and in HEK293 cells (hr-irisin) for examining its effects on cell proliferation by MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and on gene transcription profiles by qRT-PCR. R-irisin and hr-irisin both inhibited cell proliferation and activated genes related to cardiomyocyte metabolic function and differentiation, including myocardin, follistatin, smooth muscle actin, and nuclear respiratory factor-1. Signal transduction pathways affected by r-irisin in H9C2 cells and C57BL/6 mice were examined by detecting phosphorylation of PI3K-AKT, p38, ERK or STAT3. We also measured intracellular Ca2+ signaling and mitochondrial thermogenesis and energy expenditure in r-irisin-treated H9C2 cells. The results showed that r-irisin, in a certain concentration rage, could activate PI3K-AKT and intracellular Ca2+ signaling and increase cellular oxygen consumption in H9C2 cells. Our study also suggests the existence of irisin-specific receptor on the membrane of H9C2 cells. In conclusion, irisin in a certain concentration rage increased myocardial cell metabolism, inhibited cell proliferation and promoted cell differentiation. These effects might be mediated through PI3K-AKT and Ca2+ signaling, which are known to activate expression of exercise-related genes such as follistatin and myocardin. This work supports the value

  14. Activation of intracellular kinases in Xenopus oocytes by p21ras and phospholipases: a comparative study.

    PubMed

    Carnero, A; Lacal, J C

    1995-02-01

    Signal transduction induced by generations of second messengers from membrane phospholipids is a major regulatory mechanism in the control of cell proliferation. Indeed, oncogenic p21ras alters the intracellular levels of phospholipid metabolites in both mammalian cells and Xenopus oocytes. However, it is still controversial whether this alteration it is biologically significant. We have analyzed the ras-induced signal transduction pathway in Xenopus oocytes and have correlated its mechanism of activation with that of the three most relevant phospholipases (PLs). After microinjection, ras-p21 induces a rapid PLD activation followed by a late PLA2 activation. By contrast, phosphatidylcholine-specific PLC was not activated under similar conditions. When each of these PLs was studied for its ability to activate intracellular signalling kinases, all of them were found to activate maturation-promoting factor efficiently. However, only PLD was able to activate MAP kinase and S6 kinase II, a similar pattern to that induced by p21ras proteins. Thus, the comparison of activated enzymes after microinjection of p21ras or PLs indicated that only PLD microinjection mimetized p21ras signalling. Finally, inhibition of the endogenous PLD activity by neomycin substantially reduced the biological activity of p21ras. All these results suggest that PLD activation may constitute a relevant step in ras-induced germinal vesicle breakdown in Xenopus oocytes.

  15. Role of α7 nicotinic receptor in the immune system and intracellular signaling pathways

    PubMed Central

    Zdanowski, Robert; Ujazdowska, Dominika; Lewicka, Aneta; Lewicki, Sławomir

    2015-01-01

    Acetylcholine has been well known as one of the most exemplary neurotransmitters. In humans, this versatile molecule and its synthesizing enzyme, choline acetyltransferase, have been found in various non-neural tissues such as the epithelium, endothelium, mesothelium muscle, blood cells and immune cells. The non-neuronal acetylcholine is accompanied by the expression of acetylcholinesterase and nicotinic/muscarinic acetylcholine receptors. Increasing evidence of the non-neuronal acetylcholine system found throughout the last few years has indicated this neurotransmitter as one of the major cellular signaling molecules (associated e.g. with kinases and transcription factors activity). This system is responsible for maintenance and optimization of the cellular function, such as proliferation, differentiation, adhesion, migration, intercellular contact and apoptosis. Additionally, it controls proper activity of immune cells and affects differentiation, antigen presentation or cytokine production (both pro- and anti-inflammatory). The present article reviews recent findings about the non-neuronal cholinergic system in the field of immune system and intracellular signaling pathways. PMID:26648784

  16. Role of α7 nicotinic receptor in the immune system and intracellular signaling pathways.

    PubMed

    Zdanowski, Robert; Krzyżowska, Małgorzata; Ujazdowska, Dominika; Lewicka, Aneta; Lewicki, Sławomir

    2015-01-01

    Acetylcholine has been well known as one of the most exemplary neurotransmitters. In humans, this versatile molecule and its synthesizing enzyme, choline acetyltransferase, have been found in various non-neural tissues such as the epithelium, endothelium, mesothelium muscle, blood cells and immune cells. The non-neuronal acetylcholine is accompanied by the expression of acetylcholinesterase and nicotinic/muscarinic acetylcholine receptors. Increasing evidence of the non-neuronal acetylcholine system found throughout the last few years has indicated this neurotransmitter as one of the major cellular signaling molecules (associated e.g. with kinases and transcription factors activity). This system is responsible for maintenance and optimization of the cellular function, such as proliferation, differentiation, adhesion, migration, intercellular contact and apoptosis. Additionally, it controls proper activity of immune cells and affects differentiation, antigen presentation or cytokine production (both pro- and anti-inflammatory). The present article reviews recent findings about the non-neuronal cholinergic system in the field of immune system and intracellular signaling pathways.

  17. Involvement of the Tyro3 receptor and its intracellular partner Fyn signaling in Schwann cell myelination.

    PubMed

    Miyamoto, Yuki; Torii, Tomohiro; Takada, Shuji; Ohno, Nobuhiko; Saitoh, Yurika; Nakamura, Kazuaki; Ito, Akihito; Ogata, Toru; Terada, Nobuo; Tanoue, Akito; Yamauchi, Junji

    2015-10-01

    During early development of the peripheral nervous system, Schwann cell precursors proliferate, migrate, and differentiate into premyelinating Schwann cells. After birth, Schwann cells envelop neuronal axons with myelin sheaths. Although some molecular mechanisms underlying myelination by Schwann cells have been identified, the whole picture remains unclear. Here we show that signaling through Tyro3 receptor tyrosine kinase and its binding partner, Fyn nonreceptor cytoplasmic tyrosine kinase, is involved in myelination by Schwann cells. Impaired formation of myelin segments is observed in Schwann cell neuronal cultures established from Tyro3-knockout mouse dorsal root ganglia (DRG). Indeed, Tyro3-knockout mice exhibit reduced myelin thickness. By affinity chromatography, Fyn was identified as the binding partner of the Tyro3 intracellular domain, and activity of Fyn is down-regulated in Tyro3-knockout mice, suggesting that Tyro3, acting through Fyn, regulates myelination. Ablating Fyn in mice results in reduced myelin thickness. Decreased myelin formation is observed in cultures established from Fyn-knockout mouse DRG. Furthermore, decreased kinase activity levels and altered expression of myelination-associated transcription factors are observed in these knockout mice. These results suggest the involvement of Tyro3 receptor and its binding partner Fyn in Schwann cell myelination. This constitutes a newly recognized receptor-linked signaling mechanism that can control Schwann cell myelination.

  18. Anabolic androgenic steroids and intracellular calcium signaling: a mini review on mechanisms and physiological implications.

    PubMed

    Vicencio, J M; Estrada, M; Galvis, D; Bravo, R; Contreras, A E; Rotter, D; Szabadkai, G; Hill, J A; Rothermel, B A; Jaimovich, E; Lavandero, S

    2011-05-01

    Increasing evidence suggests that nongenomic effects of testosterone and anabolic androgenic steroids (AAS) operate concertedly with genomic effects. Classically, these responses have been viewed as separate and independent processes, primarily because nongenomic responses are faster and appear to be mediated by membrane androgen receptors, whereas long-term genomic effects are mediated through cytosolic androgen receptors regulating transcriptional activity. Numerous studies have demonstrated increases in intracellular Ca2+ in response to AAS. These Ca2+ mediated responses have been seen in a diversity of cell types, including osteoblasts, platelets, skeletal muscle cells, cardiac myocytes and neurons. The versatility of Ca2+ as a second messenger provides these responses with a vast number of pathophysiological implications. In cardiac cells, testosterone elicits voltage-dependent Ca2+ oscillations and IP3R-mediated Ca2+ release from internal stores, leading to activation of MAPK and mTOR signaling that promotes cardiac hypertrophy. In neurons, depending upon concentration, testosterone can provoke either physiological Ca2+ oscillations, essential for synaptic plasticity, or sustained, pathological Ca2+ transients that lead to neuronal apoptosis. We propose therefore, that Ca2+ acts as an important point of crosstalk between nongenomic and genomic AAS signaling, representing a central regulator that bridges these previously thought to be divergent responses.

  19. Intracellular Cl- as a signaling ion that potently regulates Na+/HCO3- transporters.

    PubMed

    Shcheynikov, Nikolay; Son, Aran; Hong, Jeong Hee; Yamazaki, Osamu; Ohana, Ehud; Kurtz, Ira; Shin, Dong Min; Muallem, Shmuel

    2015-01-20

    Cl(-) is a major anion in mammalian cells involved in transport processes that determines the intracellular activity of many ions and plasma membrane potential. Surprisingly, a role of intracellular Cl(-) (Cl(-) in) as a signaling ion has not been previously evaluated. Here we report that Cl(-) in functions as a regulator of cellular Na(+) and HCO3 (-) concentrations and transepithelial transport through modulating the activity of several electrogenic Na(+)-HCO3 (-) transporters. We describe the molecular mechanism(s) of this regulation by physiological Cl(-) in concentrations highlighting the role of GXXXP motifs in Cl(-) sensing. Regulation of the ubiquitous Na(+)-HCO3(-) co-transport (NBC)e1-B is mediated by two GXXXP-containing sites; regulation of NBCe2-C is dependent on a single GXXXP motif; and regulation of NBCe1-A depends on a cryptic GXXXP motif. In the basal state NBCe1-B is inhibited by high Cl(-) in interacting at a low affinity GXXXP-containing site. IP3 receptor binding protein released with IP3 (IRBIT) activation of NBCe1-B unmasks a second high affinity Cl(-) in interacting GXXXP-dependent site. By contrast, NBCe2-C, which does not interact with IRBIT, has a single high affinity N-terminal GXXP-containing Cl(-) in interacting site. NBCe1-A is unaffected by Cl(-) in between 5 and 140 mM. However, deletion of NBCe1-A residues 29-41 unmasks a cryptic GXXXP-containing site homologous with the NBCe1-B low affinity site that is involved in inhibition of NBCe1-A by Cl(-) in. These findings reveal a cellular Cl(-) in sensing mechanism that plays an important role in the regulation of Na(+) and HCO3 (-) transport, with critical implications for the role of Cl(-) in cellular ion homeostasis and epithelial fluid and electrolyte secretion.

  20. Genetic requirements for signaling from an autoactive plant NB-LRR intracellular innate immune receptor.

    PubMed

    Roberts, Melinda; Tang, Saijun; Stallmann, Anna; Dangl, Jeffery L; Bonardi, Vera

    2013-01-01

    Plants react to pathogen attack via recognition of, and response to, pathogen-specific molecules at the cell surface and inside the cell. Pathogen effectors (virulence factors) are monitored by intracellular nucleotide-binding leucine-rich repeat (NB-LRR) sensor proteins in plants and mammals. Here, we study the genetic requirements for defense responses of an autoactive mutant of ADR1-L2, an Arabidopsis coiled-coil (CC)-NB-LRR protein. ADR1-L2 functions upstream of salicylic acid (SA) accumulation in several defense contexts, and it can act in this context as a "helper" to transduce specific microbial activation signals from "sensor" NB-LRRs. This helper activity does not require an intact P-loop. ADR1-L2 and another of two closely related members of this small NB-LRR family are also required for propagation of unregulated runaway cell death (rcd) in an lsd1 mutant. We demonstrate here that, in this particular context, ADR1-L2 function is P-loop dependent. We generated an autoactive missense mutation, ADR1-L2D484V, in a small homology motif termed MHD. Expression of ADR1-L2D848V leads to dwarfed plants that exhibit increased disease resistance and constitutively high SA levels. The morphological phenotype also requires an intact P-loop, suggesting that these ADR1-L2D484V phenotypes reflect canonical activation of this NB-LRR protein. We used ADR1-L2D484V to define genetic requirements for signaling. Signaling from ADR1-L2D484V does not require NADPH oxidase and is negatively regulated by EDS1 and AtMC1. Transcriptional regulation of ADR1-L2D484V is correlated with its phenotypic outputs; these outputs are both SA-dependent and -independent. The genetic requirements for ADR1-L2D484V activity resemble those that regulate an SA-gradient-dependent signal amplification of defense and cell death signaling initially observed in the absence of LSD1. Importantly, ADR1-L2D484V autoactivation signaling is controlled by both EDS1 and SA in separable, but linked pathways

  1. Prolonged Oxaliplatin Exposure Alters Intracellular Calcium Signaling: A New Mechanism To Explain Oxaliplatin-Associated Peripheral Neuropathy

    PubMed Central

    Schulze, Christin; McGowan, Margit; Jordt, Sven; Ehrlich, Barbara E

    2012-01-01

    Oxaliplatin is a platinum based cytotoxic agent commonly used to treat colorectal cancers. Despite its effectiveness, oxaliplatin administration is associated with the development of cold-induced peripheral neuropathy. This potentially permanent side effect is provoked by cold exposure and can range from mild and self limited to severe and debilitating. Even with tumor shrinkage, these painful side effects can force dose-reduction or discontinuation of treatment. Neither the mechanism of action of oxaliplatin nor that of cold-induced neuropathy is understood. Paclitaxel, an entirely different chemotherapeutic agent used to treat a variety of malignancies, also is associated with the development of peripheral neuropathy. Unlike oxaliplatin, neurotoxicity arising from paclitaxel treatment is better understood and was found to have profound effects on intracellular calcium signaling (1,2). In this study we examined the effects of oxaliplatin on calcium signaling pathways and found that acute exposure of either a neuroblastoma cell line or primary neurons with therapeutic concentrations of oxaliplatin had no effect on intracellular calcium signaling. We also found that cellular temperature sensors (TRP channels) were also not activated by oxaliplatin. Interestingly, prolonged exposure of oxaliplatin sensitized cells to subsequent stimuli and enhanced the magnitude of intracellular calcium responses. Taken together, our results suggest that acute oxaliplatin exposure will not induce abnormal calcium signaling but oxaliplatin-primed cells do exhibit enhanced sensitivity. These findings provide new insight to the mechanism behind oxaliplatin-induced neuropathy. PMID:21859566

  2. Dictyostelium uses ether-linked inositol phospholipids for intracellular signalling

    PubMed Central

    Clark, Jonathan; Kay, Robert R; Kielkowska, Anna; Niewczas, Izabella; Fets, Louise; Oxley, David; Stephens, Len R; Hawkins, Phillip T

    2014-01-01

    Inositol phospholipids are critical regulators of membrane biology throughout eukaryotes. The general principle by which they perform these roles is conserved across species and involves binding of differentially phosphorylated inositol head groups to specific protein domains. This interaction serves to both recruit and regulate the activity of several different classes of protein which act on membrane surfaces. In mammalian cells, these phosphorylated inositol head groups are predominantly borne by a C38:4 diacylglycerol backbone. We show here that the inositol phospholipids of Dictyostelium are different, being highly enriched in an unusual C34:1e lipid backbone, 1-hexadecyl-2-(11Z-octadecenoyl)-sn-glycero-3-phospho-(1'-myo-inositol), in which the sn-1 position contains an ether-linked C16:0 chain; they are thus plasmanylinositols. These plasmanylinositols respond acutely to stimulation of cells with chemoattractants, and their levels are regulated by PIPKs, PI3Ks and PTEN. In mammals and now in Dictyostelium, the hydrocarbon chains of inositol phospholipids are a highly selected subset of those available to other phospholipids, suggesting that different molecular selectors are at play in these organisms but serve a common, evolutionarily conserved purpose. PMID:25180230

  3. Intracellular calcium signaling in the fertilized eggs of Annelida.

    PubMed

    Nakano, Takeshi; Deguchi, Ryusaku; Kyozuka, Keiichiro

    2014-08-01

    Fertilization is such a universal and indispensable step in sexual reproduction, but a high degree of variability exists in the way it takes place in the animal kingdom. As discussed in other reviews in this issue, recent works on this subject clarified many points. However, important results on the mechanisms of fertilization are obtained mainly from a few restricted model organisms. In this sense, it is utterly important to collect more information from various phyla. In this review, we have re-introduced Annelida as one of the most suitable models for the analysis of fertilization process. We have briefly reviewed the historical works on the fertilization of Annelida. Then, we have described recent findings on the two independent Ca(2+) increases in the fertilized eggs of Annelida, which arise from two different mechanisms and may have distinct physiological roles toward sperm entry and egg activation. We propose that the Ca(2+) increase in the fertilized eggs reflect the specific needs of the zygote in a given species.

  4. Human β-Cell Proliferation and Intracellular Signaling Part 2: Still Driving in the Dark Without a Road Map

    PubMed Central

    Bernal-Mizrachi, Ernesto; Kulkarni, Rohit N.; Scott, Donald K.; Mauvais-Jarvis, Franck; Stewart, Andrew F.; Garcia-Ocaña, Adolfo

    2014-01-01

    Enhancing β-cell proliferation is a major goal for type 1 and type 2 diabetes research. Unraveling the network of β-cell intracellular signaling pathways that promote β-cell replication can provide the tools to address this important task. In a previous Perspectives in Diabetes article, we discussed what was known regarding several important intracellular signaling pathways in rodent β-cells, including the insulin receptor substrate/phosphatidylinositol-3 kinase/Akt (IRS-PI3K-Akt) pathways, glycogen synthase kinase-3 (GSK3) and mammalian target of rapamycin (mTOR) S6 kinase pathways, protein kinase Cζ (PKCζ) pathways, and their downstream cell-cycle molecular targets, and contrasted that ample knowledge to the small amount of complementary data on human β-cell intracellular signaling pathways. In this Perspectives, we summarize additional important information on signaling pathways activated by nutrients, such as glucose; growth factors, such as epidermal growth factor, platelet-derived growth factor, and Wnt; and hormones, such as leptin, estrogen, and progesterone, that are linked to rodent and human β-cell proliferation. With these two Perspectives, we attempt to construct a brief summary of knowledge for β-cell researchers on mitogenic signaling pathways and to emphasize how little is known regarding intracellular events linked to human β-cell replication. This is a critical aspect in the long-term goal of expanding human β-cells for the prevention and/or cure of type 1 and type 2 diabetes. PMID:24556859

  5. Insulin signals to prenyltransferases via the Shc branch of intracellular signaling.

    PubMed

    Goalstone, M L; Leitner, J W; Berhanu, P; Sharma, P M; Olefsky, J M; Draznin, B

    2001-04-20

    We assessed the roles of insulin receptor substrate-1 (IRS-1) and Shc in insulin action on farnesyltransferase (FTase) and geranylgeranyltransferase I (GGTase I) using Chinese hamster ovary (CHO) cells that overexpress wild-type human insulin receptors (CHO-hIR-WT) or mutant insulin receptors lacking the NPEY domain (CHO-DeltaNPEY) or 3T3-L1 fibroblasts transfected with adenoviruses that express the PTB or SAIN domain of IRS-1 and Shc, the pleckstrin homology (PH) domain of IRS-1, or the Src homology 2 (SH2) domain of Shc. Insulin promoted phosphorylation of the alpha-subunit of FTase and GGTase I in CHO-hIR-WT cells, but was without effect in CHO-DeltaNPEY cells. Insulin increased FTase and GGTase I activities and the amounts of prenylated Ras and RhoA proteins in CHO-hIR-WT (but not CHO-DeltaNPEY) cells. Overexpression of the PTB or SAIN domain of IRS-1 (which blocked both IRS-1 and Shc signaling) prevented insulin-stimulated phosphorylation of the FTase and GGTase I alpha-subunit activation of FTase and GGTase I and subsequent increases in prenylated Ras and RhoA proteins. In contrast, overexpression of the IRS-1 PH domain, which impairs IRS-1 (but not Shc) signaling, did not alter insulin action on the prenyltransferases, but completely inhibited the insulin effect on the phosphorylation of IRS-1 and on the activation of phosphatidylinositol 3-kinase and Akt. Finally, overexpression of the Shc SH2 domain completely blocked the insulin effect on FTase and GGTase I activities without interfering with insulin signaling to MAPK. These data suggest that insulin signaling from its receptor to the prenyltransferases FTase and GGTase I is mediated by the Shc pathway, but not the IRS-1/phosphatidylinositol 3-kinase pathway. Shc-mediated insulin signaling to MAPK may be necessary (but not sufficient) for activation of prenyltransferase activity. An additional pathway involving the Shc SH2 domain may be necessary to mediate the insulin effect on FTase and GGTase I.

  6. THE ROLE OF INTRACELLULAR SODIUM (Na+) IN THE REGULATION OF CALCIUM (Ca2+)-MEDIATED SIGNALING AND TOXICITY

    PubMed Central

    Yu, Xian-Min; Groveman, Bradley R; Fang, Xiao-Qian; Lin, Shuang-Xiu

    2010-01-01

    It is known that activated N-methyl-D-aspartate receptors (NMDARs) are a major route of excessive calcium ion (Ca2+) entry in central neurons, which may activate degradative processes and thereby cause cell death. Therefore, NMDARs are now recognized to play a key role in the development of many diseases associated with injuries to the central nervous system (CNS). However, it remains a mystery how NMDAR activity is recruited in the cellular processes leading to excitotoxicity and how NMDAR activity can be controlled at a physiological level. The sodium ion (Na+) is the major cation in extracellular space. With its entry into the cell, Na+ can act as a critical intracellular second messenger that regulates many cellular functions. Recent data have shown that intracellular Na+ can be an important signaling factor underlying the up-regulation of NMDARs. While Ca2+ influx during the activation of NMDARs down-regulates NMDAR activity, Na+ influx provides an essential positive feedback mechanism to overcome Ca2+-induced inhibition and thereby potentiate both NMDAR activity and inward Ca2+ flow. Extensive investigations have been conducted to clarify mechanisms underlying Ca2+-mediated signaling. This review focuses on the roles of Na+ in the regulation of Ca2+-mediated NMDAR signaling and toxicity. PMID:21243124

  7. The level of intracellular glutathione is a key regulator for the induction of stress-activated signal transduction pathways including Jun N-terminal protein kinases and p38 kinase by alkylating agents.

    PubMed Central

    Wilhelm, D; Bender, K; Knebel, A; Angel, P

    1997-01-01

    Monofunctional alkylating agents like methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are potent inducers of cellular stress leading to chromosomal aberrations, point mutations, and cell killing. We show that these agents induce a specific cellular stress response program which includes the activation of Jun N-terminal kinases/stress-activated protein kinases (JNK/SAPKs), p38 mitogen-activated protein kinase, and the upstream kinase SEK1/MKK4 and which depends on the reaction mechanism of the alkylating agent in question. Similar to another inducer of cellular stress, UV irradiation, damage of nuclear DNA by alkylation is not involved in the MMS-induced response. However, in contrast to UV and other inducers of the JNK/SAPKs and p38 pathways, activation of growth factor and G-protein-coupled receptors does not play a role in the MMS response. We identified the intracellular glutathione (GSH) level as critical for JNK/SAPK activation by MMS: enhancing the GSH level by pretreatment of the cells with GSH or N-acetylcysteine inhibits, whereas depletion of the cellular GSH pool causes hyperinduction of JNK/SAPK activity by MMS. In light of the JNK/SAPK-dependent induction of c-jun and c-fos transcription, and the Jun/Fos-induced transcription of xenobiotic-metabolizing enzymes, these data provide a potential critical role of JNK/SAPK and p38 in the induction of a cellular defense program against cytotoxic xenobiotics such as MMS. PMID:9234735

  8. Methoxychlor and Vinclozolin Induce Rapid Changes in Intercellular and Intracellular Signaling in Liver Progenitor Cells.

    PubMed

    Babica, Pavel; Zurabian, Rimma; Kumar, Esha R; Chopra, Rajus; Mianecki, Maxwell J; Park, Joon-Suk; Jaša, Libor; Trosko, James E; Upham, Brad L

    2016-09-01

    Methoxychlor (MXC) and vinclozolin (VIN) are well-recognized endocrine disrupting chemicals known to alter epigenetic regulations and transgenerational inheritance; however, non-endocrine disruption endpoints are also important. Thus, we determined the effects of MXC and VIN on the dysregulation of gap junctional intercellular communication (GJIC) and activation of mitogen-activated protein kinases (MAPKs) in WB-F344 rat liver epithelial cells. Both chemicals induced a rapid dysregulation of GJIC at non-cytotoxic doses, with 30 min EC50 values for GJIC inhibition being 10 µM for MXC and 126 µM for VIN. MXC inhibited GJIC for at least 24 h, while VIN effects were transient and GJIC recovered after 4 h. VIN induced rapid hyperphosphorylation and internalization of gap junction protein connexin43, and both chemicals also activated MAPK ERK1/2 and p38. Effects on GJIC were not prevented by MEK1/2 inhibitor, but by an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), resveratrol, and in the case of VIN, also, by a p38 inhibitor. Estrogen (ER) and androgen receptor (AR) modulators (estradiol, ICI 182,780, HPTE, testosterone, flutamide, VIN M2) did not attenuate MXC or VIN effects on GJIC. Our data also indicate that the effects were elicited by the parental compounds of MXC and VIN. Our study provides new evidence that MXC and VIN dysregulate GJIC via mechanisms involving rapid activation of PC-PLC occurring independently of ER- or AR-dependent genomic signaling. Such alterations of rapid intercellular and intracellular signaling events involved in regulations of gene expression, tissue development, function and homeostasis, could also contribute to transgenerational epigenetic effects of endocrine disruptors. PMID:27413106

  9. Methoxychlor and Vinclozolin Induce Rapid Changes in Intercellular and Intracellular Signaling in Liver Progenitor Cells.

    PubMed

    Babica, Pavel; Zurabian, Rimma; Kumar, Esha R; Chopra, Rajus; Mianecki, Maxwell J; Park, Joon-Suk; Jaša, Libor; Trosko, James E; Upham, Brad L

    2016-09-01

    Methoxychlor (MXC) and vinclozolin (VIN) are well-recognized endocrine disrupting chemicals known to alter epigenetic regulations and transgenerational inheritance; however, non-endocrine disruption endpoints are also important. Thus, we determined the effects of MXC and VIN on the dysregulation of gap junctional intercellular communication (GJIC) and activation of mitogen-activated protein kinases (MAPKs) in WB-F344 rat liver epithelial cells. Both chemicals induced a rapid dysregulation of GJIC at non-cytotoxic doses, with 30 min EC50 values for GJIC inhibition being 10 µM for MXC and 126 µM for VIN. MXC inhibited GJIC for at least 24 h, while VIN effects were transient and GJIC recovered after 4 h. VIN induced rapid hyperphosphorylation and internalization of gap junction protein connexin43, and both chemicals also activated MAPK ERK1/2 and p38. Effects on GJIC were not prevented by MEK1/2 inhibitor, but by an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), resveratrol, and in the case of VIN, also, by a p38 inhibitor. Estrogen (ER) and androgen receptor (AR) modulators (estradiol, ICI 182,780, HPTE, testosterone, flutamide, VIN M2) did not attenuate MXC or VIN effects on GJIC. Our data also indicate that the effects were elicited by the parental compounds of MXC and VIN. Our study provides new evidence that MXC and VIN dysregulate GJIC via mechanisms involving rapid activation of PC-PLC occurring independently of ER- or AR-dependent genomic signaling. Such alterations of rapid intercellular and intracellular signaling events involved in regulations of gene expression, tissue development, function and homeostasis, could also contribute to transgenerational epigenetic effects of endocrine disruptors.

  10. The effect of photoinitiators on intracellular AKT signaling pathway in tissue engineering application

    PubMed Central

    Xu, Leyuan; Sheybani, Natasha; Yeudall, W. Andrew; Yang, Hu

    2015-01-01

    Free-radical photopolymerization initiated by photoinitiators is an important method to make tissue engineering scaffolds. To advance understanding of photoinitiator cytocompatibility, we examined three photoinitiators including 2,2-dimethoxy-2-phenylacetophenone (DMPA), Irgacure 2959 (I-2959), and eosin Y photoinitiating system (EY) in terms of their effects on viability of HN4 cells and expression levels of intracellular AKT and its phosphorylated form p-AKT. Our results show that the photoinitiators and their UV-exposed counterparts affect intracellular AKT signaling, which can be used in conjunction with cell viability for cytocompatibility assessment of photoinitiators. PMID:25709809

  11. Down-regulation of the P-glycoprotein relevant for multidrug resistance by intracellular acidification through the crosstalk of MAPK signaling pathways.

    PubMed

    Jin, Weina; Lu, Ying; Li, Qinghua; Wang, Jian; Zhang, Hongju; Chang, Guoqiang; Lin, Yani; Pang, Tianxiang

    2014-09-01

    In our previous study, we have found that the tumor multidrug resistance mediated by P-glycoprotein could be reversed by sustained intracellular acidification through down-regulating the multidrug resistance gene 1 mRNA and P-glycoprotein expression. However, the molecular events linking the intracellular acidification and the regulation of P-glycoprotein remain unclear. In the present study, the molecular pathways involved in the regulation of P-glycoprotein expression by the intracellular acidification were investigated. We found that the P-glycoprotein expression was down-regulated by the intracellular acidification through inhibition of p38 mitogen-activated protein kinase (MAPK) and the activation of c-Jun N-terminal kinase (JNK) in the resisitant K562/DOX cells. In the sensitive K562 and HL60 cell lines, the changes of the p38 MAPK expression after the acidification are not as obvious as that of K562/DOX cells, but the activation of extracellular signal-regulated kinase (ERK) is also observed, which indicates that the down-regulation of p38 MAPK by the intracellular acidification might be the resistant cell line specific. Blockade of ERK and JNK signaling by the inhibitors or RNA interference increased p38MAPK activities suggesting that cross-talk within MAPKs is also important for this response. Our study provides the first direct evidence that the reversal of P-glycoprotein-mediated multidrug resistance by intracellular acidification is mediated by the crosstalk of MAPK signaling pathways.

  12. Neurodegeneration in Alzheimer Disease: Role of Amyloid Precursor Protein and Presenilin 1 Intracellular Signaling

    PubMed Central

    Nizzari, Mario; Thellung, Stefano; Corsaro, Alessandro; Villa, Valentina; Pagano, Aldo; Porcile, Carola; Russo, Claudio; Florio, Tullio

    2012-01-01

    Alzheimer disease (AD) is a heterogeneous neurodegenerative disorder characterized by (1) progressive loss of synapses and neurons, (2) intracellular neurofibrillary tangles, composed of hyperphosphorylated Tau protein, and (3) amyloid plaques. Genetically, AD is linked to mutations in few proteins amyloid precursor protein (APP) and presenilin 1 and 2 (PS1 and PS2). The molecular mechanisms underlying neurodegeneration in AD as well as the physiological function of APP are not yet known. A recent theory has proposed that APP and PS1 modulate intracellular signals to induce cell-cycle abnormalities responsible for neuronal death and possibly amyloid deposition. This hypothesis is supported by the presence of a complex network of proteins, clearly involved in the regulation of signal transduction mechanisms that interact with both APP and PS1. In this review we discuss the significance of novel finding related to cell-signaling events modulated by APP and PS1 in the development of neurodegeneration. PMID:22496686

  13. Aquaporin-3 mediates hydrogen peroxide uptake to regulate downstream intracellular signaling

    PubMed Central

    Miller, Evan W.; Dickinson, Bryan C.; Chang, Christopher J.

    2010-01-01

    Hydrogen peroxide (H2O2) produced by cell-surface NADPH Oxidase (Nox) enzymes is emerging as an important signaling molecule for growth, differentiation, and migration processes. However, how cells spatially regulate H2O2 to achieve physiological redox signaling over nonspecific oxidative stress pathways is insufficiently understood. Here we report that the water channel Aquaporin-3 (AQP3) can facilitate the uptake of H2O2 into mammalian cells and mediate downstream intracellular signaling. Molecular imaging with Peroxy Yellow 1 Methyl-Ester (PY1-ME), a new chemoselective fluorescent indicator for H2O2, directly demonstrates that aquaporin isoforms AQP3 and AQP8, but not AQP1, can promote uptake of H2O2 specifically through membranes in mammalian cells. Moreover, we show that intracellular H2O2 accumulation can be modulated up or down based on endogenous AQP3 expression, which in turn can influence downstream cell signaling cascades. Finally, we establish that AQP3 is required for Nox-derived H2O2 signaling upon growth factor stimulation. Taken together, our findings demonstrate that the downstream intracellular effects of H2O2 can be regulated across biological barriers, a discovery that has broad implications for the controlled use of this potentially toxic small molecule for beneficial physiological functions. PMID:20724658

  14. Single-Molecule Imaging Reveals the Activation Dynamics of Intracellular Protein Smad3 on Cell Membrane

    NASA Astrophysics Data System (ADS)

    Li, Nan; Yang, Yong; He, Kangmin; Zhang, Fayun; Zhao, Libo; Zhou, Wei; Yuan, Jinghe; Liang, Wei; Fang, Xiaohong

    2016-09-01

    Smad3 is an intracellular protein that plays a key role in propagating transforming growth factor β (TGF-β) signals from cell membrane to nucleus. However whether the transient process of Smad3 activation occurs on cell membrane and how it is regulated remains elusive. Using advanced live-cell single-molecule fluorescence microscopy to image and track fluorescent protein-labeled Smad3, we observed and quantified, for the first time, the dynamics of individual Smad3 molecules docking to and activation on the cell membrane. It was found that Smad3 docked to cell membrane in both unstimulated and stimulated cells, but with different diffusion rates and dissociation kinetics. The change in its membrane docking dynamics can be used to study the activation of Smad3. Our results reveal that Smad3 binds with type I TGF-β receptor (TRI) even in unstimulated cells. Its activation is regulated by TRI phosphorylation but independent of receptor endocytosis. This study offers new information on TGF-β/Smad signaling, as well as a new approach to investigate the activation of intracellular signaling proteins for a better understanding of their functions in signal transduction.

  15. Single-Molecule Imaging Reveals the Activation Dynamics of Intracellular Protein Smad3 on Cell Membrane.

    PubMed

    Li, Nan; Yang, Yong; He, Kangmin; Zhang, Fayun; Zhao, Libo; Zhou, Wei; Yuan, Jinghe; Liang, Wei; Fang, Xiaohong

    2016-01-01

    Smad3 is an intracellular protein that plays a key role in propagating transforming growth factor β (TGF-β) signals from cell membrane to nucleus. However whether the transient process of Smad3 activation occurs on cell membrane and how it is regulated remains elusive. Using advanced live-cell single-molecule fluorescence microscopy to image and track fluorescent protein-labeled Smad3, we observed and quantified, for the first time, the dynamics of individual Smad3 molecules docking to and activation on the cell membrane. It was found that Smad3 docked to cell membrane in both unstimulated and stimulated cells, but with different diffusion rates and dissociation kinetics. The change in its membrane docking dynamics can be used to study the activation of Smad3. Our results reveal that Smad3 binds with type I TGF-β receptor (TRI) even in unstimulated cells. Its activation is regulated by TRI phosphorylation but independent of receptor endocytosis. This study offers new information on TGF-β/Smad signaling, as well as a new approach to investigate the activation of intracellular signaling proteins for a better understanding of their functions in signal transduction. PMID:27641076

  16. Single-Molecule Imaging Reveals the Activation Dynamics of Intracellular Protein Smad3 on Cell Membrane

    PubMed Central

    Li, Nan; Yang, Yong; He, Kangmin; Zhang, Fayun; Zhao, Libo; Zhou, Wei; Yuan, Jinghe; Liang, Wei; Fang, Xiaohong

    2016-01-01

    Smad3 is an intracellular protein that plays a key role in propagating transforming growth factor β (TGF-β) signals from cell membrane to nucleus. However whether the transient process of Smad3 activation occurs on cell membrane and how it is regulated remains elusive. Using advanced live-cell single-molecule fluorescence microscopy to image and track fluorescent protein-labeled Smad3, we observed and quantified, for the first time, the dynamics of individual Smad3 molecules docking to and activation on the cell membrane. It was found that Smad3 docked to cell membrane in both unstimulated and stimulated cells, but with different diffusion rates and dissociation kinetics. The change in its membrane docking dynamics can be used to study the activation of Smad3. Our results reveal that Smad3 binds with type I TGF-β receptor (TRI) even in unstimulated cells. Its activation is regulated by TRI phosphorylation but independent of receptor endocytosis. This study offers new information on TGF-β/Smad signaling, as well as a new approach to investigate the activation of intracellular signaling proteins for a better understanding of their functions in signal transduction. PMID:27641076

  17. Liraglutide, leptin, and their combined effects on feeding: additive intake reduction through common intracellular signaling mechanisms

    PubMed Central

    Kanoski, Scott E.; Ong, Zhi Yi; Fortin, Samantha M.; Schlessinger, Elizabeth S.; Grill, Harvey J.

    2014-01-01

    Aims Glucagon like peptide-1 receptor (GLP-1R) agonists and leptin each exert anorexigenic effects. In combination, the intake inhibitory and weight loss effects are greater than either treatment alone, however the mechanisms unclear. Materials and methods Effects of liraglutide (a long-acting GLP-1 analogue) and leptin co-treatment, delivered in low or moderate doses subcutaneously (SC) or to the 3rd ventricle respectively, on cumulative intake, meal patterns, and hypothalamic expression of intracellular signaling proteins [phosphorylated signal transducer and activator of transcription-3 (pSTAT3) and protein tyrosine phosphatase-1B (PTP1B)] were examined in lean rats. Results A low-dose combination of liraglutide (25μg/kg) and leptin (0.75μg) additively reduced cumulative food intake and body weight, a result mediated predominantly through a significant reduction in meal frequency that was not present with either drug alone. Liraglutide treatment alone also reduced meal size; an effect not enhanced with leptin co-administration. Moderate doses of liraglutide (75μg/kg) and leptin (4μg) examined separately each reduced meal frequency, cumulative food intake, and body weight; only liraglutide reduced meal size. In combination these doses did not further enhance the anorexigenic effects of either treatment alone. Ex vivo immunoblot showed elevated pSTAT3 in hypothalamic tissue following liraglutide-leptin co-treatment, an effect greater than leptin treatment alone. In addition, SC liraglutide reduced expression of PTP1B (a negative regulator of leptin receptor signaling), revealing a potential mechanism for the enhanced pSTAT3 response following liraglutide-leptin co-administration. Conclusions Collectively, these results provide novel behavioral and molecular mechanisms underlying the additive reduction in food intake and body weight following liraglutide-leptin combination treatment. PMID:25475828

  18. Tracking SERS-active nanoprobe intracellular uptake for chemical and biological sensing

    NASA Astrophysics Data System (ADS)

    Gregas, Molly K.; Yan, Fei; Scaffidi, Jonathan; Wang, Hsin-Neng; Khoury, Christopher; Zhang, Yan; Vo-Dinh, Tuan

    2007-09-01

    A critical aspect of the use of nanoprobes for intracellular studies in chemical and biological sensing involves a fundamental understanding of their uptake and trajectory in cells. In this study, we describe experiments using surface-enhanced Raman scattering (SERS) spectroscopy and mapping to track cellular uptake of plasmonics-active labeled nanoparticles. Three different Raman-active labels with positive, negative, and neutral charges were conjugated to silver colloidal nanoparticles with the aim of spatially and temporally profiling intracellular delivery and tracking of nanoprobes during uptake in single mammalian cells. 1-D Raman spectra and 2-D Raman mapping are used to identify and locate the probes via their SERS signal intensities. Because Raman spectroscopy is very specific for identification of chemical and molecular signatures, the development of functionalized plasmonics-active nanoprobes capable of exploring intracellular spaces and processes has the ability to provide specific information on the effects of biological and chemical pollutants in the intracellular environment. The results indicate that this technique will allow study of when, where, and how these substances affect cells and living organisms.

  19. Intracellular calcium signals regulate growth of hepatic stellate cells via specific effects on cell cycle progression

    PubMed Central

    Soliman, Elwy M.; Rodrigues, Michele Angela; Gomes, Dawidson Assis; Sheung, Nina; Yu, Jin; Amaya, Maria Jimina; Nathanson, Michael H.; Dranoff, Jonathan A.

    2010-01-01

    Hepatic stellate cells (HSC) are important mediators of liver fibrosis. Hormones linked to downstream intracellular Ca2+ signals upregulate HSC proliferation, but the mechanisms by which this occurs are unknown. Nuclear and cytosolic Ca2+ signals may have distinct effects on cell proliferation, so we expressed plasmid and adenoviral constructs containing the Ca2+ chelator parvalbumin (PV) linked to either a nuclear localization sequence (NLS) or a nuclear export sequence (NES) to block Ca2+ signals in distinct compartments within LX-2 immortalized human HSC and primary rat HSC. PV-NLS and PV-NES constructs each targeted to the appropriate intracellular compartment and blocked Ca2+ signals only within that compartment. PV-NLS and PV-NES constructs inhibited HSC growth. Furthermore, blockade of nuclear or cytosolic Ca2+ signals arrested growth at the G2/mitosis (G2/M) cell-cycle interface and prevented the onset of mitosis. Blockade of nuclear or cytosolic Ca2+ signals downregulated phosphorylation of the G2/M checkpoint phosphatase Cdc25C. Inhibition of calmodulin kinase II (CaMK II) had identical effects on LX-2 growth and Cdc25C phosphorylation. We propose that nuclear and cytosolic Ca2+ are critical signals that regulate HSC growth at the G2/M checkpoint via CaMK II-mediated regulation of Cdc25C phosphorylation. These data provide a new logical target for pharmacological therapy directed against progression of liver fibrosis. PMID:19131107

  20. Lipid raft-dependent uptake, signaling, and intracellular fate of Porphyromonas gingivalis in mouse macrophages

    PubMed Central

    Wang, Min; Hajishengallis, George

    2009-01-01

    Summary Lipid rafts are cholesterol-enriched microdomains involved in cellular trafficking and implicated as portals for certain pathogens. We sought to determine whether the oral pathogen Porphyromonas gingivalis enters macrophages via lipid rafts, and if so, to examine the impact of raft entry on its intracellular fate. Using J774A.1 mouse macrophages, we found that P. gingivalis colocalizes with lipid rafts in a cholesterol-dependent way. Depletion of cellular cholesterol using methyl-β-cyclodextrin resulted in about 50% inhibition of P. gingivalis uptake, although this effect was reversed by cholesterol reconstitution. The intracellular survival of P. gingivalis was dramatically inhibited in cholesterol-depleted cells relative to untreated or cholesterol-reconstituted cells, even when infections were adjusted to allow equilibration of the initial intracellular bacterial load. P. gingivalis thus appeared to exploit raft-mediated uptake for promoting its survival. Consistent with this, lipid raft disruption enhanced the colocalization of internalized P. gingivalis with lysosomes. In contrast, raft disruption did not affect the expression of host receptors interacting with P. gingivalis, although it significantly inhibited signal transduction. In summary, P. gingivalis uses macrophage lipid rafts as signaling and entry platforms, which determine its intracellular fate to the pathogen’s own advantage. PMID:18547335

  1. Intracellular Signaling Transduction Pathways Triggered by a Well-Known Anti-GHR Monoclonal Antibody, Mab263, in Vitro and in Vivo

    PubMed Central

    Lan, Hainan; Li, Wei; Jiang, Hailong; Yang, Yanhong; Zheng, Xin

    2014-01-01

    A series of studies have reported that monoclonal antibody 263 (Mab263), a monoclonal antibody against the growth hormone receptor (GHR), acts as an agonist in vitro and in vivo. However, the intracellular signaling pathways triggered by Mab263 have not yet been delineated. Therefore, we examined the intracellular signaling pathways induced by Mab263 in vivo and in vitro in the present study. The results show that this antibody activated janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), STAT1 and extracellular signal-regulated kinase 1/2 (ERK1/2), but not STAT5. The phosphorylation kinetics of JAK2, STAT3/1 and ERK1/2 induced by Mab263 were subsequently analyzed in dose-response and time course experiments. Our observations indicate that Mab263 induced different intracellular signaling pathways than GH, which indicates that Mab263 is a signal-specific molecule and that Mab263 may be a valuable biological reagent to study the mechanism(s) of GHR-mediated intracellular signaling pathways. PMID:25391041

  2. Real-time monitoring of intracellular signal transduction in PC12 cells by non-adiabatic tapered optical fiber biosensor

    NASA Astrophysics Data System (ADS)

    Zibaii, M. I.; Latifi, H.; Asadollahi, A.; Noraeipoor, Z.; Dargahi, L.

    2014-05-01

    Real-time observation of intracellular process of signal transduction is very useful for biomedical and pharmaceutical applications as well as for basic research work of cell biology. For feasible and reagentless observation of intracellular alterations in real time, we examined the use of a nonadiabatic tapered optical fiber (NATOF) biosensor for monitoring of intracellular signal transduction that was mainly translocation of protein kinase C via refractive index change in PC12 cells adhered on tapered fiber sensor without any indicator reagent. PC12 cells were stimulated with KCl . Our results suggest that complex intracellular reactions could be real-time monitored and characterized by NATOF biosensor.

  3. An enhanced functional interrogation/manipulation of intracellular signaling pathways with the peptide 'stapling' technology.

    PubMed

    He, Y; Chen, D; Zheng, W

    2015-11-12

    Specific protein-protein interactions (PPIs) constitute a key underlying mechanism for the presence of a multitude of intracellular signaling pathways, which are essential for the survival of normal and cancer cells. Specific molecular blockers for a crucial PPI would therefore be invaluable tools for an enhanced functional interrogation of the signaling pathway harboring this particular PPI. On the other hand, if a particular PPI is essential for the survival of cancer cells but is absent in or dispensable for the survival of normal cells, its specific molecular blockers could potentially be developed into effective anticancer therapeutics. Due to the flat and extended PPI interface, it would be conceivably difficult for small molecules to achieve an effective blockade, a problem which could be potentially circumvented with peptides or proteins. However, the well-documented proteolytic instability and cellular impermeability of peptides and proteins in general would make their developing into effective intracellular PPI blockers quite a challenge. With the advent of the peptide 'stapling' technology which was demonstrated to be able to stabilize the α-helical conformation of a peptide via bridging two neighboring amino-acid side chains with a 'molecular staple', a linear parent peptide could be transformed into a stronger PPI blocker with enhanced proteolytic stability and cellular permeability. This review will furnish an account on the peptide 'stapling' technology and its exploitation in efforts to achieve an enhanced functional interrogation or manipulation of intracellular signaling pathways especially those that are cancer relevant.

  4. Shock wave irradiations avoiding fluid flow evoke intracellular Ca2+ signaling

    NASA Astrophysics Data System (ADS)

    Takahashi, Toru; Tsukamoto, Akira; Tada, Shigeru

    Shock wave irradiation accelerates therapeutic effects including angiogenesis. One mechanism underlying those effects is cellular responses evoked by shock wave irradiation. Fluid flow is one of major physical phenomena induced by shock wave irradiation. Cellular responses evoked by fluid flow are similar to those evoked by shock wave irradiation. Thus, fluid flow could be responsible for cellular responses evoked by shock wave irradiation. However, it is obscure whether fluid flow is required for the cellular responses evoked by shock wave irradiation. In this study, intracellular Ca2 + signaling was observed in cells seeded in down-sized chambers. In the down-sized chambers, fluid flow was supposed to be suppressed because size of chambers (6 mm in diameter, 1 mm in thickness) was analogous to size of shock wave focus region (3mm in diameter). Dynamics of polystyrene microbeads suspended in the chambers were visualized with a CCD camera and analyzed with a particle image velocimetry (PIV) method to quantify fluid flow in the chamber. As a result, shock wave irradiation evoked intracellular Ca2 + signaling. However, fluid flow was not observed in the chamber due to shock wave irradiation. Thus, it was suggested that physical mechanics, not fluid flow, are further required for evoking intracellular Ca2 + signaling following to shock wave irradiation.

  5. Intracellular and intercellular signaling networks in cancer initiation, development and precision anti-cancer therapy: RAS acts as contextual signaling hub.

    PubMed

    Csermely, Peter; Korcsmáros, Tamás; Nussinov, Ruth

    2016-10-01

    Cancer initiation and development are increasingly perceived as systems-level phenomena, where intra- and inter-cellular signaling networks of the ecosystem of cancer and stromal cells offer efficient methodologies for outcome prediction and intervention design. Within this framework, RAS emerges as a 'contextual signaling hub', i.e. the final result of RAS activation or inhibition is determined by the signaling network context. Current therapies often 'train' cancer cells shifting them to a novel attractor, which has increased metastatic potential and drug resistance. The few therapy-surviving cancer cells are surrounded by massive cell death triggering a primordial adaptive and reparative general wound healing response. Overall, dynamic analysis of patient- and disease-stage specific intracellular and intercellular signaling networks may open new areas of anticancer therapy using multitarget drugs, drugs combinations, edgetic drugs, as well as help design 'gentler', differentiation and maintenance therapies. PMID:27395026

  6. Ehrlichia chaffeensis Exploits Canonical and Noncanonical Host Wnt Signaling Pathways To Stimulate Phagocytosis and Promote Intracellular Survival

    PubMed Central

    Luo, Tian; Dunphy, Paige S.; Lina, Taslima T.

    2015-01-01

    Ehrlichia chaffeensis invades and survives in phagocytes by modulating host cell processes and evading innate defenses, but the mechanisms are not fully defined. Recently we have determined that E. chaffeensis tandem repeat proteins (TRPs) are type 1 secreted effectors involved in functionally diverse interactions with host targets, including components of the evolutionarily conserved Wnt signaling pathways. In this study, we demonstrated that induction of host canonical and noncanonical Wnt pathways by E. chaffeensis TRP effectors stimulates phagocytosis and promotes intracellular survival. After E. chaffeensis infection, canonical and noncanonical Wnt signalings were significantly stimulated during early stages of infection (1 to 3 h) which coincided with dephosphorylation and nuclear translocation of β-catenin, a major canonical Wnt signal transducer, and NFATC1, a noncanonical Wnt transcription factor. In total, the expression of ∼44% of Wnt signaling target genes was altered during infection. Knockdown of TRP120-interacting Wnt pathway components/regulators and other critical components, such as Wnt5a ligand, Frizzled 5 receptor, β-catenin, nuclear factor of activated T cells (NFAT), and major signaling molecules, resulted in significant reductions in the ehrlichial load. Moreover, small-molecule inhibitors specific for components of canonical and noncanonical (Ca2+ and planar cell polarity [PCP]) Wnt pathways, including IWP-2, which blocks Wnt secretion, significantly decreased ehrlichial infection. TRPs directly activated Wnt signaling, as TRP-coated microspheres triggered phagocytosis which was blocked by Wnt pathway inhibitors, demonstrating a key role of TRP activation of Wnt pathways to induce ehrlichial phagocytosis. These novel findings reveal that E. chaffeensis exploits canonical and noncanonical Wnt pathways through TRP effectors to facilitate host cell entry and promote intracellular survival. PMID:26712203

  7. Rab8 modulates metabotropic glutamate receptor subtype 1 intracellular trafficking and signaling in a protein kinase C-dependent manner.

    PubMed

    Esseltine, Jessica L; Ribeiro, Fabiola M; Ferguson, Stephen S G

    2012-11-21

    Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors (GPCRs) that are activated by glutamate, the primary excitatory neurotransmitter in the CNS. Alterations in glutamate receptor signaling are implicated in neuropathologies such as Alzheimer's disease, ischemia, and Huntington's disease among others. Group 1 mGluRs (mGluR1 and mGluR5) are primarily coupled to Gα(q/11) leading to the activation of phospholipase C and the formation of diacylglycerol and inositol 1,4,5-trisphosphate, which results in the release of intracellular calcium stores and protein kinase C (PKC) activation. Desensitization, endocytosis, and recycling are major mechanisms of GPCR regulation, and the intracellular trafficking of GPCRs is linked to the Rab family of small G proteins. Rab8 is a small GTPase that is specifically involved in the regulation of secretory/recycling vesicles, modulation of the actin cytoskeleton, and cell polarity. Rab8 has been shown to regulate the synaptic delivery of AMPA receptors during long-term potentiation and during constitutive receptor recycling. We show here that Rab8 interacts with the C-terminal tail of mGluR1a in an agonist-dependent manner and plays a role in regulating of mGluR1a signaling and intracellular trafficking in human embryonic kidney 293 cells. Specifically, Rab8 expression attenuates mGluR1a-mediated inositol phosphate formation and calcium release from mouse neurons in a PKC-dependent manner, while increasing cell surface mGluR1a expression via decreased receptor endocytosis. These experiments provide us with an understanding of the role Rabs play in coordinated regulation of mGluR1a and how this impacts mGluR1a signaling.

  8. The role of intracellular calcium signals in inflammatory responses of polarised cystic fibrosis human airway epithelia.

    PubMed

    Ribeiro, Carla Maria Pedrosa

    2006-01-01

    Hyperinflammatory host responses to bacterial infection have been postulated to be a key step in the pathogenesis of cystic fibrosis (CF) lung disease. Previous studies have indicated that the CF airway epithelium itself contributes to the hyperinflammation of CF airways via an excessive inflammatory response to bacterial infection. However, it has been controversial whether the hyperinflammation of CF epithelia results from mutations in the CF transmembrane conductance regulator (CFTR) and/or is a consequence of persistent airways infection. Recent studies have demonstrated that intracellular calcium (Ca2+i) signals consequent to activation of apical G protein-coupled receptors (GPCRs) by pro-inflammatory mediators are increased in CF airway epithelia. Because of the relationship between Ca2+i mobilisation and inflammatory responses, the mechanism for the increased Ca2+i signals in CF was investigated and found to result from endoplasmic reticulum (ER) Ca2+ store expansion. The ER Ca2+ store expansion imparts a hyperinflammatory phenotype to chronically infected airway epithelia as a result of the larger Ca2+i mobilisation coupled to an excessive inflammatory response following GPCR activation. The ER expansion is not dependent on ER retention of misfolded DeltaF508 CFTR, but reflects an epithelial response acquired following persistent luminal airway infection. With respect to the mechanism of ER expansion in CF, the current view is that chronic airway epithelial infection triggers an unfolded protein response as a result of the increased flux of newly synthesised inflammatory mediators and defensive factors into the ER compartment. This unfolded protein response is coupled to X-box binding protein 1 (XBP-1) mRNA splicing and transcription of genes associated with the expansion of the protein-folding capacity of the ER (e.g. increases in ER chaperones and ER membranes). These studies have revealed a novel adaptive response in chronically infected airway epithelia

  9. Coupling of active motion and advection shapes intracellular cargo transport.

    PubMed

    Khuc Trong, Philipp; Guck, Jochen; Goldstein, Raymond E

    2012-07-13

    Intracellular cargo transport can arise from passive diffusion, active motor-driven transport along cytoskeletal filament networks, and passive advection by fluid flows entrained by such cargo-motor motion. Active and advective transport are thus intrinsically coupled as related, yet different representations of the same underlying network structure. A reaction-advection-diffusion system is used here to show that this coupling affects the transport and localization of a passive tracer in a confined geometry. For sufficiently low diffusion, cargo localization to a target zone is optimized either by low reaction kinetics and decoupling of bound and unbound states, or by a mostly disordered cytoskeletal network with only weak directional bias. These generic results may help to rationalize subtle features of cytoskeletal networks, for example as observed for microtubules in fly oocytes.

  10. Characterization of the intracellular signalling pathways that underlie growth-factor-stimulated glucose transport in Xenopus oocytes: evidence for ras- and rho-dependent pathways of phosphatidylinositol 3-kinase activation.

    PubMed Central

    Thomson, F J; Jess, T J; Moyes, C; Plevin, R; Gould, G W

    1997-01-01

    The stimulation of glucose transport is one of the early cellular responses to growth factors and is essential for cell proliferation, yet the molecular processes that underlie this response are poorly defined. The aim of this study was to characterize the role of the low-molecular-mass G-proteins, Ras and Rho, and their downstream targets, Raf protein kinase and phosphatidylinositol 3-kinase, in the regulation of glucose transport in Xenopus oocytes by two distinct growth-factor receptors: the insulin-like growth factor I (IGF-I) tyrosine kinase receptor and the heterotrimeric G-protein-coupled lysophosphatidic acid (LPA) receptor. Microinjection of a neutralizing anti-Ras antibody partially blocked IGF-I-stimulated deoxyglucose uptake but was without effect on LPA-stimulated deoxyglucose uptake. In contrast, microinjection of the C3 coenzyme of botulinum toxin, which selectively ADP-ribosylates and inactivates Rho, inhibited LPA-stimulated, but not IGF-I-stimulated, deoxyglucose uptake. Similarly, LPA- but not IGF-I-stimulated deoxyglucose uptake was attenuated in oocytes expressing a dominant negative rho construct. Cells expressing a dominant negative mutant of Raf protein kinase exhibited markedly reduced sensitivity to both LPA and IGF-I, consistent with a role for endogenous Raf in glucose uptake by both growth factors. Furthermore, expression of a constitutively activated form of raf-1 resulted in a growth-factor-independent increase in deoxyglucose uptake. Measurements of phosphatidylinositol 3-kinase activity in microinjected cells support the hypothesis that the IGF-I receptor stimulates glucose transport by a Ras-dependent activation of phosphatidylinositol 3-kinase, whereas the G-protein-coupled LPA receptor controls this response by a pathway that involves Rho-dependent activation of a distinct phosphatidylinositol 3-kinase. Thus we provide evidence for clear differences in the signalling pathways that control glucose transport by G

  11. The effect of feeding during recovery from aerobic exercise on skeletal muscle intracellular signaling.

    PubMed

    Reidy, Paul T; Konopka, Adam R; Hinkley, J Matthew; Undem, Miranda K; Harber, Matthew P

    2014-02-01

    We previously reported an increase in skeletal muscle protein synthesis during fasted and fed recovery from nonexhaustive aerobic exercise (Harber et al., 2010). The current study examined skeletal muscle intracellular signaling in the same subjects to further investigate mechanisms of skeletal muscle protein metabolism with and without feeding following aerobic exercise. Eight males (VO₂peak: 52 ± 2 ml⁻¹·kg⁻¹·min⁻¹) performed 60-min of cycle ergometry at 72 ± 1% VO₂peak on two occasions in a counter-balanced design. Exercise trials differed only in the postexercise nutritional intervention: EX-FED (5 kcal, 0.83 g carbohydrate, 0.37 g protein, 0.03 g fat per kg body weight) and EX-FAST (noncaloric, isovolumic placebo) ingested immediately and one hour after exercise. Muscle biopsies were obtained from the vastus lateralis at rest (on a separate day) and two hours postexercise to assess intracellular signaling via western blotting of p70S6K1, eEF2, 4EBP1, AMPKα and p38 MAPK. p70S6K1 phosphorylation was elevated (p < .05) in EX-FED relative to REST and EX-FAST. eEF2, 4EBP1, AMPKα and p38 MAPK signaling were unaltered at 2 h after exercise independent of feeding status when expressed as the ratio of phosphorylated to total protein normalized to actin. These data demonstrate that feeding after a nonexhaustive bout of aerobic exercise stimulates skeletal muscle p70S6K1 intracellular signaling favorable for promoting protein synthesis which may, as recent literature has suggested, better prepare the muscle for subsequent exercise bouts. These data provide further support into the role of feeding on mechanisms regulating muscle protein metabolism during recovery from aerobic exercise.

  12. A census of membrane-bound and intracellular signal transduction proteins in bacteria: Bacterial IQ, extroverts and introverts

    PubMed Central

    Galperin, Michael Y

    2005-01-01

    Background Analysis of complete microbial genomes showed that intracellular parasites and other microorganisms that inhabit stable ecological niches encode relatively primitive signaling systems, whereas environmental microorganisms typically have sophisticated systems of environmental sensing and signal transduction. Results This paper presents results of a comprehensive census of signal transduction proteins – histidine kinases, methyl-accepting chemotaxis receptors, Ser/Thr/Tyr protein kinases, adenylate and diguanylate cyclases and c-di-GMP phosphodiesterases – encoded in 167 bacterial and archaeal genomes, sequenced by the end of 2004. The data have been manually checked to avoid false-negative and false-positive hits that commonly arise during large-scale automated analyses and compared against other available resources. The census data show uneven distribution of most signaling proteins among bacterial and archaeal phyla. The total number of signal transduction proteins grows approximately as a square of genome size. While histidine kinases are found in representatives of all phyla and are distributed according to the power law, other signal transducers are abundant in certain phylogenetic groups but virtually absent in others. Conclusion The complexity of signaling systems differs even among closely related organisms. Still, it usually can be correlated with the phylogenetic position of the organism, its lifestyle, and typical environmental challenges it encounters. The number of encoded signal transducers (or their fraction in the total protein set) can be used as a measure of the organism's ability to adapt to diverse conditions, the 'bacterial IQ', while the ratio of transmembrane receptors to intracellular sensors can be used to define whether the organism is an 'extrovert', actively sensing the environmental parameters, or an 'introvert', more concerned about its internal homeostasis. Some of the microorganisms with the highest IQ, including the

  13. Diverse intracellular pathogens activate type III interferon expression from peroxisomes.

    PubMed

    Odendall, Charlotte; Dixit, Evelyn; Stavru, Fabrizia; Bierne, Helene; Franz, Kate M; Durbin, Ann Fiegen; Boulant, Steeve; Gehrke, Lee; Cossart, Pascale; Kagan, Jonathan C

    2014-08-01

    Type I interferon responses are considered the primary means by which viral infections are controlled in mammals. Despite this view, several pathogens activate antiviral responses in the absence of type I interferons. The mechanisms controlling type I interferon-independent responses are undefined. We found that RIG-I like receptors (RLRs) induce type III interferon expression in a variety of human cell types, and identified factors that differentially regulate expression of type I and type III interferons. We identified peroxisomes as a primary site of initiation of type III interferon expression, and revealed that the process of intestinal epithelial cell differentiation upregulates peroxisome biogenesis and promotes robust type III interferon responses in human cells. These findings highlight the importance of different intracellular organelles in specific innate immune responses.

  14. Enzyme-activated intracellular drug delivery with tubule clay nanoformulation

    PubMed Central

    Dzamukova, Maria R.; Naumenko, Ekaterina A.; Lvov, Yuri M.; Fakhrullin, Rawil F.

    2015-01-01

    Fabrication of stimuli-triggered drug delivery vehicle s is an important milestone in treating cancer. Here we demonstrate the selective anticancer drug delivery into human cells with biocompatible 50-nm diameter halloysite nanotube carriers. Physically-adsorbed dextrin end stoppers secure the intercellular release of brilliant green. Drug-loaded nanotubes penetrate through the cellular membranes and their uptake efficiency depends on the cells growth rate. Intercellular glycosyl hydrolases-mediated decomposition of the dextrin tube-end stoppers triggers the release of the lumen-loaded brilliant green, which allowed for preferable elimination of human lung carcinoma cells (А549) as compared with hepatoma cells (Hep3b). The enzyme-activated intracellular delivery of brilliant green using dextrin-coated halloysite nanotubes is a promising platform for anticancer treatment. PMID:25976444

  15. Enzyme-activated intracellular drug delivery with tubule clay nanoformulation

    NASA Astrophysics Data System (ADS)

    Dzamukova, Maria R.; Naumenko, Ekaterina A.; Lvov, Yuri M.; Fakhrullin, Rawil F.

    2015-05-01

    Fabrication of stimuli-triggered drug delivery vehicle s is an important milestone in treating cancer. Here we demonstrate the selective anticancer drug delivery into human cells with biocompatible 50-nm diameter halloysite nanotube carriers. Physically-adsorbed dextrin end stoppers secure the intercellular release of brilliant green. Drug-loaded nanotubes penetrate through the cellular membranes and their uptake efficiency depends on the cells growth rate. Intercellular glycosyl hydrolases-mediated decomposition of the dextrin tube-end stoppers triggers the release of the lumen-loaded brilliant green, which allowed for preferable elimination of human lung carcinoma cells (A549) as compared with hepatoma cells (Hep3b). The enzyme-activated intracellular delivery of brilliant green using dextrin-coated halloysite nanotubes is a promising platform for anticancer treatment.

  16. Identification of intracellular receptor proteins for activated protein kinase C.

    PubMed Central

    Mochly-Rosen, D; Khaner, H; Lopez, J

    1991-01-01

    Protein kinase C (PKC) translocates from the cytosol to the particulate fraction on activation. This activation-induced translocation of PKC is thought to reflect PKC binding to the membrane lipids. However, immunological and biochemical data suggest that PKC may bind to proteins in the cytoskeletal elements in the particulate fraction and in the nuclei. Here we describe evidence for the presence of intracellular receptor proteins that bind activated PKC. Several proteins from the detergent-insoluble material of the particulate fraction bound PKC in the presence of phosphatidylserine and calcium; binding was further increased with the addition of diacylglycerol. Binding of PKC to two of these proteins was concentration-dependent, saturable, and specific, suggesting that these binding proteins are receptors for activated C-kinase, termed here "RACKs." PKC binds to RACKs via a site on PKC distinct from the substrate binding site. We suggest that binding to RACKs may play a role in activation-induced translocation of PKC. Images PMID:1850844

  17. Tuning cell migration: contractility as an integrator of intracellular signals from multiple cues

    PubMed Central

    Bordeleau, Francois; Reinhart-King, Cynthia A.

    2016-01-01

    There has been immense progress in our understanding of the factors driving cell migration in both two-dimensional and three-dimensional microenvironments over the years. However, it is becoming increasingly evident that even though most cells share many of the same signaling molecules, they rarely respond in the same way to migration cues. To add to the complexity, cells are generally exposed to multiple cues simultaneously, in the form of growth factors and/or physical cues from the matrix. Understanding the mechanisms that modulate the intracellular signals triggered by multiple cues remains a challenge. Here, we will focus on the molecular mechanism involved in modulating cell migration, with a specific focus on how cell contractility can mediate the crosstalk between signaling initiated at cell-matrix adhesions and growth factor receptors. PMID:27508074

  18. BDNF-stimulated intracellular signalling mechanisms underlie exercise-induced improvement in spatial memory in the male Wistar rat.

    PubMed

    Bechara, Ranya G; Lyne, Ronan; Kelly, Áine M

    2014-12-15

    Exercise-induced improvements in learning are associated with neurotrophic and neurogenic changes in the dentate gyrus, but the intracellular signalling mechanisms that may mediate these improvements remain unknown. In the current study we investigate the effects of one week of forced exercise on spatial memory and analyse in parallel BDNF-stimulated signalling pathways in cells of the dentate gyrus. Additionally, we test whether a single intracerebroventricular (i.c.v.) injection of BDNF can mimic the observed cognitive and signalling changes. Male Wistar rats were assigned to exercised and sedentary groups and tested in a spatial task post-exercise. Tissue from the dentate gyrus was assessed for expression and release of BDNF, and for changes in expression and activation of TrkB, ERK and synapsin-1. In a separate set of experiments, male Wistar rats received a single i.c.v. injection of BDNF and were then tested in the same spatial learning task. Exercised and BDNF-treated (but not control) rats could successfully complete an object displacement task that tests spatial learning. Exercised rats and BDNF-treated rats displayed increases BDNF expression and ERK1 activation, while exercised rats showed increases in cell division, stimulated BDNF release, TrkB activation, and synapsin-1 expression in the dentate gyrus. We conclude that exercise-induced increases in BDNF in the dentate gyrus are sufficient to cause improvements in spatial memory by activating signalling cascades that enhance synaptic transmission in the hippocampus.

  19. Metabotropic glutamate receptor 5 couples cellular prion protein to intracellular signalling in Alzheimer's disease.

    PubMed

    Haas, Laura T; Salazar, Santiago V; Kostylev, Mikhail A; Um, Ji Won; Kaufman, Adam C; Strittmatter, Stephen M

    2016-02-01

    Alzheimer's disease-related phenotypes in mice can be rescued by blockade of either cellular prion protein or metabotropic glutamate receptor 5. We sought genetic and biochemical evidence that these proteins function cooperatively as an obligate complex in the brain. We show that cellular prion protein associates via transmembrane metabotropic glutamate receptor 5 with the intracellular protein mediators Homer1b/c, calcium/calmodulin-dependent protein kinase II, and the Alzheimer's disease risk gene product protein tyrosine kinase 2 beta. Coupling of cellular prion protein to these intracellular proteins is modified by soluble amyloid-β oligomers, by mouse brain Alzheimer's disease transgenes or by human Alzheimer's disease pathology. Amyloid-β oligomer-triggered phosphorylation of intracellular protein mediators and impairment of synaptic plasticity in vitro requires Prnp-Grm5 genetic interaction, being absent in transheterozygous loss-of-function, but present in either single heterozygote. Importantly, genetic coupling between Prnp and Grm5 is also responsible for signalling, for survival and for synapse loss in Alzheimer's disease transgenic model mice. Thus, the interaction between metabotropic glutamate receptor 5 and cellular prion protein has a central role in Alzheimer's disease pathogenesis, and the complex is a potential target for disease-modifying intervention.

  20. Intracellular pathways linking hypoxia to activation of c-fos and AP-1.

    PubMed

    Premkumar, D R; Adhikary, G; Overholt, J L; Simonson, M S; Cherniack, N S; Prabhakar, N R

    2000-01-01

    Organisms respond to hypoxia through detection of blood oxygen levels by sensors at peripheral chemoreceptors and by receptors in certain key cells of the body. The pathways over which peripheral chemoreceptor signals are transmitted to respiratory muscles are well established. However, the intracellular pathways that transmit hypoxic stimulus to gene activation are just being identified. Using anti-sense c-fos strategy, we have shown that c-fos is essential for the activation of activator protein-1 transcription factor complex (AP-1) and subsequent stimulation of downstream genes such as tyrosine hydroxylase (TH; Mishra et al. 1998). The purpose of the present study was to identify intracellular pathways that link hypoxia to activation of c-fos. The results of the present study show that hypoxia causes Ca2+ influx through L-type voltage gated Ca2+ channels and that hypoxia-induced c-fos gene expression is Ca2+/calmodulin dependent. We also demonstrate that hypoxia activates the extracellular-regulated kinase (ERK) and p38, but not JNK. Further, phosphorylation of ERK is essential for c-fos activation via SRE cis-element. Further characterization of nuclear signalling pathways provides evidence for the involvement of Src, a non receptor protein tyrosine kinase, and Ras, a small G protein, in the hypoxia-induced c-fos gene expression. These results suggest a possible role for non-receptor protein tyrosine kinases in propagating signals from G-protein coupled receptors to the activation of immediate early genes such as c-fos during hypoxia.

  1. Spatio-temporal PLC activation in parallel with intracellular Ca2+ wave propagation in mechanically stimulated single MDCK cells.

    PubMed

    Tsukamoto, Akira; Hayashida, Yasunori; Furukawa, Katsuko S; Ushida, Takashi

    2010-03-01

    Intracellular Ca2+ transients are evoked either by the opening of Ca2+ channels on the plasma membrane or by phospholipase C (PLC) activation resulting in IP3 production. Ca2+ wave propagation is known to occur in mechanically stimulated cells; however, it remains uncertain whether and how PLC activation is involved in intracellular Ca2+ wave propagation in mechanically stimulated cells. To answer these questions, it is indispensable to clarify the spatio-temporal relations between intracellular Ca2+ wave propagation and PLC activation. Thus, we visualized both cytosolic Ca2+ and PLC activation using a real-time dual-imaging system in individual Mardin-Darby Canine Kidney (MDCK) cells. This system allowed us to simultaneously observe intracellular Ca2+ wave propagation and PLC activation in a spatio-temporal manner in a single mechanically stimulated MDCK cell. The results showed that PLC was activated not only in the mechanically stimulated region but also in other subcellular regions in parallel with intracellular Ca2+ wave propagation. These results support a model in which PLC is involved in Ca2+ signaling amplification in mechanically stimulated cells.

  2. Effects of an EGFR-binding affibody molecule on intracellular signaling pathways.

    PubMed

    Nordberg, E; Ekerljung, L; Sahlberg, S H; Carlsson, J; Lennartsson, J; Glimelius, B

    2010-04-01

    Effects on intracellular signaling were studied in cells treated with the affibody molecule (ZEGFR:955)2 that targets the epithelial growth factor receptor (EGFR). EGFR is overexpressed in many types of cancers and plays a fundamental role in cell signaling and it is of interest to find targeting agents capable of blocking the receptor. The clinically approved antibody cetuximab (Erbitux) and the natural ligand EGF were included as reference molecules. Two EGFR-rich cell lines, A-431 and U-343, were exposed to the three targeting agents and lysed. The cell lysates were immunoprecipitated with the receptors, or directly separated by SDS-Page. Autophosphorylation of the receptors and phosphorylation of the downstream signaling proteins Erk and Akt, were evaluated by Western blotting. Although the three different agents compete for the same binding site on EGFR, they influenced the signaling differently. The affibody molecule did not induce autophosphorylation of EGFR or any other receptor in the EGFR-family but, in spite of this, induced phosphorylation of Erk in both cell lines and Akt in the A-431 cells. Thus, the results suggest that the signaling pattern induced by (ZEGFR:955)2 is only partly similar to that induced by cetuximab. This makes the affibody molecule a potentially interesting alternative to cetuximab for EGFR-targeted therapy since it might give different therapy-related effects on tumor cells and different side effects on normal tissues. PMID:20198342

  3. Platelet activating factor raises intracellular calcium ion concentration in macrophages

    PubMed Central

    1986-01-01

    Peritoneal cells from thioglycollate-stimulated mice were allowed to adhere to coverglasses for 2 h to give a dense monolayer of adherent cells greater than 95% of which were macrophages. After incubation with the tetra-acetoxymethyl ester of quin2, coverglasses were rinsed with Ca2+-free saline, oriented at a 45 degree angle in square cuvettes containing a magnetically driven stir bar, and analyzed for changes in quin2 fluorescence in a spectrofluorimeter. Such fluorescence, taken as an indication of intracellular calcium ion concentration ([Ca2+]i), increased as exogenous calcium ion concentration ([Ca2+]o) was raised to 1 mM. At [Ca2+]o approximately equal to 10 microM, [Ca2+]i = 72 +/- 14 nM (n = 26); at [Ca2+]o = 1 mM, [Ca2+]i = 140-220 nM, levels not increased by N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine, a membrane-permeant chelator of heavy metals than can quench quin2. Addition of mouse alpha + beta fibroblast interferon, lipopolysaccharide, thrombin, collagen, vasopressin, ADP, compound 48/80, or U46619 did not change [Ca2+]i. However, addition of platelet activating factor (PAF) (2-20 ng/ml) raised [Ca2+]i by 480 nM within 1 min if [Ca2+]o = 1 mM. In the presence of 5 mM EGTA, PAF raised [Ca2+]i by 25 nM. This suggests that PAF causes influx of exogenous Ca2+, as well as releasing some Ca2+ from intracellular stores. Consistent with these results, when PAF was added to 1 mM Ca2+ in the presence of 100 microM Cd2+ or Mn2+ to block Ca2+ influx, [Ca2+]i increased by only intermediate amounts; at the times of such dampened peak response, [Ca2+]i could be raised within 1 min to normal PAF-stimulated levels by chelation of the exogenous heavy metals with diethylenetriaminepentaacetic acid. Normal PAF responses were observed in the presence of indomethacin. The lowest dose of PAF observed to raise [Ca2+]i was 0.1 ng/ml. Response of [Ca2+]i to 2-20 ng/ml PAF was transient, and second applications had no effect. The PAF response also was seen in

  4. Cell- and stimulus type-specific intracellular free Ca2+ signals in Arabidopsis.

    PubMed

    Martí, María C; Stancombe, Matthew A; Webb, Alex A R

    2013-10-01

    Appropriate stimulus-response coupling requires that each signal induces a characteristic response, distinct from that induced by other signals, and that there is the potential for individual signals to initiate different downstream responses dependent on cell type. How such specificity is encoded in plant signaling is not known. One possibility is that information is encoded in signal transduction pathways to ensure stimulus- and cell type-specific responses. The calcium ion acts as a second messenger in response to mechanical stimulation, hydrogen peroxide, NaCl, and cold in plants and also in circadian timing. We use GAL4 transactivation of aequorin in enhancer trap lines of Arabidopsis (Arabidopsis thaliana) to test the hypothesis that stimulus- and cell-specific information can be encoded in the pattern of dynamic alterations in the concentration of intracellular free Ca(2+) ([Ca(2+)]i). We demonstrate that mechanically induced increases in [Ca(2+)]i are largely restricted to the epidermal pavement cells of leaves, that NaCl induces oscillatory [Ca(2+)]i signals in spongy mesophyll and vascular bundle cells, but not other cell types, and detect circadian rhythms of [Ca(2+)]i only in the spongy mesophyll. We demonstrate stimulus-specific [Ca(2+)]i dynamics in response to touch, cold, and hydrogen peroxide, which in the case of the latter two signals are common to all cell types tested. GAL4 transactivation of aequorin in specific leaf cell types has allowed us to bypass the technical limitations associated with fluorescent Ca(2+) reporter dyes in chlorophyll-containing tissues to identify the cell- and stimulus-specific complexity of [Ca(2+)]i dynamics in leaves of Arabidopsis and to determine from which tissues stress- and circadian-regulated [Ca(2+)]i signals arise.

  5. Phagocytosis and intracellular killing of MD-2 opsonized Gram-negative bacteria depend on TLR4 signaling

    PubMed Central

    Jain, Vishal; Halle, Annett; Halmen, Kristen A.; Lien, Egil; Charrel-Dennis, Marie; Ram, Sanjay; Golenbock, Douglas T.

    2008-01-01

    Both Toll-like receptor 4 (TLR4)– and MD-2–deficient mice succumb to otherwise nonfatal Gram-negative bacteria inocula, demonstrating the pivotal role played by these proteins in antibacterial defense in mammals. MD-2 is a soluble endogenous ligand for TLR4 and a receptor for lipopolysaccharide (LPS). LPS-bound MD-2 transmits an activating signal onto TLR4. In this report, we show that both recombinant and endogenous soluble MD-2 bind tightly to the surface of live Gram-negative bacteria. As a consequence, MD-2 enhances cellular activation, bacterial internalization, and intracellular killing, all in a TLR4-dependent manner. The enhanced internalization of MD-2–coated bacteria was not observed in macrophages expressing Lpsd, a signaling-incompetent mutant form of TLR4, suggesting that the enhanced phagocytosis observed is dependent on signal transduction. The data confirm the notion that soluble MD-2 is a genuine opsonin that enhances proinflammatory opsonophagocytosis by bridging live Gram-negative bacteria to the LPS transducing complex. The presented results extend our understanding of the role of the TLR4/MD-2 signaling axis in bacterial recognition by phagocytes. PMID:18203953

  6. Are Molecular Vibration Patterns of Cell Structural Elements Used for Intracellular Signalling?

    PubMed Central

    Jaross, Werner

    2016-01-01

    Background: To date the manner in which information reaches the nucleus on that part within the three-dimensional structure where specific restorative processes of structural components of the cell are required is unknown. The soluble signalling molecules generated in the course of destructive and restorative processes communicate only as needed. Hypothesis: All molecules show temperature-dependent molecular vibration creating a radiation in the infrared region. Each molecule species has in its turn a specific frequency pattern under given specific conditions. Changes in their structural composition result in modified frequency patterns of the molecules in question. The main structural elements of the cell membrane, of the endoplasmic reticulum, of the Golgi apparatus, and of the different microsomes representing the great variety of polar lipids show characteristic frequency patterns with peaks in the region characterised by low water absorption. These structural elements are very dynamic, mainly caused by the creation of signal molecules and transport containers. By means of the characteristic radiation, the area where repair or substitution services are needed could be identified; this spatial information complements the signalling of the soluble signal molecules. Based on their resonance properties receptors located on the outer leaflet of the nuclear envelope should be able to read typical frequencies and pass them into the nucleus. Clearly this physical signalling must be blocked by the cell membrane to obviate the flow of information into adjacent cells. Conclusion: If the hypothesis can be proved experimentally, it should be possible to identify and verify characteristic infrared frequency patterns. The application of these signal frequencies onto cells would open entirely new possibilities in medicine and all biological disciplines specifically to influence cell growth and metabolism. Similar to this intracellular system, an extracellular signalling system

  7. From intracellular signaling to population oscillations: bridging size- and time-scales in collective behavior

    PubMed Central

    Sgro, Allyson E; Schwab, David J; Noorbakhsh, Javad; Mestler, Troy; Mehta, Pankaj; Gregor, Thomas

    2015-01-01

    Collective behavior in cellular populations is coordinated by biochemical signaling networks within individual cells. Connecting the dynamics of these intracellular networks to the population phenomena they control poses a considerable challenge because of network complexity and our limited knowledge of kinetic parameters. However, from physical systems, we know that behavioral changes in the individual constituents of a collectively behaving system occur in a limited number of well-defined classes, and these can be described using simple models. Here, we apply such an approach to the emergence of collective oscillations in cellular populations of the social amoeba Dictyostelium discoideum. Through direct tests of our model with quantitative in vivo measurements of single-cell and population signaling dynamics, we show how a simple model can effectively describe a complex molecular signaling network at multiple size and temporal scales. The model predicts novel noise-driven single-cell and population-level signaling phenomena that we then experimentally observe. Our results suggest that like physical systems, collective behavior in biology may be universal and described using simple mathematical models. PMID:25617347

  8. Intracellular Ca(2+) mobilization and kinase activity during acylated homoserine lactone-dependent quorum sensing in Serratia liquefaciens.

    PubMed

    Werthén, M; Lundgren, T

    2001-03-01

    Quorum sensing in Gram-negative bacteria involves acylated homoserine lactones (AHLs) and a transcription factor, activated by the AHLs. In this study, a possible involvement of intracellular Ca(2+) as second messenger and/or protein kinase activity during signal transduction is analyzed. When N-hexanoyl-l-homoserine lactone was added to a suspension of Fura-2-loaded Serratia liquefaciens, there was a decline in [Ca(2+)](i), measured as a decrease in the Fura-2 fluorescence ratio. As controls, the addition of the signal molecule N-3-oxohexanoyl-l-homoserine lactone, which is not produced by S. liquefaciens, did not induce changes in [Ca(2+)](i). Using a protein kinase activity assay on AHL-stimulated cells, an increase in kinase activity after N-butanoyl-l-homoserine lactone stimulation of S. liquefaciens cells was detected, whereas the kinase activity induced by N-3-oxohexanoyl-l-homoserine lactone was not statistically significant. The conclusion from this study is that changes in [Ca(2+)](i) are involved in quorum sensing signal transduction in the Gram-negative bacteria S. liquefaciens. We also conclude that kinase activity is induced in S. liquefaciens upon AHL stimulation. We suggest that the transient intracellular [Ca(2+)] changes and kinase activity, activated by the AHL signal, are critical for the quorum-sensing signal transduction.

  9. Cell type- and activity-dependent extracellular correlates of intracellular spiking

    PubMed Central

    Perin, Rodrigo; Buzsáki, György; Markram, Henry; Koch, Christof

    2015-01-01

    Despite decades of extracellular action potential (EAP) recordings monitoring brain activity, the biophysical origin and inherent variability of these signals remain enigmatic. We performed whole cell patch recordings of excitatory and inhibitory neurons in rat somatosensory cortex slice while positioning a silicon probe in their vicinity to concurrently record intra- and extracellular voltages for spike frequencies under 20 Hz. We characterize biophysical events and properties (intracellular spiking, extracellular resistivity, temporal jitter, etc.) related to EAP recordings at the single-neuron level in a layer-specific manner. Notably, EAP amplitude was found to decay as the inverse of distance between the soma and the recording electrode with similar (but not identical) resistivity across layers. Furthermore, we assessed a number of EAP features and their variability with spike activity: amplitude (but not temporal) features varied substantially (∼30–50% compared with mean) and nonmonotonically as a function of spike frequency and spike order. Such EAP variation only partly reflects intracellular somatic spike variability and points to the plethora of processes contributing to the EAP. Also, we show that the shape of the EAP waveform is qualitatively similar to the negative of the temporal derivative to the intracellular somatic voltage, as expected from theory. Finally, we tested to what extent EAPs can impact the lowpass-filtered part of extracellular recordings, the local field potential (LFP), typically associated with synaptic activity. We found that spiking of excitatory neurons can significantly impact the LFP at frequencies as low as 20 Hz. Our results question the common assertion that the LFP acts as proxy for synaptic activity. PMID:25995352

  10. Signal focusing through active transport.

    PubMed

    Godec, Aljaž; Metzler, Ralf

    2015-07-01

    The accuracy of molecular signaling in biological cells and novel diagnostic devices is ultimately limited by the counting noise floor imposed by the thermal diffusion. Motivated by the fact that messenger RNA and vesicle-engulfed signaling molecules transiently bind to molecular motors and are actively transported in biological cells, we show here that the random active delivery of signaling particles to within a typical diffusion distance to the receptor generically reduces the correlation time of the counting noise. Considering a variety of signaling particle sizes from mRNA to vesicles and cell sizes from prokaryotic to eukaryotic cells, we show that the conditions for active focusing-faster and more precise signaling-are indeed compatible with observations in living cells. Our results improve the understanding of molecular cellular signaling and novel diagnostic devices.

  11. POSH is an intracellular signal transducer for the axon outgrowth inhibitor Nogo66

    PubMed Central

    Dickson, Heather M.; Zurawski, Jonathan; Zhang, Huanqing; Turner, David L.; Vojtek, Anne B.

    2010-01-01

    Myelin derived inhibitors limit axon outgrowth and plasticity during development and in the adult mammalian central nervous system (CNS). Nogo66, a functional domain of the myelin derived inhibitor NogoA, signals through the PirB receptor to inhibit axon outgrowth. The signaling pathway mobilized by Nogo66 engagement of PirB is not well understood. We identify a critical role for the scaffold protein Plenty of SH3s (POSH) in relaying process outgrowth inhibition downstream of Nogo66 and PirB. Blocking the function of POSH, or two POSH associated proteins, leucine zipper kinase (LZK) and Shroom3, with RNAi in cortical neurons leads to release from myelin and Nogo66 inhibition. We also observe autocrine inhibition of process outgrowth by NogoA, and suppression analysis with the POSH associated kinase LZK demonstrates that LZK operates downstream of NogoA and PirB in a POSH dependent manner. In addition, cerebellar granule neurons with an RNAi-mediated knockdown in POSH function are refractory to the inhibitory action of Nogo66, indicating that a POSH-dependent mechanism operates to inhibit axon outgrowth in different types of CNS neurons. These studies delineate an intracellular signaling pathway for process outgrowth inhibition by Nogo66, comprised of NogoA, PirB, POSH, LZK and Shroom3, and implicate the POSH complex as a potential therapeutic target to enhance axon outgrowth and plasticity in the injured CNS. PMID:20926658

  12. POSH is an intracellular signal transducer for the axon outgrowth inhibitor Nogo66.

    PubMed

    Dickson, Heather M; Zurawski, Jonathan; Zhang, Huanqing; Turner, David L; Vojtek, Anne B

    2010-10-01

    Myelin-derived inhibitors limit axon outgrowth and plasticity during development and in the adult mammalian CNS. Nogo66, a functional domain of the myelin-derived inhibitor NogoA, signals through the PirB receptor to inhibit axon outgrowth. The signaling pathway mobilized by Nogo66 engagement of PirB is not well understood. We identify a critical role for the scaffold protein Plenty of SH3s (POSH) in relaying process outgrowth inhibition downstream of Nogo66 and PirB. Blocking the function of POSH, or two POSH-associated proteins, leucine zipper kinase (LZK) and Shroom3, with RNAi in cortical neurons leads to release from myelin and Nogo66 inhibition. We also observed autocrine inhibition of process outgrowth by NogoA, and suppression analysis with the POSH-associated kinase LZK demonstrated that LZK operates downstream of NogoA and PirB in a POSH-dependent manner. In addition, cerebellar granule neurons with an RNAi-mediated knockdown in POSH function were refractory to the inhibitory action of Nogo66, indicating that a POSH-dependent mechanism operates to inhibit axon outgrowth in different types of CNS neurons. These studies delineate an intracellular signaling pathway for process outgrowth inhibition by Nogo66, comprised of NogoA, PirB, POSH, LZK, and Shroom3, and implicate the POSH complex as a potential therapeutic target to enhance axon outgrowth and plasticity in the injured CNS.

  13. Alpha-arrestins Aly1 and Aly2 regulate intracellular trafficking in response to nutrient signaling.

    PubMed

    O'Donnell, Allyson F; Apffel, Alex; Gardner, Richard G; Cyert, Martha S

    2010-10-15

    Extracellular signals regulate trafficking events to reorganize proteins at the plasma membrane (PM); however, few effectors of this regulation have been identified. β-Arrestins relay signaling cues to the trafficking machinery by controlling agonist-stimulated endocytosis of G-protein-coupled receptors. In contrast, we show that yeast α-arrestins, Aly1 and Aly2, control intracellular sorting of Gap1, the general amino acid permease, in response to nutrients. These studies are the first to demonstrate association of α-arrestins with clathrin and clathrin adaptor proteins (AP) and show that Aly1 and Aly2 interact directly with the γ-subunit of AP-1, Apl4. Aly2-dependent trafficking of Gap1 requires AP-1, which mediates endosome-to-Golgi transport, and the nutrient-regulated kinase, Npr1, which phosphorylates Aly2. During nitrogen starvation, Npr1 phosphorylation of Aly2 may stimulate Gap1 incorporation into AP-1/clathrin-coated vesicles to promote Gap1 trafficking from endosomes to the trans-Golgi network. Ultimately, increased Aly1-/Aly2-mediated recycling of Gap1 from endosomes results in higher Gap1 levels within cells and at the PM by diverting Gap away from trafficking pathways that lead to vacuolar degradation. This work defines a new role for arrestins in membrane trafficking and offers insight into how α-arrestins coordinate signaling events with protein trafficking.

  14. Engineering intracellular active transport systems as in vivo biomolecular tools.

    SciTech Connect

    Bachand, George David; Carroll-Portillo, Amanda

    2006-11-01

    Active transport systems provide essential functions in terms of cell physiology and metastasis. These systems, however, are also co-opted by invading viruses, enabling directed transport of the virus to and from the cell's nucleus (i.e., the site of virus replication). Based on this concept, fundamentally new approaches for interrogating and manipulating the inner workings of living cells may be achievable by co-opting Nature's active transport systems as an in vivo biomolecular tool. The overall goal of this project was to investigate the ability to engineer kinesin-based transport systems for in vivo applications, specifically the collection of effector proteins (e.g., transcriptional regulators) within single cells. In the first part of this project, a chimeric fusion protein consisting of kinesin and a single chain variable fragment (scFv) of an antibody was successfully produced through a recombinant expression system. The kinesin-scFv retained both catalytic and antigenic functionality, enabling selective capture and transport of target antigens. The incorporation of a rabbit IgG-specific scFv into the kinesin established a generalized system for functionalizing kinesin with a wide range of target-selective antibodies raised in rabbits. The second objective was to develop methods of isolating the intact microtubule network from live cells as a platform for evaluating kinesin-based transport within the cytoskeletal architecture of a cell. Successful isolation of intact microtubule networks from two distinct cell types was demonstrated using glutaraldehyde and methanol fixation methods. This work provides a platform for inferring the ability of kinesin-scFv to function in vivo, and may also serve as a three-dimensional scaffold for evaluating and exploiting kinesin-based transport for nanotechnological applications. Overall, the technology developed in this project represents a first-step in engineering active transport system for in vivo applications. Further

  15. Intracellular-activated Notch1 can reactivate Kaposi's sarcoma-associated herpesvirus from latency

    SciTech Connect

    Lan, Ke; Murakami, Masanao; Choudhuri, Tathagata; Kuppers, Daniel A.; Robertson, Erle S. . E-mail: erle@mail.med.upenn.edu

    2006-08-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) establishes a predominantly latent infection in the infected host. Importantly, during latency, only a small number of viral encoded genes are expressed. This viral gene expression pattern contributes to the establishment of long-term infection as well as the ability of the virus to evade the immune system. Previous studies have been shown that the replication and transcription activator (RTA) encoded by ORF50 activates it downstream genes and initiates viral lytic reactivation through functional interaction with RBP-J{kappa}, the major downstream effector of the Notch signaling pathway. This indicates that RTA can usurp the conserved Notch signaling pathway and mimic the activities of intracellular Notch1 to modulate gene expression. In this report, we show that the activated intracellular domain of Notch1 (ICN) is aberrantly accumulated in KSHV latently infected pleural effusion lymphoma (PEL) cells. ICN activated the RTA promoter in a dose-dependent manner, and forced expression of ICN in latently infected KSHV-positive cells initiated full blown lytic replication with the production of infectious viral progeny. However, latency-associated nuclear antigen (LANA) which is predominantly expressed during latency can specifically down-modulate ICN-mediated transactivation of RTA and so control KSHV for lytic reactivation. These results demonstrate that LANA can inhibit viral lytic replication by antagonizing ICN function and suggest that LANA is a critical component of the regulatory control mechanism for switching between viral latent and lytic replication by directly interacting with effectors of the conserved cellular Notch1 pathway.

  16. Co-encapsulating the fusogenic peptide INF7 and molecular imaging probes in liposomes increases intracellular signal and probe retention.

    PubMed

    Burks, Scott R; Legenzov, Eric A; Martin, Erik W; Li, Changqing; Lu, Wuyuan; Kao, Joseph P Y

    2015-01-01

    Liposomes are promising vehicles to deliver diagnostic and therapeutic agents to cells in vivo. After uptake into cells by endocytosis, liposomes are degraded in the endolysosomal system. Consequently, the encapsulated cargo molecules frequently remain sequestered in endosomal compartments; this limits their usefulness in many applications (e.g. gene delivery). To overcome this, various fusogenic peptides have been developed to facilitate delivery of liposomally-encapsulated molecules into the cytosol. One such peptide is the pH-sensitive influenza-derived peptide INF7. Liposomal delivery of imaging agents is an attractive approach for enabling cell imaging and cell tracking in vivo, but can be hampered by inadequate intracellular accumulation and retention of probes caused by exocytosis (and possible degradation) of endosome-entrapped probes. Such signal loss could be minimized by facilitating escape of probe molecules from endolysosomal compartments into the cytosol. We investigated the ability of co-encapsulated INF7 to release liposomally-delivered rhodamine fluorophores into the cytosol after endosomal acidification/maturation. We co-encapsulated INF7 and fluorescent rhodamine derivatives having vastly different transport properties to show that after endocytosis by CV1 cells, the INF7 peptide is activated by acidic endosomal pH and facilitates efficient release of the fluorescent tracers into the cytosol. Furthermore, we show that INF7-facilitated escape from endosomes markedly enhanced retention of tracers that cannot be actively extruded from the cytosol. Minimizing loss of intracellular probes improves cellular imaging by increasing the signal-to-noise ratio of images and lengthening the time window that imaging can be performed. In particular, this will enhance in vivo electron paramagnetic resonance imaging, an emergent magnetic resonance imaging modality requires exogenous paramagnetic imaging agents and is highly promising for cellular and molecular

  17. Apoptotic Cells Activate AMP-activated Protein Kinase (AMPK) and Inhibit Epithelial Cell Growth without Change in Intracellular Energy Stores*

    PubMed Central

    Patel, Vimal A.; Massenburg, Donald; Vujicic, Snezana; Feng, Lanfei; Tang, Meiyi; Litbarg, Natalia; Antoni, Angelika; Rauch, Joyce; Lieberthal, Wilfred; Levine, Jerrold S.

    2015-01-01

    Apoptosis plays an indispensable role in the maintenance and development of tissues. We have shown that receptor-mediated recognition of apoptotic target cells by viable kidney proximal tubular epithelial cells (PTECs) inhibits the proliferation and survival of PTECs. Here, we examined the effect of apoptotic targets on PTEC cell growth (cell size during G1 phase of the cell cycle). Using a cell culture model, we show that apoptotic cells potently activate AMP-activated protein kinase (AMPK), a highly sensitive sensor of intracellular energy stores. AMPK activation leads to decreased activity of its downstream target, ribosomal protein p70 S6 kinase (p70S6K), and concomitant inhibition of cell growth. Importantly, these events occur without detectable change in intracellular levels of AMP, ADP, or ATP. Inhibition of AMPK, either pharmacologically by compound C or molecularly by shRNA, diminishes the effects of apoptotic targets and largely restores p70S6K activity and cell size to normal levels. Apoptotic targets also inhibit Akt, a second signaling pathway regulating cell growth. Expression of a constitutively active Akt construct partially relieved cell growth inhibition but was less effective than inhibition of AMPK. Inhibition of cell growth by apoptotic targets is dependent on physical interaction between apoptotic targets and PTECs but independent of phagocytosis. We conclude that receptor-mediated recognition of apoptotic targets mimics the effects of intracellular energy depletion, activating AMPK and inhibiting cell growth. By acting as sentinels of environmental change, apoptotic death may enable nearby viable cells, especially nonmigratory epithelial cells, to monitor and adapt to local stresses. PMID:26183782

  18. Signal focusing through active transport

    NASA Astrophysics Data System (ADS)

    Godec, Aljaž; Metzler, Ralf

    2015-07-01

    The accuracy of molecular signaling in biological cells and novel diagnostic devices is ultimately limited by the counting noise floor imposed by the thermal diffusion. Motivated by the fact that messenger RNA and vesicle-engulfed signaling molecules transiently bind to molecular motors and are actively transported in biological cells, we show here that the random active delivery of signaling particles to within a typical diffusion distance to the receptor generically reduces the correlation time of the counting noise. Considering a variety of signaling particle sizes from mRNA to vesicles and cell sizes from prokaryotic to eukaryotic cells, we show that the conditions for active focusing—faster and more precise signaling—are indeed compatible with observations in living cells. Our results improve the understanding of molecular cellular signaling and novel diagnostic devices.

  19. Inter- and intracellular signaling induced by magnetomechanical actuation of plasma membrane channels

    NASA Astrophysics Data System (ADS)

    Vitol, Elina A.; Rozhkova, Elena A.; Novosad, Valentyn; Bader, Samuel D.

    2012-02-01

    Magnetic particles allow for non-invasive control over their spatial orientation and motion which makes them ideally suitable for studying real-time processes in living cells. Lithographically defined ferromagnetic disks with spin-vortex ground state have the advantage of zero net magnetization in remanence. This eliminates long-range magnetic forces which otherwise lead to the interaction between particles and their agglomeration. Moreover, magnetically soft permalloy particles have high magnetization of saturation thus requiring very low external field for inducing high magnetomotive force, compared to superparamagnetic particles. Our group has previously demonstrated that micron-sized permalloy disks can be used for destruction of cancer cells (D.-H. Kim, E. A. Rozhkova, I. V. Ulasov, S. D. Bader, T. Rajh, M. S. Lesniak, V. Novosad, Nat. Mater. 9, 165-171 (2010). In this work, we investigate the effects of magnetomechanical stimulation of human brain cancer cells with these particles. It will be shown that the actuation of ion channels in cell plasma membrane induces, on the one hand side, intracellular signaling triggering cell apoptosis and, on the other hand, it stimulates the energy transfer between the cells which carries the information about apoptotic signal.

  20. Intracellular signaling pathway regulation of myelination and remyelination in the CNS.

    PubMed

    Gaesser, Jenna M; Fyffe-Maricich, Sharyl L

    2016-09-01

    The restoration of myelin sheaths on demyelinated axons remains a major obstacle in the treatment of multiple sclerosis (MS). Currently approved therapies work by modulating the immune system to reduce the number and rate of lesion formation but are only partially effective since they are not able to restore lost myelin. In the healthy CNS, myelin continues to be generated throughout life and spontaneous remyelination occurs readily in response to insults. In patients with MS, however, remyelination eventually fails, at least in part as a result of a failure of oligodendrocyte precursor cell (OPC) differentiation and the subsequent production of new myelin. A better understanding of the molecular mechanisms and signaling pathways that drive the process of myelin sheath formation is therefore important in order to speed the development of novel therapeutics designed to target remyelination. Here we review data supporting critical roles for three highly conserved intracellular signaling pathways: Wnt/β-catenin, PI3K/AKT/mTOR, and ERK/MAPK in the regulation of OPC differentiation and myelination both during development and in remyelination. Potential points of crosstalk between the three pathways and important areas for future research are also discussed.

  1. Intracellular calcium signaling regulates autophagy via calcineurin-mediated TFEB dephosphorylation

    PubMed Central

    Tong, Yanju; Song, Fuyong

    2015-01-01

    The transcription-regulating activity of TFEB is dependent on its phosphorylation modification, but the phosphatase(s) involved in TFEB dephosphorylation have remained elusive. It has now become clear that lysosomal calcium signaling activates calcineurin, an endogenous serine/threonine phosphatase, which dephosphorylate TFEB leading to upregulation of autophagy. PMID:26043755

  2. Structural asymmetry in a conserved signaling system that regulates division, replication, and virulence of an intracellular pathogen

    PubMed Central

    Willett, Jonathan W.; Herrou, Julien; Briegel, Ariane; Rotskoff, Grant; Crosson, Sean

    2015-01-01

    We have functionally and structurally defined an essential protein phosphorelay that regulates expression of genes required for growth, division, and intracellular survival of the global zoonotic pathogen Brucella abortus. Our study delineates phosphoryl transfer through this molecular pathway, which initiates from the sensor kinase CckA and proceeds through the ChpT phosphotransferase to two regulatory substrates: CtrA and CpdR. Genetic perturbation of this system results in defects in cell growth and division site selection, and a specific viability deficit inside human phagocytic cells. Thus, proper control of B. abortus division site polarity is necessary for survival in the intracellular niche. We further define the structural foundations of signaling from the central phosphotransferase, ChpT, to its response regulator substrate, CtrA, and provide evidence that there are at least two modes of interaction between ChpT and CtrA, only one of which is competent to catalyze phosphoryltransfer. The structure and dynamics of the active site on each side of the ChpT homodimer are distinct, supporting a model in which quaternary structure of the 2:2 ChpT–CtrA complex enforces an asymmetric mechanism of phosphoryl transfer between ChpT and CtrA. Our study provides mechanistic understanding, from the cellular to the atomic scale, of a conserved transcriptional regulatory system that controls the cellular and infection biology of B. abortus. More generally, our results provide insight into the structural basis of two-component signal transduction, which is broadly conserved in bacteria, plants, and fungi. PMID:26124143

  3. Cyproheptadine enhances the I(K) of mouse cortical neurons through sigma-1 receptor-mediated intracellular signal pathway.

    PubMed

    He, Yan-Lin; Zhang, Chun-Lei; Gao, Xiao-Fei; Yao, Jin-Jing; Hu, Chang-Long; Mei, Yan-Ai

    2012-01-01

    Cyproheptadine (CPH) is a histamine- and serotonin-receptor antagonist, and its effects are observed recently in the modulation of multiple intracellular signals. In this study, we used cortical neurons and HEK-293 cells transfected with Kv2.1 α-subunit to address whether CPH modify neural voltage-gated K(+) channels by a mechanism independent of its serotonergic and histaminergic properties. Our results demonstrate that intracellularly delivered CPH increased the I(K) by reducing the activity of protein kinas A (PKA). Inhibition of G(i) eliminated the CPH-induced effect on both the I(K) and PKA. Blocking of 5-HT-, M-, D(2)-, H(1)- or H(2)-type GPCR receptors with relevant antagonists did not eliminate the CPH-induced effect on the I(K). Antagonists of the sigma-1 receptor, however, blocked the effect of CPH. Moreover, the inhibition of sigma-1 by siRNA knockdown significantly reduced the CPH-induced effect on the I(K). On the contrary, sigma-1 receptor agonist mimicked the effects of CPH on the induction of I(K). A ligand-receptor binding assay indicated that CPH bound to the sigma-1 receptor. Similar effect of CPH were obtained from HEK-293 cells transfected with the α-subunit of Kv2.1. In overall, we reveal for the first time that CPH enhances the I(K) by modulating activity of PKA, and that the associated activation of the sigma-1 receptor/G(i)-protein pathway might be involved. Our findings illustrate an uncharacterized effect of CPH on neuron excitability through the I(K), which is independent of histamine H(1) and serotonin receptors.

  4. Aldosterone secretion, measurements of membrane potential and intracellular potassium activity in the isolated adrenal zone glomerulosa.

    PubMed

    Wiederholt, M; Hampel, J; Belkien, L; Oelkers, W

    1984-09-01

    Cell membrane potential and intracellular potassium activity (microelectrodes filled with ion-sensitive liquid ion exchanger) were measured in the zona glomerulosa of superfused hemi-adrenals of rats kept on different diets. Simultaneously, samples of the superfusate were collected and analyzed by radioimmunoassay for aldosterone content. Cell membrane potential and intracellular potassium activity were not influenced by high sodium, low sodium or high potassium diet. However, aldosterone secretion significantly changed. These results suggest that membrane potential and intracellular potassium activity per se may not be linked to changes in aldosterone secretion.

  5. Unlocking Doors without Keys: Activation of Src by Truncated C-terminal Intracellular Receptor Tyrosine Kinases Lacking Tyrosine Kinase Activity

    PubMed Central

    Mezquita, Belén; Mezquita, Pau; Pau, Montserrat; Mezquita, Jovita; Mezquita, Cristóbal

    2014-01-01

    One of the best examples of the renaissance of Src as an open door to cancer has been the demonstration that just five min of Src activation is sufficient for transformation and also for induction and maintenance of cancer stem cells [1]. Many tyrosine kinase receptors, through the binding of their ligands, become the keys that unlock the structure of Src and activate its oncogenic transduction pathways. Furthermore, intracellular isoforms of these receptors, devoid of any tyrosine kinase activity, still retain the ability to unlock Src. This has been shown with a truncated isoform of KIT (tr-KIT) and a truncated isoform of VEGFR-1 (i21-VEGFR-1), which are intracellular and require no ligand binding, but are nonetheless able to activate Src and induce cell migration and invasion of cancer cells. Expression of the i21-VEGFR-1 is upregulated by the Notch signaling pathway and repressed by miR-200c and retinoic acid in breast cancer cells. Both Notch inhibitors and retinoic acid have been proposed as potential therapies for invasive breast cancer. PMID:24709904

  6. Modulation of Macrophage Inflammatory Nuclear Factor κB (NF-κB) Signaling by Intracellular Cryptococcus neoformans.

    PubMed

    Hayes, James B; Sircy, Linda M; Heusinkveld, Lauren E; Ding, Wandi; Leander, Rachel N; McClelland, Erin E; Nelson, David E

    2016-07-22

    Cryptococcus neoformans (Cn) is a common facultative intracellular pathogen that can cause life-threatening fungal meningitis in immunocompromised individuals. Shortly after infection, Cn is detectable as both extra- and intracellular yeast particles, with Cn being capable of establishing long-lasting latent infections within host macrophages. Although recent studies have shown that shed capsular polysaccharides and intact extracellular Cn can compromise macrophage function through modulation of NF-κB signaling, it is currently unclear whether intracellular Cn also affects NF-κB signaling. Utilizing live cell imaging and computational modeling, we find that extra- and intracellular Cn support distinct modes of NF-κB signaling in cultured murine macrophages. Specifically, in RAW 264.7 murine macrophages treated with extracellular glucuronoxylomannan (GXM), the major Cn capsular polysaccharide, LPS-induced nuclear translocation of p65 is inhibited, whereas in cells with intracellular Cn, LPS-induced nuclear translocation of p65 is both amplified and sustained. Mathematical simulations and quantification of nascent protein expression indicate that this is a possible consequence of Cn-induced "translational interference," impeding IκBα resynthesis. We also show that long term Cn infection induces stable nuclear localization of p65 and IκBα proteins in the absence of additional pro-inflammatory stimuli. In this case, nuclear localization of p65 is not accompanied by TNFα or inducible NOS (iNOS) expression. These results demonstrate that capsular polysaccharides and intact intracellular yeast manipulate NF-κB via multiple distinct mechanisms and provide new insights into how Cn might modulate cellular signaling at different stages of an infection. PMID:27231343

  7. Modeling of progesterone-induced intracellular calcium signaling in human spermatozoa.

    PubMed

    Li, Long-Fei; Xiang, Cheng; Zhu, Ya-Bing; Qin, Kai-Rong

    2014-06-21

    Calcium ion is a secondary messenger of mammalian spermatozoa. The dynamic change of its concentration plays a vital role in the process of sperm motility, capacitation, acrosome and fertilization. Progesterone released by the cumulus cells, as a potent stimulator of fertilization, can activate the calcium channels on the plasma membrane, which in turn triggers the dynamic change of intracellular calcium concentration. In this paper, a mathematical model of calcium dynamic response in mammalian spermatozoa induced by progesterone is proposed and numerical simulation of the dynamic model is conducted. The results show that the dynamic response of calcium concentration predicted by the model is in accordance with experimental evidence. The proposed dynamic model can be used to explain the phenomena observed in the experiments and predict new phenomena to be revealed by experimental investigations, which will provide the basis to quantitatively investigate the fluid mechanics and biochemistry for the sperm motility induced by progesterone.

  8. Ceramide-induced intracellular oxidant formation, iron signaling, and apoptosis in endothelial cells: protective role of endogenous nitric oxide.

    PubMed

    Matsunaga, Toshiyuki; Kotamraju, Srigiridhar; Kalivendi, Shasi V; Dhanasekaran, Anuradha; Joseph, Joy; Kalyanaraman, B

    2004-07-01

    Sphingolipid ceramide (N-acetylsphingosine), a bioactive second messenger lipid, was shown to activate reactive oxygen species (ROS), mitochondrial oxidative damage, and apoptosis in neuronal and vascular cells. The proapoptotic effects of tumor necrosis factor-alpha, hypoxia, and chemotherapeutic drugs were attributed to increased ceramide formation. Here we investigated the protective role of nitric oxide (.NO) during hydrogen peroxide (H(2)O(2))-mediated transferrin receptor (TfR)-dependent iron signaling and apoptosis in C(2)-ceramide (C(2)-cer)-treated bovine aortic endothelial cells (BAECs). Addition of C(2)-cer (5-20 microm) to BAECs enhanced .NO generation. However, at higher concentrations of C(2)-cer (> or =20 microm), .NO generation did not increase proportionately. C(2)-cer (20-50 microm) also resulted in H(2)O(2)-mediated dichlorodihydrofluorescein oxidation, reduced glutathione depletion, aconitase inactivation, TfR overexpression, TfR-dependent uptake of (55)Fe, release of cytochrome c from mitochondria into cytosol, caspase-3 activation, and DNA fragmentation. N(w)-Nitro-l-arginine methyl ester (l-NAME), a nonspecific inhibitor of nitricoxide synthases, augmented these effects in BAECs at much lower (i.e. nonapoptotic) concentrations of C(2)-cer. The 26 S proteasomal activity in BAECs was slightly elevated at lower concentrations of C(2)-cer (< or =10 microm) but was greatly suppressed at higher concentrations (>10 microm). Intracellular scavengers of H(2)O(2), cell-permeable iron chelators, anti-TfR receptor antibody, or mitochondria-targeted antioxidant greatly abrogated C(2)-cer- and/or l-NAME-induced oxidative damage, iron signaling, and apoptosis. We conclude that C(2)-cer-induced H(2)O(2) and TfR-dependent iron signaling are responsible for its prooxidant and proapoptotic effects and that .NO exerts an antioxidative and cytoprotective role.

  9. Endocannabinoid signaling enhances visual responses through modulation of intracellular chloride levels in retinal ganglion cells

    PubMed Central

    Miraucourt, Loïs S; Tsui, Jennifer; Gobert, Delphine; Desjardins, Jean-François; Schohl, Anne; Sild, Mari; Spratt, Perry; Castonguay, Annie; De Koninck, Yves; Marsh-Armstrong, Nicholas; Wiseman, Paul W; Ruthazer, Edward S

    2016-01-01

    Type 1 cannabinoid receptors (CB1Rs) are widely expressed in the vertebrate retina, but the role of endocannabinoids in vision is not fully understood. Here, we identified a novel mechanism underlying a CB1R-mediated increase in retinal ganglion cell (RGC) intrinsic excitability acting through AMPK-dependent inhibition of NKCC1 activity. Clomeleon imaging and patch clamp recordings revealed that inhibition of NKCC1 downstream of CB1R activation reduces intracellular Cl− levels in RGCs, hyperpolarizing the resting membrane potential. We confirmed that such hyperpolarization enhances RGC action potential firing in response to subsequent depolarization, consistent with the increased intrinsic excitability of RGCs observed with CB1R activation. Using a dot avoidance assay in freely swimming Xenopus tadpoles, we demonstrate that CB1R activation markedly improves visual contrast sensitivity under low-light conditions. These results highlight a role for endocannabinoids in vision and present a novel mechanism for cannabinoid modulation of neuronal activity through Cl− regulation. DOI: http://dx.doi.org/10.7554/eLife.15932.001 PMID:27501334

  10. Endocannabinoid signaling enhances visual responses through modulation of intracellular chloride levels in retinal ganglion cells.

    PubMed

    Miraucourt, Loïs S; Tsui, Jennifer; Gobert, Delphine; Desjardins, Jean-François; Schohl, Anne; Sild, Mari; Spratt, Perry; Castonguay, Annie; De Koninck, Yves; Marsh-Armstrong, Nicholas; Wiseman, Paul W; Ruthazer, Edward S

    2016-01-01

    Type 1 cannabinoid receptors (CB1Rs) are widely expressed in the vertebrate retina, but the role of endocannabinoids in vision is not fully understood. Here, we identified a novel mechanism underlying a CB1R-mediated increase in retinal ganglion cell (RGC) intrinsic excitability acting through AMPK-dependent inhibition of NKCC1 activity. Clomeleon imaging and patch clamp recordings revealed that inhibition of NKCC1 downstream of CB1R activation reduces intracellular Cl(-) levels in RGCs, hyperpolarizing the resting membrane potential. We confirmed that such hyperpolarization enhances RGC action potential firing in response to subsequent depolarization, consistent with the increased intrinsic excitability of RGCs observed with CB1R activation. Using a dot avoidance assay in freely swimming Xenopus tadpoles, we demonstrate that CB1R activation markedly improves visual contrast sensitivity under low-light conditions. These results highlight a role for endocannabinoids in vision and present a novel mechanism for cannabinoid modulation of neuronal activity through Cl(-) regulation. PMID:27501334

  11. Pervanadate activation of intracellular kinases leads to tyrosine phosphorylation and shedding of syndecan-1.

    PubMed Central

    Reiland, J; Ott, V L; Lebakken, C S; Yeaman, C; McCarthy, J; Rapraeger, A C

    1996-01-01

    Syndecan-1 is a transmembrane haparan sulphate proteoglycan that binds extracellular matrices and growth factors, making it a candidate to act between these regulatory molecules and intracellular signalling pathways. It has a highly conserved transmembrane/cytoplasmic domain that contains four conserved tyrosines. One of these is in a consensus sequence for tyrosine kinase phosphorylation. As an initial step to investigating whether or not phosphorylation of these tyrosines is part of a signal-transduction pathway, we have monitored the tyrosine phosphorylation of syndecan-1 by cytoplasmic tyrosine kinases in intact cells. Tyrosine phosphorylation of syndecan-1 is observed when NMuMG cells are treated with sodium orthovanadate or pervanadate, which have been shown to activate intracellular tyrosine kinases. Initial studies with sodium orthovanadate demonstrate a slow accumulation of phosphotyrosine on syndecan-1 over the course of several hours. Pervanadate, a more effective inhibitor of phosphatases, allows detection of phosphotyrosine on syndecan-1 within 5 min, with peak phosphorylation seen by 15 min. Concurrently, in a second process activated by pervanadate, syndecan-1 ectodomain is cleaved and released into the culture medium. Two phosphorylated fragments of syndecan-1 of apparent sizes 6 and 8 kDa remain with the cell after shedding of the ectodomain. The 8 kDa size class appears to be a highly phosphorylated form of the 6 kDa product, as it disappears if samples are dephosphorylated. These fragments contain the C-terminus of syndecan-1 and also retain at least a portion of the transmembrane domain, suggesting that they are produced by a cell surface cleavage event. Thus pervanadate treatment of cells results in two effects of syndecan-1: (i) phosphorylation of one or more of its tyrosines via the action of a cytoplasmic kinase(s) and (ii) cleavage and release of the ectodomain into the medium, producing a C-terminal fragment containing the transmembrane

  12. The effects of red ginseng saponin fraction-A (RGSF-A) on phagocytosis and intracellular signaling in Brucella abortus infected RAW 264.7 cells.

    PubMed

    Arayan, Lauren Togonon; Simborio, Hannah Leah; Reyes, Alisha Wehdnesday Bernardo; Hop, Huynh Tan; Min, WonGi; Lee, Hu Jang; Rhee, Man Hee; Chang, Hong Hee; Kim, Suk

    2015-06-01

    This study indicated that RGSF-A caused a marked reduction in the adherence, internalization and intracellular growth of Brucella abortus in RGSF-A-treated cells. Furthermore, a decline in the intensity of F-actin fluorescence was observed in RGSF-A-treated cells compared with untreated B. abortus-infected cells. In addition, an evaluation of phagocytic signaling proteins by Western blot analysis revealed an apparent reduction of ERK and p38α phosphorylation levels in B. abortus-infected RGSF-A-treated cells compared with the control. Upon intracellular trafficking of the pathogen, a higher number of B. abortus-containing phagosomes colocalized with LAMP-1 in RGSF-A-treated cells compared with control cells. These results strongly suggest that inhibition of B. abortus uptake could be mediated by suppression in the activation of MAPKs signaling proteins phospho-ERK 1/2, and p38 levels. On the other hand, inhibition of intracellular replication results from the enhancement of phagolysosome fusion in host macrophages. This study highlights the phagocytic and intracellular modulating effect of RGSF-A and its potential as an alternative remedy to control B. abortus infection.

  13. Effect of introduction of chondroitin sulfate into polymer-peptide conjugate responding to intracellular signals

    NASA Astrophysics Data System (ADS)

    Tomiyama, Tetsuro; Toita, Riki; Kang, Jeong-Hun; Koga, Haruka; Shiosaki, Shujiro; Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki

    2011-09-01

    We recently developed a novel tumor-targeted gene delivery system responding to hyperactivated intracellular signals. Polymeric carrier for gene delivery consists of hydrophilic neutral polymer as main chains and cationic peptide substrate for target enzyme as side chains, and was named polymer-peptide conjugate (PPC). Introduction of chondroitin sulfate (CS), which induces receptor-medicated endocytosis, into polymers mainly with a high cationic charge density such as polyethylenimine can increase tumor-targeted gene delivery. In the present study, we examined whether introduction of CS into PPC containing five cationic amino acids can increase gene expression in tumor cells. Size and zeta potential of plasmid DNA (pDNA)/PPC/CS complex were <200 nm and between -10 and -15 mV, respectively. In tumor cell experiments, pDNA/PPC/CS complex showed lower stability and gene regulation, compared with that of pDNA/PPC. Moreover, no difference in gene expression was identified between positive and negative polymer. These results were caused by fast disintegration of pDNA/PPC/CS complexes in the presence of serum. Thus, we suggest that introduction of negatively charged CS into polymers with a low charge density may lead to low stability and gene regulation of complexes.

  14. Identification of the nuclear export signals that regulate the intracellular localization of the mouse CMP-sialic acid synthetase

    SciTech Connect

    Fujita, Akiko; Sato, Chihiro; Kitajima, Ken. E-mail: kitajima@agr.nagoya-u.ac.jp

    2007-03-30

    The CMP-sialic acid synthetase (CSS) catalyzes the activation of sialic acid (Sia) to CMP-Sia which is a donor substrate of sialyltransferases. The vertebrate CSSs are usually localized in nucleus due to the nuclear localization signal (NLS) on the molecule. In this study, we first point out that a small, but significant population of the mouse CMP-sialic acid synthetase (mCSS) is also present in cytoplasm, though mostly in nucleus. As a mechanism for the localization in cytoplasm, we first identified two nuclear export signals (NESs) in mCSS, based on the localization studies of the potential NES-deleted mCSS mutants as well as the potential NES-tagged eGFP proteins. These two NESs are conserved among mammalian and fish CSSs, but not present in the bacterial or insect CSS. These results suggest that the intracellular localization of vertebrate CSSs is regulated by not only the NLS, but also the NES sequences.

  15. Cyclosporin A differentially inhibits multiple steps in VEGF induced angiogenesis in human microvascular endothelial cells through altered intracellular signaling

    PubMed Central

    Rafiee, Parvaneh; Heidemann, Jan; Ogawa, Hitoshi; Johnson, Nathan A; Fisher, Pamela J; Li, Mona S; Otterson, Mary F; Johnson, Christopher P; Binion, David G

    2004-01-01

    The immunosuppressive agent cyclosporin A (CsA), a calcineurin inhibitor which blocks T cell activation has provided the pharmacologic foundation for organ transplantation. CsA exerts additional effects on non-immune cell populations and may adversely effect microvascular endothelial cells, contributing to chronic rejection, a long-term clinical complication and significant cause of mortality in solid-organ transplants, including patients with small bowel allografts. Growth of new blood vessels, or angiogenesis, is a critical homeostatic mechanism in organs and tissues, and regulates vascular populations in response to physiologic requirements. We hypothesized that CsA would inhibit the angiogenic capacity of human gut microvessels. Primary cultures of human intestinal microvascular endothelial cells (HIMEC) were used to evaluate CsA's effect on four in vitro measures of angiogenesis, including endothelial stress fiber assembly, migration, proliferation and tube formation, in response to the endothelial growth factor VEGF. We characterized the effect of CsA on intracellular signaling mechanisms following VEGF stimulation. CsA affected all VEGF induced angiogenic events assessed in HIMEC. CsA differentially inhibited signaling pathways which mediated distinct steps of the angiogenic process. CsA blocked VEGF induced nuclear translocation of the transcription factor NFAT, activation of p44/42 MAPK, and partially inhibited JNK and p38 MAPK. CsA differentially affected signaling cascades in a dose dependent fashion and completely blocked expression of COX-2, which was integrally linked to HIMEC angiogenesis. These data suggest that CsA inhibits the ability of microvascular endothelial cells to undergo angiogenesis, impairing vascular homeostatic mechanisms and contributing to the vasculopathy associated with chronic rejection. PMID:15175101

  16. The effect of high pressure on the intracellular trehalose synthase activity of Thermus aquaticus.

    PubMed

    Dong, Yongsheng; Ma, Lei; Duan, Yuanliang

    2016-01-01

    To understand the effect of high pressure on the intracellular trehalose synthase activity, Thermus aquaticus (T. aquaticus) in the logarithmic growth phase was treated with high-pressure air, and its intracellular trehalose synthase (TSase) activity was determined. Our results indicated that pressure is a factor strongly affecting the cell growth. High pressure significantly attenuated the growth rate of T. aquaticus and shortened the duration of stationary phase. However, after 2 h of culture under 1.0 MPa pressure, the activity of intracellular TSase in T. aquaticus reached its maximum value, indicating that pressure can significantly increase the activity of intracellular TSase in T. aquaticus. Thus the present study provides an important guide for the enzymatic production of trehalose.

  17. Depollution potential of three macrophytes: exudated, wall-bound and intracellular peroxidase activities plus intracellular phenol concentrations.

    PubMed

    Larue, Camille; Korboulewsky, Nathalie; Wang, Runying; Mévy, Jean-Philippe

    2010-10-01

    The aim of this study was to investigate the potential role of three macrophyte species (Iris pseudacorus, Typha latifolia and Phragmites australis) for detoxication of xenobiotics, and to study their variations with seasons or concentrations of sewage sludge from the food industry. For this purpose, some aspects of the green liver concept were explored through peroxidase measurements in three compartments in roots: intracellular, cell wall and extracellular. In addition, phenol concentrations were also measured in order to assess heavy metal detoxication potential. Enzyme activities and phenol concentrations were overall lower in winter according to the phenological stages and some sludge effects occurred. Results show that P. australis roots exuded and contained more peroxidase in all seasons: 17 U/g (1373 U/g protein), 0.8 U/g (613 U/g protein) and 4.8 U/g (1329 U/g protein) in intracellular compartments, cell wall and exudates, respectively. In contrast, the highest phenol concentration was found in I. pseudacorus roots: 3.58 mg eq. [corrected] gallic acid/g. Hence, in constructed wetlands, P. australis is suitable for organic waste water treatment, while I. pseudacorus should be used in the case of waters highly charged with heavy metals.

  18. Localized intracellular calcium signaling in muscle: calcium sparks and calcium quarks.

    PubMed

    Niggli, E

    1999-01-01

    Subcellularly localized Ca2+ signals in cardiac and skeletal muscle have recently been identified as elementary Ca2+ signaling events. The signals, termed Ca2+ sparks and Ca2+ quarks, represent openings of Ca2+ release channels located in the membrane of the sarcoplasmic reticulum (SR). In cardiac muscle, the revolutionary discovery of Ca2+ sparks has allowed the development of a fundamentally different concept for the amplification of Ca2+ signals by Ca(2+)-induced Ca2+ release. In such a system, a graded amplification of the triggering Ca2+ signal entering the myocyte via L-type Ca2+ channels is accomplished by a recruitment process whereby individual SR Ca2+ release units are locally controlled by L-type Ca2+ channels. In skeletal muscle, the initial SR Ca2+ release is governed by voltage-sensors but subsequently activates additional Ca2+ sparks by Ca(2+)-induced Ca2+ release from the SR. Results from studies on elementary Ca2+ release events will improve our knowledge of muscle Ca2+ signaling at all levels of complexity, from the molecule to normal cellular function, and from the regulation of cardiac and skeletal muscle force to the pathophysiology of excitation-contraction coupling.

  19. Intracellular signal triggered by cholera toxin in Saccharomyces boulardii and Saccharomyces cerevisiae.

    PubMed

    Brandão, R L; Castro, I M; Bambirra, E A; Amaral, S C; Fietto, L G; Tropia, M J; Neves, M J; Dos Santos, R G; Gomes, N C; Nicoli, J R

    1998-02-01

    As is the case for Saccharomyces boulardii, Saccharomyces cerevisiae W303 protects Fisher rats against cholera toxin (CT). The addition of glucose or dinitrophenol to cells of S. boulardii grown on a nonfermentable carbon source activated trehalase in a manner similar to that observed for S.cerevisiae. The addition of CT to the same cells also resulted in trehalase activation. Experiments performed separately on the A and B subunits of CT showed that both are necessary for activation. Similarly, the addition of CT but not of its separate subunits led to a cyclic AMP (cAMP) signal in both S. boulardii and S. cerevisiae. These data suggest that trehalase stimulation by CT probably occurred through the cAMP-mediated protein phosphorylation cascade. The requirement of CT subunit B for both the cAMP signal and trehalase activation indicates the presence of a specific receptor on the yeasts able to bind to the toxin, a situation similar to that observed for mammalian cells. This hypothesis was reinforced by experiments with 125I-labeled CT showing specific binding of the toxin to yeast cells. The adhesion of CT to a receptor on the yeast surface through the B subunit and internalization of the A subunit (necessary for the cAMP signal and trehalase activation) could be one more mechanism explaining protection against the toxin observed for rats treated with yeasts.

  20. Intracellular Signal Triggered by Cholera Toxin in Saccharomyces boulardii and Saccharomyces cerevisiae

    PubMed Central

    Brandão, Rogelio L.; Castro, Ieso M.; Bambirra, Eduardo A.; Amaral, Sheila C.; Fietto, Luciano G.; Tropia, Maria José M.; Neves, Maria José; Dos Santos, Raquel G.; Gomes, Newton C. M.; Nicoli, Jacques R.

    1998-01-01

    As is the case for Saccharomyces boulardii, Saccharomyces cerevisiae W303 protects Fisher rats against cholera toxin (CT). The addition of glucose or dinitrophenol to cells of S. boulardii grown on a nonfermentable carbon source activated trehalase in a manner similar to that observed for S. cerevisiae. The addition of CT to the same cells also resulted in trehalase activation. Experiments performed separately on the A and B subunits of CT showed that both are necessary for activation. Similarly, the addition of CT but not of its separate subunits led to a cyclic AMP (cAMP) signal in both S. boulardii and S. cerevisiae. These data suggest that trehalase stimulation by CT probably occurred through the cAMP-mediated protein phosphorylation cascade. The requirement of CT subunit B for both the cAMP signal and trehalase activation indicates the presence of a specific receptor on the yeasts able to bind to the toxin, a situation similar to that observed for mammalian cells. This hypothesis was reinforced by experiments with 125I-labeled CT showing specific binding of the toxin to yeast cells. The adhesion of CT to a receptor on the yeast surface through the B subunit and internalization of the A subunit (necessary for the cAMP signal and trehalase activation) could be one more mechanism explaining protection against the toxin observed for rats treated with yeasts. PMID:9464394

  1. Role of CheW Protein in Coupling Membrane Receptors to the Intracellular Signaling System of Bacterial Chemotaxis

    NASA Astrophysics Data System (ADS)

    Liu, Jingdong; Parkinson, John S.

    1989-11-01

    Chemotactic behavior in Escherichia coli is mediated by membrane-associated chemoreceptors that transmit sensory signals to the flagellar motors through an intracellular signaling system, which appears to involve a protein phosphorylation cascade. This study concerns the role of CheW, a cytoplasmic protein, in coupling methyl-accepting chemotaxis proteins (MCPs), the major class of membrane receptors, to the intracellular signaling system. Steady-state flagellar rotation behavior was examined in a series of strains with different combinations and relative amounts of CheW, MCPs, and other signaling components. At normal expression levels, CheW stimulated clockwise rotation, and receptors appeared to enhance this stimulatory effect. At high expression levels, MCPs inhibited clockwise rotation, and CheW appeared to augment this inhibitory effect. Since overexpression of CheW or MCP molecules had the same behavioral effect as their absence, chemoreceptors probably use CheW to modulate two distinct signals, one that stimulates and one that inhibits the intracellular phosphorylation cascade.

  2. Intracellular Complement Activation Sustains T Cell Homeostasis and Mediates Effector Differentiation

    PubMed Central

    Liszewski, M. Kathryn; Kolev, Martin; Le Friec, Gaelle; Leung, Marilyn; Bertram, Paula G.; Fara, Antonella F.; Subias, Marta; Pickering, Matthew C.; Drouet, Christian; Meri, Seppo; Arstila, T. Petteri; Pekkarinen, Pirkka T.; Ma, Margaret; Cope, Andrew; Reinheckel, Thomas; Rodriguez de Cordoba, Santiago; Afzali, Behdad; Atkinson, John P.; Kemper, Claudia

    2013-01-01

    Summary Complement is viewed as a critical serum-operative component of innate immunity, with processing of its key component, C3, into activation fragments C3a and C3b confined to the extracellular space. We report here that C3 activation also occurred intracellularly. We found that the T cell-expressed protease cathepsin L (CTSL) processed C3 into biologically active C3a and C3b. Resting T cells contained stores of endosomal and lysosomal C3 and CTSL and substantial amounts of CTSL-generated C3a. While “tonic” intracellular C3a generation was required for homeostatic T cell survival, shuttling of this intracellular C3-activation-system to the cell surface upon T cell stimulation induced autocrine proinflammatory cytokine production. Furthermore, T cells from patients with autoimmune arthritis demonstrated hyperactive intracellular complement activation and interferon-γ production and CTSL inhibition corrected this deregulated phenotype. Importantly, intracellular C3a was observed in all examined cell populations, suggesting that intracellular complement activation might be of broad physiological significance. PMID:24315997

  3. Intracellular complement activation sustains T cell homeostasis and mediates effector differentiation.

    PubMed

    Liszewski, M Kathryn; Kolev, Martin; Le Friec, Gaelle; Leung, Marilyn; Bertram, Paula G; Fara, Antonella F; Subias, Marta; Pickering, Matthew C; Drouet, Christian; Meri, Seppo; Arstila, T Petteri; Pekkarinen, Pirkka T; Ma, Margaret; Cope, Andrew; Reinheckel, Thomas; Rodriguez de Cordoba, Santiago; Afzali, Behdad; Atkinson, John P; Kemper, Claudia

    2013-12-12

    Complement is viewed as a critical serum-operative component of innate immunity, with processing of its key component, C3, into activation fragments C3a and C3b confined to the extracellular space. We report here that C3 activation also occurred intracellularly. We found that the T cell-expressed protease cathepsin L (CTSL) processed C3 into biologically active C3a and C3b. Resting T cells contained stores of endosomal and lysosomal C3 and CTSL and substantial amounts of CTSL-generated C3a. While "tonic" intracellular C3a generation was required for homeostatic T cell survival, shuttling of this intracellular C3-activation-system to the cell surface upon T cell stimulation induced autocrine proinflammatory cytokine production. Furthermore, T cells from patients with autoimmune arthritis demonstrated hyperactive intracellular complement activation and interferon-γ production and CTSL inhibition corrected this deregulated phenotype. Importantly, intracellular C3a was observed in all examined cell populations, suggesting that intracellular complement activation might be of broad physiological significance. PMID:24315997

  4. Evaluation of cytotoxicity and DNA damage response with analysis of intracellular ATM signaling pathways.

    PubMed

    Bandi, Sriram; Viswanathan, Preeti; Gupta, Sanjeev

    2014-06-01

    Maintenance of genome integrity by preventing and overcoming DNA damage is critical for cell survival. Deficiency or aberrancy in the DNA damage response, for example, through ataxia telangiectasia mutated (ATM) signaling, lead to pathophysiological perturbations in organs throughout the body. Therefore, control of DNA damage is of major interest for development of therapeutic agents. Such efforts will greatly benefit from convenient and simple diagnostic and/or drug development tools to demonstrate whether ATM and related genes have been activated and to then determine whether these have been returned to normal levels of activity because pathway members sense and also repair DNA damage. To overcome difficulties in analyzing differences in multitudinous ATM pathway members following DNA damage, we measured ATM promoter activity with a fluorescent td-Tomato reporter gene to interrogate the global effects of ATM signaling pathways. In cultured HuH-7 cell line derived from human hepatocellular carcinoma, cis-platinum, acetaminophen, or hydrogen peroxide caused DNA strand breaks and ATM pathway activation as shown by γH2AX expression, which in turn, led to rapid and sustained increases in ATM promoter activity. This assay of ATM promoter activity identified biological agents capable of controlling cellular DNA damage in toxin-treated HuH-7 cells and in mice after onset of drug-induced acute liver failure. Therefore, the proposed assay of ATM promoter activity in HuH-7 cells was appropriately informative for treating DNA damage. High-throughput screens using ATM promoter activation will be helpful for therapeutic development in DNA damage-associated abnormal ATM signaling in various cell types and organs. PMID:24927134

  5. Intracellular disassembly and activity of pertussis toxin require interaction with ATP.

    PubMed

    Plaut, Roger D; Scanlon, Karen M; Taylor, Michael; Teter, Ken; Carbonetti, Nicholas H

    2016-08-01

    The active subunit (S1) of pertussis toxin (PT), a major virulence factor of Bordetella pertussis, ADP-ribosylates Gi proteins in the mammalian cell cytosol to inhibit GPCR signaling. The intracellular pathway of PT includes endocytosis and retrograde transport to the trans-Golgi network (TGN) and endoplasmic reticulum (ER). Subsequent translocation of S1 to the cytosol is presumably preceded by dissociation from the holotoxin. In vitro, such dissociation is stimulated by interaction of PT with ATP. To investigate the role of this interaction in cellular events, we engineered a form of PT (PTDM) with changes to two amino acids involved in the interaction with ATP. PTDM was reduced in (1) binding to ATP, (2) dissociability by interaction with ATP, (3) in vitro enzymatic activity and (4) cellular ADP-ribosylation activity. In cells treated with PTDM carrying target sequences for organelle-specific modifications, normal transport to the TGN and ER occurred, but N-glycosylation patterns of the S1 and S4 subunits were consistent with an inability of PTDM to dissociate in the ER. These results indicate a requirement for interaction with ATP for PT dissociation in the ER and cellular activity. They also indicate that the retrograde transport route is the cellular intoxication pathway for PT.

  6. Gene set analysis of survival following ovarian cancer implicates macrolide binding and intracellular signaling genes

    PubMed Central

    Fridley, Brooke L.; Jenkins, Gregory D.; Tsai, Ya-Yu; Song, Honglin; Bolton, Kelly L.; Fenstermacher, David; Tyrer, Jonathan; Ramus, Susan J.; Cunningham, Julie M.; Vierkant, Robert A.; Chen, Zhihua; Chen, Y. Ann; Iversen, Ed; Menon, Usha; Gentry-Maharaj, Aleksandra; Schildkraut, Joellen; Sutphen, Rebecca; Gayther, Simon A.; Hartmann, Lynn C.; Pharoah, Paul D. P.; Sellers, Thomas A.; Goode, Ellen L.

    2012-01-01

    Background Genome-wide association studies (GWAS) for epithelial ovarian cancer (EOC), the most lethal gynecologic malignancy, have identified novel susceptibility loci. GWAS for survival after EOC have had more limited success. The association of each single nucleotide polymorphism (SNP) individually may not be well-suited to detect small effects of multiple SNPs, such as those operating within the same biological pathway. Gene set analysis (GSA) overcomes this limitation by assessing overall evidence for association of a phenotype with all measured variation in a set of genes. Methods To determine gene sets associated with EOC overall survival, we conducted GSA using data from two large GWASes (N cases = 2,813, N deaths = 1,116), with a novel Principal Component – Gamma GSA method. Analysis was completed for all cases and then separately for high grade serous (HGS) histological subtype. Results Analysis of the HGS subjects resulted in 43 gene sets with p<0.005 (1.7%); of these, 21 gene sets had p < 0.10 in both GWASes, including intracellular signaling pathway (p = 7.3 × 10−5) and macrolide binding (p = 6.2 ×10−4) gene sets. The top gene sets in analysis of all cases were meiotic mismatch repair (p=6.3 ×10−4) and macrolide binding (p=1.0×10−3). Of 18 gene sets with p<0.005 (0.7%), eight had p < 0.10 in both GWASes. Conclusion This research detected novel gene sets associated with EOC survival. Impact Novel gene sets associated with EOC survival might lead to new insights and avenues for development of novel therapies for EOC and pharmacogenomic studies. PMID:22302016

  7. Near-membrane ensemble elongation in the proline-rich LRP6 intracellular domain may explain the mysterious initiation of the Wnt signaling pathway

    PubMed Central

    2011-01-01

    Background LRP6 is a membrane protein crucial in the initiation of canonical Wnt/β-catenin signalling. Its function is dependent on its proline-serine rich intracellular domain. LRP6 has five PPP(S/T)P motifs that are phosphorylated during activation, starting with the site closest to the membrane. Like all long proline rich regions, there is no stable 3D structure for this isolated, contiguous region. Results In our study, we use a computational simulation tool to sample the conformational space of the LRP6 intracellular domain, under the spatial constraints imposed by (a) the membrane and (b) the close approach of the neighboring intracellular molecular complex, which is assembled on Frizzled when Wnt binds to both LRP6 and Frizzled on the opposite side of the membrane. We observe that an elongated form dominates in the LRP6 intracellular domain structure ensemble. This elongation could relieve conformational auto-inhibition of the PPP(S/T)PX(S/T) motif binding sites and allow GSK3 and CK1 to approach their phosphorylation sites, thereby activating LRP6 and the downstream pathway. Conclusions We propose a model in which the conformation of the LRP6 intracellular domain is elongated before activation. This is based on the intrusion of the Frizzled complex into the ensemble space of the proline rich region of LRP6, which alters the shape of its available ensemble space. To test whether this observed ensemble conformational change is sequence dependent, we did a control simulation with a hypothetical sequence with 50% proline and 50% serine in alternating residues. We confirm that this ensemble neighbourhood-based conformational change is independent of sequence and conclude that it is likely found in all proline rich sequences. These observations help us understand the nature of proline rich regions which are both unstructured and which seem to evolve at a higher rate of mutation, while maintaining sequence composition. PMID:22372892

  8. Effect of N-acetyl cysteine on enterocyte apoptosis and intracellular signalling pathways' response to oxidative stress in weaned piglets.

    PubMed

    Zhu, Lihui; Cai, Xuan; Guo, Qi; Chen, Xiaolian; Zhu, Suwen; Xu, Jianxiong

    2013-12-14

    N-Acetyl cysteine (NAC) has been widely used for preventing reactive oxygen species-induced damage. However, little is known as to whether dietary NAC supplementation would alleviate intestinal injury in weaned piglets. The present study evaluated the effect of NAC on enterocyte apoptosis and intracellular signalling pathways' response to weaning stress. The control piglets were normally suckling, and piglets in the weaning and NAC groups were fed the basal diet and basal+NAC diet from 14 to 25 d of age, respectively. Compared with the control piglets, weaning increased cortisol concentrations (P< 0·05), decreased superoxide dismutase and glutathione peroxidase activities (P< 0·05), increased malondialdehyde content (P< 0·05) in serum and enhanced enterocyte apoptosis index (AI) and concentrations of caspase-3, caspase-8 and caspase-9 (P< 0·05). Gene expression analyses indicated that weaning induced apoptosis via Fas signalling and mitochondrial pathways in weaned piglets. Dietary NAC supplementation decreased (P< 0·05) cortisol concentrations and the AI, increased (P< 0·05) antioxidant status in serum and alleviated histopathological changes in the intestine. It also inhibited Fas, caspase-3, caspase-8 and integrin αvβ6 (αvβ6) gene expressions in the NAC-treated piglets. However, no significant decrease (P>0·10) in caspase-3, caspase-8 and caspase-9 concentrations was observed in the NAC group compared with the weaning group. In conclusion, weaning may induce enterocyte apoptosis via the activation of Fas-dependent and mitochondria-dependent apoptosis. Although NAC had no effect on caspase concentrations, it was clearly beneficial for preserving morphological integrity in weaned piglets via the regulation of cell apoptosis and the inhibition of Fas-dependent apoptosis and αvβ6 expression.

  9. Tissue Plasminogen Activator Alters Intracellular Sequestration of Zinc through Interaction with the Transporter ZIP4

    SciTech Connect

    Emmetsberger, Jaime; Mirrione, Martine M.; Zhou, Chun; Fernandez-Monreal, Monica; Siddiq, Mustafa M.; Ji, Kyungmin; Tsirka, Stella E.

    2010-09-17

    Glutamatergic neurons contain free zinc packaged into neurotransmitter-loaded synaptic vesicles. Upon neuronal activation, the vesicular contents are released into the synaptic space, whereby the zinc modulates activity of postsynaptic neurons though interactions with receptors, transporters and exchangers. However, high extracellular concentrations of zinc trigger seizures and are neurotoxic if substantial amounts of zinc reenter the cells via ion channels and accumulate in the cytoplasm. Tissue plasminogen activator (tPA), a secreted serine protease, is also proepileptic and excitotoxic. However, tPA counters zinc toxicity by promoting zinc import back into the neurons in a sequestered form that is nontoxic. Here, we identify the zinc influx transporter, ZIP4, as the pathway through which tPA mediates the zinc uptake. We show that ZIP4 is upregulated after excitotoxin stimulation of the mouse, male and female, hippocampus. ZIP4 physically interacts with tPA, correlating with an increased intracellular zinc influx and lysosomal sequestration. Changes in prosurvival signals support the idea that this sequestration results in neuroprotection. These experiments identify a mechanism via which neurons use tPA to efficiently neutralize the toxic effects of excessive concentrations of free zinc.

  10. [Studies of interaction of intracellular signalling and metabolic pathways under inhibition of mitochondrial aconitase with fluoroacetate].

    PubMed

    Zinchenko, V P; Goncharov, N V; Teplova, V V; Kasymov, V A; Petrova, O I; Berezhnov, A V; Senchenkov, E V; Mindukshev, I V; Jenkins, R O; Radilov, A S

    2007-01-01

    Mitochondrial aconitase has been shown to be inactivated by a spectrum of substances or critical states. Fluoroacetate (FA) is the most known toxic agent inhibiting aconitase. The biochemistry of toxic action of FA is rather well understood, though no effective therapy has been proposed for the past six decades. In order to reveal novel approaches for possible antidotes to be developed, experiments were performed with rat liver mitochondria, Ehrlich ascite tumor cells and cardiomyocytes, exposed to FA or fluorocitrate in vitro. The effect of FA developed at much higher concentrations in comparison with fluorocitrate and was dependent upon respiratory substrates in experiments with mitochondria: with pyruvate, FA induced a slow oxidation and/or leak of pyridine nucleotides and inhibition of respiration. Oxidation of pyridine nucleotides was prevented by incubation of mitochondria with cyclosporin A. Studies of the pyridine nucleotides level and calcium response generated in Ehrlich ascite tumor cells under activation with ATP also revealed a loss of pyridine nucleotides from mitochondria resulting in a shift in the balance of mitochondrial and cytosolic NAD(P)H under exposure to FA. An increase of cytosolic [Ca2+] was observed in the cell lines exposed to FA and is explained by activation of plasma membrane calcium channels; this mechanism, could have an impact on amplitude and rate of Ca2+ waves in cardiomyocytes. Highlighting the reciprocal relationship between intracellular pyridine nucleotides and calcium balance, we discuss metabolic pathway modulation in the context of probable development of an effective therapy for FA poisoning and other inhibitors of aconitase. PMID:18318221

  11. Intracellular Delivery and Antibacterial Activity of Gentamicin Encapsulated in pH-Sensitive Liposomes

    PubMed Central

    Lutwyche, Peter; Cordeiro, Carol; Wiseman, David J.; St-Louis, Maryse; Uh, Mitchell; Hope, Michael J.; Webb, Murray S.; Finlay, B. Brett

    1998-01-01

    Cell membranes are relatively impermeable to the antibiotic gentamicin, a factor that, along with the toxicity of gentamicin, precludes its use against many important intracellular bacterial infections. Liposomal encapsulation of this drug was used in order to achieve intracellular antibiotic delivery and therefore increase the drug’s therapeutic activity against intracellular pathogens. Gentamicin encapsulation in several dipalmitoylphosphatidylcholine (DPPC) and pH-sensitive dioleoylphosphatidylethanolamine (DOPE)-based carrier systems was characterized. To systematically test the antibacterial efficacies of these formulations, a tissue culture assay system was developed wherein murine macrophage-like J774A.1 cells were infected with bacteria and were then treated with encapsulated drug. Of these formulations, DOPE–N-succinyl-DOPE and DOPE–N-glutaryl-DOPE (70:30;mol:mol) containing small amounts of polyethyleneglycol-ceramide showed appreciable antibacterial activities, killing greater than 75% of intracellular vacuole-resident wild-type Salmonella typhimurium compared to the level of killing of the control formulations. These formulations also efficiently eliminated intracellular infections caused by a recombinant hemolysin-expressing S. typhimurium strain and a Listeria monocytogenes strain, both of which escape the vacuole and reside in the cytoplasm. Control non-pH-sensitive liposomal formulations of gentamicin had poor antibacterial activities. A fluorescence resonance energy transfer assay indicated that the efficacious formulations undergo a pH-dependent lipid mixing and fusion event. Intracellular delivery of the fluorescent molecules encapsulated in these formulations was confirmed by confocal fluorescence microscopy and was shown to be dependent on endosomal acidification. This work shows that encapsulation of membrane-impermeative antibiotics in appropriately designed lipid-based delivery systems can enable their use in treating intracellular

  12. Cell-type-specific modelling of intracellular calcium signalling: a urothelial cell model.

    PubMed

    Appleby, Peter A; Shabir, Saqib; Southgate, Jennifer; Walker, Dawn

    2013-09-01

    Calcium signalling plays a central role in regulating a wide variety of cell processes. A number of calcium signalling models exist in the literature that are capable of reproducing a variety of experimentally observed calcium transients. These models have been used to examine in more detail the mechanisms underlying calcium transients, but very rarely has a model been directly linked to a particular cell type and experimentally verified. It is important to show that this can be achieved within the general theoretical framework adopted by these models. Here, we develop a framework designed specifically for modelling cytosolic calcium transients in urothelial cells. Where possible, we draw upon existing calcium signalling models, integrating descriptions of components known to be important in this cell type from a number of studies in the literature. We then add descriptions of several additional pathways that play a specific role in urothelial cell signalling, including an explicit ionic influx term and an active pumping mechanism that drives the cytosolic calcium concentration to a target equilibrium. The resulting one-pool model of endoplasmic reticulum (ER)-dependent calcium signalling relates the cytosolic, extracellular and ER calcium concentrations and can generate a wide range of calcium transients, including spikes, bursts, oscillations and sustained elevations in the cytosolic calcium concentration. Using single-variate robustness and multivariate sensitivity analyses, we quantify how varying each of the parameters of the model leads to changes in key features of the calcium transient, such as initial peak amplitude and the frequency of bursting or spiking, and in the transitions between bursting- and plateau-dominated modes. We also show that, novel to our urothelial cell model, the ionic and purinergic P2Y pathways make distinct contributions to the calcium transient. We then validate the model using human bladder epithelial cells grown in monolayer cell

  13. Calcium-Oxidant Signaling Network Regulates AMP-activated Protein Kinase (AMPK) Activation upon Matrix Deprivation*

    PubMed Central

    Sundararaman, Ananthalakshmy; Amirtham, Usha; Rangarajan, Annapoorni

    2016-01-01

    The AMP-activated protein kinase (AMPK) has recently been implicated in anoikis resistance. However, the molecular mechanisms that activate AMPK upon matrix detachment remain unexplored. In this study, we show that AMPK activation is a rapid and sustained phenomenon upon matrix deprivation, whereas re-attachment to the matrix leads to its dephosphorylation and inactivation. Because matrix detachment leads to loss of integrin signaling, we investigated whether integrin signaling negatively regulates AMPK activation. However, modulation of focal adhesion kinase or Src, the major downstream components of integrin signaling, failed to cause a corresponding change in AMPK signaling. Further investigations revealed that the upstream AMPK kinases liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) contribute to AMPK activation upon detachment. In LKB1-deficient cells, we found AMPK activation to be predominantly dependent on CaMKKβ. We observed no change in ATP levels under detached conditions at early time points suggesting that rapid AMPK activation upon detachment was not triggered by energy stress. We demonstrate that matrix deprivation leads to a spike in intracellular calcium as well as oxidant signaling, and both these intracellular messengers contribute to rapid AMPK activation upon detachment. We further show that endoplasmic reticulum calcium release-induced store-operated calcium entry contributes to intracellular calcium increase, leading to reactive oxygen species production, and AMPK activation. We additionally show that the LKB1/CaMKK-AMPK axis and intracellular calcium levels play a critical role in anchorage-independent cancer sphere formation. Thus, the Ca2+/reactive oxygen species-triggered LKB1/CaMKK-AMPK signaling cascade may provide a quick, adaptable switch to promote survival of metastasizing cancer cells. PMID:27226623

  14. Intracellular reorganization and ionic signaling of the Phycomyces stage I sporangiophore in response to gravity and touch

    PubMed Central

    Živanović, Branka D.

    2013-01-01

    Unicellular zygomycete fungus Phycomyces blakesleeanus exhibits a typical apical growth that depends on a complex interaction of different physiological processes. Sensitivity to the light, wind, barriers, touch and gravity of Phycomyces sporangiophore implicate the existence of the cross-talk between different signaling pathways in the same cell. Touch and gravity in Phycomyces sporangiophore seem to share some common elements of Ca2+ and H+ ion-based signal transduction systems. Apoplastic Ca2+ and H+ ionic fluxes are important for establishing cell polarity in tip growing sporangiophore both in vertical and tilted position. Upon gravistimulation sporangiophores display asymmetric ionic distribution and intracellular reorganization leading to change in the growth pattern. PMID:23650568

  15. Delineation of Platelet Activation Pathway of Scutellarein Revealed Its Intracellular Target as Protein Kinase C.

    PubMed

    Tian, Xiaoxuan; Chang, Lianying; Ma, Guangyin; Wang, Taiyi; Lv, Ming; Wang, Zhilong; Chen, Liping; Wang, Yuefei; Gao, Xiumei; Zhu, Yan

    2016-01-01

    Erigeron breviscapus has been widely used in traditional Chinese medicine (TCM) and its total flavonoid component is commonly used to treat ischemic stroke, coronary heart disease, diabetes and hypertension. Scutellarin is the major ingredient of E. breviscapus and scutellarein is one of the main bioactive metabolites of scutellarin in vivo, but the latter's pharmacological activities have not been fully characterized. Provided evidence that could inhibit platelet aggregation, the effect of scutellarein on rat washed platelets and its underlying mechanisms were evaluated in our research. Scutellarein inhibited platelet adhesion and aggregation induced by multiple G protein coupled receptor agonists such as thrombin, U46619 and ADP, in a concentration-dependent manner. Furthermore, the mild effect of scutellarein on intracellular Ca(2+) mobilization and cyclic AMP (cAMP) level was observed. On the other hand, the role of scutellarein as potential protein kinase C (PKC) inhibitor was confirmed by PKC activity analysis and molecular docking. The phorbol myristate acetate-induced platelets aggregation assay with or without ADP implied that the scutellarein takes PKC(s) as its primary target(s), and acts on it in a reversible way. Finally, scutellarein as a promising agent exhibited a high inhibition effect on ADP-induced platelet aggregation among its analogues. This study clarifies the PKC-related signaling pathway involved in antiplatelet action of scutellarein, and may be beneficial for the treatment of cardiovascular diseases. PMID:26581323

  16. PKCζ regulates Notch receptor routing and activity in a Notch signaling-dependent manner.

    PubMed

    Sjöqvist, Marika; Antfolk, Daniel; Ferraris, Saima; Rraklli, Vilma; Haga, Cecilia; Antila, Christian; Mutvei, Anders; Imanishi, Susumu Y; Holmberg, Johan; Jin, Shaobo; Eriksson, John E; Lendahl, Urban; Sahlgren, Cecilia

    2014-04-01

    Activation of Notch signaling requires intracellular routing of the receptor, but the mechanisms controlling the distinct steps in the routing process is poorly understood. We identify PKCζ as a key regulator of Notch receptor intracellular routing. When PKCζ was inhibited in the developing chick central nervous system and in cultured myoblasts, Notch-stimulated cells were allowed to undergo differentiation. PKCζ phosphorylates membrane-tethered forms of Notch and regulates two distinct routing steps, depending on the Notch activation state. When Notch is activated, PKCζ promotes re-localization of Notch from late endosomes to the nucleus and enhances production of the Notch intracellular domain, which leads to increased Notch activity. In the non-activated state, PKCζ instead facilitates Notch receptor internalization, accompanied with increased ubiquitylation and interaction with the endosomal sorting protein Hrs. Collectively, these data identify PKCζ as a key regulator of Notch trafficking and demonstrate that distinct steps in intracellular routing are differentially modulated depending on Notch signaling status.

  17. Intracellular localization of mevalonate-activating enzymes in plant cells

    PubMed Central

    Rogers, L. J.; Shah, S. P. J.; Goodwin, T. W.

    1966-01-01

    Mevalonate-activating enzymes are shown to be present in the chloroplasts of French-bean leaves. The chloroplast membrane is impermeable to mevalonic acid. Mevalonate-activating enzymes also appear to be found outside the chloroplast. These results support the view that terpenoid biosynthesis in the plant cell is controlled by a combination of enzyme segregation and specific membrane permeability. ImagesFig. 1.Fig. 2. PMID:5947149

  18. Dysregulated intracellular signaling in the striatum in a pathophysiologically grounded model of Tourette syndrome.

    PubMed

    Rapanelli, Maximiliano; Frick, Luciana R; Pogorelov, Vladimir; Ota, Kristie T; Abbasi, Eeman; Ohtsu, Hiroshi; Pittenger, Christopher

    2014-12-01

    Tic disorders produce substantial morbidity, but their pathophysiology remains poorly understood. Convergent evidence suggests that dysregulation of the cortico-basal ganglia circuitry is central to the pathogenesis of tics. Tourette syndrome (TS), the most severe end of the continuum of tic disorders, is substantially genetic, but causative mutations have been elusive. We recently described a mouse model, the histidine decarboxylase (Hdc) knockout mouse, that recapitulates a rare, highly penetrant mutation found in a single family; these mice exhibit TS-like phenomenology. These animals have a global deficit in brain histamine and a consequent dysregulation of DA in the basal ganglia. Histamine modulation of DA effects is increasingly appreciated, but the mechanisms underlying this modulation remain unclear; the consequences of modest DA elevation in the context of profound HA deficiency are difficult to predict, but understanding them in the Hdc knockout mouse may provide generalizable insights into the pathophysiology of TS. Here we characterized signaling pathways in striatal cells in this model system, at baseline and after amphetamine challenge. In vivo microdialysis confirms elevated DA in Hdc-KO mice. We find dephosphorylation of Akt and its target GSK3β and activation of the MAPK signaling cascade and its target rpS6; these are characteristic of the effects of DA on D2- and D1-expressing striatal neurons, respectively. Strikingly, there is no alteration in mTOR signaling, which can be regulated by DA in both cell types. These cellular effects help elucidate striatal signaling abnormalities in a uniquely validated mouse model of TS and move towards the identification of new potential therapeutic targets for tic disorders. PMID:25464894

  19. Dysregulated intracellular signaling in the striatum in a pathophysiologically grounded model of Tourette syndrome

    PubMed Central

    Rapanelli, Maximiliano; Frick, Luciana R.; Pogorelov, Vladimir; Ota, Kristie T.; Abbasi, Eeman; Ohtsu, Hiroshi; Pittenger, Christopher

    2015-01-01

    Tic disorders produce substantial morbidity, but their pathophysiology remains poorly understood. Convergent evidence suggests that dysregulation of the cortico-basal ganglia circuitry is central to the pathogenesis of tics. Tourette syndrome (TS), the most severe end of the continuum of tic disorders, is substantially genetic, but causative mutations have been elusive. We recently described a mouse model, the histidine decarboxylase (Hdc) knockout mouse, that recapitulates a rare, highly penetrant mutation found in a single family; these mice exhibit TS-like phenomenology. These animals have a global deficit in brain histamine and a consequent dysregulation of DA in the basal ganglia. Histamine modulation of DA effects is increasingly appreciated, but the mechanisms underlying this modulation remain unclear; the consequences of modest DA elevation in the context of profound HA deficiency are difficult to predict, but understanding them in the Hdc knockout mouse may provide generalizable insights into the pathophysiology of TS. Here we characterized signaling pathways in striatal cells in this model system, at baseline and after amphetamine challenge. In vivo microdialysis confirms elevated DA in Hdc-KO mice. We find dephosphorylation of Akt and its target GSK3β and activation of the MAPK signaling cascade and its target rpS6; these are characteristic of the effects of DA on D2- and D1-expressing striatal neurons, respectively. Strikingly, there is no alteration in mTOR signaling, which can be regulated by DA in both cell types. These cellular effects help elucidate striatal signaling abnormalities in a uniquely validated mouse model of TS and move towards the identification of new potential therapeutic targets for tic disorders. PMID:25464894

  20. Ursolic Acid Activates Intracellular Killing Effect of Macrophages During Mycobacterium tuberculosis Infection.

    PubMed

    Podder, Biswajit; Jang, Woong Sik; Nam, Kung-Woo; Lee, Byung-Eui; Song, Ho-Yeon

    2015-05-01

    Tuberculosis is one of the most threatening infectious diseases to public health all over the world, for which Mycobacterium tuberculosis (MTB) is the etiological agent of pathogenesis. Ursolic acid (UA) has immunomodulatory function and exhibits antimycobacterial activity. However, the intracellular killing effect of UA has yet to be elucidated. The aim of this study was to evaluate the intracellular killing effect of UA during mycobacterial infection. The intracellular killing activity of UA was evaluated in the macrophage cell line THP-1 by the MGIT 960 system as well as by CFU count. The production of reactive oxygen species (ROS) and the level of nitric oxide (NO) were measured using DCF-DA and Griess reagent, respectively. Phagocytosis was observed by a fluorescence-based staining method, and the colony forming units were enumerated on 7H11 agar medium following infection. In addition, MRP8 mRNA expression was measured by qRT-PCR. UA significantly decreased the number of intracellular Mycobacterium through generation of ROS and NO. In addition, it profoundly activated the phagocytosis process of THP-1 cells during MTB-infection. Furthermore, our data demonstrated that UA activated the phagocytosis process in human monocyte cells through MRP8 induction. These data suggest that UA firmly contributes to the intracellular killing effect of macrophages during mycobacterial infection.

  1. Leptin differentially regulates NPY secretion in hypothalamic cell lines through distinct intracellular signal transduction pathways.

    PubMed

    Dhillon, Sandeep S; Belsham, Denise D

    2011-04-11

    Leptin acts as a key peripheral hormone in distinct neurons in the hypothalamus to modulate both reproductive function and energy homeostasis. The control of neuropeptide Y (NPY) secretion is an example of a process that can be differentially regulated by leptin. In order to further understand these distinct modulatory effects, we have used immortalized, neuronal hypothalamic cell lines expressing NPY, mHypoE-38 and mHypoE-46. We found that these cell lines express the endogenous leptin receptor, ObRb, and secrete detectable levels of NPY. We exposed the neurons to 100nM leptin for 1h and determined that the basal levels of NPY in the cell lines were differentially regulated: NPY secretion was inhibited in mHypoE-46 neurons, whereas NPY secretion was induced in the mHypoE-38 neurons. In order to determine the mechanisms involved in the divergent regulation of NPY release, we analyzed the activity of a number of signaling components using phospho-specific antibodies directed towards specific proteins in the MAP kinase, PI3K, and AMPK pathways, among others. We found that leptin activated a different combination of second messengers in each cell line. Importantly, we could link the regulation of NPY secretion to different signaling pathways, AMPK in the mHypoE-46 and both MAPK and PI3K in the mHypoE-38 neurons. This is the first demonstration that leptin can specifically regulate individual NPY neuron secretory responses through distinct signaling pathways.

  2. Direct activation of cardiac pacemaker channels by intracellular cyclic AMP.

    PubMed

    DiFrancesco, D; Tortora, P

    1991-05-01

    Cyclic AMP acts as a second messenger in the modulation of several ion channels that are typically controlled by a phosphorylation process. In cardiac pacemaker cells, adrenaline and acetylcholine regulate the hyperpolarization-activated current (if), but in opposite ways; this current is involved in the generation and modulation of pacemaker activity. These actions are mediated by cAMP and underlie control of spontaneous rate by neurotransmitters. Whether the cAMP modulation of if is mediated by channel phosphorylation is, however, still unknown. Here we investigate the action of cAMP on if in excised patches of cardiac pacemaker cells and find that cAMP activates if by a mechanism independent of phosphorylation, involving a direct interaction with the channels at their cytoplasmic side. Cyclic AMP activates if by shifting its activation curve to more positive voltages, in agreement with whole-cell results. This is the first evidence of an ion channel whose gating is dually regulated by voltage and direct cAMP binding.

  3. Single-cell codetection of metabolic activity, intracellular functional proteins, and genetic mutations from rare circulating tumor cells.

    PubMed

    Zhang, Yu; Tang, Yin; Sun, Shuai; Wang, Zhihua; Wu, Wenjun; Zhao, Xiaodong; Czajkowsky, Daniel M; Li, Yan; Tian, Jianhui; Xu, Ling; Wei, Wei; Deng, Yuliang; Shi, Qihui

    2015-10-01

    The high glucose uptake and activation of oncogenic signaling pathways in cancer cells has long made these features, together with the mutational spectrum, prime diagnostic targets of circulating tumor cells (CTCs). Further, an ability to characterize these properties at a single cell resolution is widely believed to be essential, as the known extensive heterogeneity in CTCs can obscure important correlations in data obtained from cell population-based methods. However, to date, it has not been possible to quantitatively measure metabolic, proteomic, and genetic data from a single CTC. Here we report a microchip-based approach that allows for the codetection of glucose uptake, intracellular functional proteins, and genetic mutations at the single-cell level from rare tumor cells. The microchip contains thousands of nanoliter grooves (nanowells) that isolate individual CTCs and allow for the assessment of their glucose uptake via imaging of a fluorescent glucose analog, quantification of a panel of intracellular signaling proteins using a miniaturized antibody barcode microarray, and retrieval of the individual cell nuclei for subsequent off-chip genome amplification and sequencing. This approach integrates molecular-scale information on the metabolic, proteomic, and genetic status of single cells and permits the inference of associations between genetic signatures, energy consumption, and phosphoproteins oncogenic signaling activities in CTCs isolated from blood samples of patients. Importantly, this microchip chip-based approach achieves this multidimensional molecular analysis with minimal cell loss (<20%), which is the bottleneck of the rare cell analysis. PMID:26378744

  4. Activation of the recombinant human alpha 7 nicotinic acetylcholine receptor significantly raises intracellular free calcium.

    PubMed

    Delbono, O; Gopalakrishnan, M; Renganathan, M; Monteggia, L M; Messi, M L; Sullivan, J P

    1997-01-01

    The alpha 7 nicotinic acetylcholine receptor (nAChR) subtype, unlike other neuronal nicotinic receptors, exhibits a relatively high permeability to Ca++ ions. Although Ca++ entry through this receptor subtype has been implicated in various Ca(++)-dependent processes in the central nervous system, little is known about how this receptor modulates mammalian intracellular Ca++ dynamics. Intracellular Ca++ responses evoked by activation of the human alpha 7 nAChRs stably expressed in HEK-293 (human embryonic kidney) cells were studied. Inward current and intracellular Ca++ transients were recorded simultaneously in response to a fast drug application system. Current recordings under whole-cell voltage-clamp and fast ratiometric intracellular Ca++ imaging acquisition were synchronized to drug pulses. The mean peak [Ca++]i observed with 100 microM (-)-nicotine was 356 +/- 48 nM (n = 8). The magnitude of the intracellular Ca++ elevation corresponds to a 20% fractional current carried by Ca++ ions. The EC50 of the intracellular Ca++ responses for (-)-nicotine, (+/-)-epibatidine, 1,1 dimethyl-4-phenyl-piperazinium and acetylcholine were 51, 3.5, 75 and 108 microM, respectively. These EC50 values strongly correlate with those recorded for the cationic inward current through alpha 7 nAChR. alpha-Bungarotoxin, methyllcaconitine or extracellular Ca++ chelation ablated (-)-nicotine-evoked increase in intracellular Ca++ concentration. This study provides evidence that cation influx through the human alpha 7 nAChR is sufficient to mediate a significant, transient, rise in intracellular Ca++ concentration.

  5. Endolysosomes Are the Principal Intracellular Sites of Acid Hydrolase Activity.

    PubMed

    Bright, Nicholas A; Davis, Luther J; Luzio, J Paul

    2016-09-12

    The endocytic delivery of macromolecules from the mammalian cell surface for degradation by lysosomal acid hydrolases requires traffic through early endosomes to late endosomes followed by transient (kissing) or complete fusions between late endosomes and lysosomes. Transient or complete fusion results in the formation of endolysosomes, which are hybrid organelles from which lysosomes are re-formed. We have used synthetic membrane-permeable cathepsin substrates, which liberate fluorescent reporters upon proteolytic cleavage, as well as acid phosphatase cytochemistry to identify which endocytic compartments are acid hydrolase active. We found that endolysosomes are the principal organelles in which acid hydrolase substrates are cleaved. Endolysosomes also accumulated acidotropic probes and could be distinguished from terminal storage lysosomes, which were acid hydrolase inactive and did not accumulate acidotropic probes. Using live-cell microscopy, we have demonstrated that fusion events, which form endolysosomes, precede the onset of acid hydrolase activity. By means of sucrose and invertase uptake experiments, we have also shown that acid-hydrolase-active endolysosomes and acid-hydrolase-inactive, terminal storage lysosomes exist in dynamic equilibrium. We conclude that the terminal endocytic compartment is composed of acid-hydrolase-active, acidic endolysosomes and acid hydrolase-inactive, non-acidic, terminal storage lysosomes, which are linked and function in a lysosome regeneration cycle. PMID:27498570

  6. A Novel Intracellular Isoform of Matrix Metalloproteinase-2 Induced by Oxidative Stress Activates Innate Immunity

    PubMed Central

    Lovett, David H.; Mahimkar, Rajeev; Raffai, Robert L.; Cape, Leslie; Maklashina, Elena; Cecchini, Gary; Karliner, Joel S.

    2012-01-01

    Background Experimental and clinical evidence has pinpointed a critical role for matrix metalloproteinase-2 (MMP-2) in ischemic ventricular remodeling and systolic heart failure. Prior studies have demonstrated that transgenic expression of the full-length, 68 kDa, secreted form of MMP-2 induces severe systolic failure. These mice also had unexpected and severe mitochondrial structural abnormalities and dysfunction. We hypothesized that an additional intracellular isoform of MMP-2, which affects mitochondrial function is induced under conditions of systolic failure-associated oxidative stress. Methodology and Principal Findings Western blots of cardiac mitochondria from the full length MMP-2 transgenics, ageing mice and a model of accelerated atherogenesis revealed a smaller 65 kDa MMP-2 isoform. Cultured cardiomyoblasts subjected to transient oxidative stress generated the 65 kDa MMP-2 isoform. The 65 kDa MMP-2 isoform was also induced by hypoxic culture of cardiomyoblasts. Genomic database analysis of the MMP-2 gene mapped transcriptional start sites and RNA transcripts induced by hypoxia or epigenetic modifiers within the first intron of the MMP-2 gene. Translation of these transcripts yields a 65 kDa N-terminal truncated isoform beginning at M77, thereby deleting the signal sequence and inhibitory prodomain. Cellular trafficking studies demonstrated that the 65 kDa MMP-2 isoform is not secreted and is present in cytosolic and mitochondrial fractions, while the full length 68 kDa isoform was found only in the extracellular space. Expression of the 65 kDa MMP-2 isoform induced mitochondrial-nuclear stress signaling with activation of the pro-inflammatory NF-κB, NFAT and IRF transcriptional pathways. By microarray, the 65 kDa MMP-2 induces an innate immunity transcriptome, including viral stress response genes, innate immunity transcription factor IRF7, chemokines and pro-apoptosis genes. Conclusion A novel N-terminal truncated intracellular isoform of MMP-2 is

  7. Endosomal receptor kinetics determine the stability of intracellular growth factor signalling complexes

    PubMed Central

    Tzafriri, A. Rami; Edelman, Elazer R.

    2006-01-01

    There is an emerging paradigm that growth factor signalling continues in the endosome and that cell response to a growth factor is defined by the integration of cell surface and endosomal events. As activated receptors in the endosome are exposed to a different set of binding partners, they probably elicit differential signals compared with when they are at the cell surface. As such, complete appreciation of growth factor signalling requires understanding of growth factor–receptor binding and trafficking kinetics both at the cell surface and in endosomes. Growth factor binding to surface receptors is well characterized, and endosomal binding is assumed to follow surface kinetics if one accounts for changes in pH. Yet, specific binding kinetics within the endosome has not been examined in detail. To parse the factors governing the binding state of endosomal receptors we analysed a whole-cell mathematical model of epidermal growth factor receptor trafficking and binding. We discovered that the stability of growth factor–receptor complexes within endosomes is governed by three primary independent factors: the endosomal dissociation constant, total endosomal volume and the number of endosomal receptors. These factors were combined into a single dimensionless parameter that determines the endosomal binding state of the growth factor–receptor complex and can distinguish different growth factors from each other and different cell states. Our findings indicate that growth factor binding within endosomal compartments cannot be appreciated solely on the basis of the pH-dependence of the dissociation constant and that the concentration of receptors in the endosomal compartment must also be considered. PMID:17117924

  8. The Role of the Francisella Tularensis Pathogenicity Island in Type VI Secretion, Intracellular Survival, and Modulation of Host Cell Signaling

    PubMed Central

    Bröms, Jeanette E.; Sjöstedt, Anders; Lavander, Moa

    2010-01-01

    Francisella tularensis is a highly virulent gram-negative intracellular bacterium that causes the zoonotic disease tularemia. Essential for its virulence is the ability to multiply within host cells, in particular monocytic cells. The bacterium has developed intricate means to subvert host immune mechanisms and thereby facilitate its intracellular survival by preventing phagolysosomal fusion followed by escape into the cytosol, where it multiplies. Moreover, it targets and manipulates numerous host cell signaling pathways, thereby ameliorating the otherwise bactericidal capacity. Many of the underlying molecular mechanisms still remain unknown but key elements, directly or indirectly responsible for many of the aforementioned mechanisms, rely on the expression of proteins encoded by the Francisella pathogenicity island (FPI), suggested to constitute a type VI secretion system. We here describe the current knowledge regarding the components of the FPI and the roles that have been ascribed to them. PMID:21687753

  9. Inhibitory effect of red ginseng acidic polysaccharide from Korean red ginseng on phagocytic activity and intracellular replication of Brucella abortus in RAW 264.7 cells.

    PubMed

    Reyes, Alisha Wehdnesday Bernardo; Simborio, Hannah Leah Tadeja; Hop, Huynh Tan; Arayan, Lauren Togonon; Min, Won Gi; Lee, Hu Jang; Rhee, Man Hee; Chang, Hong Hee; Kim, Suk

    2016-09-30

    Korean red ginseng (KRG) has long been used in traditional Korean and Oriental medicine. However, the anti-bacterial mechanism and therapeutic efficiency of KGR for intracellular Brucella infection are still unclear. In this study, the bactericidal activity of Korean red ginseng acidic polysaccharide (RGAP) on Brucella (B.) abortus and its cytotoxic effects on RAW 264.7 cells were evaluated. In addition, B. abortus internalization and intracellular replication in macrophages were investigated after RGAP treatment. RGAP-incubated cells displayed a marked reduction in the adherence, internalization and intracellular growth of B. abortus in macrophages. Furthermore, decreased F-actin fluorescence was observed relative to untreated B. abortus-infected cells. Western blot analysis of intracellular signaling proteins revealed reduced ERK, JNK and p38α phosphorylation levels in B. abortus-infected RGAP-treated cells compared to the control. Moreover, elevated co-localization of B. abortus-containing phagosomes with lysosome-associated membrane protein 1 (LAMP-1) were observed in RGAP-treated cells compared with the control. Overall, the results of this study suggest that RGAP can disrupt phagocytic activity of B. abortus via suppression of mitogen-activated protein kinases (MAPKs) signaling proteins ERK, JNK and p38 levels and inhibit intracellular replication of B. abortus by enhancing phagolysosome fusion, which may provide an alternative control of brucellosis.

  10. Inhibitory effect of red ginseng acidic polysaccharide from Korean red ginseng on phagocytic activity and intracellular replication of Brucella abortus in RAW 264.7 cells

    PubMed Central

    Bernardo Reyes, Alisha Wehdnesday; Simborio, Hannah Leah Tadeja; Hop, Huynh Tan; Arayan, Lauren Togonon; Min, Won Gi; Lee, Hu Jang; Rhee, Man Hee; Chang, Hong Hee

    2016-01-01

    Korean red ginseng (KRG) has long been used in traditional Korean and Oriental medicine. However, the anti-bacterial mechanism and therapeutic efficiency of KGR for intracellular Brucella infection are still unclear. In this study, the bactericidal activity of Korean red ginseng acidic polysaccharide (RGAP) on Brucella (B.) abortus and its cytotoxic effects on RAW 264.7 cells were evaluated. In addition, B. abortus internalization and intracellular replication in macrophages were investigated after RGAP treatment. RGAP-incubated cells displayed a marked reduction in the adherence, internalization and intracellular growth of B. abortus in macrophages. Furthermore, decreased F-actin fluorescence was observed relative to untreated B. abortus-infected cells. Western blot analysis of intracellular signaling proteins revealed reduced ERK, JNK and p38α phosphorylation levels in B. abortus-infected RGAP-treated cells compared to the control. Moreover, elevated co-localization of B. abortus-containing phagosomes with lysosome-associated membrane protein 1 (LAMP-1) were observed in RGAP-treated cells compared with the control. Overall, the results of this study suggest that RGAP can disrupt phagocytic activity of B. abortus via suppression of mitogen-activated protein kinases (MAPKs) signaling proteins ERK, JNK and p38 levels and inhibit intracellular replication of B. abortus by enhancing phagolysosome fusion, which may provide an alternative control of brucellosis. PMID:26726017

  11. Diverse intracellular pathogens activate Type III Interferon expression from peroxisomes

    PubMed Central

    Odendall, Charlotte; Dixit, Evelyn; Stavru, Fabrizia; Bierne, Helene; Franz, Kate M.; Fiegen, Ann; Boulant, Steeve; Gehrke, Lee; Cossart, Pascale; Kagan, Jonathan C.

    2014-01-01

    Type I Interferon (IFN) responses are considered the primary means by which viral infections are controlled in mammals. Despite this view, several pathogens activate antiviral responses in the absence of Type I IFNs. The mechanisms controlling Type I IFN-independent responses are undefined. We have found that RIG-I like Receptors (RLRs) induce Type III IFN expression in a variety of human cell types, and identified factors that differentially regulate Type I and III IFN expression. We identified peroxisomes as a primary site that initiates Type III IFN expression, and revealed that the process of intestinal epithelial cell differentiation upregulates peroxisome biogenesis and promotes robust Type III IFN responses in human cells. These findings highlight the interconnections between innate immunity and cell biology. PMID:24952503

  12. Glial potassium channels activated by neuronal firing or intracellular cyclic AMP in Helix.

    PubMed Central

    Gommerat, I; Gola, M

    1996-01-01

    1. Cell-attached and whole cell patch clamp experiments were performed on satellite glial cells adhering to the cell body of neurones in situ within the nervous system of the snail Helix pomatia. The underlying neurone was under current or voltage-clamp control. 2. Neuronal firing induced a delayed (20-30 s) persistent (3-4 min) increase in the opening probability of glial K+ channels. The channels were also activated by perfusing the ganglion with a depolarizing high-K+ saline, except when the underlying neurone was prevented from depolarizing under voltage-clamp conditions. 3. Two K(+)-selective channels were detected in the glial membrane. The channel responding to neuronal firing was present in 95% of the patches (n = 393). It had a unitary conductance of 56 pS, a Na+ :K+ permeability ratio < 0.02 and displayed slight inward rectification in symmetrical [K+] conditions. It was sensitive to TEA, Ba2+ and Cs+. The following results refer to this channel as studied in the cell-attached configuration. 4. The glial K+ channel was activated by bath application of the membrane-permeant cyclic AMP derivatives 8-bromo-cAMP and dibutyryl-cAMP, the adenylyl cyclase activator forskolin and the diesterase inhibitors IBMX, theophylline and caffeine. It was insensitive to cyclic GMP activators and to conditions that might alter the intracellular [Ca2+] (ionomycin, low-Ca2+ saline and Ca2+ channel blockers). 5. The forskolin-induced changes in channel behaviour (open and closed time distributions, burst duration, short and long gaps within bursts) could be accounted for by a four-state model (3 closed states, 1 open state) by simply changing one of the six rate parameters. 6. The present results suggest that the signal sent by an active neurone to satellite glial cells is confined to the glial cells round that neurone. The effect of this signal on the class of glial K+ channels studied can be mimicked by an increase in glial cAMP concentration. The subsequent delayed opening

  13. Shigella IpaD has a dual role: signal transduction from the type III secretion system needle tip and intracellular secretion regulation.

    PubMed

    Roehrich, A Dorothea; Guillossou, Enora; Blocker, Ariel J; Martinez-Argudo, Isabel

    2013-02-01

    Type III secretion systems (T3SSs) are protein injection devices essential for the interaction of many Gram-negative bacteria with eukaryotic cells. While Shigella assembles its T3SS when the environmental conditions are appropriate for invasion, secretion is only activated after physical contact with a host cell. First, the translocators are secreted to form a pore in the host cell membrane, followed by effectors which manipulate the host cell. Secretion activation is tightly controlled by conserved T3SS components: the needle tip proteins IpaD and IpaB, the needle itself and the intracellular gatekeeper protein MxiC. To further characterize the role of IpaD during activation, we combined random mutagenesis with a genetic screen to identify ipaD mutant strains unable to respond to host cell contact. Class II mutants have an overall defect in secretion induction. They map to IpaD's C-terminal helix and likely affect activation signal generation or transmission. The Class I mutant secretes translocators prematurely and is specifically defective in IpaD secretion upon activation. A phenotypically equivalent mutant was found in mxiC. We show that IpaD and MxiC act in the same intracellular pathway. In summary, we demonstrate that IpaD has a dual role and acts at two distinct locations during secretion activation.

  14. Intracellular Ca(2+) signaling and Ca(2+) microdomains in the control of cell survival, apoptosis and autophagy.

    PubMed

    La Rovere, Rita M L; Roest, Gemma; Bultynck, Geert; Parys, Jan B

    2016-08-01

    The endoplasmic reticulum (ER), mitochondria and lysosomes are physically and/or functionally linked, establishing close contact sites between these organelles. As a consequence, Ca(2+) release events from the ER, the major intracellular Ca(2+)-storage organelle, have an immediate effect on the physiological function of mitochondria and lysosomes. Also, the lysosomes can act as a Ca(2+) source for Ca(2+) release into the cytosol, thereby influencing ER-based Ca(2+) signaling. Given the important role for mitochondria and lysosomes in cell survival, cell death and cell adaptation processes, it has become increasingly clear that Ca(2+) signals from or towards these organelles impact these processes. In this review, we discuss the most recent insights in the emerging role of Ca(2+) signaling in cellular survival by controlling basal mitochondrial bioenergetics and by regulating apoptosis, a mitochondrial process, and autophagy, a lysosomal process, in response to cell damage and cell stress.

  15. Intracellular Ca(2+) signaling and Ca(2+) microdomains in the control of cell survival, apoptosis and autophagy.

    PubMed

    La Rovere, Rita M L; Roest, Gemma; Bultynck, Geert; Parys, Jan B

    2016-08-01

    The endoplasmic reticulum (ER), mitochondria and lysosomes are physically and/or functionally linked, establishing close contact sites between these organelles. As a consequence, Ca(2+) release events from the ER, the major intracellular Ca(2+)-storage organelle, have an immediate effect on the physiological function of mitochondria and lysosomes. Also, the lysosomes can act as a Ca(2+) source for Ca(2+) release into the cytosol, thereby influencing ER-based Ca(2+) signaling. Given the important role for mitochondria and lysosomes in cell survival, cell death and cell adaptation processes, it has become increasingly clear that Ca(2+) signals from or towards these organelles impact these processes. In this review, we discuss the most recent insights in the emerging role of Ca(2+) signaling in cellular survival by controlling basal mitochondrial bioenergetics and by regulating apoptosis, a mitochondrial process, and autophagy, a lysosomal process, in response to cell damage and cell stress. PMID:27157108

  16. TGF-β-Mediated Sustained ERK1/2 Activity Promotes the Inhibition of Intracellular Growth of Mycobacterium avium in Epithelioid Cells Surrogates

    PubMed Central

    L'Abbate, Carolina; Cipriano, Ivone; Pérez-Hurtado, Elizabeth Cristina; Leão, Sylvia Cardoso; Carneiro, Célia Regina Whitaker; Machado, Joel

    2011-01-01

    Transforming growth factor beta (TGF-β) has been implicated in the pathogenesis of several diseases including infection with intracellular pathogens such as the Mycobacterium avium complex. Infection of macrophages with M. avium induces TGF-β production and neutralization of this cytokine has been associated with decreased intracellular bacterial growth. We have previously demonstrated that epithelioid cell surrogates (ECs) derived from primary murine peritoneal macrophages through a process of differentiation induced by IL-4 overlap several features of epithelioid cells found in granulomas. In contrast to undifferentiated macrophages, ECs produce larger amounts of TGF-β and inhibit the intracellular growth of M. avium. Here we asked whether the levels of TGF-β produced by ECs are sufficient to induce a self-sustaining autocrine TGF-β signaling controlling mycobacterial replication in infected-cells. We showed that while exogenous addition of increased concentration of TGF-β to infected-macrophages counteracted M. avium replication, pharmacological blockage of TGF-β receptor kinase activity with SB-431542 augmented bacterial load in infected-ECs. Moreover, the levels of TGF-β produced by ECs correlated with high and sustained levels of ERK1/2 activity. Inhibition of ERK1/2 activity with U0126 increased M. avium replication in infected-cells, suggesting that modulation of intracellular bacterial growth is dependent on the activation of ERK1/2. Interestingly, blockage of TGF-β receptor kinase activity with SB-431542 in infected-ECs inhibited ERK1/2 activity, enhanced intracellular M. avium burden and these effects were followed by a severe decrease in TGF-β production. In summary, our findings indicate that the amplitude of TGF-β signaling coordinates the strength and duration of ERK1/2 activity that is determinant for the control of intracellular mycobacterial growth. PMID:21731758

  17. The phosphatidylethanolamine derivative diDCP-LA-PE mimics intracellular insulin signaling.

    PubMed

    Nishizaki, Tomoyuki; Gotoh, Akinobu; Shimizu, Tadashi; Tanaka, Akito

    2016-06-02

    Insulin facilitates glucose uptake into cells by translocating the glucose transporter GLUT4 towards the cell surface through a pathway along an insulin receptor (IR)/IR substrate 1 (IRS-1)/phosphatidylinositol 3 kinase (PI3K)/3-phosphoinositide-dependent protein kinase-1 (PDK1)/Akt axis. The newly synthesized phosphatidylethanolamine derivative 1,2-O-bis-[8-{2-(2-pentyl-cyclopropylmethyl)-cyclopropyl}-octanoyl]-sn-glycero-3-phosphatidylethanolamine (diDCP-LA-PE) has the potential to inhibit protein tyrosine phosphatase 1B (PTP1B) and to directly activate PKCζ, an atypical isozyme, and PKCε, a novel isozyme. PTP1B inhibition enhanced insulin signaling cascades downstream IR/IRS-1 by preventing tyrosine dephosphorylation. PKCζ and PKCε directly activated Akt2 by phosphorylating at Thr309 and Ser474, respectively. diDCP-LA-PE increased cell surface localization of GLUT4 and stimulated glucose uptake into differentiated 3T3-L1 adipocytes, still with knocking-down IR or in the absence of insulin. Moreover, diDCP-LA-PE effectively reduced serum glucose levels in type 1 diabetes (DM) model mice. diDCP-LA-PE, thus, may enable type 1 DM therapy without insulin injection.

  18. The phosphatidylethanolamine derivative diDCP-LA-PE mimics intracellular insulin signaling

    PubMed Central

    Nishizaki, Tomoyuki; Gotoh, Akinobu; Shimizu, Tadashi; Tanaka, Akito

    2016-01-01

    Insulin facilitates glucose uptake into cells by translocating the glucose transporter GLUT4 towards the cell surface through a pathway along an insulin receptor (IR)/IR substrate 1 (IRS-1)/phosphatidylinositol 3 kinase (PI3K)/3-phosphoinositide-dependent protein kinase-1 (PDK1)/Akt axis. The newly synthesized phosphatidylethanolamine derivative 1,2-O-bis-[8-{2-(2-pentyl-cyclopropylmethyl)-cyclopropyl}-octanoyl]-sn-glycero-3-phosphatidylethanolamine (diDCP-LA-PE) has the potential to inhibit protein tyrosine phosphatase 1B (PTP1B) and to directly activate PKCζ, an atypical isozyme, and PKCε, a novel isozyme. PTP1B inhibition enhanced insulin signaling cascades downstream IR/IRS-1 by preventing tyrosine dephosphorylation. PKCζ and PKCε directly activated Akt2 by phosphorylating at Thr309 and Ser474, respectively. diDCP-LA-PE increased cell surface localization of GLUT4 and stimulated glucose uptake into differentiated 3T3-L1 adipocytes, still with knocking-down IR or in the absence of insulin. Moreover, diDCP-LA-PE effectively reduced serum glucose levels in type 1 diabetes (DM) model mice. diDCP-LA-PE, thus, may enable type 1 DM therapy without insulin injection. PMID:27251941

  19. ARE MACROPHAGES ACTIVATED AND INDUCE PULMONARY INJURY BY INTRACELLULARLY BIOAVAILABLE IRON?

    EPA Science Inventory

    ARE MACROPHAGES ACTIVATED AND INDUCE PULMONARY INJURY BY INTRACELLULARLY BIOAVAILABLE IRON? UP Kodavanti1, MCJ Schladweiler1, S Becker2, DL Costa1, P Mayer3, A Ziesenis3, WG Kreyling3, 1ETD, 2HSDivision, NHEERL, USEPA, Research Triangle Park, NC, USA, and 3GSF, Inhalation Biology...

  20. Intracellular activity of tedizolid phosphate and ACH-702 versus Mycobacterium tuberculosis infected macrophages

    PubMed Central

    2014-01-01

    Background Due to the emergency of multidrug-resistant strains of Mycobacterium tuberculosis, is necessary the evaluation of new compounds. Findings Tedizolid, a novel oxazolidinone, and ACH-702, a new isothiazoloquinolone, were tested against M. tuberculosis infected THP-1 macrophages. These two compounds significantly decreased the number of intracellular mycobacteria at 0.25X, 1X, 4X and 16X the MIC value. The drugs were tested either in nanoparticules or in free solution. Conclusion Tedizolid and ACH-702 have a good intracellular killing activity comparable to that of rifampin or moxifloxacin. PMID:24708819

  1. The Analysis of Intracellular and Intercellular Calcium Signaling in Human Anterior Lens Capsule Epithelial Cells with Regard to Different Types and Stages of the Cataract

    PubMed Central

    Gosak, Marko; Markovič, Rene; Fajmut, Aleš; Marhl, Marko; Hawlina, Marko; Andjelić, Sofija

    2015-01-01

    In this work we investigated how modifications of the Ca2+ homeostasis in anterior lens epithelial cells (LECs) are associated with different types of cataract (cortical or nuclear) and how the progression of the cataract (mild or moderate) affects the Ca2+ signaling. We systematically analyzed different aspects of intra- and inter-cellular Ca2+ signaling in the human LECs, which are attached to surgically isolated lens capsule (LC), obtained during cataract surgery. We monitored the temporal and spatial changes in intracellular Ca2+ concentration after stimulation with acetylcholine by means of Fura-2 fluorescence captured with an inverted microscope. In our analysis we compared the features of Ca2+ signals in individual cells, synchronized activations, spatio-temporal grouping and the nature of intercellular communication between LECs. The latter was assessed by using the methodologies of the complex network theory. Our results point out that at the level of individual cells there are no significant differences when comparing the features of the signals with regard either to the type or the stage of the cataract. On the other hand, noticeable differences are observed at the multicellular level, despite inter-capsule variability. LCs associated with more developed cataracts were found to exhibit a slower collective response to stimulation, a less pronounced spatio-temporal clustering of LECs with similar signaling characteristics. The reconstructed intercellular networks were found to be sparser and more segregated than in LCs associated with mild cataracts. Moreover, we show that spontaneously active LECs often operate in localized groups with quite well aligned Ca2+ activity. The presence of spontaneous activity was also found to affect the stimulated Ca2+ responses of individual cells. Our findings indicate that the cataract progression entails the impairment of intercellular signaling thereby suggesting the functional importance of altered Ca2+ signaling of

  2. Dextran sulfate sodium upregulates MAPK signaling for the uptake and subsequent intracellular survival of Brucella abortus in murine macrophages.

    PubMed

    Reyes, Alisha Wehdnesday Bernardo; Arayan, Lauren Togonon; Simborio, Hannah Leah Tadeja; Hop, Huynh Tan; Min, WonGi; Lee, Hu Jang; Kim, Dong Hee; Chang, Hong Hee; Kim, Suk

    2016-02-01

    Brucellosis is one of the major zoonoses worldwide that inflicts important health problems in animal and human. Here, we demonstrated that dextran sulfate sodium (DSS) significantly increased adhesion of Brucella (B.) abortus in murine macrophages compared to untreated cells. Even without infection, Brucella uptake into macrophages increased and F-actin reorganization was induced compared with untreated cells. Furthermore, DSS increased the phosphorylation of MAPKs (ERK1/2 and p38α) in Brucella-infected, DSS-treated cells compared with the control cells. Lastly, DSS markedly increased the intracellular survival of Brucella abortus in macrophages by up to 48 h. These results suggest that DSS enhanced the adhesion and phagocytosis of B. abortus into murine macrophages by stimulating the MAPK signaling proteins phospho-ERK1/2 and p38α and that DSS increased the intracellular survival of B. abortus by inhibiting colocalization of Brucella-containing vacuoles (BCVs) with the late endosome marker LAMP-1. This study emphasizes the enhancement of the phagocytic and intracellular modulatory effects of DSS, which may suppress the innate immune system and contribute to prolonged Brucella survival and chronic infection.

  3. Intracellular acidification is required for full activation of the sweet taste receptor by miraculin

    PubMed Central

    Sanematsu, Keisuke; Kitagawa, Masayuki; Yoshida, Ryusuke; Nirasawa, Satoru; Shigemura, Noriatsu; Ninomiya, Yuzo

    2016-01-01

    Acidification of the glycoprotein, miraculin (MCL), induces sweet taste in humans, but not in mice. The sweet taste induced by MCL is more intense when acidification occurs with weak acids as opposed to strong acids. MCL interacts with the human sweet receptor subunit hTAS1R2, but the mechanisms by which the acidification of MCL activates the sweet taste receptor remain largely unexplored. The work reported here speaks directly to this activation by utilizing a sweet receptor TAS1R2 + TAS1R3 assay. In accordance with previous data, MCL-applied cells displayed a pH dependence with citric acid (weak acid) being right shifted to that with hydrochloric acid (strong acid). When histidine residues in both the intracellular and extracellular region of hTAS1R2 were exchanged for alanine, taste-modifying effect of MCL was reduced or abolished. Stronger intracellular acidification of HEK293 cells was induced by citric acid than by HCl and taste-modifying effect of MCL was proportional to intracellular pH regardless of types of acids. These results suggest that intracellular acidity is required for full activation of the sweet taste receptor by MCL. PMID:26960429

  4. Involvement of intracellular labile zinc in suppression of DEVD-caspase activity in human neuroblastoma cells.

    PubMed

    Ho, L H; Ratnaike, R N; Zalewski, P D

    2000-02-01

    Age-related tissue Zn deficiency may contribute to neuronal and glial cell death by apoptosis in Alzheimer's dementia. To investigate this, we studied the effects of increasing or decreasing the levels of intracellular labile Zn on apoptosis of human neuroblastoma BE(2)-C cells in vitro. BE(2)-C cells were primed for 18 h with butyrate (1 mM) before addition of staurosporine (1 microM), an effector enzyme of apoptosis, for a further 3 h to induce DEVD-caspase activity. An increase in intracellular Zn using Zn ionophore pyrithione suppressed DEVD-caspase activity, while a decrease in intracellular Zn induced by Zn chelator TPEN mimicked staurosporine by activating DEVD-caspase in butyrate-primed cells. The distribution of intracellular Zn in the cells was demonstrated with the UV-excitable Zn-specific fluorophore Zinquin. Confocal images showed distinct cytoplasmic and cytoskeletal fluorescence. We propose that Zn decreases the level of apoptosis in neuronal cells exposed to toxins, possibly by stabilizing their cytoskeleton.

  5. Intracellular reduction/activation of a disulfide switch in thiosemicarbazone iron chelators

    PubMed Central

    Akam, Eman A.; Chang, Tsuhen M.; Astashkin, Andrei V.

    2014-01-01

    Iron scavengers (chelators) offer therapeutic opportunities in anticancer drug design by targeting the increased demand for iron in cancer cells as compared to normal cells. Prochelation approaches are expected to avoid systemic iron depletion as chelators are liberated under specific intracellular conditions. In the strategy described herein, a disulfide linkage is employed as a redox-directed switch within the binding unit of an antiproliferative thiosemicarbazone prochelator, which is activated for iron coordination following reduction to the thiolate chelator. In glutathione redox buffer, this reduction event occurs at physiological concentrations and half-cell potentials. Consistent with concurrent reduction and activation, higher intracellular thiol concentrations increase cell susceptibility to prochelator toxicity in cultured cancer cells. The reduction of the disulfide switch and intracellular iron chelation are confirmed in cell-based assays using calcein as a fluorescent probe for paramagnetic ions. The resulting low-spin Fe(III) complex is identified in intact Jurkat cells by EPR spectroscopy measurements, which also document a decreased concentration of active ribonucleotide reductase following exposure to the prochelator. Cell viability and fluorescence-based assays show that the iron complex presents low cytotoxicity and does not participate in intracellular redox chemistry, indicating that this antiproliferative chelation strategy does not rely on the generation of reactive oxygen species. PMID:25100578

  6. Intracellular acidification is required for full activation of the sweet taste receptor by miraculin.

    PubMed

    Sanematsu, Keisuke; Kitagawa, Masayuki; Yoshida, Ryusuke; Nirasawa, Satoru; Shigemura, Noriatsu; Ninomiya, Yuzo

    2016-01-01

    Acidification of the glycoprotein, miraculin (MCL), induces sweet taste in humans, but not in mice. The sweet taste induced by MCL is more intense when acidification occurs with weak acids as opposed to strong acids. MCL interacts with the human sweet receptor subunit hTAS1R2, but the mechanisms by which the acidification of MCL activates the sweet taste receptor remain largely unexplored. The work reported here speaks directly to this activation by utilizing a sweet receptor TAS1R2 + TAS1R3 assay. In accordance with previous data, MCL-applied cells displayed a pH dependence with citric acid (weak acid) being right shifted to that with hydrochloric acid (strong acid). When histidine residues in both the intracellular and extracellular region of hTAS1R2 were exchanged for alanine, taste-modifying effect of MCL was reduced or abolished. Stronger intracellular acidification of HEK293 cells was induced by citric acid than by HCl and taste-modifying effect of MCL was proportional to intracellular pH regardless of types of acids. These results suggest that intracellular acidity is required for full activation of the sweet taste receptor by MCL. PMID:26960429

  7. Enhanced hypoxic preconditioning by isoflurane: signaling gene expression and requirement of intracellular Ca2+ and inositol triphosphate receptors

    PubMed Central

    Bickler, Philip E.; Fahlman, Christian S.

    2012-01-01

    Neurons preconditioned with non-injurious hypoxia or the anesthetic isoflurane express different genes but are equally protected against severe hypoxia/ischemia. We hypothesized that neuroprotection would be augmented when preconditioning with isoflurane and hypoxic preconditioning are combined. We also tested if preconditioning requires intracellular Ca2+ and the inositol triphosphate receptor, and if gene expression is similar in single agent and combined preconditioning. Hippocampal slice cultures prepared from 9 day-old rats were preconditioned with hypoxia (95% N2, 5% CO2 for 15 min, HPC), 1% isoflurane for 15 min (APC) or their combination (CPC) for 15 min. A day later cultures were deprived of O2 and glucose (OGD) to produce neuronal injury. Cell death was assessed 48 hr after OGD. mRNA encoding 119 signal transduction genes was quantified with cDNA micro arrays. Intracellular Ca2+ in CA1 region was measured with fura-2 during preconditioning. The cell-permeable Ca2+ buffer BAPTA-AM, the IP3 receptor antagonist Xestospongin C and RNA silencing were used to investigate preconditioning mechanisms. CPC decreased CA1, CA3 and dentate region death by 64–86% following OGD, more than HPC or APC alone (P<0.01). Gene expression following CPC was an amalgam of gene expression in HPC and APC, with simultaneous increases in growth/development and survival/apoptosis regulation genes. Intracellular Ca2+ chelation and RNA silencing of IP3 receptors prevented preconditioning neuroprotection and gene responses. We conclude that combined isoflurane-hypoxia preconditioning augments neuroprotection compared to single agents in immature rat hippocampal slice cultures. The mechanism involves genes for growth, development, apoptosis regulation and cell survival as well as IP3 receptors and intracellular Ca2+. PMID:20434434

  8. Enhanced hypoxic preconditioning by isoflurane: signaling gene expression and requirement of intracellular Ca2+ and inositol triphosphate receptors.

    PubMed

    Bickler, Philip E; Fahlman, Christian S

    2010-06-22

    Neurons preconditioned with non-injurious hypoxia or the anesthetic isoflurane express different genes but are equally protected against severe hypoxia/ischemia. We hypothesized that neuroprotection would be augmented when preconditioning with isoflurane and hypoxic preconditioning are combined. We also tested if preconditioning requires intracellular Ca(2+) and the inositol triphosphate receptor, and if gene expression is similar in single agent and combined preconditioning. Hippocampal slice cultures prepared from 9 day old rats were preconditioned with hypoxia (95% N(2), 5% CO(2) for 15 min, HPC), 1% isoflurane for 15 min (APC) or their combination (CPC) for 15 min. A day later cultures were deprived of O(2) and glucose (OGD) to produce neuronal injury. Cell death was assessed 48 h after OGD. mRNA encoding 119 signal transduction genes was quantified with cDNA micro arrays. Intracellular Ca(2+) in CA1 region was measured with fura-2 during preconditioning. The cell-permeable Ca(2+) buffer BAPTA-AM, the IP(3) receptor antagonist Xestospongin C and RNA silencing were used to investigate preconditioning mechanisms. CPC decreased CA1, CA3 and dentate region death by 64-86% following OGD, more than HPC or APC alone (P<0.01). Gene expression following CPC was an amalgam of gene expression in HPC and APC, with simultaneous increases in growth/development and survival/apoptosis regulation genes. Intracellular Ca(2+) chelation and RNA silencing of IP(3) receptors prevented preconditioning neuroprotection and gene responses. We conclude that combined isoflurane-hypoxia preconditioning augments neuroprotection compared to single agents in immature rat hippocampal slice cultures. The mechanism involves genes for growth, development, apoptosis regulation and cell survival as well as IP(3) receptors and intracellular Ca(2+).

  9. Intracellular lipid flux and membrane microdomains as organizing principles in inflammatory cell signaling.

    PubMed

    Fessler, Michael B; Parks, John S

    2011-08-15

    Lipid rafts and caveolae play a pivotal role in organization of signaling by TLR4 and several other immune receptors. Beyond the simple cataloguing of signaling events compartmentalized by these membrane microdomains, recent studies have revealed the surprisingly central importance of dynamic remodeling of membrane lipid domains to immune signaling. Simple interventions upon membrane lipid, such as changes in cholesterol loading or crosslinking of raft lipids, are sufficient to induce micrometer-scale reordering of membranes and their protein cargo with consequent signal transduction. In this review, using TLR signaling in the macrophage as a central focus, we discuss emerging evidence that environmental and genetic perturbations of membrane lipid regulate protein signaling, illustrate how homeostatic flow of cholesterol and other lipids through rafts regulates the innate immune response, and highlight recent attempts to harness these insights toward therapeutic development.

  10. Intracellular Shigella remodels its LPS to dampen the innate immune recognition and evade inflammasome activation.

    PubMed

    Paciello, Ida; Silipo, Alba; Lembo-Fazio, Luigi; Curcurù, Laura; Zumsteg, Anna; Noël, Gaëlle; Ciancarella, Valeria; Sturiale, Luisa; Molinaro, Antonio; Bernardini, Maria Lina

    2013-11-12

    LPS is a potent bacterial effector triggering the activation of the innate immune system following binding with the complex CD14, myeloid differentiation protein 2, and Toll-like receptor 4. The LPS of the enteropathogen Shigella flexneri is a hexa-acylated isoform possessing an optimal inflammatory activity. Symptoms of shigellosis are produced by severe inflammation caused by the invasion process of Shigella in colonic and rectal mucosa. Here we addressed the question of the role played by the Shigella LPS in eliciting a dysregulated inflammatory response of the host. We unveil that (i) Shigella is able to modify the LPS composition, e.g., the lipid A and core domains, during proliferation within epithelial cells; (ii) the LPS of intracellular bacteria (iLPS) and that of bacteria grown in laboratory medium differ in the number of acyl chains in lipid A, with iLPS being the hypoacylated; (iii) the immunopotential of iLPS is dramatically lower than that of bacteria grown in laboratory medium; (iv) both LPS forms mainly signal through the Toll-like receptor 4/myeloid differentiation primary response gene 88 pathway; (v) iLPS down-regulates the inflammasome-mediated release of IL-1β in Shigella-infected macrophages; and (vi) iLPS exhibits a reduced capacity to prime polymorfonuclear cells for an oxidative burst. We propose a working model whereby the two forms of LPS might govern different steps of the invasive process of Shigella. In the first phases, the bacteria, decorated with hypoacylated LPS, are able to lower the immune system surveillance, whereas, in the late phases, shigellae harboring immunopotent LPS are fully recognized by the immune system, which can then successfully resolve the infection.

  11. Intracellular Shigella remodels its LPS to dampen the innate immune recognition and evade inflammasome activation

    PubMed Central

    Paciello, Ida; Silipo, Alba; Lembo-Fazio, Luigi; Curcurù, Laura; Zumsteg, Anna; Noël, Gaëlle; Ciancarella, Valeria; Sturiale, Luisa; Molinaro, Antonio; Bernardini, Maria Lina

    2013-01-01

    LPS is a potent bacterial effector triggering the activation of the innate immune system following binding with the complex CD14, myeloid differentiation protein 2, and Toll-like receptor 4. The LPS of the enteropathogen Shigella flexneri is a hexa-acylated isoform possessing an optimal inflammatory activity. Symptoms of shigellosis are produced by severe inflammation caused by the invasion process of Shigella in colonic and rectal mucosa. Here we addressed the question of the role played by the Shigella LPS in eliciting a dysregulated inflammatory response of the host. We unveil that (i) Shigella is able to modify the LPS composition, e.g., the lipid A and core domains, during proliferation within epithelial cells; (ii) the LPS of intracellular bacteria (iLPS) and that of bacteria grown in laboratory medium differ in the number of acyl chains in lipid A, with iLPS being the hypoacylated; (iii) the immunopotential of iLPS is dramatically lower than that of bacteria grown in laboratory medium; (iv) both LPS forms mainly signal through the Toll-like receptor 4/myeloid differentiation primary response gene 88 pathway; (v) iLPS down-regulates the inflammasome-mediated release of IL-1β in Shigella-infected macrophages; and (vi) iLPS exhibits a reduced capacity to prime polymorfonuclear cells for an oxidative burst. We propose a working model whereby the two forms of LPS might govern different steps of the invasive process of Shigella. In the first phases, the bacteria, decorated with hypoacylated LPS, are able to lower the immune system surveillance, whereas, in the late phases, shigellae harboring immunopotent LPS are fully recognized by the immune system, which can then successfully resolve the infection. PMID:24167293

  12. Haemorrhagic shock in mice--intracellular signalling and immunomodulation of peritoneal macrophages' LPS response.

    PubMed

    Rani, Meenakshi; Husain, Baher; Lendemans, Sven; Schade, Fritz U; Flohé, Sascha

    2006-01-01

    Haemorrhagic shock leads to decreased proinflammatory cytokine response which is associated with an increased susceptibility to bacterial infections. In the present study, the effect of GM-CSF on lipopolysaccharide (LPS)-induced TNF-alpha release and MAPkinase activation was analysed on the background of a possible immunostimulating activity of this substance. Male BALB/c mice were bled to a mean arterial blood pressure of 50 mmHg for 45 min followed by resuscitation. Peritoneal macrophages were isolated 20 h after haemorrhage and incubated with 10 ng/ml GM-CSF for 6h before LPS stimulation. TNF-alpha synthesis was studied in the culture supernatants using ELISA. Phosphorylation of ERK, p38MAPK and IkappaBalpha was detected by Western blotting. LPS-induced TNF-alpha production of peritoneal macrophages was significantly decreased 20 h after haemorrhage in comparison to the corresponding cells of sham-operated mice. In parallel the phosphorylation of IkappaBalpha was less in LPS-stimulated peritoneal macrophages from haemorrhagic mice. LPS-induced phosphorylation of ERK1/2 was also decreased in peritoneal macrophages isolated after haemorrhagic shock. In contrast, p38MAPK was phosphorylated more intensely after LPS-stimulation in macrophages collected from shocked mice. GM-CSF incubation elevated LPS-induced TNF-alpha response of macrophages from both sham-operated and shocked mice which was accompanied by an elevated IkappaB and ERK phosphorylation. In general, GM-CSF treatment in vitro enhanced peritoneal macrophages LPS-response both in terms of TNF-alpha synthesis and IkappaB and MAPK signalling, but the levels always stayed lower than those of GM-CSF-treated cells from sham-operated animals. In conclusion, GM-CSF preincubation could partly reactivate the depressed functions of peritoneal macrophages and may therefore exert immunostimulating properties after shock or trauma.

  13. LL37:DNA complexes provide antimicrobial activity against intracellular bacteria in human macrophages.

    PubMed

    Stephan, Alexander; Batinica, Marina; Steiger, Julia; Hartmann, Pia; Zaucke, Frank; Bloch, Wilhelm; Fabri, Mario

    2016-08-01

    As part of the innate host response neutrophils release neutrophil extracellular traps (NETs), protein:DNA complexes that contain a number of antimicrobial peptides (AMPs), such as cathelicidin. Human cathelicidin in its active form, LL37, has potent antimicrobial activity against bacteria. However, whether LL37 derived from NETs contributes to antimicrobial activity against intracellular pathogens remains unclear. Here, we report that NETs induced by mycobacteria contain cathelicidin. Human macrophages internalized NET-bound cathelicidin, which is transported to lysosomal compartments. Furthermore, using a model of in vitro-generated LL37:DNA complexes we found that LL37 derived from such complexes attacks mycobacteria in macrophage phagolysosomes resulting in antimicrobial activity. Taken together, our results suggest a mechanism by which LL37 in complex with DNA contributes to host defence against intracellular bacteria in human macrophages.

  14. Dynamics of free intracellular Ca2+ during synaptic and spike activity of cricket tibial motoneurons.

    PubMed

    Baden, Tom; Hedwig, Berthold

    2009-04-01

    For all nervous systems, motoneurons are the main output pathway. They are involved in generating episodic motor activity as well as enduring motor rhythms. To determine whether changes in cytosolic Ca(2+) correlate with motor performance, we studied the spatiotemporal dynamics, mode of entry and role of free intracellular Ca(2+) in cricket (Gryllus bimaculatus) front leg tibial extensor and flexor motoneurons. Synaptic activation or intracellular depolarising current injection uniformly increased Ca(2+) with the same dynamics throughout the primary and secondary branches of the dendritic tree of all motoneurons. Ca(2+) rise times (mean tau(rise), 233-295 ms) were lower than decay times (mean tau(decay), 1927-1965 ms), and resulted in a Ca(2+) plateau during repetitive activation, such as during walking. The neurons therefore operate with a different Ca(2+) level during walking than during episodic leg movements. Ca(2+) enters the dendritic processes of motoneurons via a voltage-activated mechanism. Entry is driven by subthreshold excitation, and is largely independent of the neurons' spiking activity. To what extent ligand-activated mechanisms of Ca(2+) entry operate remains uncertain. We found no evidence for any prominent Ca(2+)-activated secondary currents in these motoneurons. Excitatory postsynaptic potentials evoked by extracellular stimulation of descending neurons were unaffected by the level of free intracellular Ca(2+). The activity of tibial motoneurons therefore appears to be only weakly dependent on the level of free intracellular Ca(2+) in dendrites. This is different to what has been found for many other neurons studied, and may represent an essential prerequisite for insect motoneurons to support a wide range of both episodic and rhythmic motor sequences underlying behaviour.

  15. Intracellular Activation of Tenofovir Alafenamide and the Effect of Viral and Host Protease Inhibitors.

    PubMed

    Birkus, Gabriel; Bam, Rujuta A; Willkom, Madeleine; Frey, Christian R; Tsai, Luong; Stray, Kirsten M; Yant, Stephen R; Cihlar, Tomas

    2016-01-01

    Tenofovir alafenamide fumarate (TAF) is an oral phosphonoamidate prodrug of the HIV reverse transcriptase nucleotide inhibitor tenofovir (TFV). Previous studies suggested a principal role for the lysosomal serine protease cathepsin A (CatA) in the intracellular activation of TAF. Here we further investigated the role of CatA and other human hydrolases in the metabolism of TAF. Overexpression of CatA or liver carboxylesterase 1 (Ces1) in HEK293T cells increased intracellular TAF hydrolysis 2- and 5-fold, respectively. Knockdown of CatA expression with RNA interference (RNAi) in HeLa cells reduced intracellular TAF metabolism 5-fold. Additionally, the anti-HIV activity and the rate of CatA hydrolysis showed good correlation within a large set of TFV phosphonoamidate prodrugs. The covalent hepatitis C virus (HCV) protease inhibitors (PIs) telaprevir and boceprevir potently inhibited CatA-mediated TAF activation (50% inhibitory concentration [IC50] = 0.27 and 0.16 μM, respectively) in vitro and also reduced its anti-HIV activity in primary human CD4(+) T lymphocytes (21- and 3-fold, respectively) at pharmacologically relevant concentrations. In contrast, there was no inhibition of CatA or any significant effect on anti-HIV activity of TAF observed with cobicistat, noncovalent HIV and HCV PIs, or various prescribed inhibitors of host serine proteases. Collectively, these studies confirm that CatA plays a pivotal role in the intracellular metabolism of TAF, whereas the liver esterase Ces1 likely contributes to the hepatic activation of TAF. Moreover, this work demonstrates that a wide range of viral and host PIs, with the exception of telaprevir and boceprevir, do not interfere with the antiretroviral activity of TAF. PMID:26503655

  16. Monitoring intracellular calcium in response to GPCR activation using thin-film silicon photodiodes with integrated fluorescence filters.

    PubMed

    Martins, S A M; Moulas, G; Trabuco, J R C; Monteiro, G A; Chu, V; Conde, J P; Prazeres, D M F

    2014-02-15

    G-protein coupled receptor (GPCRs) drug discovery is a thriving strategy in the pharmaceutical industry. The standard approach uses living cells to test millions of compounds in a high-throughput format. Typically, changes in the intracellular levels of key elements in the signaling cascade are monitored using fluorescence or luminescence read-out systems, which require external equipment for signal acquisition. In this work, thin-film amorphous silicon photodiodes with an integrated fluorescence filter were developed to capture the intracellular calcium dynamics in response to the activation of the endogenous muscarinic M1 GPCR of HEK 293T cells. Using the new device it was possible to characterize the potency of carbachol (EC50=10.5 µM) and pirenzepine (IC50=4.2 μM), with the same accuracy as standard microscopy optical systems. The smaller foot-print provided by the detection system makes it an ideal candidate for the future integration in microfluidic devices for drug discovery. PMID:24055937

  17. CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil

    NASA Astrophysics Data System (ADS)

    Almqvist, Helena; Axelsson, Hanna; Jafari, Rozbeh; Dan, Chen; Mateus, André; Haraldsson, Martin; Larsson, Andreas; Molina, Daniel Martinez; Artursson, Per; Lundbäck, Thomas; Nordlund, Pär

    2016-03-01

    Target engagement is a critical factor for therapeutic efficacy. Assessment of compound binding to native target proteins in live cells is therefore highly desirable in all stages of drug discovery. We report here the first compound library screen based on biophysical measurements of intracellular target binding, exemplified by human thymidylate synthase (TS). The screen selected accurately for all the tested known drugs acting on TS. We also identified TS inhibitors with novel chemistry and marketed drugs that were not previously known to target TS, including the DNA methyltransferase inhibitor decitabine. By following the cellular uptake and enzymatic conversion of known drugs we correlated the appearance of active metabolites over time with intracellular target engagement. These data distinguished a much slower activation of 5-fluorouracil when compared with nucleoside-based drugs. The approach establishes efficient means to associate drug uptake and activation with target binding during drug discovery.

  18. CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil.

    PubMed

    Almqvist, Helena; Axelsson, Hanna; Jafari, Rozbeh; Dan, Chen; Mateus, André; Haraldsson, Martin; Larsson, Andreas; Martinez Molina, Daniel; Artursson, Per; Lundbäck, Thomas; Nordlund, Pär

    2016-01-01

    Target engagement is a critical factor for therapeutic efficacy. Assessment of compound binding to native target proteins in live cells is therefore highly desirable in all stages of drug discovery. We report here the first compound library screen based on biophysical measurements of intracellular target binding, exemplified by human thymidylate synthase (TS). The screen selected accurately for all the tested known drugs acting on TS. We also identified TS inhibitors with novel chemistry and marketed drugs that were not previously known to target TS, including the DNA methyltransferase inhibitor decitabine. By following the cellular uptake and enzymatic conversion of known drugs we correlated the appearance of active metabolites over time with intracellular target engagement. These data distinguished a much slower activation of 5-fluorouracil when compared with nucleoside-based drugs. The approach establishes efficient means to associate drug uptake and activation with target binding during drug discovery. PMID:27010513

  19. Raster image cross-correlation analysis for spatiotemporal visualization of intracellular degradation activities against exogenous DNAs.

    PubMed

    Sasaki, Akira; Yamamoto, Johtaro; Jin, Takashi; Kinjo, Masataka

    2015-09-24

    Reducing intracellular DNA degradation is critical to enhance the efficiency of gene therapy. Exogenous DNA incorporation into cells is strictly blocked by the defense machinery of intracellular nuclease activity. Raster image correlation spectroscopy (RICS) and raster image cross-correlation spectroscopy (cross-correlation RICS; ccRICS) are image-based correlation methods. These powerful tools allow the study of spatiotemporal molecular dynamics. Here we performed spatiotemporal ccRICS analyses of fluorescent DNA and directly monitored the process of exogenous DNA degradation in living cell cytoplasm. Such direct monitors of DNA degradation allow us to determine the fate of the exogenous DNA in living cells. On comparing the process in living cells, our study shows that cytoplasmic nuclease activity differs between cell lines; therefore, we propose that the difference of nuclease activity in cytoplasm dictates a different resistance to exogenous DNA incorporation. New insight on efficient gene delivery can be provided with our study.

  20. CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil

    PubMed Central

    Almqvist, Helena; Axelsson, Hanna; Jafari, Rozbeh; Dan, Chen; Mateus, André; Haraldsson, Martin; Larsson, Andreas; Molina, Daniel Martinez; Artursson, Per; Lundbäck, Thomas; Nordlund, Pär

    2016-01-01

    Target engagement is a critical factor for therapeutic efficacy. Assessment of compound binding to native target proteins in live cells is therefore highly desirable in all stages of drug discovery. We report here the first compound library screen based on biophysical measurements of intracellular target binding, exemplified by human thymidylate synthase (TS). The screen selected accurately for all the tested known drugs acting on TS. We also identified TS inhibitors with novel chemistry and marketed drugs that were not previously known to target TS, including the DNA methyltransferase inhibitor decitabine. By following the cellular uptake and enzymatic conversion of known drugs we correlated the appearance of active metabolites over time with intracellular target engagement. These data distinguished a much slower activation of 5-fluorouracil when compared with nucleoside-based drugs. The approach establishes efficient means to associate drug uptake and activation with target binding during drug discovery. PMID:27010513

  1. Raster image cross-correlation analysis for spatiotemporal visualization of intracellular degradation activities against exogenous DNAs.

    PubMed

    Sasaki, Akira; Yamamoto, Johtaro; Jin, Takashi; Kinjo, Masataka

    2015-01-01

    Reducing intracellular DNA degradation is critical to enhance the efficiency of gene therapy. Exogenous DNA incorporation into cells is strictly blocked by the defense machinery of intracellular nuclease activity. Raster image correlation spectroscopy (RICS) and raster image cross-correlation spectroscopy (cross-correlation RICS; ccRICS) are image-based correlation methods. These powerful tools allow the study of spatiotemporal molecular dynamics. Here we performed spatiotemporal ccRICS analyses of fluorescent DNA and directly monitored the process of exogenous DNA degradation in living cell cytoplasm. Such direct monitors of DNA degradation allow us to determine the fate of the exogenous DNA in living cells. On comparing the process in living cells, our study shows that cytoplasmic nuclease activity differs between cell lines; therefore, we propose that the difference of nuclease activity in cytoplasm dictates a different resistance to exogenous DNA incorporation. New insight on efficient gene delivery can be provided with our study. PMID:26400011

  2. Modelling intracellular competition for calcium: kinetic and thermodynamic control of different molecular modes of signal decoding

    PubMed Central

    Antunes, Gabriela; Roque, Antonio C.; Simoes de Souza, Fabio M.

    2016-01-01

    Frequently, a common chemical entity triggers opposite cellular processes, which implies that the components of signalling networks must detect signals not only through their chemical natures, but also through their dynamic properties. To gain insights on the mechanisms of discrimination of the dynamic properties of cellular signals, we developed a computational stochastic model and investigated how three calcium ion (Ca2+)-dependent enzymes (adenylyl cyclase (AC), phosphodiesterase 1 (PDE1), and calcineurin (CaN)) differentially detect Ca2+ transients in a hippocampal dendritic spine. The balance among AC, PDE1 and CaN might determine the occurrence of opposite Ca2+-induced forms of synaptic plasticity, long-term potentiation (LTP) and long-term depression (LTD). CaN is essential for LTD. AC and PDE1 regulate, indirectly, protein kinase A, which counteracts CaN during LTP. Stimulations of AC, PDE1 and CaN with artificial and physiological Ca2+ signals demonstrated that AC and CaN have Ca2+ requirements modulated dynamically by different properties of the signals used to stimulate them, because their interactions with Ca2+ often occur under kinetic control. Contrarily, PDE1 responds to the immediate amplitude of different Ca2+ transients and usually with the same Ca2+ requirements observed under steady state. Therefore, AC, PDE1 and CaN decode different dynamic properties of Ca2+ signals. PMID:27033299

  3. Variation in human cancer cell external phosphatidylserine is regulated by flippase activity and intracellular calcium

    PubMed Central

    Vallabhapurapu, Subrahmanya D.; Blanco, Víctor M.; Sulaiman, Mahaboob K.; Vallabhapurapu, Swarajya Lakshmi; Chu, Zhengtao; Franco, Robert S.; Qi, Xiaoyang

    2015-01-01

    Viable cancer cells expose elevated levels of phosphatidylserine (PS) on the exoplasmic face of the plasma membrane. However, the mechanisms leading to elevated PS exposure in viable cancer cells have not been defined. We previously showed that externalized PS may be used to monitor, target and kill tumor cells. In addition, PS on tumor cells is recognized by macrophages and has implications in antitumor immunity. Therefore, it is important to understand the molecular details of PS exposure on cancer cells in order to improve therapeutic targeting. Here we explored the mechanisms regulating the surface PS exposure in human cancer cells and found that differential flippase activity and intracellular calcium are the major regulators of surface PS exposure in viable human cancer cells. In general, cancer cell lines with high surface PS exhibited low flippase activity and high intracellular calcium, whereas cancer cells with low surface PS exhibited high flippase activity and low intracellular calcium. High surface PS cancer cells also had higher total cellular PS than low surface PS cells. Together, our results indicate that the amount of external PS in cancer cells is regulated by calcium dependent flippase activity and may also be influenced by total cellular PS. PMID:26462157

  4. Mitochondrial reactive oxygen species (ROS) as signaling molecules of intracellular pathways triggered by the cardiac renin-angiotensin II-aldosterone system (RAAS)

    PubMed Central

    De Giusti, V. C.; Caldiz, C. I.; Ennis, I. L.; Pérez, N. G.; Cingolani, H. E.; Aiello, E. A.

    2013-01-01

    Mitochondria represent major sources of basal reactive oxygen species (ROS) production of the cardiomyocyte. The role of ROS as signaling molecules that mediate different intracellular pathways has gained increasing interest among physiologists in the last years. In our lab, we have been studying the participation of mitochondrial ROS in the intracellular pathways triggered by the renin-angiotensin II-aldosterone system (RAAS) in the myocardium during the past few years. We have demonstrated that acute activation of cardiac RAAS induces mitochondrial ATP-dependent potassium channel (mitoKATP) opening with the consequent enhanced production of mitochondrial ROS. These oxidant molecules, in turn, activate membrane transporters, as sodium/hydrogen exchanger (NHE-1) and sodium/bicarbonate cotransporter (NBC) via the stimulation of the ROS-sensitive MAPK cascade. The stimulation of such effectors leads to an increase in cardiac contractility. In addition, it is feasible to suggest that a sustained enhanced production of mitochondrial ROS induced by chronic cardiac RAAS, and hence, chronic NHE-1 and NBC stimulation, would also result in the development of cardiac hypertrophy. PMID:23755021

  5. Cellular Metabolic Activity and the Oxygen and Hydrogen Stable Isotope Composition of Intracellular Water and Metabolites

    NASA Astrophysics Data System (ADS)

    Kreuzer-Martin, H. W.; Hegg, E. L.

    2008-12-01

    Intracellular water is an important pool of oxygen and hydrogen atoms for biosynthesis. Intracellular water is usually assumed to be isotopically identical to extracellular water, but an unexpected experimental result caused us to question this assumption. Heme O isolated from Escherichia coli cells grown in 95% H218O contained only a fraction of the theoretical value of labeled oxygen at a position where the O atom was known to be derived from water. In fact, fewer than half of the oxygen atoms were labeled. In an effort to explain this surprising result, we developed a method to determine the isotope ratios of intracellular water in cultured cells. The results of our experiments showed that during active growth, up to 70% of the oxygen atoms and 50% of the hydrogen atoms in the intracellular water of E. coli are generated during metabolism and can be isotopically distinct from extracellular water. The fraction of isotopically distinct atoms was substantially less in stationary phase and chilled cells, consistent with our hypothesis that less metabolically-generated water would be present in cells with lower metabolic activity. Our results were consistent with and explained the result of the heme O labeling experiment. Only about 40% of the O atoms on the heme O molecule were labeled because, presumably, only about 40% of the water inside the cells was 18O water that had diffused in from the culture medium. The rest of the intracellular water contained 16O atoms derived from either nutrients or atmospheric oxygen. To test whether we could also detect metabolically-derived hydrogen atoms in cellular constituents, we isolated fatty acids from log-phase and stationary phase E. coli and determined the H isotope ratios of individual fatty acids. The results of these experiments showed that environmental water contributed more H atoms to fatty acids isolated in stationary phase than to the same fatty acids isolated from log-phase cells. Stable isotope analyses of

  6. Activation of cell signaling via optical manipulation of gold-coated liposomes encapsulating signaling molecules

    NASA Astrophysics Data System (ADS)

    Orsinger, Gabriel V.; Leung, Sarah J.; Romanowski, Marek

    2013-02-01

    Many diseases involve changes in cell signaling cascades, as seen commonly in drug resistant cancers. To better understand these intricate signaling events in diseased cells and tissues, experimental methods of probing cellular communication at a single to multi-cell level are required. We recently introduced a general platform for activation of selected signaling pathways by optically controlled delivery and release of water soluble factors using gold-coated liposomes. In the example presented here, we encapsulated inositol trisphosphate (IP3), a ubiquitous intracellular secondary messenger involved in GPCR and Akt signaling cascades, within 100 nm gold-coated liposomes. The high polarizability of the liposome's unique gold pseudo-shell allows stable optical trapping for subcellular manipulation in the presence of cells. We take this optical manipulation further by optically injecting IP3-containing liposomes into the cytosol of a single cell to initiate localized cell signaling. Upon optical injection of liposomal IP3 into a single ovarian carcinoma cell, we observed localized activation as reported by changes in Indo-1 fluorescence intensity. With established gap junctions between the injected cell and neighboring cells, we monitored propagation of this signaling to and through nearby cells.

  7. Release of plasminogen activator inhibitor-1 from human astrocytes is regulated by intracellular ceramide.

    PubMed

    Kimura, M; Soeda, S; Oda, M; Ochiai, T; Kihara, T; Ono, N; Shimeno, H

    2000-12-15

    The present study underscores a regulatory role of intracellular ceramide in astrocytes for the release of an extracellular serine protease, tissue-type plasminogen activator (t-PA), and its inhibitor, plasminogen activator inhibitor-1 (PAI-1). Treatment of cultured human astrocytes with N-acetylsphingosine, a cell-permeable short-chain ceramide analogue or daunorubicin that could increase intracellular ceramide via activation of ceramide synthase or sphingomyelin hydrolysis increased the release of t-PA and conversely decreased the PAI-1 release. Interestingly, treatment of the astrocytes with tumor necrosis factor (TNF)-alpha also increased the intracellular ceramide levels but caused the elevation of PAI-1 release without altering the t-PA release. These data suggest that the generation of ceramide in astrocytes is linked at least with the regulation of PAI-1 release. We also demonstrate that the suppression of PAI-1 release with daunorubicin accelerates the cell death of neuronally differentiated PC12 cells and suggest an antiapoptotic role of PAI-1 in the nervous system.

  8. Active intracellular transport in metastatic cells studied by spatial light interference microscopy

    NASA Astrophysics Data System (ADS)

    Ceballos, Silvia; Kandel, Mikhail; Sridharan, Shamira; Majeed, Hassaan; Monroy, Freddy; Popescu, Gabriel

    2015-11-01

    Spatiotemporal patterns of intracellular transport are very difficult to quantify and, consequently, continue to be insufficiently understood. While it is well documented that mass trafficking inside living cells consists of both random and deterministic motions, quantitative data over broad spatiotemporal scales are lacking. We studied the intracellular transport in live cells using spatial light interference microscopy, a high spatiotemporal resolution quantitative phase imaging tool. The results indicate that in the cytoplasm, the intracellular transport is mainly active (directed, deterministic), while inside the nucleus it is both active and passive (diffusive, random). Furthermore, we studied the behavior of the two-dimensional mass density over 30 h in HeLa cells and focused on the active component. We determined the standard deviation of the velocity distribution at the point of cell division for each cell and compared the standard deviation velocity inside the cytoplasm and the nucleus. We found that the velocity distribution in the cytoplasm is consistently broader than in the nucleus, suggesting mechanisms for faster transport in the cytosol versus the nucleus. Future studies will focus on improving phase measurements by applying a fluorescent tag to understand how particular proteins are transported inside the cell.

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

    PubMed

    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

  10. Active intracellular transport in metastatic cells studied by spatial light interference microscopy.

    PubMed

    Ceballos, Silvia; Kandel, Mikhail; Sridharan, Shamira; Majeed, Hassaan; Monroy, Freddy; Popescu, Gabriel

    2015-01-01

    Spatiotemporal patterns of intracellular transport are very difficult to quantify and, consequently, continue to be insufficiently understood. While it is well documented that mass trafficking inside living cells consists of both random and deterministic motions, quantitative data over broad spatiotemporal scales are lacking. We studied the intracellular transport in live cells using spatial light interference microscopy, a high spatiotemporal resolution quantitative phase imaging tool. The results indicate that in the cytoplasm, the intracellular transport is mainly active (directed, deterministic), while inside the nucleus it is both active and passive (diffusive, random). Furthermore, we studied the behavior of the two-dimensional mass density over 30 h in HeLa cells and focused on the active component. We determined the standard deviation of the velocity distribution at the point of cell division for each cell and compared the standard deviation velocity inside the cytoplasm and the nucleus. We found that the velocity distribution in the cytoplasm is consistently broader than in the nucleus, suggesting mechanisms for faster transport in the cytosol versus the nucleus. Future studies will focus on improving phase measurements by applying a fluorescent tag to understand how particular proteins are transported inside the cell.

  11. Activity Dependent Signal Transduction in Skeletal Muscle

    NASA Technical Reports Server (NTRS)

    Hamilton, Susan L.

    1999-01-01

    The overall goals of this project are: 1) to define the initial signal transduction events whereby the removal of gravitational load from antigravity muscles, such as the soleus, triggers muscle atrophy, and 2) to develop countermeasures to prevent this from happening. Our rationale for this approach is that, if countermeasures can be developed to regulate these early events, we could avoid having to deal with the multiple cascades of events that occur downstream from the initial event. One of our major findings is that hind limb suspension causes an early and sustained increase in intracellular Ca(2+) concentration ([Ca (2+)](sub i)). In most cells the consequences of changes in ([Ca (2+)](sub i))depend on the amplitude, frequency and duration of the Ca(2+) signal and on other factors in the intracellular environment. We propose that muscle remodeling in microgravity represents a change in the balance among several CA(2+) regulated signal transduction pathways, in particular those involving the transcription factors NFAT and NFkB and the pro-apoptotic protein BAD. Other Ca(2+) sensitive pathways involving PKC, ras, rac, and CaM kinase II may also contribute to muscle remodeling.

  12. Microspectroscopic imaging tracks the intracellular processing of a signal transduction protein: fluorescent-labeled protein kinase C beta I.

    PubMed Central

    Bastiaens, P I; Jovin, T M

    1996-01-01

    We have devised a microspectroscopic strategy for assessing the intracellular (re)distribution and the integrity of the primary structure of proteins involved in signal transduction. The purified proteins are fluorescent-labeled in vitro and reintroduced into the living cell. The localization and molecular state of fluorescent-labeled protein kinase C beta I isozyme were assessed by a combination of quantitative confocal laser scanning microscopy, fluorescence lifetime imaging microscopy, and novel determinations of fluorescence resonance energy transfer based on photobleaching digital imaging microscopy. The intensity and fluorescence resonance energy transfer efficiency images demonstrate the rapid nuclear translocation and ensuing fragmentation of protein kinase C beta I in BALB/c3T3 fibroblasts upon phorbol ester stimulation, and suggest distinct, compartmentalized roles for the regulatory and catalytic fragments. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8710884

  13. Localization of the Intracellular Activity Domain of Pasteurella multocida Toxin to the N Terminus

    PubMed Central

    Wilson, Brenda A.; Ponferrada, Virgilio G.; Vallance, Jefferson E.; Ho, Mengfei

    1999-01-01

    We have shown that Pasteurella multocida toxin (PMT) directly causes transient activation of Gqα protein that is coupled to phosphatidylinositol-specific phospholipase Cβ1 in Xenopus oocytes (B. A. Wilson, X. Zhu, M. Ho, and L. Lu, J. Biol. Chem. 272:1268–1275, 1997). We found that antibodies directed against an N-terminal peptide of PMT inhibited the toxin-induced response in Xenopus oocytes, but antibodies against a C-terminal peptide did not. To test whether the intracellular activity domain of PMT is localized to the N terminus, we conducted a deletion mutational analysis of the PMT protein, using the Xenopus oocyte system as a means of screening for toxin activity. Using PCR and conventional cloning techniques, we cloned from a toxinogenic strain of P. multocida the entire toxA gene, encoding the 1,285-amino-acid PMT protein, and expressed the recombinant toxin as a His-tagged fusion protein in Escherichia coli. We subsequently generated a series of N-terminal and C-terminal deletion mutants and expressed the His-tagged PMT fragments in E. coli. These proteins were screened for cytotoxic activity on cultured Vero cells and for intracellular activity in the Xenopus oocyte system. Only the full-length protein without the His tag exhibited activity on Vero cells. The full-length PMT and N-terminal fragments containing the first 500 residues elicited responses in oocytes, but the C-terminal 780 amino acid fragment did not. Our results confirm that the intracellular activity domain of PMT is localized to the N-terminal 500 amino acids of the protein and that the C terminus is required for entry into cells. PMID:9864199

  14. Activation of intracellular serine proteinase in Bacillus subtilis cells during sporulation.

    PubMed Central

    Burnett, T J; Shankweiler, G W; Hageman, J H

    1986-01-01

    Cells of Bacillus subtilis 168 (trpC2) growing and sporulating in a single chemically defined medium carried out intracellular protein degradation and increased their levels of intracellular serine protease-1 in a manner very similar to what had previously been reported for cells sporulating in nutrient broth. The results were interpreted to mean that these processes are intrinsic to sporulation rather than medium dependent. To determine the cause of these increases in specific activity of proteinases, we purified the protease, prepared rabbit immunoglobulins directed against it, and monitored changes in protease antigen levels by performing rocket immunoelectrophoresis. In cells sporulating in nutrient broth, the protease antigen levels increased about 7-fold, whereas the specific activity increased about 150-fold, for an activation of about 20-fold. In cells sporulating in the single chemically defined sporulation medium, the protease antigen increased about 10-fold, whereas the specific activity increased at least 400-fold, for an activation of about 40-fold. These results were interpreted to mean that a posttranslational event activated the protease in vivo; a previously described endogenous proteinase inhibitor was confirmed to be present in the strain used. Chloramphenicol added to the cultures inhibited both the increases in antigen levels and in the specific activity of the proteinase. PMID:3079745

  15. Calcium-dependent intracellular signal pathways in primary cultured adipocytes and ANK3 gene variation in patients with bipolar disorder and healthy controls.

    PubMed

    Hayashi, A; Le Gal, K; Södersten, K; Vizlin-Hodzic, D; Ågren, H; Funa, K

    2015-08-01

    Bipolar disorder (BD) is a chronic psychiatric disorder of public health importance affecting >1% of the Swedish population. Despite progress, patients still suffer from chronic mood switches with potential severe consequences. Thus, early detection, diagnosis and initiation of correct treatment are critical. Cultured adipocytes from 35 patients with BD and 38 healthy controls were analysed using signal pathway reporter assays, that is, protein kinase C (PKC), protein kinase A (PKA), mitogen-activated protein kinases (extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK)), Myc, Wnt and p53. The levels of activated target transcriptional factors were measured in adipocytes before and after stimulation with lithium and escitalopram. Variations were analysed in the loci of 25 different single-nucleotide polymorphisms (SNPs). Activation of intracellular signals in several pathways analysed were significantly higher in patients than in healthy controls upon drug stimulation, especially with escitalopram stimulation of PKC, JNK and Myc, as well as lithium-stimulated PKC, whereas no meaningful difference was observed before stimulation. Univariate analyses of contingency tables for 80 categorical SNP results versus diagnoses showed a significant link with the ANK3 gene (rs10761482; likelihood ratio χ(2)=4.63; P=0.031). In a multivariate ordinal logistic fit for diagnosis, a backward stepwise procedure selected ANK3 as the remaining significant predictor. Comparison of the escitalopram-stimulated PKC activity and the ANK3 genotype showed them to add their share of the diagnostic variance, with no interaction (15% of variance explained, P<0.002). The study is cross-sectional with no longitudinal follow-up. Cohorts are relatively small with no medication-free patients, and there are no 'ill patient' controls. It takes 3 to 4 weeks of culture to expand adipocytes that may change epigenetic profiles but remove the possibility of medication effects

  16. Screening of new antileukemic agents from essential oils of algae extracts and computational modeling of their interactions with intracellular signaling nodes.

    PubMed

    Atasever-Arslan, Belkis; Yilancioglu, Kaan; Kalkan, Zeynep; Timucin, Ahmet Can; Gür, Hazal; Isik, Fatma Busra; Deniz, Emre; Erman, Batu; Cetiner, Selim

    2016-02-15

    Microalgae are very rich in bioactive compounds, minerals, polysaccharides, poly-unsaturated fatty acids and vitamins, and these rich constituents make microalgae an important resource for the discovery of new bioactive compounds with applications in biotechnology. In this study, we studied the antileukemic activity of several chosen microalgae species at the molecular level and assessed their potential for drug development. Here we identified Stichococcus bacillaris, Phaeodactylum tricornutum, Microcystis aeruginosa and Nannochloropsis oculata microalgae extracts with possible antileukemic agent potentials. Specifically we studied the effects of these extracts on intracellular signal nodes and apoptotic pathways. We characterized the composition of essential oils of these fifteen different algae extracts using gas chromatography-mass spectrometry (GC-MS). Finally, to identify potential molecular targets causing the phenotypic changes in leukemic cell lines, we docked a selected group of these essential oils to several key intracellular proteins. According to results of rank score algorithm, five of these essential oils analyzed might be considered as in silico plausible candidates to be used as antileukemic agents. PMID:26709080

  17. Intracellular chromium reduction.

    PubMed

    Arslan, P; Beltrame, M; Tomasi, A

    1987-10-22

    Two steps are involved in the uptake of Cr(VI): (1) the diffusion of the anion CrO4(2-) through a facilitated transport system, presumably the non-specific anion carrier and (2) the intracellular reduction of Cr(VI) to Cr(III). The intracellular reduction of Cr(VI), keeping the cytoplasmic concentration of Cr(VI) low, facilitates accumulation of chromate from extracellular medium into the cell. In the present paper, a direct demonstration of intracellular chromium reduction is provided by means of electron paramagnetic (spin) resonance (EPR) spectroscopy. Incubation of metabolically active rat thymocytes with chromate originates a signal which can be attributed to a paramagnetic species of chromium, Cr(V) or Cr(III). The EPR signal is originated by intracellular reduction of chromium since: (1) it is observed only when cells are incubated with chromate, (2) it is present even after extensive washings of the cells in a chromium-free medium; (3) it is abolished when cells are incubated with drugs able to reduce the glutathione pool, i.e., diethylmaleate or phorone; and (4) it is abolished when cells are incubated in the presence of a specific inhibitor of the anion carrier, 4-acetamido-4'-isothiocyanatostilbene-2-2'-disulfonic acid. PMID:2820507

  18. Intracellular ATP Decrease Mediates NLRP3 Inflammasome Activation upon Nigericin and Crystal Stimulation.

    PubMed

    Nomura, Johji; So, Alexander; Tamura, Mizuho; Busso, Nathalie

    2015-12-15

    Activation of the nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome initiates an inflammatory response, which is associated with host defense against pathogens and the progression of chronic inflammatory diseases such as gout and atherosclerosis. The NLRP3 inflammasome mediates caspase-1 activation and subsequent IL-1β processing in response to various stimuli, including extracellular ATP, although the roles of intracellular ATP (iATP) in NLRP3 activation remain unclear. In this study, we found that in activated macrophages artificial reduction of iATP by 2-deoxyglucose, a glycolysis inhibitor, caused mitochondrial membrane depolarization, leading to IL-1β secretion via NLRP3 and caspase-1 activation. Additionally, the NLRP3 activators nigericin and monosodium urate crystals lowered iATP through K(+)- and Ca(2+)-mediated mitochondrial dysfunction, suggesting a feedback loop between iATP loss and lowering of mitochondrial membrane potential. These results demonstrate the fundamental roles of iATP in the maintenance of mitochondrial function and regulation of IL-1β secretion, and they suggest that maintenance of the intracellular ATP pools could be a strategy for countering NLRP3-mediated inflammation. PMID:26546608

  19. Fluorescent protein pair emit intracellular FRET signal suitable for FACS screening

    SciTech Connect

    Johansson, Daniel X.; Brismar, Hjalmar . E-mail: mats.persson@ki.se

    2007-01-12

    The fluorescent proteins ECFP and HcRed were shown to give an easily resolved FRET-signal when expressed as a fusion inside mammalian cells. HeLa-tat cells expressing ECFP, pHcRed, or the fusion protein pHcRed-ECFP were analyzed by flow cytometry after excitation of ECFP. Cells expressing HcRed-ECFP, or ECFP and HcRed, were mixed and FACS-sorted for FRET positive cells: HcRed-ECFP cells were greatly enriched (72 times). Next, cloned human antibodies were fused with ECFP and expressed anchored to the ER membrane. Their cognate antigens (HIV-1 gp120 or gp41) were fused to HcRed and co-expressed in the ER. An increase of 13.5 {+-} 1.5% (mean {+-} SEM) and 8.0 {+-} 0.7% in ECFP fluorescence for the specific antibodies reacting with gp120 or gp41, respectively, was noted after photobleaching. A positive control (HcRed-ECFP) gave a 14.8 {+-} 2.6% increase. Surprisingly, the unspecific antibody (anti-TT) showed 12.1 {+-} 1.1% increase, possibly because overexpression in the limited ER compartment gave false FRET signals.

  20. Crosstalk between intracellular and extracellular signals regulating interneuron production, migration and integration into the cortex

    PubMed Central

    Peyre, Elise; Silva, Carla G.; Nguyen, Laurent

    2015-01-01

    During embryogenesis, cortical interneurons are generated by ventral progenitors located in the ganglionic eminences of the telencephalon. They travel along multiple tangential paths to populate the cortical wall. As they reach this structure they undergo intracortical dispersion to settle in their final destination. At the cellular level, migrating interneurons are highly polarized cells that extend and retract processes using dynamic remodeling of microtubule and actin cytoskeleton. Different levels of molecular regulation contribute to interneuron migration. These include: (1) Extrinsic guidance cues distributed along migratory streams that are sensed and integrated by migrating interneurons; (2) Intrinsic genetic programs driven by specific transcription factors that grant specification and set the timing of migration for different subtypes of interneurons; (3) Adhesion molecules and cytoskeletal elements/regulators that transduce molecular signalings into coherent movement. These levels of molecular regulation must be properly integrated by interneurons to allow their migration in the cortex. The aim of this review is to summarize our current knowledge of the interplay between microenvironmental signals and cell autonomous programs that drive cortical interneuron porduction, tangential migration, and intergration in the developing cerebral cortex. PMID:25926769

  1. The Pseudomonas aeruginosa Chp Chemosensory System Regulates Intracellular cAMP Levels by Modulating Adenylate Cyclase Activity

    PubMed Central

    Fulcher, Nanette B.; Holliday, Phillip M.; Klem, Erich; Cann, Martin J.; Wolfgang, Matthew C.

    2010-01-01

    Summary Multiple virulence systems in the opportunistic pathogen Pseudomonas aeruginosa are regulated by the second messenger signaling molecule adenosine 3’, 5’-cyclic monophosphate (cAMP). Production of cAMP by the putative adenylate cyclase enzyme CyaB represents a critical control point for virulence gene regulation. To identify regulators of CyaB, we screened a transposon insertion library for mutants with reduced intracellular cAMP. The majority of insertions resulting in reduced cAMP mapped to the Chp gene cluster encoding a putative chemotaxis-like chemosensory system. Further genetic analysis of the Chp system revealed that it has both positive and negative effects on intracellular cAMP and that it regulates cAMP levels by modulating CyaB activity. The Chp system was previously implicated in the production and function of type IV pili (TFP). Given that cAMP and the cAMP-dependent transcriptional regulator Vfr control TFP biogenesis gene expression, we explored the relationship between cAMP, the Chp system and TFP regulation. We discovered that the Chp system controls TFP production through modulation of cAMP while control of TFP-dependent twitching motility is cAMP-independent. Overall, our data define a novel function for a chemotaxis-like system in controlling cAMP production and establish a regulatory link between the Chp system, TFP and other cAMP-dependent virulence systems. PMID:20345659

  2. Superoxide dismutase activity of Mycobacterium avium, M. intracellulare, and M. scrofulaceum.

    PubMed Central

    Mayer, B K; Falkinham, J O

    1986-01-01

    Superoxide dismutase (EC 1.15.1.1) (SOD) activity has been detected in crude cell extracts of representative strains of the Mycobacterium avium, M. intracellulare, and M. scrofulaceum (MAIS) group. Polyacrylamide gel electrophoresis demonstrated a single SOD activity band for each of the MAIS strains, though there were differences in mobility. All M. avium and M. intracellulare and two of five M. scrofulaceum strains demonstrated a single activity band of identical mobility (Rf = 0.83), while the SOD activity band for the three remaining M. scrofulaceum strains migrated farther (Rf = 0.85). The differences in mobility correlated with differences in sensitivity to NaN3 and H2O2. The SOD activities of the majority of the MAIS strains which displayed the slower-migrating activity band were inhibited 22 to 81% after 15 min of exposure to 5 mM H2O2, suggesting that both iron and manganese may be present in a single enzyme. The SOD activities of the three M. scrofulaceum strains which had the faster-migrating activity band were inhibited 100% after only 5 min of exposure to 5 mM H2O2 and exhibited greater sensitivity to 5 and 10 mM NaN3, characteristics of an iron-containing SOD. A concentration of 1 mM KCN did not cause inhibition of enzyme activity in any of the MAIS strains tested. Extracellular SOD activity was detected in four of six MAIS strains and was shown to be identical in mobility to the SOD activity of the crude extracts. Images PMID:3744555

  3. The satiety signaling neuropeptide perisulfakinin inhibits the activity of central neurons promoting general activity.

    PubMed

    Wicher, Dieter; Derst, Christian; Gautier, Hélène; Lapied, Bruno; Heinemann, Stefan H; Agricola, Hans-Jürgen

    2007-01-01

    The metabolic state is one of the determinants of the general activity level. Satiety is related to resting or sleep whereas hunger correlates to wakefulness and activity. The counterpart to the mammalian satiety signal cholecystokinin (CCK) in insects are the sulfakinins. The aim of this study was to resolve the mechanism by which the antifeedant activity of perisulfakinin (PSK) in Periplaneta americana is mediated. We identified the sources of PSK which is used both as hormone and as paracrine messenger. PSK is found in the neurohemal organ of the brain and in nerve endings throughout the central nervous system. To correlate the distributions of PSK and its receptor (PSKR), we cloned the gene coding for PSKR and provide evidence for its expression within the nervous system. It occurs only in a few neurons, among them are the dorsal unpaired median (DUM) neurons which release octopamine thereby regulating the general level of activity. Application of PSK to DUM neurons attenuated the spiking frequency (EC(50)=11pM) due to reduction of a pacemaker Ca(2+) current through cAMP-inhibited pTRPgamma channels. PSK increased the intracellular cAMP level while decreasing the intracellular Ca(2+) concentration in DUM neurons. Thus, the satiety signal conferred by PSK acts antagonistically to the hunger signal, provided by the adipokinetic hormone (AKH): PSK depresses the electrical activity of DUM neurons by inhibiting the pTRPgamma channel that is activated by AKH under conditions of food shortage. PMID:18946521

  4. The Satiety Signaling Neuropeptide Perisulfakinin Inhibits the Activity of Central Neurons Promoting General Activity

    PubMed Central

    Wicher, Dieter; Derst, Christian; Gautier, Hélène; Lapied, Bruno; Heinemann, Stefan H.; Agricola, Hans-Jürgen

    2007-01-01

    The metabolic state is one of the determinants of the general activity level. Satiety is related to resting or sleep whereas hunger correlates to wakefulness and activity. The counterpart to the mammalian satiety signal cholecystokinin (CCK) in insects are the sulfakinins. The aim of this study was to resolve the mechanism by which the antifeedant activity of perisulfakinin (PSK) in Periplaneta americana is mediated. We identified the sources of PSK which is used both as hormone and as paracrine messenger. PSK is found in the neurohemal organ of the brain and in nerve endings throughout the central nervous system. To correlate the distributions of PSK and its receptor (PSKR), we cloned the gene coding for PSKR and provide evidence for its expression within the nervous system. It occurs only in a few neurons, among them are the dorsal unpaired median (DUM) neurons which release octopamine thereby regulating the general level of activity. Application of PSK to DUM neurons attenuated the spiking frequency (EC50=11pM) due to reduction of a pacemaker Ca2+ current through cAMP-inhibited pTRPγ channels. PSK increased the intracellular cAMP level while decreasing the intracellular Ca2+ concentration in DUM neurons. Thus, the satiety signal conferred by PSK acts antagonistically to the hunger signal, provided by the adipokinetic hormone (AKH): PSK depresses the electrical activity of DUM neurons by inhibiting the pTRPγ channel that is activated by AKH under conditions of food shortage. PMID:18946521

  5. Glucose enhances insulin promoter activity in MIN6 beta-cells independently of changes in intracellular Ca2+ concentration and insulin secretion.

    PubMed Central

    Kennedy, H J; Rafiq, I; Pouli, A E; Rutter, G A

    1999-01-01

    Recent studies have suggested that glucose may activate insulin gene transcription through increases in intracellular Ca(2+) concentration, possibly acting via the release of stored insulin. We have investigated this question by dynamic photon-counting imaging of insulin- and c-fos-promoter-firefly luciferase reporter construct activity. Normalized to constitutive viral promoter activity, insulin promoter activity in MIN6 beta-cells was increased 1.6-fold after incubation at 30 mM compared with 3 mM glucose, but was unaltered at either glucose concentration by the presence of insulin (100 nM) or the Ca(2+) channel inhibitor, verapamil (100 microM). Increases in intracellular [Ca(2+)] achieved by plasma membrane depolarization with KCl failed to enhance either insulin or c-fos promoter activity in MIN6 cells, but increased c-fos promoter activity 5-fold in AtT20 cells. Together, these results demonstrate that glucose can exert a direct effect on insulin promoter activity in islet beta-cells, via a signalling pathway which does not require increases in intracellular [Ca(2+)] nor insulin release and insulin receptor activation. PMID:10455011

  6. Anti-infective Activity of 2-Cyano-3-Acrylamide Inhibitors with Improved Drug-Like Properties against Two Intracellular Pathogens.

    PubMed

    Passalacqua, Karla D; Charbonneau, Marie-Eve; Donato, Nicholas J; Showalter, Hollis D; Sun, Duxin; Wen, Bo; He, Miao; Sun, Hanshi; O'Riordan, Mary X D; Wobus, Christiane E

    2016-07-01

    Due to the rise of antibiotic resistance and the small number of effective antiviral drugs, new approaches for treating infectious diseases are urgently needed. Identifying targets for host-based therapies represents an emerging strategy for drug discovery. The ubiquitin-proteasome system is a central mode of signaling in the eukaryotic cell and may be a promising target for therapies that bolster the host's ability to control infection. Deubiquitinase (DUB) enzymes are key regulators of the host inflammatory response, and we previously demonstrated that a selective DUB inhibitor and its derivative promote anti-infective activities in host cells. To find compounds with anti-infective efficacy but improved toxicity profiles, we tested a library of predominantly 2-cyano-3-acrylamide small-molecule DUB inhibitors for anti-infective activity in macrophages against two intracellular pathogens: murine norovirus (MNV) and Listeria monocytogenes We identified compound C6, which inhibited DUB activity in human and murine cells and reduced intracellular replication of both pathogens with minimal toxicity in cell culture. Treatment with C6 did not significantly affect the ability of macrophages to internalize virus, suggesting that the anti-infective activity interferes with postentry stages of the MNV life cycle. Metabolic stability and pharmacokinetic assays showed that C6 has a half-life in mouse liver microsomes of ∼20 min and has a half-life of approximately 4 h in mice when administered intravenously. Our results provide a framework for targeting the host ubiquitin system in the development of host-based therapies for infectious disease. Compound C6 represents a promising tool with which to elucidate the role of DUBs in the macrophage response to infection. PMID:27139470

  7. [The regulation of the activation in BCR signaling by ZIP9 transporter].

    PubMed

    Taniguchi, Masanari; Enomoto, Shuichi; Hiromura, Makoto

    2014-01-01

    Zinc is the essential trace element and important for all living organisms. Zinc functions not only as a nutritional factor, but also as a second messenger. However, the effects of intracellular zinc on the B cell-receptor (BCR) signaling pathway are not poorly understood. Here, we indicate that the ZIP9 induces increase in intracellular zinc level and plays an important role in the phosphorylation of Akt and Erk in response to BCR activation. In DT40 cells, the enhancement of Akt and Erk phosphorylation requires intracellular zinc. To clarify this event, we used chicken Zip9-knockout DT40 (cZip9KO) cells. The levels of Akt and Erk phosphorylation significantly decreased in cZip9KO cells treated with zinc pyrithione (ZnPy), and overexpressing the human Zip9 gene restored these biochemical events. Moreover, we found that the increase in intracellular zinc level depends on the expression of ZIP9. Additionally, intracellular zinc was localized at the Golgi, even if it was treated with ZnPy in cZip9KO cells. We concluded that ZIP9 regulates cytosolic zinc level, resulting in the enhancement of Akt and Erk phosphorylation. Our observations provide new mechanistic insights into the BCR signaling pathway underlying the regulation of intracellular zinc level by ZIP9 in response to the BCR activation. PMID:24989471

  8. Aroclor 1254, a developmental neurotoxicant, alters energy metabolism- and intracellular signaling-associated protein networks in rat cerebellum and hippocampus

    SciTech Connect

    Kodavanti, Prasada Rao S.; Osorio, Cristina; Royland, Joyce E.; Ramabhadran, Ram; Alzate, Oscar

    2011-11-15

    The vast literature on the mode of action of polychlorinated biphenyls (PCBs) indicates that PCBs are a unique model for understanding the mechanisms of toxicity of environmental mixtures of persistent chemicals. PCBs have been shown to adversely affect psychomotor function and learning and memory in humans. Although the molecular mechanisms for PCB effects are unclear, several studies indicate that the disruption of Ca{sup 2+}-mediated signal transduction plays significant roles in PCB-induced developmental neurotoxicity. Culminating events in signal transduction pathways include the regulation of gene and protein expression, which affects the growth and function of the nervous system. Our previous studies showed changes in gene expression related to signal transduction and neuronal growth. In this study, protein expression following developmental exposure to PCB is examined. Pregnant rats (Long Evans) were dosed with 0.0 or 6.0 mg/kg/day of Aroclor-1254 from gestation day 6 through postnatal day (PND) 21, and the cerebellum and hippocampus from PND14 animals were analyzed to determine Aroclor 1254-induced differential protein expression. Two proteins were found to be differentially expressed in the cerebellum following PCB exposure while 18 proteins were differentially expressed in the hippocampus. These proteins are related to energy metabolism in mitochondria (ATP synthase, sub unit {beta} (ATP5B), creatine kinase, and malate dehydrogenase), calcium signaling (voltage-dependent anion-selective channel protein 1 (VDAC1) and ryanodine receptor type II (RyR2)), and growth of the nervous system (dihydropyrimidinase-related protein 4 (DPYSL4), valosin-containing protein (VCP)). Results suggest that Aroclor 1254-like persistent chemicals may alter energy metabolism and intracellular signaling, which might result in developmental neurotoxicity. -- Highlights: Black-Right-Pointing-Pointer We performed brain proteomic analysis of rats exposed to the neurotoxicant

  9. AMP-activated Protein Kinase Suppresses Biosynthesis of Glucosylceramide by Reducing Intracellular Sugar Nucleotides*

    PubMed Central

    Ishibashi, Yohei; Hirabayashi, Yoshio

    2015-01-01

    The membrane glycolipid glucosylceramide (GlcCer) plays a critical role in cellular homeostasis. Its intracellular levels are thought to be tightly regulated. How cells regulate GlcCer levels remains to be clarified. AMP-activated protein kinase (AMPK), which is a crucial cellular energy sensor, regulates glucose and lipid metabolism to maintain energy homeostasis. Here, we investigated whether AMPK affects GlcCer metabolism. AMPK activators (5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside and metformin) decreased intracellular GlcCer levels and synthase activity in mouse fibroblasts. AMPK inhibitors or AMPK siRNA reversed these effects, suggesting that GlcCer synthesis is negatively regulated by an AMPK-dependent mechanism. Although AMPK did not affect the phosphorylation or expression of GlcCer synthase, the amount of UDP-glucose, an activated form of glucose required for GlcCer synthesis, decreased under AMPK-activating conditions. Importantly, the UDP-glucose pyrophosphatase Nudt14, which degrades UDP-glucose, generating UMP and glucose 1-phosphate, was phosphorylated and activated by AMPK. On the other hand, suppression of Nudt14 by siRNA had little effect on UDP-glucose levels, indicating that mammalian cells have an alternative UDP-glucose pyrophosphatase that mainly contributes to the reduction of UDP-glucose under AMPK-activating conditions. Because AMPK activators are capable of reducing GlcCer levels in cells from Gaucher disease patients, our findings suggest that reducing GlcCer through AMPK activation may lead to a new strategy for treating diseases caused by abnormal accumulation of GlcCer. PMID:26048992

  10. Nicotine enhances murine airway contractile responses to kinin receptor agonists via activation of JNK- and PDE4-related intracellular pathways

    PubMed Central

    2010-01-01

    Background Nicotine plays an important role in cigarette-smoke-associated airway disease. The present study was designed to examine if nicotine could induce airway hyperresponsiveness through kinin receptors, and if so, explore the underlying mechanisms involved. Methods Murine tracheal segments were cultured for 1, 2 or 4 days in serum-free DMEM medium in presence of nicotine (1 and 10 μM) or vehicle (DMSO). Contractile responses induced by kinin B1 receptor agonist, des-Arg9-bradykinin, and B2 receptor agonist, bradykinin, were monitored with myographs. The B1 and B2 receptor mRNA expressions were semi-quantified using real-time PCR and their corresponding protein expressions assessed with confocal-microscopy-based immunohistochemistry. Various pharmacological inhibitors were used for studying intracellular signaling pathways. Results Four days of organ culture with nicotine concentration-dependently increased kinin B1 and B2 receptor-mediated airway contractions, without altering the kinin receptor-mediated relaxations. No such increase was seen at day 1 or day 2. The airway contractile responses to 5-HT, acetylcholine and endothelin receptor agonists remained unaffected by nicotine. Two different neuronal nicotinic receptor antagonists MG624 and hexamethonium blocked the nicotine-induced effects. The enhanced contractile responses were accompanied by increased mRNA and protein expression for both kinin receptors, suggesting the involvement of transcriptional mechanisms. Confocal-microscopy-based immunohistochemistry showed that 4 days of nicotine treatment induced activation (phosphorylation) of c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase 1 and 2 (ERK1/2) and p38. Inhibition of JNK with its specific inhibitor SP600125 abolished the nicotine-induced effects on kinin receptor-mediated contractions and reverted the enhanced receptor mRNA expression. Administration of phosphodiesterase inhibitors (YM976 and theophylline

  11. Characteristics of intracellular Ca2+ signals consisting of two successive peaks in hepatocytes during liver regeneration after 70% partial hepatectomy in rats

    PubMed Central

    Taira, Zenei; Ueda, Yukari; Monmasu, Hiroshi; Yamase, Daisuke; Miyake, Sayaka; Shiraishi, Maya

    2016-01-01

    Two specific signals for regulating liver regeneration were found after 70% partial hepatectomy (PH) in rats. The first finding was a sustained increasing signal of intracellular Ca2+ ([Ca2+]i) in hepatocytes, consisting of two successive peaks with the first narrow peak at 1 hour and the second broad peak increasing by day 3 and then returning to normal by day 4. The second finding was an abnormal peak in the restoring ratio (Rr) curve of liver regeneration after 70% PH at day 4, where the Rr exceeded 100% temporarily, returned to a lower level, and then proceeded to a termination phase of liver regeneration. For 4 days around the two successive [Ca2+]i peaks and abnormal peak, various physiological activities were induced to promote liver regeneration after 70% PH. mRNA expression of genes encoding Ca2+-binding proteins S100A4 and calpain was induced between the two Ca2+ peaks. Hepatocytes underwent synchronous cell proliferation as the liver was restored from 30% to 70% at day 4, and significant expression of VEGF mRNA at around day 4 promoted angiogenesis to remodel the sinusoidal system. Cytochrome P450 activity levels in microsomes and alanine aminotransferase values at 24 hours after CCl4 administration were decreased after 70% PH, which recovered transiently to the control level at day 4, returned to the decreased level, and then slowly recovered by day 10. Thus, these results indicate that day 4 is important during liver regeneration after 70% PH. PMID:27757054

  12. The Peptide Microarray-Based Resonance Light Scattering Assay for Sensitively Detecting Intracellular Kinase Activity.

    PubMed

    Li, Tao; Liu, Xia; Liu, Dianjun; Wang, Zhenxin

    2016-01-01

    The peptide microarray technology is a robust, reliable, and efficient technique for large-scale determination of enzyme activities, and high-throughput profiling of substrate/inhibitor specificities of enzymes. Here, the activities of cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) in different cell lysates have been detected by a peptide microarray-based resonance light scattering (RLS) assay with gold nanoparticle (GNP) probes. Highly sensitive detection of PKA activity in 0.1 μg total cell proteins of SHG-44 (human glioma cell) cell lysate (corresponding to 200 cells) is achieved by a selected peptide substrate. The experimental results also demonstrate that the RLS assay can be employed to evaluate the chemical regulation of intracellular kinase activity. PMID:26490469

  13. Biosynthesis of B2-integrin, intracellular calcium signalling and functional responses of normal and CD18-deficient bovine neutrophils.

    PubMed

    Nagahata, H; Higuchi, H; Nochi, H; Tamoto, K; Araiso, T; Noda, H; Kociba, G J

    1996-09-01

    1Biosynthesis of CD11/CD18 in bovine leucocytes, intracellular Ca2+ ([Ca2+]i) signalling, chemiluminescent responses and membrane fluidity of neutrophils and the effects of D-mannose on neutrophils from control heifers and a heifer with bovine leucocyte adhesion deficiency (BLAD) were measured. The synthesis of CD11/CD18 complex was clearly detected in leucocytes from a normal heifer, but not in a BLAD-affected heifer. The transient phase of increased [Ca2+]i was clearly detected in neutrophils from a heifer with BLAD stimulated with opsonised zymosan, aggregated bovine immunoglobulin G or concanavalin A, whereas the sustained phase was deficient or significantly decreased compared with control heifers. [Ca2+]i signalling of neutrophils from control heifers and a heifer with BLAD stimulated with phorbol myristate acetate via an 11b/CD18-independent pathway showed no transient phase, and the subsequent increase in [Ca2+]i was almost identical in neutrophils from affected and control heifers. [Ca2+]i concentration and chemiluminescent responses of neutrophils from a control heifer were clearly decreased by treatment with anti-CD18 and anti-IgG antibodies. No differences in membrane fluidity were detected between neutrophils derived from control and CD18-deficient cattle. D-mannose binds mainly to Fc rather than CD18 receptors, and decreased Agg-IgG induced [Ca2+]i and the chemiluminescent response of neutrophils. The [Ca2+]i responses and Agg-IgG induced chemiluminescent responses of neutrophils from control heifers and a BLAD-affected heifer were inhibited by D-mannose. The characteristic changes of [Ca2+]i signalling and functional responses of B2-integrin-deficient neutrophils were demonstrated. PMID:8880976

  14. Delineation of Diverse Macrophage Activation Programs in Response to Intracellular Parasites and Cytokines

    PubMed Central

    Zhang, Shuyi; Kim, Charles C.; Batra, Sajeev; McKerrow, James H.; Loke, P'ng

    2010-01-01

    Background The ability to reside and proliferate in macrophages is characteristic of several infectious agents that are of major importance to public health, including the intracellular parasites Trypanosoma cruzi (the etiological agent of Chagas disease) and Leishmania species (etiological agents of Kala-Azar and cutaneous leishmaniasis). Although recent studies have elucidated some of the ways macrophages respond to these pathogens, the relationships between activation programs elicited by these pathogens and the macrophage activation programs elicited by bacterial pathogens and cytokines have not been delineated. Methodology/Principal Findings To provide a global perspective on the relationships between macrophage activation programs and to understand how certain pathogens circumvent them, we used transcriptional profiling by genome-wide microarray analysis to compare the responses of mouse macrophages following exposure to the intracellular parasites T. cruzi and Leishmania mexicana, the bacterial product lipopolysaccharide (LPS), and the cytokines IFNG, TNF, IFNB, IL-4, IL-10, and IL-17. We found that LPS induced a classical activation state that resembled macrophage stimulation by the Th1 cytokines IFNG and TNF. However, infection by the protozoan pathogen L. mexicana produced so few transcriptional changes that the infected macrophages were almost indistinguishable from uninfected cells. T. cruzi activated macrophages produced a transcriptional signature characterized by the induction of interferon-stimulated genes by 24 h post-infection. Despite this delayed IFN response by T. cruzi, the transcriptional response of macrophages infected by the kinetoplastid pathogens more closely resembled the transcriptional response of macrophages stimulated by the cytokines IL-4, IL-10, and IL-17 than macrophages stimulated by Th1 cytokines. Conclusions/Significance This study provides global gene expression data for a diverse set of biologically significant pathogens and

  15. Platelet Activating Factor Enhances Synaptic Vesicle Exocytosis Via PKC, Elevated Intracellular Calcium, and Modulation of Synapsin 1 Dynamics and Phosphorylation

    PubMed Central

    Hammond, Jennetta W.; Lu, Shao-Ming; Gelbard, Harris A.

    2016-01-01

    Platelet activating factor (PAF) is an inflammatory phospholipid signaling molecule implicated in synaptic plasticity, learning and memory and neurotoxicity during neuroinflammation. However, little is known about the intracellular mechanisms mediating PAF’s physiological or pathological effects on synaptic facilitation. We show here that PAF receptors are localized at the synapse. Using fluorescent reporters of presynaptic activity we show that a non-hydrolysable analog of PAF (cPAF) enhances synaptic vesicle release from individual presynaptic boutons by increasing the size or release of the readily releasable pool and the exocytosis rate of the total recycling pool. cPAF also activates previously silent boutons resulting in vesicle release from a larger number of terminals. The underlying mechanism involves elevated calcium within presynaptic boutons and protein kinase C activation. Furthermore, cPAF increases synapsin I phosphorylation at sites 1 and 3, and increases dispersion of synapsin I from the presynaptic compartment during stimulation, freeing synaptic vesicles for subsequent release. These findings provide a conceptual framework for how PAF, regardless of its cellular origin, can modulate synapses during normal and pathologic synaptic activity. PMID:26778968

  16. Smart vesicle kit for in situ monitoring of intracellular telomerase activity using a telomerase-responsive probe.

    PubMed

    Qian, Ruocan; Ding, Lin; Yan, Liwen; Lin, Manfei; Ju, Huangxian

    2014-09-01

    A smart vesicle kit was designed for in situ imaging and detection of cytoplasmic telomerase activity. The vesicle kit contained a telomerase primer (TSP) and a Cy5-tagged molecular beacon (MB) functionalized gold nanoparticle probe, which were encapsulated in liposome for intracellular delivery. After the vesicle kit was transfected into cytoplasm, the released TSP could be extended in the presence of telomerase to produce a telomeric repeated sequence at the 3' end, which was just complementary with the loop of MB assembled on probe surface. Thus, the MB was opened upon hybridization to switch the fluorescent state from "off" to "on". The fluorescence signal depended on telomerase activity, leading to a novel strategy for in situ imaging and quantitative detection of the cytoplasmic telomerase activity. The cytoplasmic telomerase activity was estimated to be 3.2 × 10(-11), 2.4 × 10(-11), and 8.6 × 10(-13) IU in each HeLa, BEL tumor and QSG normal cell, respectively, demonstrating the capability of this approach to distinguish tumor from normal cells. The proposed method could be employed for dynamic monitoring of the cytoplasmic telomerase activity in response to a telomerase-based drug, suggesting the potential application in discovery and screening of telomerase-targeted anticancer drugs.

  17. Intracellular Modulation, Extracellular Disposal and Serum Increase of MiR-150 Mark Lymphocyte Activation

    PubMed Central

    de Candia, Paola; Torri, Anna; Gorletta, Tatiana; Fedeli, Maya; Bulgheroni, Elisabetta; Cheroni, Cristina; Marabita, Francesco; Crosti, Mariacristina; Moro, Monica; Pariani, Elena; Romanò, Luisa; Esposito, Susanna; Mosca, Fabio; Rossetti, Grazisa; Rossi, Riccardo L.; Geginat, Jens; Casorati, Giulia; Dellabona, Paolo; Pagani, Massimiliano; Abrignani, Sergio

    2013-01-01

    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. PMID:24205408

  18. Intracellular pH regulates basolateral K+ and Cl- conductances in colonic epithelial cells by modulating Ca2+ activation

    PubMed Central

    1991-01-01

    The role of intracellular pH as a modulator of basolateral K+ and Cl- conductances in epithelial cells was studied using digitonin- permeabilized colonic cell layers so that cytosolic pH could be clamped at specific values, while basolateral K+ and Cl- conductances were activated by stepwise increases in intracellular free Ca2+. Increasing the intracellular pH from 6.6 to 8.0 enhanced the sensitivity of both ionic conductances to intracellular Ca2+, but changing extracellular pH had no effect. Maximal K+ and Cl- currents activated by Ca2+ were not affected by changes in intracellular pH, suggesting that protons do not alter the conduction properties of the channels. Hill analysis of the Ca2+ activation process revealed that raising the cytosolic pH from 6.6 to 8.0 reduced the K1/2 for Ca2+ activation. In the absence of Ca2+, changes in intracellular pH did not have a significant effect on the basolateral K+ and Cl- conductances. These results are consistent with the notion that changes in cytosolic pH can modulate basolateral conductances by modifying the action of calcium, perhaps by acting at or near the activation site to provide a mechanism of variable "gain control." PMID:1719125

  19. Cleaved CD44 intracellular domain supports activation of stemness factors and promotes tumorigenesis of breast cancer.

    PubMed

    Cho, Yunhee; Lee, Hyun-Woo; Kang, Hyeok-Gu; Kim, Hye-Young; Kim, Seok-Jun; Chun, Kyung-Hee

    2015-04-20

    CD44 plays a role in the progression of tumors and is expressed in cancer stem cells (CSCs). However, the mechanisms underlying the crosstalk of CD44 with stemness genes in CSC maintenance remains unclear. In this study, we demonstrated how the cleaved intracellular domain of CD44 (CD44ICD) activates stemness factors such as Nanog, Sox2 and Oct4, and contributes to the tumorigenesis of breast cancer. We have found that the overexpression of CD44ICD increased mammosphere formation in breast cancer cells. Treatment with a γ-secretase inhibitor (GSI), which blocks the cleavage of CD44ICD, interfered with mammosphere formation. Interestingly, CD44ICD decreased the expression levels and nuclear localization of stemness factors, but overexpression of CD44ICD reversed these effects. In addition, we showed that nuclear localization of CD44ICD is important for transcriptional activation of the stemness factors. Furthermore, CD44ICD-overexpressed cells exhibited strong tumorigenecity and greater metastatic potential than did the control cells or CD44-depleted cells in vivo in mice models. Taken together, it was supposed that CD44 promotes tumorigenesis through the interaction and nuclear-translocation of its intracellular domain and stemness factors. We suggest that the prevention of cleavage and nuclear-translocation of CD44ICD is a potential target in treating breast cancer. PMID:25909162

  20. Intracellular Oxidant Activity, Antioxidant Enzyme Defense System, and Cell Senescence in Fibroblasts with Trisomy 21

    PubMed Central

    Rodríguez-Sureda, Víctor; Vilches, Ángel; Sánchez, Olga; Audí, Laura; Domínguez, Carmen

    2015-01-01

    Down's syndrome (DS) is characterized by a complex phenotype associated with chronic oxidative stress and mitochondrial dysfunction. Overexpression of genes on chromosome-21 is thought to underlie the pathogenesis of the major phenotypic features of DS, such as premature aging. Using cultured fibroblasts with trisomy 21 (T21F), this study aimed to ascertain whether an imbalance exists in activities, mRNA, and protein expression of the antioxidant enzymes SOD1, SOD2, glutathione-peroxidase, and catalase during the cell replication process in vitro. T21F had high SOD1 expression and activity which led to an interenzymatic imbalance in the antioxidant defense system, accentuated with replicative senescence. Intracellular ROS production and oxidized protein levels were significantly higher in T21F compared with control cells; furthermore, a significant decline in intracellular ATP content was detected in T21F. Cell senescence was found to appear prematurely in DS cells as shown by SA-β-Gal assay and p21 assessment, though not apoptosis, as neither p53 nor the proapoptotic proteins cytochrome c and caspase 9 were altered in T21F. These novel findings would point to a deleterious role of oxidatively modified molecules in early cell senescence of T21F, thereby linking replicative and stress-induced senescence in cultured cells to premature aging in DS. PMID:25852816

  1. Intracellular oxidant activity, antioxidant enzyme defense system, and cell senescence in fibroblasts with trisomy 21.

    PubMed

    Rodríguez-Sureda, Víctor; Vilches, Ángel; Sánchez, Olga; Audí, Laura; Domínguez, Carmen

    2015-01-01

    Down's syndrome (DS) is characterized by a complex phenotype associated with chronic oxidative stress and mitochondrial dysfunction. Overexpression of genes on chromosome-21 is thought to underlie the pathogenesis of the major phenotypic features of DS, such as premature aging. Using cultured fibroblasts with trisomy 21 (T21F), this study aimed to ascertain whether an imbalance exists in activities, mRNA, and protein expression of the antioxidant enzymes SOD1, SOD2, glutathione-peroxidase, and catalase during the cell replication process in vitro. T21F had high SOD1 expression and activity which led to an interenzymatic imbalance in the antioxidant defense system, accentuated with replicative senescence. Intracellular ROS production and oxidized protein levels were significantly higher in T21F compared with control cells; furthermore, a significant decline in intracellular ATP content was detected in T21F. Cell senescence was found to appear prematurely in DS cells as shown by SA-β-Gal assay and p21 assessment, though not apoptosis, as neither p53 nor the proapoptotic proteins cytochrome c and caspase 9 were altered in T21F. These novel findings would point to a deleterious role of oxidatively modified molecules in early cell senescence of T21F, thereby linking replicative and stress-induced senescence in cultured cells to premature aging in DS.

  2. Merlin, a “Magic” Linker Between the Extracellular Cues and Intracellular Signaling Pathways that Regulate Cell Motility, Proliferation, and Survival

    PubMed Central

    Stamenkovic, Ivan; Yu, Qin

    2010-01-01

    Genetic alterations of neurofibromatosis type 2 (NF2) gene lead to the development of schwannomas, meningiomas, and ependymomas. Mutations of NF2 gene were also found in thyroid cancer, mesothelioma, and melanoma, suggesting that it functions as a tumor suppressor in a wide spectrum of cells. The product of NF2 gene is merlin (moesinezrin-radixin-like protein), a member of the Band 4.1 superfamily proteins. Merlin shares significant sequence homology with the ERM (Ezrin-Radixin-Moesin) family proteins and serves as a linker between transmembrane proteins and the actin-cytoskeleton. Merlin is a multifunctional protein and involved in integrating and regulating the extracellular cues and intracellular signaling pathways that control cell fate, shape, proliferation, survival, and motility. Recent studies showed that merlin regulates the cell-cell and cell-matrix adhesions and functions of the cell surface adhesion/extracellular matrix receptors including CD44 and that merlin and CD44 antagonize each other's function and work upstream of the mammalian Hippo signaling pathway. Furthermore, merlin plays important roles in stabilizing the contact inhibition of proliferation and in regulating activities of several receptor tyrosine kinases. Accumulating data also suggested an emerging role of merlin as a negative regulator of growth and progression of several non-NF2 associated cancer types. Together, these recent advances have improved our basic understanding about merlin function, its regulation, and the major signaling pathways regulated by merlin and provided the foundation for future translation of these findings into the clinic for patients bearing the cancers in which merlin function and/or its downstream signaling pathways are impaired or altered. PMID:20491622

  3. Activation of α7 nicotinic acetylcholine receptors increases intracellular cAMP levels via activation of AC1 in hippocampal neurons

    PubMed Central

    Cheng, Qing; Yakel, Jerrel L.

    2015-01-01

    The activation of α7 nAChRs has been shown to improve hippocampal-dependent learning and memory. However, the molecular mechanism of α7 nAChRs’ action remains elusive. We previously reported that activation of α7 nAChRs induced a prolonged enhancement of glutamatergic synaptic transmission in a PKA-dependent manner. Here, we investigated any connection between the activation of the α7 nAChR and cAMP signaling in hippocampal neurons. To address this question, we employed a FRET-based biosensor to measure the intracellular cAMP levels directly via live cell imaging. We found that application of the α7 nAChR-selective agonist choline, in the presence of the α7 nAChR positive allosteric modulator PNU-120596, induced a significant change in emission ratio of F535/F470, which indicated an increase in intracellular cAMP levels. This choline-induced increase was abolished by the α7 nAChR antagonist MLA and the calcium chelator BAPTA, suggesting that the cAMP increase depends on the α7 nAChR activation and subsequent intracellular calcium rise. The selective AC1 inhibitor CB-6673567 and siRNA-mediated deletion of AC1 both blocked the choline-induced cAMP increase, suggesting that calcium-dependent AC1 is required for choline’s action. Furthermore, α7 nAChR activation stimulated the phosphorylation of synapsin, which serves as a downstream effector to regulate neurotransmitter release. Our findings provide the first direct evidence to link activation of α7 nAChRs to a cAMP rise via AC1, which defines a new signaling pathway employed by α7 nAChRs. Our study sheds light into potential molecular mechanisms of the positive cognitive actions of α7 nAChR agonists and development of therapeutic treatments for cognitive impairments. PMID:25937212

  4. Inositol-Requiring Enzyme 1-Dependent Activation of AMPK Promotes Brucella abortus Intracellular Growth

    PubMed Central

    Liu, Ning; Li, Yingying; Dong, Chunyan; Xu, Xiaohan; Wei, Pan; Sun, Wanchun

    2016-01-01

    ABSTRACT AMP-activated protein kinase (AMPK) is a serine/threonine kinase that is well conserved during evolution. AMPK activation inhibits production of reactive oxygen species (ROS) in cells via suppression of NADPH oxidase. However, the role of AMPK during the process of Brucella infection remains unknown. Our data demonstrate that B. abortus infection induces AMPK activation in HeLa cells in a time-dependent manner. The known AMPK kinases LKB1, CAMKKβ, and TAK1 are not required for the activation of AMPK by B. abortus infection. Instead, this activation is dependent on the RNase activity of inositol-requiring enzyme 1 (IRE1). Moreover, we also found that B. abortus infection-induced IRE1-dependent activation of AMPK promotes B. abortus intracellular growth with peritoneal macrophages via suppression of NADPH-derived ROS production. IMPORTANCE Previous studies showed that B. abortus infection does not promote any oxidative burst regulated by NADPH oxidase. However, the underlying mechanism remains elusive. We report for the first time that AMPK activation caused by B. abortus infection plays important role in NADPH oxidase-derived ROS production. PMID:26755628

  5. Studies on the bivalent-cation-activated ATPase activities of highly purified human platelet surface and intracellular membranes.

    PubMed

    Hack, N; Croset, M; Crawford, N

    1986-02-01

    Membrane-bound Ca2+-ATPases are responsible for the energy-dependent transport of Ca2+ across membrane barriers against concentration gradients. Such enzymes have been identified in sarcoplasmic reticulum of muscle tissues and in non-muscle cells in both surface membranes and endoplasmic-reticulum-like intracellular membrane complexes. In a previous study using membrane fractionation by density-gradient and free-flow electrophoresis, we reported that the intracellular membranes of human blood platelets were a major storage site for Ca2+ and involved in maintaining low cytosol [Ca2+] in the unactivated cell. In the present report we demonstrated that the intracellular membranes also exhibit a high-affinity Ca2+-ATPase which appears to be kinetically associated with the Ca2+-sequestering process. We found that both the surface membrane and the intracellular membrane exhibited a basal Mg2+-ATPase activity, but Ca2+ activation of this enzyme was confined only to the intracellular membrane. Use of Ca2+-EGTA buffers to control the extravesicle [Ca2+] allowed a direct comparison of the Ca2+-ATPase and the Ca2+-uptake process over a Ca2+ range of 0.01 microM to 1.0 mM, and it was found that both properties were maximally expressed in the range of external [Ca2+] 1-50 microM, with concentrations greater than 100 microM showing substantial inhibition. Double-reciprocal plots for the Ca2+-ATPase activity and Ca2+ uptake gave apparent Km values for Ca2+ of 0.15 and 0.13 microM respectively. However, similar plots for ATP with the enzyme revealed a discontinuity (two affinity sites, with Km 20 and 145 microM), whereas plots for the Ca2+ uptake gave a single Km value for Ca2+, 1.1 microM. Phosphorylation studies during Ca2+ uptake using [gamma-32P]ATP revealed two components of 90 and 95 kDa phosphorylated at extravesicle [Ca2+] of 3 microM. The Ca2+-ATPase activity, Ca2+ uptake and phosphorylation were all almost completely inhibited in the presence of 500 microM-Ca2+. Similar

  6. Nesfatin-1 increases intracellular calcium concentration by protein kinase C activation in cultured rat dorsal root ganglion neurons.

    PubMed

    Ozcan, Mete; Gok, Zeynep Betul; Kacar, Emine; Serhatlioglu, Ihsan; Kelestimur, Haluk

    2016-04-21

    Nesfatin-1 is a recently identified anorexigenic hypothalamic polypeptide derived from the posttranslational processing of nucleobindin 2 (NUCB2). Several studies have indicated that this neuropeptide may be participated in somatosensory and visceral transmission including pain signals in addition to energy metabolism. The aim of this study was to explore the possible role of nesfatin-1 in the transmission of peripheral neural signals by investigating the effects of nesfatin-1 on intracellular free calcium levels ([Ca(2+)]i) in cultured neonatal rat dorsal root ganglion (DRG) neurons. The effects of nesfatin-1 on [Ca(2+)]i in DRG neurons were investigated by using an in vitro calcium imaging system. DRG neurons were grown in primary culture following enzymatic and mechanical dissociation of ganglia from 1-or 2-day-old neonatal Wistar rats. Using the fura-2-based calcium imaging technique, the effects of nesfatin-1 on [Ca(2+)]i and role of the protein kinase C (PKC)-mediated pathway in nesfatin-1 effect were assessed. Nesfatin-1 elevated [Ca(2+)]i in cultured DRG neurons. The response was prevented by pretreating the cells with pertussis toxin. The protein kinase C inhibitor chelerythrine chloride suppressed nesfatin-1-induced rise in [Ca(2+)]i. The result shows that nesfatin-1 interacts with a G protein-coupled receptor, leading to an increase of [Ca(2+)]i, which is linked to protein kinase C activation in cultured rat DRG neurons.

  7. Intracellular coenzymes as natural biomarkers for metabolic activities and mitochondrial anomalies.

    PubMed

    Heikal, Ahmed A

    2010-04-01

    Mitochondria play a pivotal role in energy metabolism, programmed cell death and oxidative stress. Mutated mitochondrial DNA in diseased cells compromises the structure of key enzyme complexes and, therefore, mitochondrial function, which leads to a myriad of health-related conditions such as cancer, neurodegenerative diseases, diabetes and aging. Early detection of mitochondrial and metabolic anomalies is an essential step towards effective diagnoses and therapeutic intervention. Reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) play important roles in a wide range of cellular oxidation-reduction reactions. Importantly, NADH and FAD are naturally fluorescent, which allows noninvasive imaging of metabolic activities of living cells and tissues. Furthermore, NADH and FAD autofluorescence, which can be excited using distinct wavelengths for complementary imaging methods and is sensitive to protein binding and local environment. This article highlights recent developments concerning intracellular NADH and FAD as potential biomarkers for metabolic and mitochondrial activities.

  8. Regulator of G-protein signaling 18 integrates activating and inhibitory signaling in platelets.

    PubMed

    Gegenbauer, Kristina; Elia, Giuliano; Blanco-Fernandez, Alfonso; Smolenski, Albert

    2012-04-19

    Regulator of G-protein signaling 18 (RGS18) is a GTPase-activating protein for the G-α-q and G-α-i subunits of heterotrimeric G-proteins that turns off signaling by G-protein coupled receptors. RGS18 is highly expressed in platelets. In the present study, we show that the 14-3-3γ protein binds to phosphorylated serines 49 and 218 of RGS18. Platelet activation by thrombin, thromboxane A2, or ADP stimulates the association of 14-3-3 and RGS18, probably by increasing the phosphorylation of serine 49. In contrast, treatment of platelets with prostacyclin and nitric oxide, which trigger inhibitory cyclic nucleotide signaling involving cyclic AMP-dependent protein kinase A (PKA) and cyclic GMP-dependent protein kinase I (PKGI), induces the phosphorylation of serine 216 of RGS18 and the detachment of 14-3-3. Serine 216 phosphorylation is able to block 14-3-3 binding to RGS18 even in the presence of thrombin, thromboxane A2, or ADP. 14-3-3-deficient RGS18 is more active compared with 14-3-3-bound RGS18, leading to a more pronounced inhibition of thrombin-induced release of calcium ions from intracellular stores. Therefore, PKA- and PKGI-mediated detachment of 14-3-3 activates RGS18 to block Gq-dependent calcium signaling. These findings indicate cross-talk between platelet activation and inhibition pathways at the level of RGS18 and Gq. PMID:22234696

  9. Prolonged Intracellular Na+ Dynamics Govern Electrical Activity in Accessory Olfactory Bulb Mitral Cells.

    PubMed

    Zylbertal, Asaph; Kahan, Anat; Ben-Shaul, Yoram; Yarom, Yosef; Wagner, Shlomo

    2015-12-01

    Persistent activity has been reported in many brain areas and is hypothesized to mediate working memory and emotional brain states and to rely upon network or biophysical feedback. Here, we demonstrate a novel mechanism by which persistent neuronal activity can be generated without feedback, relying instead on the slow removal of Na+ from neurons following bursts of activity. We show that mitral cells in the accessory olfactory bulb (AOB), which plays a major role in mammalian social behavior, may respond to a brief sensory stimulation with persistent firing. By combining electrical recordings, Ca2+ and Na+ imaging, and realistic computational modeling, we explored the mechanisms underlying the persistent activity in AOB mitral cells. We found that the exceptionally slow inward current that underlies this activity is governed by prolonged dynamics of intracellular Na+ ([Na+]i), which affects neuronal electrical activity via several pathways. Specifically, elevated dendritic [Na+]i reverses the Na+-Ca2+ exchanger activity, thus modifying the [Ca2+]i set-point. This process, which relies on ubiquitous membrane mechanisms, is likely to play a role in other neuronal types in various brain regions. PMID:26674618

  10. Prolonged Intracellular Na+ Dynamics Govern Electrical Activity in Accessory Olfactory Bulb Mitral Cells.

    PubMed

    Zylbertal, Asaph; Kahan, Anat; Ben-Shaul, Yoram; Yarom, Yosef; Wagner, Shlomo

    2015-12-01

    Persistent activity has been reported in many brain areas and is hypothesized to mediate working memory and emotional brain states and to rely upon network or biophysical feedback. Here, we demonstrate a novel mechanism by which persistent neuronal activity can be generated without feedback, relying instead on the slow removal of Na+ from neurons following bursts of activity. We show that mitral cells in the accessory olfactory bulb (AOB), which plays a major role in mammalian social behavior, may respond to a brief sensory stimulation with persistent firing. By combining electrical recordings, Ca2+ and Na+ imaging, and realistic computational modeling, we explored the mechanisms underlying the persistent activity in AOB mitral cells. We found that the exceptionally slow inward current that underlies this activity is governed by prolonged dynamics of intracellular Na+ ([Na+]i), which affects neuronal electrical activity via several pathways. Specifically, elevated dendritic [Na+]i reverses the Na+-Ca2+ exchanger activity, thus modifying the [Ca2+]i set-point. This process, which relies on ubiquitous membrane mechanisms, is likely to play a role in other neuronal types in various brain regions.

  11. Prolonged Intracellular Na+ Dynamics Govern Electrical Activity in Accessory Olfactory Bulb Mitral Cells

    PubMed Central

    Zylbertal, Asaph; Kahan, Anat; Ben-Shaul, Yoram; Yarom, Yosef; Wagner, Shlomo

    2015-01-01

    Persistent activity has been reported in many brain areas and is hypothesized to mediate working memory and emotional brain states and to rely upon network or biophysical feedback. Here, we demonstrate a novel mechanism by which persistent neuronal activity can be generated without feedback, relying instead on the slow removal of Na+ from neurons following bursts of activity. We show that mitral cells in the accessory olfactory bulb (AOB), which plays a major role in mammalian social behavior, may respond to a brief sensory stimulation with persistent firing. By combining electrical recordings, Ca2+ and Na+ imaging, and realistic computational modeling, we explored the mechanisms underlying the persistent activity in AOB mitral cells. We found that the exceptionally slow inward current that underlies this activity is governed by prolonged dynamics of intracellular Na+ ([Na+]i), which affects neuronal electrical activity via several pathways. Specifically, elevated dendritic [Na+]i reverses the Na+-Ca2+ exchanger activity, thus modifying the [Ca2+]i set-point. This process, which relies on ubiquitous membrane mechanisms, is likely to play a role in other neuronal types in various brain regions. PMID:26674618

  12. Arabinosylated lipoarabinomannan (Ara-LAM) mediated intracellular mechanisms against tuberculosis infection: involvement of protein kinase C (PKC) mediated signaling.

    PubMed

    Das, Shibali; Bhattacharjee, Oindrila; Goswami, Avranil; Pal, Nishith K; Majumdar, Subrata

    2015-03-01

    Tuberculosis causes severe immunosuppression thereby ensuring the loss of the host protective immune responses. During Mycobacterium tuberculosis infection, the pathogen modulates TLR-2 receptor down-stream signaling, indicating the possible involvement of TLR-2 in the regulation of the host immune response. Moreover, different PKC isoforms are also involved in the course of infection. Arabinosylated lipoarabinomannan (Ara-LAM) possesses immuno-modulatory properties which induce the pro-inflammatory responses via induction of TLR-2-mediated signaling. Here, we found that pretreatment of M. tuberculosis-infected macrophages with Ara-LAM caused a significant increase in the conventional PKC expression along with their active association with TLR-2. This association activated the TLR-2 -mediated downstream signaling, facilitating the activation of MAP kinase P38. All these events culminated in the up-regulation of proinflammatory response, which was abrogated by treatment with PKC-α and P38 inhibitors. Moreover, pretreatment of macrophages with Ara-LAM abrogated the IL-10 production while restored MHC-II expression in the infected macrophages. This study demonstrates that Ara-LAM confers protection against tuberculosis via TLR-2/PKC signaling crosstalk which is responsible for the induction of host protective immune response against tuberculosis.

  13. Expression of the potential therapeutic target CXXC5 in primary acute myeloid leukemia cells - high expression is associated with adverse prognosis as well as altered intracellular signaling and transcriptional regulation

    PubMed Central

    Bruserud, Øystein; Reikvam, Håkon; Fredly, Hanne; Skavland, Jørn; Hagen, Karen-Marie; van Hoang, Tuyen Thy; Brenner, Annette K.; Kadi, Amir; Astori, Audrey; Gjertsen, Bjørn Tore; Pendino, Frederic

    2015-01-01

    The CXXC5 gene encodes a transcriptional activator with a zinc-finger domain, and high expression in human acute myeloid leukemia (AML) cells is associated with adverse prognosis. We now characterized the biological context of CXXC5 expression in primary human AML cells. The global gene expression profile of AML cells derived from 48 consecutive patients was analyzed; cells with high and low CXXC5 expression then showed major differences with regard to extracellular communication and intracellular signaling. We observed significant differences in the phosphorylation status of several intracellular signaling mediators (CREB, PDK1, SRC, STAT1, p38, STAT3, rpS6) that are important for PI3K-Akt-mTOR signaling and/or transcriptional regulation. High CXXC5 expression was also associated with high mRNA expression of several stem cell-associated transcriptional regulators, the strongest associations being with WT1, GATA2, RUNX1, LYL1, DNMT3, SPI1, and MYB. Finally, CXXC5 knockdown in human AML cell lines caused significantly increased expression of the potential tumor suppressor gene TSC22 and genes encoding the growth factor receptor KIT, the cytokine Angiopoietin 1 and the selenium-containing glycoprotein Selenoprotein P. Thus, high CXXC5 expression seems to affect several steps in human leukemogenesis, including intracellular events as well as extracellular communication. PMID:25605239

  14. T Cell Receptor (TCR) Interacting Molecule (TRIM), A Novel Disulfide-linked Dimer Associated with the TCR–CD3–ζ Complex, Recruits Intracellular Signaling Proteins to the Plasma Membrane

    PubMed Central

    Bruyns, Eddy; Marie-Cardine, Anne; Kirchgessner, Henning; Sagolla, Karin; Shevchenko, Andrej; Mann, Matthias; Autschbach, Frank; Bensussan, Armand; Meuer, Stefan; Schraven, Burkhart

    1998-01-01

    The molecular mechanisms regulating recruitment of intracellular signaling proteins like growth factor receptor–bound protein 2 (Grb2), phospholipase Cγ1, or phosphatidylinositol 3-kinase (PI3-kinase) to the plasma membrane after stimulation of the T cell receptor (TCR)– CD3–ζ complex are not very well understood. We describe here purification, tandem mass spectrometry sequencing, molecular cloning, and biochemical characterization of a novel transmembrane adaptor protein which associates and comodulates with the TCR–CD3–ζ complex in human T lymphocytes and T cell lines. This protein was termed T cell receptor interacting molecule (TRIM). TRIM is a disulfide-linked homodimer which is comprised of a short extracellular domain of 8 amino acids, a 19–amino acid transmembrane region, and a 159–amino acid cytoplasmic tail. In its intracellular domain, TRIM contains several tyrosine-based signaling motifs that could be involved in SH2 domain–mediated protein–protein interactions. Indeed, after T cell activation, TRIM becomes rapidly phosphorylated on tyrosine residues and then associates with the 85-kD regulatory subunit of PI3-kinase via an YxxM motif. Thus, TRIM represents a TCR-associated transmembrane adaptor protein which is likely involved in targeting of intracellular signaling proteins to the plasma membrane after triggering of the TCR. PMID:9687533

  15. Neuropeptide Y regulates intracellular calcium through different signalling pathways linked to a Y1-receptor in rat mesenteric small arteries

    PubMed Central

    Prieto, Dolores; Buus, Carsten L; Mulvany, Michael J; Nilsson, Holger

    2000-01-01

    Simultaneous measurements of intracellular calcium concentration ([Ca2+]i) and tension were performed to clarify whether the mechanisms which cause the neuropeptide Y (NPY)-elicited contraction and potentiation of noradrenaline contractions, and the NPY inhibition of forskolin responses are linked to a single or different NPY receptor(s) in rat mesenteric small arteries.In resting arteries, NPY moderately elevated [Ca2+]i and tension. These effects were antagonized by the selective Y1 receptor antagonist, (R)-N2-(diphenacetyl)-N-[(4-hydroxyphenyl)methyl]-D-arginineamide (BIBP 3226) (apparent pKB values of 8.54±0.25 and 8.27±0.17, respectively).NPY (0.1 μM) caused a near 3 fold increase in sensitivity to noradrenaline but did not significantly modify the tension-[Ca2+]i relationship for this agonist. BIBP 3226 competitively antagonized the contractile response to NPY in arteries submaximally preconstricted with noradrenaline (pA2 7.87±0.20).In arteries activated by vasopressin, the adenylyl cyclase activator forskolin (3 μM) induced a maximum relaxation and a return of [Ca2+]i to resting levels. NPY completely inhibited these effects. The contractile responses to NPY in arteries maximally relaxed with either sodium nitroprusside (SNP) or nifedipine were not significantly higher than those evoked by the peptide at resting tension, in contrast to the contractions to NPY in forskolin-relaxed arteries. BIBP 3226 competitively antagonized the contraction to NPY in forskolin-relaxed arteries with a pA2 of 7.92±0.29.Electrical field stimulation (EFS) at 8–32 Hz caused large contractions in arteries relaxed with either forskolin or noradrenaline in the presence of phentolamine. These responses to EFS were inhibited by BIBP 3226. Similar EFS in resting, non-activated arteries did not produce any response.The present results suggest that different intracellular pathways are linked to a single NPY Y1 receptor in intact rat mesenteric small arteries, and provide

  16. Neuropeptide Y regulates intracellular calcium through different signalling pathways linked to a Y(1)-receptor in rat mesenteric small arteries.

    PubMed

    Prieto, D; Buus, C L; Mulvany, M J; Nilsson, H

    2000-04-01

    Simultaneous measurements of intracellular calcium concentration ([Ca(2+)](i)) and tension were performed to clarify whether the mechanisms which cause the neuropeptide Y (NPY)-elicited contraction and potentiation of noradrenaline contractions, and the NPY inhibition of forskolin responses are linked to a single or different NPY receptor(s) in rat mesenteric small arteries. In resting arteries, NPY moderately elevated [Ca(2+)](i) and tension. These effects were antagonized by the selective Y(1) receptor antagonist, (R)-N(2)-(diphenacetyl)-N-[(4-hydroxyphenyl)methyl]-D-argininea mide (BIBP 3226) (apparent pK(B) values of 8.54+/-0.25 and 8.27+/-0.17, respectively). NPY (0.1 microM) caused a near 3 fold increase in sensitivity to noradrenaline but did not significantly modify the tension-[Ca(2+)](i) relationship for this agonist. BIBP 3226 competitively antagonized the contractile response to NPY in arteries submaximally preconstricted with noradrenaline (pA(2) 7.87+/-0.20). In arteries activated by vasopressin, the adenylyl cyclase activator forskolin (3 microM) induced a maximum relaxation and a return of [Ca(2+)](i) to resting levels. NPY completely inhibited these effects. The contractile responses to NPY in arteries maximally relaxed with either sodium nitroprusside (SNP) or nifedipine were not significantly higher than those evoked by the peptide at resting tension, in contrast to the contractions to NPY in forskolin-relaxed arteries. BIBP 3226 competitively antagonized the contraction to NPY in forskolin-relaxed arteries with a pA(2) of 7.92+/-0.29. Electrical field stimulation (EFS) at 8-32 Hz caused large contractions in arteries relaxed with either forskolin or noradrenaline in the presence of phentolamine. These responses to EFS were inhibited by BIBP 3226. Similar EFS in resting, non-activated arteries did not produce any response. The present results suggest that different intracellular pathways are linked to a single NPY Y(1) receptor in intact rat

  17. Biased Signaling of Protease-Activated Receptors

    PubMed Central

    Zhao, Peishen; Metcalf, Matthew; Bunnett, Nigel W.

    2014-01-01

    In addition to their role in protein degradation and digestion, proteases can also function as hormone-like signaling molecules that regulate vital patho-physiological processes, including inflammation, hemostasis, pain, and repair mechanisms. Certain proteases can signal to cells by cleaving protease-activated receptors (PARs), a family of four G protein-coupled receptors. PARs are expressed by almost all cell types, control important physiological and disease-relevant processes, and are an emerging therapeutic target for major diseases. Most information about PAR activation and function derives from studies of a few proteases, for example thrombin in the case of PAR1, PAR3, and PAR4, and trypsin in the case of PAR2 and PAR4. These proteases cleave PARs at established sites with the extracellular N-terminal domains, and expose tethered ligands that stabilize conformations of the cleaved receptors that activate the canonical pathways of G protein- and/or β-arrestin-dependent signaling. However, a growing number of proteases have been identified that cleave PARs at divergent sites to activate distinct patterns of receptor signaling and trafficking. The capacity of these proteases to trigger distinct signaling pathways is referred to as biased signaling, and can lead to unique patho-physiological outcomes. Given that a different repertoire of proteases are activated in various patho-physiological conditions that may activate PARs by different mechanisms, signaling bias may account for the divergent actions of proteases and PARs. Moreover, therapies that target disease-relevant biased signaling pathways may be more effective and selective approaches for the treatment of protease- and PAR-driven diseases. Thus, rather than mediating the actions of a few proteases, PARs may integrate the biological actions of a wide spectrum of proteases in different patho-physiological conditions. PMID:24860547

  18. Intracellular mechanisms modulating gamma band activity in the pedunculopontine nucleus (PPN).

    PubMed

    Luster, Brennon R; Urbano, Francisco J; Garcia-Rill, Edgar

    2016-06-01

    The pedunculopontine nucleus is a part of the reticular activating system, and is active during waking and REM sleep. Previous results showed that all PPN cells tested fired maximally at gamma frequencies when depolarized. This intrinsic membrane property was shown to be mediated by high-threshold N- and P/Q-type Ca(2+) channels. Recent studies show that the PPN contains three independent populations of neurons which can generate gamma band oscillations through only N-type channels, only P/Q-type channels, or both N- and P/Q-type channels. This study investigated the intracellular mechanisms modulating gamma band activity in each population of neurons. We performed in vitro patch-clamp recordings of PPN neurons from Sprague-Dawley rat pups, and applied 1-sec ramps to induce intrinsic membrane oscillations. Our results show that there are two pathways modulating gamma band activity in PPN neurons. We describe populations of neurons mediating gamma band activity through only N-type channels and the cAMP/PKA pathway (presumed "REM-on" neurons), through only P/Q-type channels and the CaMKII pathway (presumed "Wake-on" neurons), and a third population which can mediate gamma activity through both N-type channels and cAMP/PK and P/Q-type channels and CaMKII (presumed "Wake/REM-on" neurons). These novel results suggest that PPN gamma oscillations are modulated by two independent pathways related to different Ca(2+) channel types.

  19. Nerve Growth Factor Mediates a Switch in Intracellular Signaling for PGE2-Induced Sensitization of Sensory Neurons from Protein Kinase A to Epac

    PubMed Central

    Vasko, Michael R.; Habashy Malty, Ramy; Guo, Chunlu; Duarte, Djane B.; Zhang, Yihong; Nicol, Grant D.

    2014-01-01

    We examined whether nerve growth factor (NGF), an inflammatory mediator that contributes to chronic hypersensitivity, alters the intracellular signaling that mediates the sensitizing actions of PGE2 from activation of protein kinase A (PKA) to exchange proteins directly activated by cAMP (Epacs). When isolated sensory neurons are grown in the absence of added NGF, but not in cultures grown with 30 ng/ml NGF, inhibiting protein kinase A (PKA) activity blocks the ability of PGE2 to augment capsaicin-evoked release of the neuropeptide CGRP and to increase the number of action potentials (APs) evoked by a ramp of current. Growing sensory neurons in culture in the presence of increasing concentrations of NGF increases the expression of Epac2, but not Epac1. An intradermal injection of complete Freund's adjuvant into the rat hindpaw also increases the expression of Epac2, but not Epac1 in the dorsal root ganglia and spinal cord: an effect blocked by intraplantar administration of NGF antibodies. Treating cultures grown in the presence of 30 ng/ml NGF with Epac1siRNA significantly reduced the expression of Epac1, but not Epac2, and did not block the ability of PGE2 to augment capsaicin-evoked release of CGRP from sensory neurons. Exposing neuronal cultures grown in NGF to Epac2siRNAreduced the expression of Epac2, but not Epac1 and prevented the PGE2-induced augmentation of capsaicin and potassium-evoked CGRP release in sensory neurons and the PGE2-induced increase in the number of APs generated by a ramp of current. In neurons grown with no added NGF, Epac siRNAs did not attenuate PGE2-induced sensitization. These results demonstrate that NGF, through increasing Epac2 expression, alters the signaling cascade that mediates PGE2-induced sensitization of sensory neurons, thus providing a novel mechanism for maintaining PGE2-induced hypersensitivity during inflammation. PMID:25126967

  20. Inhibition of intracellular growth of Histoplasma capsulatum yeast cells by cytokine-activated human monocytes and macrophages.

    PubMed Central

    Newman, S L; Gootee, L; Bucher, C; Bullock, W E

    1991-01-01

    Human monocytes/macrophages (M psi) were infected with Histoplasma capsulatum yeast cells, and intracellular growth was quantified after 24 h of incubation in medium alone or in medium containing cytokines. Yeast cells multiplied within freshly isolated monocytes, cultured M psi, and alveolar M psi with intracellular generation times of 14.2 +/- 1.4, 18.5 +/- 2.1, and 19.9 +/- 1.9 h (mean +/- standard error of the mean), respectively. Monocytes and M psi inhibited the intracellular growth of yeast cells in response to cytokine supernatant; maximum inhibition was obtained when cytokines were added to cell monolayers immediately after infection. Opsonization of yeast cells in normal serum or in H. capsulatum-immune serum did not affect the intracellular generation time of yeast cells in either control M psi or cytokine-activated M psi. PMID:1898916

  1. Signal integration by Ca2+ regulates intestinal stem cell activity

    PubMed Central

    Deng, Hansong; Gerencser, Akos A.; Jasper, Heinrich

    2015-01-01

    Summary Somatic stem cells (SCs) maintain tissue homeostasis by dynamically adjusting proliferation and differentiation in response to stress and metabolic cues. Here, we identify Ca2+ signaling as a central regulator of intestinal SC (ISC) activity in Drosophila. We find that dietary L-glutamate stimulates ISC division and gut growth. The metabotropic glutamate receptor (mGluR) is required in ISCs for this response and for an associated modulation of cytosolic Ca2+ oscillations that results in sustained high cytosolic Ca2+ concentrations. High cytosolic Ca2+ induces ISC proliferation by regulating Calcineurin and CREB - regulated transcriptional co-activator (CRTC). In response to a wide range of dietary and stress stimuli, ISCs reversibly transition between Ca2+ oscillation states that represent poised or activated modes of proliferation, respectively. We propose that the dynamic regulation of intracellular Ca2+ levels allows effective integration of diverse mitogenic signals in ISCs to tailor their proliferative activity to the needs of the tissue. PMID:26633624

  2. PKCζ regulates Notch receptor routing and activity in a Notch signaling-dependent manner

    PubMed Central

    Sjöqvist, Marika; Antfolk, Daniel; Ferraris, Saima; Rraklli, Vilma; Haga, Cecilia; Antila, Christian; Mutvei, Anders; Imanishi, Susumu Y; Holmberg, Johan; Jin, Shaobo; Eriksson, John E; Lendahl, Urban; Sahlgren, Cecilia

    2014-01-01

    Activation of Notch signaling requires intracellular routing of the receptor, but the mechanisms controlling the distinct steps in the routing process is poorly understood. We identify PKCζ as a key regulator of Notch receptor intracellular routing. When PKCζ was inhibited in the developing chick central nervous system and in cultured myoblasts, Notch-stimulated cells were allowed to undergo differentiation. PKCζ phosphorylates membrane-tethered forms of Notch and regulates two distinct routing steps, depending on the Notch activation state. When Notch is activated, PKCζ promotes re-localization of Notch from late endosomes to the nucleus and enhances production of the Notch intracellular domain, which leads to increased Notch activity. In the non-activated state, PKCζ instead facilitates Notch receptor internalization, accompanied with increased ubiquitylation and interaction with the endosomal sorting protein Hrs. Collectively, these data identify PKCζ as a key regulator of Notch trafficking and demonstrate that distinct steps in intracellular routing are differentially modulated depending on Notch signaling status. PMID:24662486

  3. Oxidative stress induces increase in intracellular amyloid beta-protein production and selective activation of betaI and betaII PKCs in NT2 cells.

    PubMed

    Paola, D; Domenicotti, C; Nitti, M; Vitali, A; Borghi, R; Cottalasso, D; Zaccheo, D; Odetti, P; Strocchi, P; Marinari, U M; Tabaton, M; Pronzato, M A

    2000-02-16

    Amyloid beta-protein (Abeta) aggregation produces an oxidative stress in neuronal cells that, in turn, may induce an amyloidogenic shift of neuronal metabolism. To investigate this hypothesis, we analyzed intra- and extracellular Abeta content in NT2 differentiated cells incubated with 4-hydroxy-2,3-nonenal (HNE), a major product of lipid peroxidation. In parallel, we evaluated protein kinase C (PKC) isoenzymes activity, a signaling system suspected to modulate amyloid precursor protein (APP) processing. Low HNE concentrations (0.1-1 microM) induced a 2-6 fold increase of intracellular Abeta production that was concomitant with selective activation of betaI and betaII PKC isoforms, without affecting either cell viability or APP full-length expression. Selective activation of the same PKC isoforms was observed following NT2 differentiation. Our findings suggest that PKC beta isoenzymes are part of cellular mechanisms that regulate production of the intracellular Abeta pool. Moreover, they indicate that lipid peroxidation fosters intracellular Abeta accumulation, creating a vicious neurodegenerative loop. PMID:10679257

  4. Intracellular cytoplasm-specific delivery of SH3 and SH2 domains of SLAP inhibits TcR-mediated signaling.

    PubMed

    Kim, Jung-Ho; Moon, Jae-Seung; Yu, JiSang; Lee, Sang-Kyou

    2015-05-01

    Signaling events triggered by T cell receptor (TcR) stimulation are important targets for the development of common therapeutics for various autoimmune diseases. SLAP is a negative regulator of TcR-mediated signaling cascade via targeting TcR zeta chain for degradation through recruiting the ubiquitin ligase c-Cbl. In this study, we generated a transducible form of SH3 and SH2 domains of SLAP (ctSLAPΔC) which can be specifically targeted to the cytoplasm of a cell. ctSLAPΔC inhibited tyrosine phosphorylation of signaling mediators such as ZAP-70 and LAT involved in T cell activation, and effectively suppressed transcriptional activity of NFAT and NFκB upon TcR stimulation. The transduced ctSLAPΔC in T cells blocked the secretion of T cell-specific cytokines such as IL-2, IFNγ, IL-17A, and IL-4 and induced the expression of CD69 and CD25 on effector T cells without influencing the cell viability. Inhibition of TcR-mediated signaling via SLAP blocked the differentiation of naïve T cells into Th1, Th2 or Treg cells with different sensitivity, suggesting that qualitative and quantitative intensity of TcR-mediated signaling in the context of polarizing cytokines environment may be a critical factor to determine the differentiation fate of naïve T cells. These results suggest that cytoplasm-specific transduction of the SH3 and SH2 domains of SLAP has a therapeutic potential of being an immunosuppressive reagent for the treatment of various autoimmune diseases. PMID:25800872

  5. Intracellular cytoplasm-specific delivery of SH3 and SH2 domains of SLAP inhibits TcR-mediated signaling.

    PubMed

    Kim, Jung-Ho; Moon, Jae-Seung; Yu, JiSang; Lee, Sang-Kyou

    2015-05-01

    Signaling events triggered by T cell receptor (TcR) stimulation are important targets for the development of common therapeutics for various autoimmune diseases. SLAP is a negative regulator of TcR-mediated signaling cascade via targeting TcR zeta chain for degradation through recruiting the ubiquitin ligase c-Cbl. In this study, we generated a transducible form of SH3 and SH2 domains of SLAP (ctSLAPΔC) which can be specifically targeted to the cytoplasm of a cell. ctSLAPΔC inhibited tyrosine phosphorylation of signaling mediators such as ZAP-70 and LAT involved in T cell activation, and effectively suppressed transcriptional activity of NFAT and NFκB upon TcR stimulation. The transduced ctSLAPΔC in T cells blocked the secretion of T cell-specific cytokines such as IL-2, IFNγ, IL-17A, and IL-4 and induced the expression of CD69 and CD25 on effector T cells without influencing the cell viability. Inhibition of TcR-mediated signaling via SLAP blocked the differentiation of naïve T cells into Th1, Th2 or Treg cells with different sensitivity, suggesting that qualitative and quantitative intensity of TcR-mediated signaling in the context of polarizing cytokines environment may be a critical factor to determine the differentiation fate of naïve T cells. These results suggest that cytoplasm-specific transduction of the SH3 and SH2 domains of SLAP has a therapeutic potential of being an immunosuppressive reagent for the treatment of various autoimmune diseases.

  6. Enhanced glucose-induced intracellular signaling promotes insulin hypersecretion: pancreatic beta-cell functional adaptations in a model of genetic obesity and prediabetes.

    PubMed

    Irles, Esperanza; Ñeco, Patricia; Lluesma, Mónica; Villar-Pazos, Sabrina; Santos-Silva, Junia Carolina; Vettorazzi, Jean F; Alonso-Magdalena, Paloma; Carneiro, Everardo M; Boschero, Antonio C; Nadal, Ángel; Quesada, Ivan

    2015-03-15

    Obesity is associated with insulin resistance and is known to be a risk factor for type-2 diabetes. In obese individuals, pancreatic beta-cells try to compensate for the increased insulin demand in order to maintain euglycemia. Most studies have reported that this adaptation is due to morphological changes. However, the involvement of beta-cell functional adaptations in this process needs to be clarified. For this purpose, we evaluated different key steps in the glucose-stimulated insulin secretion (GSIS) in intact islets from female ob/ob obese mice and lean controls. Obese mice showed increased body weight, insulin resistance, hyperinsulinemia, glucose intolerance and fed hyperglycemia. Islets from ob/ob mice exhibited increased glucose-induced mitochondrial activity, reflected by enhanced NAD(P)H production and mitochondrial membrane potential hyperpolarization. Perforated patch-clamp examination of beta-cells within intact islets revealed several alterations in the electrical activity such as increased firing frequency and higher sensitivity to low glucose concentrations. A higher intracellular Ca(2+) mobilization in response to glucose was also found in ob/ob islets. Additionally, they displayed a change in the oscillatory pattern and Ca(2+) signals at low glucose levels. Capacitance experiments in intact islets revealed increased exocytosis in individual ob/ob beta-cells. All these up-regulated processes led to increased GSIS. In contrast, we found a lack of beta-cell Ca(2+) signal coupling, which could be a manifestation of early defects that lead to beta-cell malfunction in the progression to diabetes. These findings indicate that beta-cell functional adaptations are an important process in the compensatory response to obesity.

  7. α/β-Peptide Foldamers Targeting Intracellular Protein-Protein Interactions with Activity in Living Cells.

    PubMed

    Checco, James W; Lee, Erinna F; Evangelista, Marco; Sleebs, Nerida J; Rogers, Kelly; Pettikiriarachchi, Anne; Kershaw, Nadia J; Eddinger, Geoffrey A; Belair, David G; Wilson, Julia L; Eller, Chelcie H; Raines, Ronald T; Murphy, William L; Smith, Brian J; Gellman, Samuel H; Fairlie, W Douglas

    2015-09-01

    Peptides can be developed as effective antagonists of protein-protein interactions, but conventional peptides (i.e., oligomers of l-α-amino acids) suffer from significant limitations in vivo. Short half-lives due to rapid proteolytic degradation and an inability to cross cell membranes often preclude biological applications of peptides. Oligomers that contain both α- and β-amino acid residues ("α/β-peptides") manifest decreased susceptibility to proteolytic degradation, and when properly designed these unnatural oligomers can mimic the protein-recognition properties of analogous "α-peptides". This report documents an extension of the α/β-peptide approach to target intracellular protein-protein interactions. Specifically, we have generated α/β-peptides based on a "stapled" Bim BH3 α-peptide, which contains a hydrocarbon cross-link to enhance α-helix stability. We show that a stapled α/β-peptide can structurally and functionally mimic the parent stapled α-peptide in its ability to enter certain types of cells and block protein-protein interactions associated with apoptotic signaling. However, the α/β-peptide is nearly 100-fold more resistant to proteolysis than is the parent stapled α-peptide. These results show that backbone modification, a strategy that has received relatively little attention in terms of peptide engineering for biomedical applications, can be combined with more commonly deployed peripheral modifications such as side chain cross-linking to produce synergistic benefits.

  8. α/β-Peptide Foldamers Targeting Intracellular Protein-Protein Interactions with Activity in Living Cells

    PubMed Central

    Checco, James W.; Lee, Erinna F.; Evangelista, Marco; Sleebs, Nerida J.; Rogers, Kelly; Pettikiriarachchi, Anne; Kershaw, Nadia J.; Eddinger, Geoffrey A.; Belair, David G.; Wilson, Julia L.; Eller, Chelcie H.; Raines, Ronald T.; Murphy, William L.; Smith, Brian J.; Gellman, Samuel H.; Fairlie, W. Douglas

    2015-01-01

    Peptides can be developed as effective antagonists of protein-protein interactions, but conventional peptides (i.e., oligomers of L-α-amino acids) suffer from significant limitations in vivo. Short half-lives due to rapid proteolytic degradation and an inability to cross cell membranes often preclude biological applications of peptides. Oligomers that contain both α- and β-amino acid residues (“α/β-peptides”) manifest decreased susceptibility to proteolytic degradation, and when properly designed these unnatural oligomers can mimic the protein-recognition properties of analogous “α-peptides”. This report documents an extension of the α/β-peptide approach to target intracellular protein-protein interactions. Specifically, we have generated α/β-peptides based on a “stapled” Bim BH3 α-peptide, which contains a hydrocarbon crosslink to enhance α-helix stability. We show that a stapled α/β-peptide can structurally and functionally mimic the parent stapled α-peptide in its ability to enter certain types of cells and block protein-protein interactions associated with apoptotic signaling. However, the α/β-peptide is nearly 100-fold more resistant to proteolysis than is the parent α-peptide. These results show that backbone modification, a strategy that has received relatively little attention in terms of peptide engineering for biomedical applications, can be combined with more commonly deployed peripheral modifications such as side chain crosslinking to produce synergistic benefits. PMID:26317395

  9. Ribavirin-induced intracellular GTP depletion activates transcription elongation in coagulation factor VII gene expression.

    PubMed

    Suzuki, Atsuo; Miyawaki, Yuhri; Okuyama, Eriko; Murata, Moe; Ando, Yumi; Kato, Io; Takagi, Yuki; Takagi, Akira; Murate, Takashi; Saito, Hidehiko; Kojima, Tetsuhito

    2013-01-01

    Coagulation FVII (Factor VII) is a vitamin K-dependent glycoprotein synthesized in hepatocytes. It was reported previously that FVII gene (F7) expression was up-regulated by ribavirin treatment in hepatitis C virus-infected haemophilia patients; however, its precise mechanism is still unknown. In the present study, we investigated the molecular mechanism of ribavirin-induced up-regulation of F7 expression in HepG2 (human hepatoma cell line). We found that intracellular GTP depletion by ribavirin as well as other IMPDH (inosine-5'-monophosphate dehydrogenase) inhibitors, such as mycophenolic acid and 6-mercaptopurine, up-regulated F7 expression. FVII mRNA transcription was mainly enhanced by accelerated transcription elongation, which was mediated by the P-TEFb (positive-transcription elongation factor b) complex, rather than by promoter activation. Ribavirin unregulated ELL (eleven-nineteen lysine-rich leukaemia) 3 mRNA expression before F7 up-regulation. We observed that ribavirin enhanced ELL3 recruitment to F7, whereas knockdown of ELL3 diminished ribavirin-induced FVII mRNA up-regulation. Ribavirin also enhanced recruitment of CDK9 (cyclin-dependent kinase 9) and AFF4 to F7. These data suggest that ribavirin-induced intracellular GTP depletion recruits a super elongation complex containing P-TEFb, AFF4 and ELL3, to F7, and modulates FVII mRNA transcription elongation. Collectively, we have elucidated a basal mechanism for ribavirin-induced FVII mRNA up-regulation by acceleration of transcription elongation, which may be crucial in understanding its pleiotropic functions in vivo.

  10. The Drosophila FTZ-F1 Nuclear Receptor Mediates Juvenile Hormone Activation of E75A Gene Expression through an Intracellular Pathway*

    PubMed Central

    Dubrovsky, Edward B.; Dubrovskaya, Veronica A.; Bernardo, Travis; Otte, Valerie; DiFilippo, Robert; Bryan, Heather

    2011-01-01

    Juvenile hormone (JH) regulates a wide variety of biological activities in holometabolous insects, ranging from vitellogenesis and caste determination in adults to the timing of metamorphosis in larvae. The mechanism of JH signaling in such a diverse array of processes remains either unknown or contentious. We previously found that the nuclear receptor gene E75A is activated in S2 cells as a primary response to JH. Here, by expressing an intracellular form of JH esterase, we demonstrate that JH must enter the cell in order to activate E75A. To find intracellular receptors involved in the JH response, we performed an RNAi screen against nuclear receptor genes expressed in this cell line and identified the orphan receptor FTZ-F1. Removal of FTZ-F1 prevents JH activation of E75A, whereas overexpression enhances activation, implicating FTZ-F1 as a critical component of the JH response. FTZ-F1 is bound in vivo to multiple enhancers upstream of E75A, suggesting that it participates in direct JH-mediated gene activation. To better define the role of FTZ-F1 in JH signaling, we investigated interactions with candidate JH receptors and found that the bHLH-PAS proteins MET and GCE both interact with FTZ-F1 and can activate transcription through the FTZ-F1 response element. Removal of endogenous GCE, but not MET, prevents JH activation of E75A. We propose that FTZ-F1 functions as a competence factor by loading JH signaling components to the promoter, thus facilitating the direct regulation of E75A gene expression by JH. PMID:21832074

  11. Cyclic AMP directs inositol (1,4,5)-trisphosphate-evoked Ca2+ signalling to different intracellular Ca2+ stores

    PubMed Central

    Tovey, Stephen C.; Taylor, Colin W.

    2013-01-01

    Summary Cholesterol depletion reversibly abolishes carbachol-evoked Ca2+ release from inositol (1,4,5)-trisphosphate (IP3)-sensitive stores, without affecting the distribution of IP3 receptors (IP3R) or endoplasmic reticulum, IP3 formation or responses to photolysis of caged IP3. Receptors that stimulate cAMP formation do not alone evoke Ca2+ signals, but they potentiate those evoked by carbachol. We show that these potentiated signals are entirely unaffected by cholesterol depletion and that, within individual cells, different IP3-sensitive Ca2+ stores are released by carbachol alone and by carbachol combined with receptors that stimulate cAMP formation. We suggest that muscarinic acetylcholine receptors in lipid rafts deliver IP3 at high concentration to associated IP3R, stimulating them to release Ca2+. Muscarinic receptors outside rafts are less closely associated with IP3R and provide insufficient local IP3 to activate IP3R directly. These IP3R, probably type 2 IP3R within a discrete Ca2+ store, are activated only when their sensitivity is increased by cAMP. Sensitization of IP3R by cAMP extends the effective range of signalling by phospholipase C, allowing muscarinic receptors that are otherwise ineffective to recruit additional IP3-sensitive Ca2+ stores. PMID:23525004

  12. Activity of Medicinal Plant Extracts on Multiplication of Mycobacterium tuberculosis under Reduced Oxygen Conditions Using Intracellular and Axenic Assays.

    PubMed

    Bhatter, Purva D; Gupta, Pooja D; Birdi, Tannaz J

    2016-01-01

    Aim. Test the activity of selected medicinal plant extracts on multiplication of Mycobacterium tuberculosis under reduced oxygen concentration which represents nonreplicating conditions. Material and Methods. Acetone, ethanol and aqueous extracts of the plants Acorus calamus L. (rhizome), Ocimum sanctum L. (leaf), Piper nigrum L. (seed), and Pueraria tuberosa DC. (tuber) were tested on Mycobacterium tuberculosis H37Rv intracellularly using an epithelial cell (A549) infection model. The extracts found to be active intracellularly were further studied axenically under reducing oxygen concentrations. Results and Conclusions. Intracellular multiplication was inhibited ≥60% by five of the twelve extracts. Amongst these 5 extracts, in axenic culture, P. nigrum (acetone) was active under aerobic, microaerophilic, and anaerobic conditions indicating presence of multiple components acting at different levels and P. tuberosa (aqueous) showed bactericidal activity under microaerophilic and anaerobic conditions implying the influence of anaerobiosis on its efficacy. P. nigrum (aqueous) and A. calamus (aqueous and ethanol) extracts were not active under axenic conditions but only inhibited intracellular growth of Mycobacterium tuberculosis, suggesting activation of host defense mechanisms to mediate bacterial killing rather than direct bactericidal activity. PMID:26941797

  13. Activity of Medicinal Plant Extracts on Multiplication of Mycobacterium tuberculosis under Reduced Oxygen Conditions Using Intracellular and Axenic Assays

    PubMed Central

    Bhatter, Purva D.; Gupta, Pooja D.; Birdi, Tannaz J.

    2016-01-01

    Aim. Test the activity of selected medicinal plant extracts on multiplication of Mycobacterium tuberculosis under reduced oxygen concentration which represents nonreplicating conditions. Material and Methods. Acetone, ethanol and aqueous extracts of the plants Acorus calamus L. (rhizome), Ocimum sanctum L. (leaf), Piper nigrum L. (seed), and Pueraria tuberosa DC. (tuber) were tested on Mycobacterium tuberculosis H37Rv intracellularly using an epithelial cell (A549) infection model. The extracts found to be active intracellularly were further studied axenically under reducing oxygen concentrations. Results and Conclusions. Intracellular multiplication was inhibited ≥60% by five of the twelve extracts. Amongst these 5 extracts, in axenic culture, P. nigrum (acetone) was active under aerobic, microaerophilic, and anaerobic conditions indicating presence of multiple components acting at different levels and P. tuberosa (aqueous) showed bactericidal activity under microaerophilic and anaerobic conditions implying the influence of anaerobiosis on its efficacy. P. nigrum (aqueous) and A. calamus (aqueous and ethanol) extracts were not active under axenic conditions but only inhibited intracellular growth of Mycobacterium tuberculosis, suggesting activation of host defense mechanisms to mediate bacterial killing rather than direct bactericidal activity. PMID:26941797

  14. Activation of Src and release of intracellular calcium by phosphatidic acid during Xenopus laevis fertilization

    PubMed Central

    Bates, Ryan C.; Fees, Colby P.; Holland, William L.; Winger, Courtney C.; Batbayar, Khulan; Ancar, Rachel; Bergren, Todd; Petcoff, Douglas; Stith, Bradley J.

    2014-01-01

    We report a new step in the fertilization in Xenopus laevis which has been found to involve activation of Src tyrosine kinase to stimulate phospholipase C-γ (PLC- γ) which increases inositol 1,4,5-trisphosphate (IP3) to release intracellular calcium ([Ca]i). Molecular species analysis and mass measurements suggested that sperm activate phospholipase D (PLD) to elevate phosphatidic acid (PA). We now report that PA mass increased 2.7 fold by 1 minute after insemination and inhibition of PA production by two methods inhibited activation of Src and PLCγ, increased [Ca]i and other fertilization events. As compared to 14 other lipids, PA strongly bound Xenopus Src but not PLCγ. Addition of synthetic PA activated egg Src (an action requiring intact lipid rafts) and PLCγ as well as doubling the amount of PLCγ in rafts. In the absence of elevated [Ca]i, PA addition elevated IP3 mass to levels equivalent to that induced by sperm (but twice that achieved by calcium ionophore). Finally, PA induced [Ca]i release that was blocked by an IP3 receptor inhibitor. As only PLD1b message was detected, and Western blotting did not detect PLD2, we suggest that sperm activate PLD1b to elevate PA which then binds to and activates Src leading to PLCγ stimulation, IP3 elevation and [Ca]i release. Due to these and other studies, PA may also play a role in membrane fusion events such as sperm-egg fusion, cortical granule exocytosis, the elevation of phosphatidylinositol 4,5-bisphosphate and the large, late increase in sn 1,2-diacylglycerol in fertilization. PMID:24269904

  15. Intracellular mechanisms modulating gamma band activity in the pedunculopontine nucleus (PPN).

    PubMed

    Luster, Brennon R; Urbano, Francisco J; Garcia-Rill, Edgar

    2016-06-01

    The pedunculopontine nucleus is a part of the reticular activating system, and is active during waking and REM sleep. Previous results showed that all PPN cells tested fired maximally at gamma frequencies when depolarized. This intrinsic membrane property was shown to be mediated by high-threshold N- and P/Q-type Ca(2+) channels. Recent studies show that the PPN contains three independent populations of neurons which can generate gamma band oscillations through only N-type channels, only P/Q-type channels, or both N- and P/Q-type channels. This study investigated the intracellular mechanisms modulating gamma band activity in each population of neurons. We performed in vitro patch-clamp recordings of PPN neurons from Sprague-Dawley rat pups, and applied 1-sec ramps to induce intrinsic membrane oscillations. Our results show that there are two pathways modulating gamma band activity in PPN neurons. We describe populations of neurons mediating gamma band activity through only N-type channels and the cAMP/PKA pathway (presumed "REM-on" neurons), through only P/Q-type channels and the CaMKII pathway (presumed "Wake-on" neurons), and a third population which can mediate gamma activity through both N-type channels and cAMP/PK and P/Q-type channels and CaMKII (presumed "Wake/REM-on" neurons). These novel results suggest that PPN gamma oscillations are modulated by two independent pathways related to different Ca(2+) channel types. PMID:27354537

  16. An NB-LRR protein required for HR signalling mediated by both extra- and intracellular resistance proteins.

    PubMed

    Gabriëls, Suzan H E J; Vossen, Jack H; Ekengren, Sophia K; van Ooijen, Gerben; Abd-El-Haliem, Ahmed M; van den Berg, Grardy C M; Rainey, Daphne Y; Martin, Gregory B; Takken, Frank L W; de Wit, Pierre J G M; Joosten, Matthieu H A J

    2007-04-01

    Tomato (Solanum lycopersicum) Cf resistance genes confer hypersensitive response (HR)-associated resistance to strains of the pathogenic fungus Cladosporium fulvum that express the matching avirulence (Avr) gene. Previously, we identified an Avr4-responsive tomato (ART) gene that is required for Cf-4/Avr4-induced HR in Nicotiana benthamiana as demonstrated by virus-induced gene silencing (VIGS). The gene encodes a CC-NB-LRR type resistance (R) protein analogue that we have designated NRC1 (NB-LRR protein required for HR-associated cell death 1). Here we describe that knock-down of NRC1 in tomato not only affects the Cf-4/Avr4-induced HR but also compromises Cf-4-mediated resistance to C. fulvum. In addition, VIGS using NRC1 in N. benthamiana revealed that this protein is also required for the HR induced by the R proteins Cf-9, LeEix, Pto, Rx and Mi. Transient expression of NRC1(D481V), which encodes a constitutively active NRC1 mutant protein, triggers an elicitor-independent HR. Subsequently, we transiently expressed this auto-activating protein in N. benthamiana silenced for genes known to be involved in HR signalling, thereby allowing NRC1 to be positioned in an HR signalling pathway. We found that NRC1 requires RAR1 and SGT1 to be functional, whereas it does not require NDR1 and EDS1. As the Cf-4 protein requires EDS1 for its function, we hypothesize that NRC1 functions downstream of EDS1. We also found that NRC1 acts upstream of a MAP kinase pathway. We conclude that Cf-mediated resistance signalling requires a downstream NB-LRR protein that also functions in cell death signalling pathways triggered by other R proteins.

  17. The role of substrate specificity and metal binding in defining the activity and structure of an intracellular subtilisin.

    PubMed

    Gamble, Michael; Künze, Georg; Brancale, Andrea; Wilson, Keith S; Jones, D Dafydd

    2012-01-01

    The dimeric intracellular subtilisin proteases (ISPs) found throughout Gram-positive bacteria are a structurally distinct class of the subtilase family. Unlike the vast majority of subtilisin-like proteases, the ISPs function exclusively within the cell, contributing the majority of observed cellular proteolytic activity. Given that they are active within the cell, little is known about substrate specificity and the role of stress signals such as divalent metal ions in modulating ISP function. We demonstrate that both play roles in defining the proteolytic activity of Bacillus clausii ISP and propose the molecular basis of their effects. Enzyme kinetics reveal that one particular synthetic tetrapeptide substrate, Phe-Ala-Ala-Phe-pNA, is hydrolysed with a catalytic efficiency ∼100-fold higher than any other tested. Heat-denatured whole proteins were found to be better substrates for ISP than the native forms. Substrate binding simulations suggest that the S1, S2 and S4 sites form defined binding pockets. The deep S1 cavity and wide S4 site are fully occupied by the hydrophobic aromatic side-chains of Phe. Divalent metal ions, probably Ca(2+), are proposed to be important for ISP activity through structural changes. The presence of >0.01 mM EDTA inactivates ISP, with CD and SEC suggesting that the protein becomes less structured and potentially monomeric. Removal of Ca(2+) at sites close to the dimer interface and the S1 pocket are thought to be responsible for the effect. These studies provide a new insight into the potential physiological function of ISPs, by reconciling substrate specificity and divalent metal binding to associate ISP with the unfolded protein response under stress conditions. PMID:23650602

  18. Inhibition of spontaneous network activity in neonatal hippocampal slices by energy substrates is not correlated with intracellular acidification.

    PubMed

    Mukhtarov, Marat; Ivanov, Anton; Zilberter, Yuri; Bregestovski, Piotr

    2011-01-01

    Several energy substrates complementary to glucose, including lactate, pyruvate and β-hydroxybutyrate, serve as a fuel for neurons. It was reported recently that these substrates can substantially modulate cortical excitability in neonatal slices. However, complementary energy substrates (CES) can also induce an intracellular acidification when added exogenously. Therefore, action of CES on the neuronal properties governing excitability in neonatal brain slices may be underlain by a change in the cell energy status or by intracellular acidification, or both. Here, we attempt to elucidate these possibilities in neonatal hippocampus by recording neuronal population activity and monitoring intracellular pH. We show that a spontaneous network activity pattern, giant depolarizing potentials (GDPs), characteristic for the neonatal hippocampal slices exposed to artificial cerebrospinal fluid, is strongly inhibited by CES and this effect is unlikely to be caused by a subtle intracellular acidification induced by these compounds. Indeed, a much stronger intracellular acidification in the HCO(3) -free solution inhibited neither the GDP frequency nor the GDP amplitude. Therefore, modulation of neuronal energy homeostasis is the most likely factor underlying the effect of lactate, pyruvate and β-hydroxybutyrate on network excitability in neonatal brain slices.

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

    PubMed

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

    2016-01-01

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

  20. In vivo optical microprobe imaging for intracellular Ca2+ dynamics in response to dopaminergic signaling in deep brain evoked by cocaine

    NASA Astrophysics Data System (ADS)

    Luo, Zhongchi; Pan, Yingtian; Du, Congwu

    2012-02-01

    Ca2+ plays a vital role as second messenger in signal transduction and the intracellular Ca2+ ([Ca2+]i) change is an important indicator of neuronal activity in the brain, including both cortical and subcortical brain regions. Due to the highly scattering and absorption of brain tissue, it is challenging to optically access the deep brain regions (e.g., striatum at >3mm under the brain surface) and image [Ca2+]i changes with cellular resolutions. Here, we present two micro-probe approaches (i.e., microlens, and micro-prism) integrated with a fluorescence microscope modified to permit imaging of neuronal [Ca2+]i signaling in the striatum using a calcium indicator Rhod2(AM). While a micro-prism probe provides a larger field of view to image neuronal network from cortex to striatum, a microlens probe enables us to track [Ca2+]i dynamic change in individual neurons within the brain. Both techniques are validated by imaging neuronal [Ca2+]i changes in transgenic mice with dopamine receptors (D1R, D2R) expressing EGFP. Our results show that micro-prism images can map the distribution of D1R- and D2R-expressing neurons in various brain regions and characterize their different mean [Ca2+]i changes induced by an intervention (e.g., cocaine administration, 8mg/kg., i.p). In addition, microlens images can characterize the different [Ca2+]i dynamics of D1 and D2 neurons in response to cocaine, including new mechanisms of these two types of neurons in striatum. These findings highlight the power of the optical micro-probe imaging for dissecting the complex cellular and molecular insights of cocaine in vivo.

  1. Antagonism of the STING Pathway via Activation of the AIM2 Inflammasome by Intracellular DNA.

    PubMed

    Corrales, Leticia; Woo, Seng-Ryong; Williams, Jason B; McWhirter, Sarah M; Dubensky, Thomas W; Gajewski, Thomas F

    2016-04-01

    Recent evidence has indicated that innate immune sensing of cytosolic DNA in dendritic cells via the host STING pathway is a major mechanism leading to spontaneous T cell responses against tumors. However, the impact of the other major pathway triggered by intracellular DNA, the absent in melanoma 2 (AIM2) inflammasome, on the functional output from the stimulator of IFN genes (STING) pathway is poorly understood. We found that dendritic cells and macrophages deficient in AIM2, apoptosis-associated specklike protein, or caspase-1 produced markedly higher IFN-β in response to DNA. Biochemical analyses showed enhanced generation of cyclic GMP-AMP, STING aggregation, and TANK-binding kinase 1 and IFN regulatory factor 3 phosphorylation in inflammasome-deficient cells. Induction of pyroptosis by the AIM2 inflammasome was a major component of this effect, and inhibition of caspase-1 reduced cell death, augmenting phosphorylation of TANK-binding kinase 1/IFN regulatory factor 3 and production of IFN-β. Our data suggest that in vitro activation of the AIM2 inflammasome in murine macrophages and dendritic cells leads to reduced activation of the STING pathway, in part through promoting caspase-1-dependent cell death.

  2. α-Galactosidase-A Loaded-Nanoliposomes with Enhanced Enzymatic Activity and Intracellular Penetration.

    PubMed

    Cabrera, Ingrid; Abasolo, Ibane; Corchero, José L; Elizondo, Elisa; Gil, Pilar Rivera; Moreno, Evelyn; Faraudo, Jordi; Sala, Santi; Bueno, Dolores; González-Mira, Elisabet; Rivas, Merche; Melgarejo, Marta; Pulido, Daniel; Albericio, Fernando; Royo, Miriam; Villaverde, Antonio; García-Parajo, Maria F; Schwartz, Simó; Ventosa, Nora; Veciana, Jaume

    2016-04-01

    Lysosomal storage disorders (LSD) are caused by lysosomal dysfunction usually as a consequence of deficiency of a single enzyme required for the metabolism of macromolecules, such as lipids, glycoproteins, and mucopolysaccharides. For instance, the lack of α-galactosidase A (GLA) activity in Fabry disease patients causes the accumulation of glycosphingolipids in the vasculature leading to multiple organ pathology. Enzyme replacement therapy, which is the most common treatment of LSD, exhibits several drawbacks mainly related to the instability and low efficacy of the exogenously administered therapeutic enzyme. In this work, the unprecedented increased enzymatic activity and intracellular penetration achieved by the association of a human recombinant GLA to nanoliposomes functionalized with Arginine-Glycine-Aspartic acid (RGD) peptides is reported. Moreover, these new GLA loaded nanoliposomes lead to a higher efficacy in the reduction of the GLA substrate named globotriasylceramide in a cellular model of Fabry disease, than that achieved by the same concentration of the free enzyme. The preparation of these new liposomal formulations by DELOS-SUSP, based on the depressurization of a CO2 -expanded liquid organic solution, shows the great potential of this CO2 -based methodology for the one-step production of protein-nanoliposome conjugates as bioactive nanomaterials with therapeutic interest. PMID:26890358

  3. α-Galactosidase-A Loaded-Nanoliposomes with Enhanced Enzymatic Activity and Intracellular Penetration.

    PubMed

    Cabrera, Ingrid; Abasolo, Ibane; Corchero, José L; Elizondo, Elisa; Gil, Pilar Rivera; Moreno, Evelyn; Faraudo, Jordi; Sala, Santi; Bueno, Dolores; González-Mira, Elisabet; Rivas, Merche; Melgarejo, Marta; Pulido, Daniel; Albericio, Fernando; Royo, Miriam; Villaverde, Antonio; García-Parajo, Maria F; Schwartz, Simó; Ventosa, Nora; Veciana, Jaume

    2016-04-01

    Lysosomal storage disorders (LSD) are caused by lysosomal dysfunction usually as a consequence of deficiency of a single enzyme required for the metabolism of macromolecules, such as lipids, glycoproteins, and mucopolysaccharides. For instance, the lack of α-galactosidase A (GLA) activity in Fabry disease patients causes the accumulation of glycosphingolipids in the vasculature leading to multiple organ pathology. Enzyme replacement therapy, which is the most common treatment of LSD, exhibits several drawbacks mainly related to the instability and low efficacy of the exogenously administered therapeutic enzyme. In this work, the unprecedented increased enzymatic activity and intracellular penetration achieved by the association of a human recombinant GLA to nanoliposomes functionalized with Arginine-Glycine-Aspartic acid (RGD) peptides is reported. Moreover, these new GLA loaded nanoliposomes lead to a higher efficacy in the reduction of the GLA substrate named globotriasylceramide in a cellular model of Fabry disease, than that achieved by the same concentration of the free enzyme. The preparation of these new liposomal formulations by DELOS-SUSP, based on the depressurization of a CO2 -expanded liquid organic solution, shows the great potential of this CO2 -based methodology for the one-step production of protein-nanoliposome conjugates as bioactive nanomaterials with therapeutic interest.

  4. Crystal structure of TRAF1 TRAF domain and its implications in the TRAF1-mediated intracellular signaling pathway

    PubMed Central

    Kim, Chang Min; Choi, Jae Young; Bhat, Eijaz Ahmed; Jeong, Jae-Hee; Son, Young-Jin; Kim, Sunghwan; Park, Hyun Ho

    2016-01-01

    TNF-receptor associated factor (TRAF) proteins are key adaptor molecules containing E3 ubiquitin ligase activity that play a critical role in immune cell signaling. TRAF1 is a unique family of TRAF lacking the N-terminal RING finger domain. TRAF1 is an important scaffold protein that participates in TNFR2 signaling in T cells as a negative or positive regulator via direct interaction with TRAF2, which has recently been identified as a pro-apoptotic regulator in neuronal cell death. Here, we report the first crystal structure of the TRAF1 TRAF domain containing both the TRAF-N coiled-coil domain and the TRAF-C domain. Our structure reveals both similarities and differences with other TRAF family members, which may be functionally relevant to TRAFs. We also found that the TRAF-N coiled-coil domain of TRAF1 is critical for the trimer formation and stability of the protein. Finally, we found that conserved surface residues on the TRAF1 TRAF domain that might be binding hot spots that are critical for interaction with signaling molecules. PMID:27151821

  5. Carbon dioxide, membrane potential and intracellular potassium activity in frog skeletal muscle.

    PubMed Central

    Huguenin, F; Reber, W; Zeuthen, T

    1980-01-01

    1. The membrane potential of isolated frog muscle fibres has been measured in absence and in presence of CO2, at constant external pH. 2. At a normal external Cl concentration, CO2 (PCO2 = 97 mmHg; pH = 7.0) applied for 10 min caused a highly variable depolarization, the average potential change being 8 mV after 5 min. The effect was reversible 3. In Cl-free solutions, CO2 (PCO2 = 97 mmHg; pH = 7.0) caused a biphasic depolarization of 20 mV after 5 min. The effect was fully reversible on CO2 removal. 4. The same effect appeared at a lower partial pressure (PCO2 = 38 mmHg; pH 7.3) in the presence of tetrodotoxin (10(-7) M). 5. In order to investigate the cause of the CO2-induced depolarization, membrane potential and intracellular K activity (ai/K,)( of surface muscle fibres were measured with voltage and ion-sensitive micro-electrodes. 6. At a normal external Cl concentration, CO2(PCO2 approximately equal to 97 mmHg; pH = 7.0) decreased ai/K by 5 mM after 5 min. 7. The same effect appeared at low external Cl concentration (11 mM). 8. At high partial pressure (PCO2 approximately equal to 588 mmHg; pH = 6.8), CO2 reduced ai/K by 19 mM in 10 min. 9. In long-term experiments performed over 4 h with a normal external Cl concentration, CO2 (PCO2 approximately equal to 97 mmHg; pH 5.8 or 7) changed practically neither membrane potential, nor ai/K. 10. It is concluded that increasing the PCO2 when keeping the external pH constant causes an early depolarization of muscle. This effect is particularly marked in the absence of chloride. It can be explained partly, in surface muscle fibres, by a decrease of the intracellular K activity. PMID:6776259

  6. Diethyldithiocarbamate induces apoptosis in neuroblastoma cells by raising the intracellular copper level, triggering cytochrome c release and caspase activation.

    PubMed

    Matias, Andreza C; Manieri, Tânia M; Cipriano, Samantha S; Carioni, Vivian M O; Nomura, Cassiana S; Machado, Camila M L; Cerchiaro, Giselle

    2013-02-01

    Dithiocarbamates are nitrogen- and sulfur-containing compounds commonly used in pharmacology, medicine and agriculture. The molecular effects of dithiocarbamates on neuronal cell systems are not fully understood, especially in terms of their ability to accumulate copper ions inside the cell. In this work, the molecular effects of N,N-diethyldithiocarbamate (DEDTC) were studied in human SH-SY5Y neuroblastoma cells to determine the role of copper in the DEDTC toxicity and the pathway trigged in cell by the complex Cu-DEDTC. From concentration-dependent studies, we found that 5 μM of this compound induced a drastic decrease in viable cells with a concomitant accumulation in intracellular copper resulted from complexation with DEDTC, measured by atomic absorption spectroscopy. The mechanism of DEDTC-induced apoptosis in neuronal model cells is thought to occur through the death receptor signaling triggered by DEDTC-copper complex in low concentration that is associated with the activation of caspase 8. Our results indicated that the mechanism of cell death involves cytochrome c release forming the apoptosome together with Apaf-1 and caspase 9, converting the caspase 9 into its active form, allowing it to activate caspase 3 as observed by immunofluorescence. This pathway is induced by the cytotoxic effects that occur when DEDTC forms a complex with the copper ions present in the culture medium and transports them into the cell, suggesting that the DEDTC by itself was not able to cause cell death and the major effect is from its copper-complex in neuroblastoma cells. The present study suggests a role for the influence of copper by low concentrations of DEDTC in the extracellular media, the absorption and accumulation of copper in the cell and apoptotic events, induced by the cytotoxic effects that occur when DEDTC forms a complex with the copper ions. PMID:22951949

  7. Signal transduction activated by cannabinoid receptors.

    PubMed

    Díaz-Laviada, Inés; Ruiz-Llorente, Lidia

    2005-07-01

    Since the discovery that cannabinoids exert biological actions through binding to specific receptors, signal mechanisms triggered by these receptors have been focus of extensive study. This review summarizes the current knowledge of the signalling events produced by cannabinoids from membrane receptors to downstream regulators. Two types of cannabinoid receptors have been identified to date: CB(1) and CB(2) both belonging to the heptahelichoidal receptor family but with different tissue distribution and signalling mechanisms. Coupling to inhibitory guanine nucleotide-binding protein and thus inhibition of adenylyl cyclase has been observed in both receptors but other signal transduction pathways that are regulated or not by these G proteins are differently activated upon ligand-receptor binding including ion channels, sphingomyelin hydrolysis, ceramide generation, phospholipases activation and downstream targets as MAP kinase cascade, PI3K, FAK or NOS regulation. Cannabinoids may also act independently of CB(1)or CB(2) receptors. The existence of new unidentified putative cannabinoid receptors has been claimed by many investigators. Endocannabinoids activate vanilloid TRPV1 receptors that may mediate some of the cannabinoid effects. Other actions of cannabinoids can occur through non-receptor-mediated mechanisms.

  8. Depletion of Intracellular Thiols and Increased Production of 4-Hydroxynonenal that Occur During Cryopreservation of Stallion Spermatozoa Lead to Caspase Activation, Loss of Motility, and Cell Death.

    PubMed

    Martin Muñoz, Patricia; Ortega Ferrusola, Cristina; Vizuete, Guillermo; Plaza Dávila, Maria; Rodriguez Martinez, Heriberto; Peña, Fernando J

    2015-12-01

    Oxidative stress has been linked to sperm death and the accelerated senescence of cryopreserved spermatozoa. However, the molecular mechanisms behind this phenomenon remain poorly understood. Reactive oxygen species (ROS) are considered relevant signaling molecules for sperm function, only becoming detrimental when ROS homeostasis is lost. We hereby hypothesize that a major component of the alteration of ROS homeostasis in cryopreserved spermatozoa is the exhaustion of intrinsic antioxidant defense mechanisms. To test this hypothesis, semen from seven stallions was frozen using a standard technique. The parameters of sperm quality (motility, velocity, and membrane integrity) and markers of sperm senescence (caspase 3, 4-hydroxynonenal, and mitochondrial membrane potential) were assessed before and after cryopreservation. Changes in the intracellular thiol content were also monitored. Cryopreservation caused significant increases in senescence markers as well as dramatic depletion of intracellular thiols to less than half of the initial values (P < 0.001) postthaw. Interestingly, very high and positive correlations were observed among thiol levels with sperm functionality postthaw: total motility (r = 0.931, P < 0.001), progressive motility (r = 0.904, P < 0.001), and percentage of live spermatozoa without active caspase 3 (r = 0.996, P < 0.001). In contrast, negative correlations were detected between active caspase 3 and thiol content both in living (r = -0.896) and dead (r = -0.940) spermatozoa; additionally, 4-hydroxynonenal levels were negatively correlated with thiol levels (r = -0.856). In conclusion, sperm functionality postthaw correlates with the maintenance of adequate levels of intracellular thiols. The accelerated senescence of thawed spermatozoa is related to oxidative and electrophilic stress induced by increased production of 4-hydroxynoneal in thawed samples once intracellular thiols are depleted.

  9. Differentiation Between Intracellular and Cell Surface Glycosyl Transferases: Galactosyl Transferase Activity in Intact Cells and in Cell Homogenate

    PubMed Central

    Deppert, Wolfgang; Werchau, Hermann; Walter, Gernot

    1974-01-01

    Intact BHK (baby hamster kidney) cells catalyze the hydrolysis of UDP-galactose to free galactose. The generation of galactose from UDP-galactose and its intracellular utilization impede the detection of possible galactosyl transferases on the cell surface of intact cells. Several independent procedures have been used to distinguish between intracellular and cell surface glycosyl transferases. With these procedures, no evidence was obtained for the presence of detectable amounts of galactosyl transferase activity on the surface of BHK cells. The data suggest that galactosyl transferases do not play a general role in the phenomena of cell adhesion and contact inhibition. PMID:4528509

  10. Diquafosol promotes corneal epithelial healing via intracellular calcium-mediated ERK activation.

    PubMed

    Byun, Yong-Soo; Yoo, Young-Sik; Kwon, Ji-Young; Joo, Jong-Soo; Lim, Sung-A; Whang, Woong-Joo; Mok, Jee-Won; Choi, Jun-Sub; Joo, Choun-Ki

    2016-02-01

    Diquafosol is known as a purinergic P2Y2 receptor (P2Y2R) agonist that stimulates water and mucin secretion from conjunctival epithelial cells and goblet cells, leading to tear film stability in dry eye. However, its effect on corneal epithelial healing has not yet been elucidated. The aim of the present study was to evaluate the effect of diquafosol on corneal epithelial healing in vivo and on P2Y2R-related downstream signaling pathways in vitro. We administered 3% diquafosol ophthalmic solution on 3 mm-diameter epithelial defects made in rat corneas and assessed the wound closure over time. Corneal epithelial healing was significantly accelerated in diquafosol-treated eyes compared to control eyes at 12 and 24 h. During wound healing, P2Y2R staining appeared stronger in the re-epithelized margin near the wound defect. To evaluate whether diquafosol stimulates epidermal growth factor receptor/extracellular-signal-regulated kinase (EGFR/ERK)-related cell proliferation and migration, simian virus 40-transfected human corneal epithelial (THCE) cells were used for in vitro experiments. Cell proliferation was accelerated by diquafosol at concentrations from 20 to 200 μM during 48 h, but inhibited at concentrations over 2000 μM. The intracellular calcium ([Ca(2+)]i) elevation was measured in diquafosol (100 μM)-stimulated cells using Fluo-4/AM ([Ca(2+)]i indicator). [Ca(2+)]i elevation was observed in diquafosol-stimulated cells regardless of the presence of calcium in media, and suramin pretreatment inhibited the calcium response. The effect of diquafosol on phosphorylation of EGFR, ERK and Akt, and cell migration was determined by western blotting and in vitro cell migration assay. Diquafosol induced phosphorylation of EGFR at 2 min post-stimulation, and phosphorylation of ERK at 5 min post-stimulation. Phosphorylation of ERK was attenuated in cells pretreated with suramin or BAPTA/AM ([Ca(2+)]i chelator), and partially with AG1478 (EGFR inhibitor

  11. Nuclease Activity of Legionella pneumophila Cas2 Promotes Intracellular Infection of Amoebal Host Cells

    PubMed Central

    Gunderson, Felizza F.; Mallama, Celeste A.; Fairbairn, Stephanie G.

    2014-01-01

    Legionella pneumophila, the primary agent of Legionnaires' disease, flourishes in both natural and man-made environments by growing in a wide variety of aquatic amoebae. Recently, we determined that the Cas2 protein of L. pneumophila promotes intracellular infection of Acanthamoeba castellanii and Hartmannella vermiformis, the two amoebae most commonly linked to cases of disease. The Cas2 family of proteins is best known for its role in the bacterial and archeal clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein (Cas) system that constitutes a form of adaptive immunity against phage and plasmid. However, the infection event mediated by L. pneumophila Cas2 appeared to be distinct from this function, because cas2 mutants exhibited infectivity defects in the absence of added phage or plasmid and since mutants lacking the CRISPR array or any one of the other cas genes were not impaired in infection ability. We now report that the Cas2 protein of L. pneumophila has both RNase and DNase activities, with the RNase activity being more pronounced. By characterizing a catalytically deficient version of Cas2, we determined that nuclease activity is critical for promoting infection of amoebae. Also, introduction of Cas2, but not its catalytic mutant form, into a strain of L. pneumophila that naturally lacks a CRISPR-Cas locus caused that strain to be 40- to 80-fold more infective for amoebae, unequivocally demonstrating that Cas2 facilitates the infection process independently of any other component encoded within the CRISPR-Cas locus. Finally, a cas2 mutant was impaired for infection of Willaertia magna but not Naegleria lovaniensis, suggesting that Cas2 promotes infection of most but not all amoebal hosts. PMID:25547789

  12. Intracellular mediators of Na -K pump activity in guinea pig pancreatic acinar cells

    SciTech Connect

    Hootman, S.R.; Ochs, D.L.; Williams, J.A.

    1985-10-01

    The involvement of CaS and cyclic nucleotides in neurohormonal regulation of Na -K -ATPase (Na -K pump) activity in guinea pig pancreatic acinar cells was investigated. Changes in Na+-K+ pump activity elicited by secretagogues were assessed by (3H)ouabain binding and by ouabain-sensitive YWRb uptake. Carbachol (CCh) and cholecystokinin octapeptide (CCK-8) each stimulated both ouabain-sensitive 86Rb+ uptake and equilibrium binding of (TH)ouabain by approximately 60%. Secretin increased both indicators of Na+-K+ pump activity by approximately 40% as did forskolin, 8-bromo- and dibutyryl cAMP, theophylline, and isobutylmethylxanthine. Incubation of acinar cells in CaS -free HEPES-buffered Ringer (HR) with 0.5 mM EGTA reduced the stimulatory effects of CCh and CCK-8 by up to 90% but caused only a small reduction in the effects of secretin, forskolin, and cAMP analogues. In addition, CCh, CCK-8, secretin, and forskolin each stimulated ouabain-insensitive 86Rb+ uptake by acinar cells. The increase elicited by CCh and CCK-8 was greatly reduced in the absence of extracellular CaS , while that caused by the latter two agents was not substantially altered. The effects of secretagogues on free CaS levels in pancreatic acinar cells also were investigated with quin-2, a fluorescent CaS chelator. Basal intracellular CaS concentration ((CaS )i) was 161 nM in resting cells and increased to 713 and 803 nM within 15 s after addition of 100 microM CCh or 10 nM CCK-8, respectively.

  13. Ziram and Sodium N,N-Dimethyldithiocarbamate Inhibit Ubiquitin Activation through Intracellular Metal Transport and Increased Oxidative Stress in HEK293 Cells

    PubMed Central

    2015-01-01

    Ubiquitin activating enzyme E1 plays a pivotal role in ubiquitin based protein signaling through regulating the initiating step of the cascade. Previous studies demonstrated that E1 is inhibited by covalent modification of reactive cysteines contained within the ubiquitin-binding groove and by conditions that increase oxidative stress and deplete cellular antioxidants. In this study, we determined the relative contribution of covalent adduction and oxidative stress to E1 inhibition produced by ziram and sodium N,N-dimethyldithiocarbamate (DMDC) in HEK293 cells. Although no dithiocarbamate-derived E1 adducts were identified on E1 using shotgun LC/MS/MS for either ziram or DMDC, both dithiocarbamates significantly decreased E1 activity, with ziram demonstrating greater potency. Ziram increased intracellular levels of zinc and copper, DMDC increased intracellular levels of only copper, and both dithiocarbamates enhanced oxidative injury evidenced by elevated levels of protein carbonyls and expression of heme oxygenase-1. To assess the contribution of intracellular copper transport to E1 inhibition, coincubations were performed with the copper chelator triethylenetetramine hydrochloride (TET). TET significantly protected E1 activity for both of the dithiocarbamates and decreased the associated oxidative injury in HEK293 cells as well as prevented dithiocarbamate-mediated lipid peroxidation assayed using an ethyl aracidonate micelle system. Because TET did not completely ameliorate intracellular transport of copper or zinc for ziram, TET apparently maintained E1 activity through its ability to diminish dithiocarbamate-mediated oxidative stress. Experiments to determine the relative contribution of elevated intracellular zinc and copper were performed using a metal free incubation system and showed that increases in either metal were sufficient to inhibit E1. To evaluate the utility of the HEK293 in vitro system for screening environmental agents, a series of additional

  14. Ziram and sodium N,N-dimethyldithiocarbamate inhibit ubiquitin activation through intracellular metal transport and increased oxidative stress in HEK293 cells.

    PubMed

    Dennis, Kathleen E; Valentine, William M

    2015-04-20

    Ubiquitin activating enzyme E1 plays a pivotal role in ubiquitin based protein signaling through regulating the initiating step of the cascade. Previous studies demonstrated that E1 is inhibited by covalent modification of reactive cysteines contained within the ubiquitin-binding groove and by conditions that increase oxidative stress and deplete cellular antioxidants. In this study, we determined the relative contribution of covalent adduction and oxidative stress to E1 inhibition produced by ziram and sodium N,N-dimethyldithiocarbamate (DMDC) in HEK293 cells. Although no dithiocarbamate-derived E1 adducts were identified on E1 using shotgun LC/MS/MS for either ziram or DMDC, both dithiocarbamates significantly decreased E1 activity, with ziram demonstrating greater potency. Ziram increased intracellular levels of zinc and copper, DMDC increased intracellular levels of only copper, and both dithiocarbamates enhanced oxidative injury evidenced by elevated levels of protein carbonyls and expression of heme oxygenase-1. To assess the contribution of intracellular copper transport to E1 inhibition, coincubations were performed with the copper chelator triethylenetetramine hydrochloride (TET). TET significantly protected E1 activity for both of the dithiocarbamates and decreased the associated oxidative injury in HEK293 cells as well as prevented dithiocarbamate-mediated lipid peroxidation assayed using an ethyl aracidonate micelle system. Because TET did not completely ameliorate intracellular transport of copper or zinc for ziram, TET apparently maintained E1 activity through its ability to diminish dithiocarbamate-mediated oxidative stress. Experiments to determine the relative contribution of elevated intracellular zinc and copper were performed using a metal free incubation system and showed that increases in either metal were sufficient to inhibit E1. To evaluate the utility of the HEK293 in vitro system for screening environmental agents, a series of additional

  15. Ziram and sodium N,N-dimethyldithiocarbamate inhibit ubiquitin activation through intracellular metal transport and increased oxidative stress in HEK293 cells.

    PubMed

    Dennis, Kathleen E; Valentine, William M

    2015-04-20

    Ubiquitin activating enzyme E1 plays a pivotal role in ubiquitin based protein signaling through regulating the initiating step of the cascade. Previous studies demonstrated that E1 is inhibited by covalent modification of reactive cysteines contained within the ubiquitin-binding groove and by conditions that increase oxidative stress and deplete cellular antioxidants. In this study, we determined the relative contribution of covalent adduction and oxidative stress to E1 inhibition produced by ziram and sodium N,N-dimethyldithiocarbamate (DMDC) in HEK293 cells. Although no dithiocarbamate-derived E1 adducts were identified on E1 using shotgun LC/MS/MS for either ziram or DMDC, both dithiocarbamates significantly decreased E1 activity, with ziram demonstrating greater potency. Ziram increased intracellular levels of zinc and copper, DMDC increased intracellular levels of only copper, and both dithiocarbamates enhanced oxidative injury evidenced by elevated levels of protein carbonyls and expression of heme oxygenase-1. To assess the contribution of intracellular copper transport to E1 inhibition, coincubations were performed with the copper chelator triethylenetetramine hydrochloride (TET). TET significantly protected E1 activity for both of the dithiocarbamates and decreased the associated oxidative injury in HEK293 cells as well as prevented dithiocarbamate-mediated lipid peroxidation assayed using an ethyl aracidonate micelle system. Because TET did not completely ameliorate intracellular transport of copper or zinc for ziram, TET apparently maintained E1 activity through its ability to diminish dithiocarbamate-mediated oxidative stress. Experiments to determine the relative contribution of elevated intracellular zinc and copper were performed using a metal free incubation system and showed that increases in either metal were sufficient to inhibit E1. To evaluate the utility of the HEK293 in vitro system for screening environmental agents, a series of additional

  16. Cationic triblock copolymer micelles enhance antioxidant activity, intracellular uptake and cytotoxicity of curcumin.

    PubMed

    Yoncheva, Krassimira; Kamenova, Katya; Perperieva, Teodora; Hadjimitova, Vera; Donchev, Petar; Kaloyanov, Kaloyan; Konstantinov, Spiro; Kondeva-Burdina, Magdalena; Tzankova, Virginia; Petrov, Petar

    2015-07-25

    The aim of the present study was to develop curcumin loaded cationic polymeric micelles and to evaluate their loading, preservation of curcumin antioxidant activity and intracellular uptake ability. The micelles were prepared from a triblock copolymer consisting of poly(ϵ-caprolactone) and very short poly(2-(dimethylamino) ethyl methacrylate) segments (PDMAEMA9-PCL70-PDMAEMA9). The micelles showed monomodal size distribution, mean diameter of 145 nm, positive charge (+72 mV), critical micellar concentration around 0.05 g/l and encapsulation efficiency of 87%. The ability of the micellar curcumin to scavenge the ABTS radical and hypochlorite ions was higher than that of the free curcumin. Confocal microscopy revealed that the uptake of curcumin by chronic myeloid leukemia derived K-562 cells and human multiple myeloma cells U-266 was more intensive when curcumin was loaded into the micelles. These results correlated with the higher cytotoxicity of the micellar curcumin compared to free curcumin. Intraperitoneal treatment of Wistar rats indicated that PDMAEMA-PCL-PDMAEMA copolymer, comprising very short cationic chains, did not change the levels of malondialdehyde and glutathione in livers indicating an absence of oxidative stress. Thus, PDMAEMA-PCL-PDMAEMA triblock micelles could be considered efficient and safe platform for curcumin delivery. PMID:26026253

  17. Electrogenic glutamate uptake in glial cells is activated by intracellular potassium

    NASA Astrophysics Data System (ADS)

    Barbour, Boris; Brew, Helen; Attwell, David

    1988-09-01

    Uptake of glutamate into glial cells in the CNS maintains the extracellular glutamate concentration below neurotoxic levels and helps terminate its action as a neurotransmitter 1. The co-transport of two sodium ions on the glutamate carrier is thought to provide the energy needed to transport glutamate into cells2,3. We have shown recently that glutamate uptake can be detected electrically because the excess of Na+ ions transported with each glutamate anion results in a net current flow into the cell4. We took advantage of the control of the environment, both inside and outside the cell, provided by whole-cell patch-clamping and now report that glutamate uptake is activated by intracellular potassium and inhibited by extracellular potassium. Our results indicate that one K+ ion is transported out of the cell each time a glutamate anion and three Na+ ions are transported in. A carrier with this stoichiometry can accumulate glutamate against a much greater concentration gradient than a carrier co-transporting one glutamate anion and two Na+ ions. Pathological rises in extracellular potassium concentration will inhibit glutamate uptake by depolarizing glial cells and by preventing the loss of K+ from the glutamate carrier. This will facilitate a rise in the extracellular glutamate concentration to neurotoxic levels and contribute to the neuronal death occurring in brain anoxia and ischaemia.

  18. Anti-angiogenic activity and intracellular distribution of epigallocatechin-3-gallate analogs.

    PubMed

    Piyaviriyakul, Suratsawadee; Shimizu, Kosuke; Asakawa, Tomohiro; Kan, Toshiyuki; Siripong, Pongpun; Oku, Naoto

    2011-01-01

    Angiogenesis, a process of construction of new blood capillaries, is crucial for tumor progression and metastasis. Our previous studies demonstrated that a component of green tea, epigallocatechin-3-gallate (EGCG), suppressed angiogenesis and subsequent tumor growth. In this study, to elucidate the detailed mechanism of the anti-angiogenic effect of EGCG and to enhance the antiangiogenic activity of EGCG, we designed and synthesized EGCG derivatives and examined their biological effect and intracellular localization in human umbilical vein endothelial cells (HUVECs). EGCG derivatives aminopentyl dideoxyEGCG and aminopentyl dideoxygallocatechin-3-gallate (cis-APDOEGCG and trans-APDOEGCG) had an enhanced inhibitory effect on the proliferation when used at more than 30 µM. To elucidate antiangiogenic effect of EGCG, we used a 1 µM concentration for subsequent experiments where no effect on proliferation was observed. These EGCG derivatives showed a stronger inhibitory effect on migration, invasion, and tube formation by HUVECs than the non-derivatized EGCG. Furthermore, the derivatives induced a change in the distribution of F-actin and subsequent morphology of the HUVECs. Next, we synthesized fluorescent TokyoGreen-conjugated EGCG derivative (EGCG-TG) and observed the distribution in HUVECs under a confocal laser scanning microscope. Abundant fluorescence was observed in the cells after a 3-h incubation, and was localized in mitochondria as well as in cytoplasm. These results suggest that EGCG was incorporated into the HUVECs, that a portion of it entered into their mitochondria. PMID:21372391

  19. Parathyroid hormone inhibition of Na{sup +}/H{sup +} exchanger 3 transcription: Intracellular signaling pathways and transcription factor expression

    SciTech Connect

    Neri, Elida Adalgisa; Bezerra, Camila Nogueira Alves Queiroz-Leite, Gabriella Duarte; Polidoro, Juliano Zequini; Rebouças, Nancy Amaral

    2015-06-12

    The main transport mechanism of reabsorption of sodium bicarbonate and fluid in the renal proximal tubules involves Na{sup +}/H{sup +} exchanger 3 (NHE3), which is acutely and chronically downregulated by parathyroid hormone (PTH). Although PTH is known to exert an inhibitory effect on NHE3 expression and transcription, the molecular mechanisms involved remain unclear. Here, we demonstrated that, in opossum kidney proximal tubule (OKP) cells, PTH-induced inhibition of Nhe3 gene promoter occurs even in the core promoter that controls expression of the reporter gene. We found that inhibition of the protein kinase A (PKA) and Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways transformed PTH from an inhibitor of promoter activity into an activator of that same activity, as did point mutations in the EGR1, Sp1, and Sp3 binding consensus elements in the promoter. In nuclear extracts of PTH-treated OKP cells, we also observed increased expression of EGR1 mRNA and of some Sp3 isoforms. Electrophoretic mobility shift assay showed a supershift of the −61 to −42-bp probe with an anti-EGR1 antibody in PTH-treated cells, suggesting that EGR1 binding is relevant for the inhibitory activity of PTH. We conclude that PTH-induced inhibition of NHE3 transcription is related to higher EGR1 expression; to EGR1 binding to the proximal and core promoters; and to PKA and JAK/STAT pathway activation. This mechanism might be responsible, at least in part, for lower NHE3 expression and sodium reabsorption in renal proximal tubules in the presence of high PTH levels. - Highlights: • PTH regulation of Nhe3 promoter depends on EGR1 binding. • EGR1, PKA and JAK/STAT are involved in PTH inhibition of the Nhe3 promoter. • PTH alters expression of EGR1 and Sp3. • PTH inhibits the Nhe3 promoter by regulating PKA and JAK/STAT signaling.

  20. Methylmercury-induced toxicity is mediated by enhanced intracellular calcium through activation of phosphatidylcholine-specific phospholipase C

    SciTech Connect

    Kang, Mi Sun; Jeong, Ju Yeon; Seo, Ji Heui; Jeon, Hyung Jun; Jung, Kwang Mook; Chin, Mi-Reyoung; Moon, Chang-Kiu; Bonventre, Joseph V.; Jung, Sung Yun; Kim, Dae Kyong . E-mail: proteinlab@hanmail.net

    2006-10-15

    Methylmercury (MeHg) is a ubiquitous environmental toxicant to which humans can be exposed by ingestion of contaminated food. MeHg has been suggested to exert its toxicity through its high reactivity to thiols, generation of arachidonic acid and reactive oxygen species (ROS), and elevation of free intracellular Ca{sup 2+} levels ([Ca{sup 2+}]{sub i}). However, the precise mechanism has not been fully defined. Here we show that phosphatidylcholine-specific phospholipase C (PC-PLC) is a critical pathway for MeHg-induced toxicity in MDCK cells. D609, an inhibitor of PC-PLC, significantly reversed the toxicity in a time- and dose-dependent manner with concomitant inhibition of the diacylglycerol (DAG) generation and the phosphatidylcholine (PC)-breakdown. MeHg activated the group IV cytosolic phospholipase A{sub 2} (cPLA{sub 2}) and acidic form of sphingomyelinase (A-SMase) downstream of PC-PLC, but these enzymes as well as protein kinase C (PKC) were not linked to the toxicity by MeHg. Furthermore, MeHg produced ROS, which did not affect the toxicity. Addition of EGTA to culture media resulted in partial decrease of [Ca{sup 2+}]{sub i} and partially blocked the toxicity. In contrast, when the cells were treated with MeHg in the presence of Ca{sup 2+} in the culture media, D609 completely prevented cell death with parallel decrease in [Ca{sup 2+}]{sub i}. Our results demonstrated that MeHg-induced toxicity was linked to elevation of [Ca{sup 2+}]{sub i} through activation of PC-PLC, but not attributable to the signaling pathways such as cPLA{sub 2}, A-SMase, and PKC, or to the generation of ROS.

  1. Enhanced multistatic active sonar signal processing.

    PubMed

    Zhao, Kexin; Liang, Junli; Karlsson, Johan; Li, Jian

    2013-07-01

    Multistatic active sonar systems involve the transmission and reception of multiple probing sequences and can achieve significantly enhanced performance of target detection and localization through exploiting spatial diversity. This paper mainly focuses on two signal processing aspects of such systems, namely, enhanced range-Doppler imaging and improved target parameter estimation. The main contributions of this paper are (1) a hybrid dense-sparse method is proposed to generate range-Doppler images with both low sidelobe levels and high accuracy; (2) a generalized K-Means clustering (GKC) method for target association is developed to associate the range measurements from different transmitter-receiver pairs, which is actually a range fitting procedure; (3) the extended invariance principle-based weighted least-squares method is developed for accurate target position and velocity estimation. The effectiveness of the proposed multistatic active sonar signal processing techniques is verified using numerical examples.

  2. An arsenate-activated glutaredoxin from the arsenic hyperaccumulator fern Pteris vittata L. regulates intracellular arsenite.

    PubMed

    Sundaram, Sabarinath; Rathinasabapathi, Bala; Ma, Lena Q; Rosen, Barry P

    2008-03-01

    To elucidate the mechanisms of arsenic resistance in the arsenic hyperaccumulator fern Pteris vittata L., a cDNA for a glutaredoxin (Grx) Pv5-6 was isolated from a frond expression cDNA library based on the ability of the cDNA to increase arsenic resistance in Escherichia coli. The deduced amino acid sequence of Pv5-6 showed high homology with an Arabidopsis chloroplastic Grx and contained two CXXS putative catalytic motifs. Purified recombinant Pv5-6 exhibited glutaredoxin activity that was increased 1.6-fold by 10 mm arsenate. Site-specific mutation of Cys(67) to Ala(67) resulted in the loss of both GRX activity and arsenic resistance. PvGrx5 was expressed in E. coli mutants in which the arsenic resistance genes of the ars operon were deleted (strain AW3110), a deletion of the gene for the ArsC arsenate reductase (strain WC3110), and a strain in which the ars operon was deleted and the gene for the GlpF aquaglyceroporin was disrupted (strain OSBR1). Expression of PvGrx5 increased arsenic tolerance in strains AW3110 and WC3110, but not in OSBR1, suggesting that PvGrx5 had a role in cellular arsenic resistance independent of the ars operon genes but dependent on GlpF. AW3110 cells expressing PvGrx5 had significantly lower levels of arsenite when compared with vector controls when cultured in medium containing 2.5 mm arsenate. Our results are consistent with PvGrx5 having a role in regulating intracellular arsenite levels, by either directly or indirectly modulating the aquaglyceroporin. To our knowledge, PvGrx5 is the first plant Grx implicated in arsenic metabolism.

  3. Intracellular Signal Transduction and Modification of the Tumor Microenvironment Induced by RET/PTCs in Papillary Thyroid Carcinoma

    PubMed Central

    Menicali, Elisa; Moretti, Sonia; Voce, Pasquale; Romagnoli, Serena; Avenia, Nicola; Puxeddu, Efisio

    2012-01-01

    RET gene rearrangements (RET/PTCs) represent together with BRAF point mutations the two major groups of mutations involved in papillary thyroid carcinoma (PTC) initiation and progression. In this review, we will examine the mechanisms involved in RET/PTC-induced thyroid cell transformation. In detail, we will summarize the data on the molecular mechanisms involved in RET/PTC formation and in its function as a dominant oncogene, on the activated signal transduction pathways and on the induced gene expression modifications. Moreover, we will report on the effects of RET/PTCs on the tumor microenvironment. Finally, a short review of the literature on RET/PTC prognostic significance will be presented. PMID:22661970

  4. Evaluation of potential implication of membrane estrogen binding sites on ERE-dependent transcriptional activity and intracellular estrogen receptor-alpha regulation in MCF-7 breast cancer cells.

    PubMed

    Seo, Hye Sook; Leclercq, Guy

    2002-01-01

    The potential involvement of membrane estrogen binding sites in the induction of ERE-dependent transcriptional activity as well as in the regulation of intracellular estrogen receptor alpha (ER-alpha) level under estradiol (E2) stimulation was investigated. Our approach relied upon the use of two DCC-treated E2-BSA (bovine serum albumin) solutions (E2-6-BSA and E2-17-BSA). The absence of detectable free E2 in these solutions was established. Both E2-BSA conjugates led to a transient dose-dependent stimulation of the expression of ERE-luciferase (LUC) reporter gene in MVLN cells (MCF-7 cells stably transfected with a pVit-tk-LUC reporter plasmid), a property not recorded with free E2, which maintained enhanced transcriptional activity during the whole experiment. A very low concentration of E2 (10 pM) synergistically acted with E2-BSA conjugates. Hence, ERE-dependent transcriptional activity induced by these conjugates appeared to result from their known interactions with membrane estrogen binding sites. Anti-estrogens (AEs: 4-OH-TAM and RU 58,668), which antagonize genomic ER responses, abrogated the luciferase activity induced by E2-BSA conjugates, confirming a potential relationship between membrane-related signals and intracellular ER. Moreover, induction of luciferase was recorded when the cells were exposed to IBMX (3-isobutyl-1-methylxanthine) and cyclic nucleotides (cAMP/cGMP), suggesting the implication of the latter in the signal transduction pathway leading to the expression of the reporter gene. Growth factors (IGF-I, EGF and TGF-alpha) also slightly stimulated luciferase and synergistically acted with 10 pM E2, or 1 microM E2-BSA conjugates, in agreement with the concept of a cross-talk between steroids and peptides acting on the cell membrane. Remarkably, E2-BSA conjugates, IBMX and all investigated growth factors failed to down-regulate intracellular ER in MCF-7 cells, indicating the need for a direct intracellular interaction of the ligand with the

  5. Lack of regulation by intracellular Ca2+ of the hyperpolarization-activated cation current in rat thalamic neurones.

    PubMed Central

    Budde, T; Biella, G; Munsch, T; Pape, H C

    1997-01-01

    1. The regulation of the hyperpolarization-activated cation current, Ih, in thalamocortical neurones by intracellular calcium ions has been implemented in a number of mathematical models on the waxing and waning behaviour of synchronized rhythmic activity in thalamocortical circuits. In the present study, the Ca2+ dependence of Ih in thalamocortical neurones was experimentally investigated by combining Ca2+ imaging and patch-clamp techniques in the ventrobasal thalamic complex (VB) in vitro. 2. Properties of Ih were analysed before and during rhythmic stimulation of Ca2+ entry by trains of depolarizing voltage pulses. Despite a significant increase in intracellular Ca2+ concentration ([Ca2+]i) from resting levels of 74 +/- 23 nM to 251 +/- 78 nM upon rhythmic stimulation, significant differences in the voltage dependence of Ih activation did not occur (half-maximal activation at -86.4 +/- 1.3 mV vs. -85.2 +/- 2.9 mV; slope of the activation curve, 11.2 +/- 2.4 mV vs. 12.5 +/- 2.5 mV). Recording of Ih with predefined values of [Ca2+]i (13.2 nM or 10.01 microM in the patch pipette) revealed no significant differences in the activation curve or the fully activated I-V relationship of Ih. 3. In comparison, stimulation of the intracellular cyclic adenosine monophosphate (cAMP) pathway induced a significantly positive shift in Ih voltage dependence of +5.1 +/- 1.9 mV, with no alteration in the fully activated I-V relationship. 4. These data argue against a direct regulation of Ih by intracellular Ca2+, and particularly do not support a primary role of Ca(2+)-dependent modulation of the Ih channels in the waxing and waning of sleep spindle oscillations in thalamocortical neurones. Images Figure 1 PMID:9288676

  6. Detection of Intracellular Selenol-Containing Molecules Using a Fluorescent Probe with Near-Zero Background Signal.

    PubMed

    Sun, Qi; Yang, Shu-Hou; Wu, Lei; Dong, Qing-Jian; Yang, Wen-Chao; Yang, Guang-Fu

    2016-06-01

    Selenocysteine (Sec), encoded as the 21st amino acid, is the predominant chemical form of selenium that is closely related to various human diseases. Thus, it is of high importance to identify novel probes for sensitive and selective recognition of Sec and Sec-containing proteins. Although a few probes have been reported to detect artificially introduced selenols in cells or tissues, none of them has been shown to be sensitive enough to detect endogenous selenols. We report the characterization and application of a new fluorogenic molecular probe for the detection of intracellular selenols. This probe exhibits near-zero background fluorescence but produces remarkable fluorescence enhancement upon reacting with selenols in a fast chemical reaction. It is highly specific and sensitive for intracellular selenium-containing molecules such as Sec and selenoproteins. When combined with flow cytometry, this probe is able to detect endogenous selenols in various human cancer cells. It is also able to image endogenous selenol-containing molecules in zebrafish under a fluorescence microscope. These results demonstrate that this molecular probe can function as a useful molecular tool for intracellular selenol sensing, which is valuable in the clinical diagnosis for human diseases associated with Sec-deficiency or overdose. PMID:27161304

  7. Disentangling between stellar activity and planetary signals

    NASA Astrophysics Data System (ADS)

    Boisse, I.; Bouchy, F.; Hébrard, G.; Bonfils, X.; Santos, N.; Vauclair, S.

    2011-04-01

    Photospheric stellar activity (i.e. dark spots or bright plages) might be an important source of noise and confusion in stellar radial-velocity (RV) measurements. Radial-velocimetry planet search surveys as well as follow-up of photometric transit surveys require a deeper understanding and characterization of the effects of stellar activities to differentiate them from planetary signals. We simulate dark spots on a rotating stellar photosphere. The variations in the photometry, RV, and spectral line shapes are characterized and analyzed according to the stellar inclination, the latitude, and the number of spots. We show that the anti-correlation between RV and bisector span, known to be a signature of activity, requires a good sampling to be resolved when there are several spots on the photosphere. The Lomb-Scargle periodograms of the RV variations induced by activity present power at the rotational period Prot of the star and its two first harmonics Prot/2 and Prot/3. Three adjusted sinusoids fixed at the fundamental period and its two-first harmonics allow us to remove about 90% of the RV jitter amplitude. We apply and validate our approach on four known active planet-host stars: HD 189733, GJ 674, CoRoT-7, and ι Hor. We succeed in fitting simultaneously activity and planetary signals on GJ674 and CoRoT-7. This simultaneous modeling of the activity and planetary parameters leads to slightly higher masses of CoRoT-7b and c of respectively, 5.7 ± 2.5 MEarth and 13.2 ± 4.1 MEarth. The larger uncertainties properly take into account the stellar active jitter. We exclude short-period low-mass exoplanets around ι Hor. For data with realistic time-sampling and white Gaussian noise, we use simulations to show that our approach is effective in distinguishing reflex-motion due to a planetary companion and stellar-activity-induced RV variations provided that 1) the planetary orbital period is not close to that of the stellar rotation or one of its two first harmonics; 2

  8. Cadmium Induces Apoptosis in Freshwater Crab Sinopotamon henanense through Activating Calcium Signal Transduction Pathway

    PubMed Central

    Wang, Jinxiang; Zhang, Pingping; Liu, Na; Wang, Qian; Luo, Jixian; Wang, Lan

    2015-01-01

    Calcium ion (Ca2+) is one of the key intracellular signals, which is implicated in the regulation of cell functions such as impregnation, cell proliferation, differentiation and death. Cadmium (Cd) is a toxic environmental pollutant that can disturb cell functions and even lead to cell death. Recently, we have found that Cd induced apoptosis in gill cells of the freshwater crab Sinopotamon henanense via caspase activation. In the present study, we further investigated the role of calcium signaling in the Cd-induced apoptosis in the animals. Our data showed that Cd triggered gill cell apoptosis which is evidenced by apoptotic DNA fragmentation, activations of caspases-3, -8 and -9 and the presence of apoptotic morphological features. Moreover, Cd elevated the intracellular concentration of Ca2+, the protein concentration of calmodulin (CaM) and the activity of Ca2+-ATPase in the gill cells of the crabs. Pretreatment of the animals with ethylene glycol-bis-(b-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), Ca2+ chelator, inhibited Cd-induced activation of caspases-3, -8 and -9 as well as blocked the Cd-triggered apoptotic DNA fragmentation. The apoptotic morphological features were no longer observed in gill cells pretreated with the Ca2+ signaling inhibitors before Cd treatment. Our results indicate that Cd evokes gill cell apoptosis through activating Ca2+-CaM signaling transduction pathway. PMID:26714174

  9. The roles of serine protease, intracellular and extracellular phenoloxidase in activation of prophenoloxidase system, and characterization of phenoloxidase from shrimp haemocytes induced by lipopolysaccharide or dopamine

    NASA Astrophysics Data System (ADS)

    Xie, Peng; Pan, Luqing; Xu, Wujie; Yue, Feng

    2013-09-01

    We investigated the effects of lipopolysaccharide (LPS) and dopamine (DA) on the activation of the prophenoloxidase (proPO) system of Litopenaeus vannamei. LPS and DA were shown with a negative dose-dependent effect on hyalne cells (HC), semi-granular cells (SGC), large granular cells (LGC), and total haemocyte count (THC). When haemocytes were treated with LPS or DA, serine proteinase activity and intracellular phenoloxidase (PO) activity were significantly reduced, but extracellular PO activity increased significantly. These findings indicated that the reduction in haemocyte counts was mainly because of the degranulation and activation of the proPO system from semi-granule and large granule cells. The PKC inhibitor, chelerythrine, and the TPK inhibitor, genistein, had an inhibitory effect on extracellular PO activity, while serine proteinase and intracellular PO activity increased. This suggests that the LPS and DA induce the activation of proPO in haemocytes via PKC and TPK-related signaling pathways, but serine proteinase may be activated only by PKC, as the genistein effects were not statistically significant. Electrophoresis analysis revealed that POs induced by LPS or DA have the same molecular mass and high diphenolase activity. Two PO bands at 526 kDa and 272 kDa were observed in PAGE, while in the haemocyte lysate supernatant (HLS), only a 272-kDa band was observed. This band was resolved after SDS-PAGE under non-reducing and reducing conditions into two groups of POs, 166 kDa and 126 kDa, and 78.1 kDa and 73.6 kDa, respectively, suggesting that PO in L. vannamei is an oligomer, which may have different compositions intra- and extracellularly.

  10. The cytotoxic and proapoptotic activities of hypnophilin are associated with calcium signaling in UACC-62 cells.

    PubMed

    Pinto, Mauro C X; Cota, Betania B; Rodrigues, Michele A; Leite, Maria F; de Souza-Fagundes, Elaine M

    2013-11-01

    Hypnophilin (HNP) is a sesquiterpene that is isolated from Lentinus cf. strigosus and has cytotoxic activities. Here, we studied the calcium signaling and cytotoxic effects of HNP in UACC-62 cells, a human skin melanoma cell line. HNP was able to increase the intracellular calcium concentration in UACC-62 cells, which was blocked in cells stimulated in Ca(2+) -free media. HNP treatment with BAPTA-AM, an intracellular Ca(2+) chelator, caused an increase in calcium signals. HNP showed cytotoxicity against UACC-62 cells in which it induced DNA fragmentation and morphological alterations, including changes in the nuclear chromatin profile and increased cytoplasmatic vacuolization, but it had no effect on the plasma membrane integrity. These data suggest that cytotoxicity in UACC-62 cells, after treatment with HNP, is associated with Ca(2+) influx. Together, these findings suggest that HNP is a relevant tool for the further investigation of new anticancer approaches.

  11. Intracellular staining and detection of cytokines by fluorescence-activated flow cytometry.

    PubMed

    Freer, Giulia

    2014-01-01

    The detection of cytokines inside cells producing them has made a tremendous impact on the way immune reactivity is measured. Intracellular cytokine staining is the only immunological technique allowing determination of antigen-specific T cell function and phenotype at the same time; for this reason, it is one of the most popular methods to measure antigenicity in the evaluation of vaccine efficacy and in the study of infectious diseases. It is a flow cytometric technique based on staining of intracellular cytokines and cell markers (surface or cytoplasmic) with fluorescent antibodies after short term culture of stimulated immune cells in the presence of a protein secretion inhibitor, followed by fixation and permeabilization. Most experiments involve detection of five to ten different colors but many more can be detected by modern flow cytometers. Here, we discuss our experience using a standard protocol for intracellular cytokine staining. PMID:24908309

  12. Unique intracellular activation of the potent anti-human immunodeficiency virus agent 1592U89.

    PubMed Central

    Faletto, M B; Miller, W H; Garvey, E P; St Clair, M H; Daluge, S M; Good, S S

    1997-01-01

    The anabolism of 1592U89, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclo pentene-1-methanol, a selective inhibitor of human immunodeficiency virus (HIV), was characterized in human T-lymphoblastoid CD4+ CEM cells. 1592U89 was ultimately anabolized to the triphosphate (TP) of the guanine analog (-)-carbovir (CBV), a potent inhibitor of HIV reverse transcriptase. However, less than 2% of intracellular 1592U89 was converted to CBV, an amount insufficient to account for the CBV-TP levels observed. 1592U89 was anabolized to its 5'-monophosphate (MP) by the recently characterized enzyme adenosine phosphotransferase, but neither its diphosphate (DP) nor its TP was detected. The MP, DP, and TP of CBV were found in cells incubated with either 1592U89 or CBV, with CBV-TP being the major phosphorylated species. We confirmed that CBV is phosphorylated by 5'-nucleotidase and that mycophenolic acid increased the formation of CBV-TP from CBV 75-fold. However, mycophenolic acid did not stimulate 1592U89 anabolism to CBV-TP. The adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) did not inhibit CBV-TP formation from CBV or 1592U89, whereas the adenylate deaminase inhibitor 2'-deoxycoformycin selectively inhibited 1592U89 anabolism to CBV-TP and reversed the antiviral activity of 1592U89. 1592U89-MP was not a substrate for adenylate deaminase but was a substrate for a distinct cytosolic deaminase that was inhibited by 2'-deoxycoformycin-5'-MP. Thus, 1592U89 is phosphorylated by adenosine phosphotransferase to 1592U89-MP, which is converted by a novel cytosolic enzyme to CBV-MP. CBV-MP is then further phosphorylated to CBV-TP by cellular kinases. This unique activation pathway enables 1592U89 to overcome the pharmacokinetic and toxicological deficiencies of CBV while maintaining potent and selective anti-HIV activity. PMID:9145876

  13. Evaluating the anti Mycobacterium tuberculosis activity of Alpinia galanga (L.) Willd. axenically under reducing oxygen conditions and in intracellular assays

    PubMed Central

    2014-01-01

    Background In tuberculosis (TB), the steadily increasing bacterial resistance to existing drugs and latent TB continue to be major concerns. A combination of conventional drugs and plant derived therapeutics can serve to expand the antimicrobial spectrum, prevent the emergence of drug resistant mutants and minimize toxicity. Alpinia galanga, used in various traditional medicines, possesses broad spectrum antibacterial properties. The study was undertaken to assess the antimycobacterial potential of A. galanga in axenic (under aerobic and anaerobic conditions) and intracellular assays. Methods Phytochemical analysis was done using HPTLC. The acetone, aqueous and ethanolic extracts (1, 10, 25, 50 and 100 μg/ml) of A. galanga were tested axenically using Microplate Alamar Blue Assay (MABA) against Mycobacterium tuberculosis (M.tb) H37Rv and three drug sensitive and three multi drug resistant clinical isolates. The activity of the extracts was also evaluated intracellularly in A549 cell line against these strains. The extracts active under intracellular conditions were further tested in an axenic setup under reducing oxygen concentrations using only H37Rv. Results 1´ acetoxychavicol acetate, the reference standard used, was present in all the three extracts. The acetone and ethanolic extracts were active in axenic (aerobic and anaerobic) and intracellular assays. The aqueous extract did not demonstrate activity under the defined assay parameters. Conclusion A. galanga exhibits anti M.tb activity with multiple modes of action. Since the activity of the extracts was observed under reducing oxygen concentrations, it may be effective in treating the dormant and non-replicating bacteria of latent TB. Though the hypothesis needs further testing, A. galanga being a regular dietary component may be utilized in combination with the conventional TB therapy for enhanced efficacy. PMID:24592852

  14. Supramolecular organizing centers (SMOCs) as signaling machines in innate immune activation

    PubMed Central

    Qi, QIAO; Hao, WU

    2016-01-01

    Innate immunity offers the first line of defense against infections and other types of danger such as tumorigenesis. Its discovery provides tremendous therapeutic opportunities for numerous human diseases. Delving into the structural basis of signal transduction by innate immune receptors, our lab has recently helped to establish the new paradigm in which innate immune receptors transduce ligand-binding signals through formation of higher-order assemblies containing intracellular adapters, signaling enzymes and their substrates. These large signalosome assemblies may be visible under light microscopy as punctate structures in the μm scale, connecting to the underlying molecular structures in the nm scale. They drive proximity-induced enzyme activation, and provide a mechanism for signaling amplification by nucleated polymerization. These supramolecular signaling complexes also open new questions on their cellular organization and mode of regulation, pose challenges to our methodology, and afford valuable implications in drug discovery against these medically important pathways. PMID:26511517

  15. Mathematical modeling of the intracellular protein dynamics: the importance of active transport along microtubules.

    PubMed

    Szymańska, Zuzanna; Parisot, Martin; Lachowicz, Mirosław

    2014-12-21

    In this paper we propose a mathematical model of protein and mRNA transport inside a cell. The spatio-temporal model takes into account the active transport along microtubules in the cytoplasm as well as diffusion and is able to reproduce the oscillatory changes in protein concentration observed in many experimental data. In the model the protein and the mRNA interact with each other that allows us to classify the model as a simple gene regulatory network. The proposed model is generic and may be adapted to specific signaling pathways. On the basis of numerical simulations, we formulate a new hypothesis that the oscillatory dynamics is allowed by the mRNA active transport along microtubules from the nucleus to distant locations.

  16. Benzoxazolone Carboxamides: Potent and Systemically Active Inhibitors of Intracellular Acid Ceramidase**

    PubMed Central

    Pizzirani, Daniela; Bach, Anders; Realini, Natalia; Armirotti, Andrea; Mengatto, Luisa; Bauer, Inga; Girotto, Stefania; Pagliuca, Chiara; De Vivo, Marco; Summa, Maria; Ribeiro, Alison; Piomelli, Daniele

    2015-01-01

    The ceramides are a family of bioactive lipid-derived messengers involved in the control of cellular senescence, inflammation, and apoptosis. Ceramide hydrolysis by acid ceramidase (AC) stops the biological activity of these substances and influences survival and function of normal and neoplastic cells. Because of its central role in the ceramide metabolism, AC may offer a novel molecular target in disorders with dysfunctional ceramide-mediated signaling. Here, a class of benzoxazolone carboxamides is identified as the first potent and systemically active inhibitors of AC. Prototype members of this class inhibit AC with low nanomolar potency by covalent binding to the catalytic cysteine. Their metabolic stability and high in vivo efficacy suggest that these compounds may be used as probes to investigate the roles of ceramide in health and disease, and that this scaffold may represent a promising starting point for the development of novel therapeutic agents. PMID:25395373

  17. Bile acid signaling through FXR induces intracellular adhesion molecule-1 expression in mouse liver and human hepatocytes.

    PubMed

    Qin, Pu; Borges-Marcucci, Lisa A; Evans, Mark J; Harnish, Douglas C

    2005-08-01

    Previous studies have demonstrated a dramatic induction of inflammatory gene expression in livers from mice fed a high-fat, high-cholesterol diet containing cholate after 3-5 wk. To determine the contribution of cholate in mediating these inductions, C57BL/6 mice were fed a chow diet supplemented with increasing concentrations of cholic acid (CA) for 5 days. A dose-dependent induction in the hepatic levels of TNF-alpha, VCAM-1, ICAM-1, and SAA-2 mRNA were observed. As positive controls, a dose-dependent repression of cholesterol 7alpha-hydroxylase and a dose-dependent induction of small heterodimer partner (SHP) expression were also observed, suggesting that farnesoid X receptor (FXR) was activated. In addition, ICAM-1 and SHP mRNA levels were also induced in primary human hepatocytes when treated with chenodeoxycholic acid or GW4064, a FXR-selective agonist. The involvement of FXR in CA-induced inflammatory gene expression was further investigated in the human hepatic cell line HepG2. Both ICAM-1 and SHP expression were induced in a dose- and time-dependent manner by treatment with the FXR-selective agonist GW4064. Moreover, the induction of ICAM-1 by GW4064 was inhibited by the FXR antagonist guggulsterone or with transfection of FXR siRNA. Finally, the activity of FXR was mapped to a retinoic acid response element (RARE) site containing an imbedded farnesoid X response element (FXRE) on the human ICAM-1 promoter and FXR and retinoid X receptor were demonstrated to bind to this site. Finally, FXR-mediated activation of ICAM-1 could be further enhanced by TNF-alpha cotreatment in hepatocytes, suggesting a potential cooperation between cytokine and bile acid-signaling pathways during hepatic inflammatory events.

  18. Efficient intracellular delivery of molecules with high cell viability using nanosecond-pulsed laser-activated carbon nanoparticles.

    PubMed

    Sengupta, Aritra; Kelly, Sean C; Dwivedi, Nishant; Thadhani, Naresh; Prausnitz, Mark R

    2014-03-25

    Conventional physical and chemical methods that efficiently deliver molecules into cells are often associated with low cell viability. In this study, we evaluated the cellular effects of carbon nanoparticles believed to emit photoacoustic waves due to nanosecond-pulse laser activation to test the hypothesis that this method could achieve efficient intracellular delivery while maintaining high cell viability. Suspensions of DU145 human prostate carcinoma cells, carbon black (CB) nanoparticles, and calcein were exposed to 5-9 ns long laser pulses of near-infrared (1064 nm wavelength) light and then analyzed by flow cytometry for intracellular uptake of calcein and cell viability by propidium iodide staining. We found that intracellular uptake increased and in some cases saturated at high levels with only small losses in cell viability as a result of increasing laser fluence, laser exposure time, and as a unifying parameter, the total laser energy. Changing interpulse spacing between 0.1 and 10 s intervals showed no significant change in bioeffects, suggesting that the effects of each pulse were independent when spaced by at least 0.1 s intervals. Pretreatment of CB nanoparticles to intense laser exposure followed by mixing with cells also had no significant effect on uptake or viability. Similar uptake and viability were seen when CB nanoparticles were substituted with India ink, when DU145 cells were substituted with H9c2 rat cardiomyoblast cells, and when calcein was substituted with FITC-dextran. The best laser exposure conditions tested led to 88% of cells with intracellular uptake and close to 100% viability, indicating that nanosecond-pulse laser-activated carbon nanoparticles can achieve efficient intracellular delivery while maintaining high cell viability.

  19. Efficient Intracellular Delivery of Molecules with High Cell Viability Using Nanosecond-Pulsed Laser-Activated Carbon Nanoparticles

    PubMed Central

    2015-01-01

    Conventional physical and chemical methods that efficiently deliver molecules into cells are often associated with low cell viability. In this study, we evaluated the cellular effects of carbon nanoparticles believed to emit photoacoustic waves due to nanosecond-pulse laser activation to test the hypothesis that this method could achieve efficient intracellular delivery while maintaining high cell viability. Suspensions of DU145 human prostate carcinoma cells, carbon black (CB) nanoparticles, and calcein were exposed to 5–9 ns long laser pulses of near-infrared (1064 nm wavelength) light and then analyzed by flow cytometry for intracellular uptake of calcein and cell viability by propidium iodide staining. We found that intracellular uptake increased and in some cases saturated at high levels with only small losses in cell viability as a result of increasing laser fluence, laser exposure time, and as a unifying parameter, the total laser energy. Changing interpulse spacing between 0.1 and 10 s intervals showed no significant change in bioeffects, suggesting that the effects of each pulse were independent when spaced by at least 0.1 s intervals. Pretreatment of CB nanoparticles to intense laser exposure followed by mixing with cells also had no significant effect on uptake or viability. Similar uptake and viability were seen when CB nanoparticles were substituted with India ink, when DU145 cells were substituted with H9c2 rat cardiomyoblast cells, and when calcein was substituted with FITC-dextran. The best laser exposure conditions tested led to 88% of cells with intracellular uptake and close to 100% viability, indicating that nanosecond-pulse laser-activated carbon nanoparticles can achieve efficient intracellular delivery while maintaining high cell viability. PMID:24547946

  20. Short inter-set rest blunts resistance exercise-induced increases in myofibrillar protein synthesis and intracellular signalling in young males.

    PubMed

    McKendry, James; Pérez-López, Alberto; McLeod, Michael; Luo, Dan; Dent, Jessica R; Smeuninx, Benoit; Yu, Jinglei; Taylor, Angela E; Philp, Andrew; Breen, Leigh

    2016-07-01

    phosphorylation of eEF2(Thr56) , TSC2(Thr1462) , AMPK(Thr172) and REDD1 protein were greater for 1 compared with 5 min inter-set rest. Serum testosterone was greater at 20-40 min postexercise and plasma lactate greater immediately postexercise for 1 versus 5 min inter-set rest. Resistance exercise with short (1 min) inter-set rest duration attenuated myofibrillar protein synthesis during the early postexercise recovery period compared with longer (5 min) rest duration, potentially through compromised activation of intracellular signalling.

  1. Disentangling between stellar activity and planetary signals

    NASA Astrophysics Data System (ADS)

    Boisse, Isabelle; Bouchy, François; Hébrard, Guillaume; Bonfils, Xavier; Santos, Nuno; Vauclair, Sylvie

    2011-08-01

    Photospheric stellar activity (i.e. dark spots or bright plages) might be an important source of noise and confusion in the radial-velocity (RV) measurements. Radial-velocimetry planet search surveys as well as follow-up of photometric transit surveys require a deeper understanding and characterization of the effects of stellar activities to disentangle it from planetary signals. We simulate dark spots on a rotating stellar photosphere. The variations of the RV are characterized and analyzed according to the stellar inclination, the latitude and the number of spots. The Lomb-Scargle periodograms of the RV variations induced by activity present power at the rotational period Prot of the star and its two-first harmonics Prot/2 and Prot/3. Three adjusted sinusoids fixed at the fundamental period and its two-first harmonics allow to remove about 90% of the RV jitter amplitude. We apply and validate our approach on four known active planet-host stars: HD 189733, GJ 674, CoRoT-7 and ι Hor. We succeed in fitting simultaneously activity and planetary signals on GJ674 and CoRoT-7. We excluded short-period low-mass exoplanets around ι Hor. Our approach is efficient to disentangle reflex-motion due to a planetary companion and stellar-activity induced-RV variations provided that 1) the planetary orbital period is not close to that of the stellar rotation or one of its two-first harmonics, 2) the rotational period of the star is accurately known, 3) the data cover more than one stellar rotational period.

  2. ß-Adrenergic stimulation increases RyR2 activity via intracellular Ca2+ and Mg2+ regulation.

    PubMed

    Li, Jiao; Imtiaz, Mohammad S; Beard, Nicole A; Dulhunty, Angela F; Thorne, Rick; vanHelden, Dirk F; Laver, Derek R

    2013-01-01

    Here we investigate how ß-adrenergic stimulation of the heart alters regulation of ryanodine receptors (RyRs) by intracellular Ca(2+) and Mg(2+) and the role of these changes in SR Ca(2+) release. RyRs were isolated from rat hearts, perfused in a Langendorff apparatus for 5 min and subject to 1 min perfusion with 1 µM isoproterenol or without (control) and snap frozen in liquid N2 to capture their phosphorylation state. Western Blots show that RyR2 phosphorylation was increased by isoproterenol, confirming that RyR2 were subject to normal ß-adrenergic signaling. Under basal conditions, S2808 and S2814 had phosphorylation levels of 69% and 15%, respectively. These levels were increased to 83% and 60%, respectively, after 60 s of ß-adrenergic stimulation consistent with other reports that ß-adrenergic stimulation of the heart can phosphorylate RyRs at specific residues including S2808 and S2814 causing an increase in RyR activity. At cytoplasmic [Ca(2+)] <1 µM, ß-adrenergic stimulation increased luminal Ca(2+) activation of single RyR channels, decreased luminal Mg(2+) inhibition and decreased inhibition of RyRs by mM cytoplasmic Mg(2+). At cytoplasmic [Ca(2+)] >1 µM, ß-adrenergic stimulation only decreased cytoplasmic Mg(2+) and Ca(2+) inhibition of RyRs. The Ka and maximum levels of cytoplasmic Ca(2+) activation site were not affected by ß-adrenergic stimulation. Our RyR2 gating model was fitted to the single channel data. It predicted that in diastole, ß-adrenergic stimulation is mediated by 1) increasing the activating potency of Ca(2+) binding to the luminal Ca(2+) site and decreasing its affinity for luminal Mg(2+) and 2) decreasing affinity of the low-affinity Ca(2+)/Mg(2+) cytoplasmic inhibition site. However in systole, ß-adrenergic stimulation is mediated mainly by the latter.

  3. Impact of intracellular domain flexibility upon properties of activated human 5-HT3 receptors*

    PubMed Central

    Kozuska, J L; Paulsen, I M; Belfield, W J; Martin, I L; Cole, D J; Holt, A; Dunn, S M J

    2014-01-01

    Background and Purpose It has been proposed that arginine residues lining the intracellular portals of the homomeric 5-HT3A receptor cause electrostatic repulsion of cation flow, accounting for a single-channel conductance substantially lower than that of the 5-HT3AB heteromer. However, comparison of receptor homology models for wild-type pentamers suggests that salt bridges in the intracellular domain of the homomer may impart structural rigidity, and we hypothesized that this rigidity could account for the low conductance. Experimental Approach Mutations were introduced into the portal region of the human 5-HT3A homopentamer, such that putative salt bridges were broken by neutralizing anionic partners. Single-channel and whole cell currents were measured in transfected tsA201 cells and in Xenopus oocytes respectively. Computational simulations of protein flexibility facilitated comparison of wild-type and mutant receptors. Key Results Single-channel conductance was increased substantially, often to wild-type heteromeric receptor values, in most 5-HT3A mutants. Conversely, introduction of arginine residues to the portal region of the heteromer, conjecturally creating salt bridges, decreased conductance. Gating kinetics varied significantly between different mutant receptors. EC50 values for whole-cell responses to 5-HT remained largely unchanged, but Hill coefficients for responses to 5-HT were usually significantly smaller in mutants. Computational simulations suggested increased flexibility throughout the protein structure as a consequence of mutations in the intracellular domain. Conclusions and Implications These data support a role for intracellular salt bridges in maintaining the quaternary structure of the 5-HT3 receptor and suggest a role for the intracellular domain in allosteric modulation of cooperativity and agonist efficacy. Linked Article This article is commented on by Vardy and Kenakin, pp. 1614–1616 of volume 171 issue 7. To view this commentary

  4. Nandrolone reduces activation of Notch signaling in denervated muscle associated with increased Numb expression

    SciTech Connect

    Liu, Xin-Hua; Yao, Shen; Qiao, Rui-Fang; Levine, Alice C.; Kirschenbaum, Alexander; Pan, Jiangping; Wu, Yong; Qin, Weiping; Bauman, William A.; Cardozo, Christopher P.

    2011-10-14

    Highlights: {yields} Nerve transection increased Notch signaling in paralyzed muscle. {yields} Nandrolone prevented denervation-induced Notch signaling. {yields} Nandrolone induced the expression of an inhibitor of the Notch signaling, Numb. {yields} Reduction of denervation-induced Notch signaling by nandrolone is likely through upregulation of Numb. -- Abstract: Nandrolone, an anabolic steroid, slows denervation-atrophy in rat muscle. The molecular mechanisms responsible for this effect are not well understood. Androgens and anabolic steroids activate Notch signaling in animal models of aging and thereby mitigate sarcopenia. To explore the molecular mechanisms by which nandrolone prevents denervation-atrophy, we investigated the effects of nandrolone on Notch signaling in denervated rat gastrocnemius muscle. Denervation significantly increased Notch activity reflected by elevated levels of nuclear Notch intracellular domain (NICD) and expression of Hey1 (a Notch target gene). Activation was greatest at 7 and 35 days after denervation but remained present at 56 days after denervation. Activation of Notch in denervated muscle was prevented by nandrolone associated with upregulated expression of Numb mRNA and protein. These data demonstrate that denervation activates Notch signaling, and that nandrolone abrogates this response associated with increased expression of Numb, suggesting a potential mechanism by which nandrolone reduces denervation-atrophy.

  5. Comparative activities of new fluoroquinolones, alone or in combination with amoxicillin, trimethoprim-sulfamethoxazole, or rifampin, against intracellular Listeria monocytogenes.

    PubMed Central

    Michelet, C; Avril, J L; Arvieux, C; Jacquelinet, C; Vu, N; Cartier, F

    1997-01-01

    We studied the activities of the new fluoroquinolones clinafloxacin, levofloxacin, ofloxacin, and sparfloxacin alone or in combination on the intracellular growth of Listeria monocytogenes. Against intracellular growth of the four strains tested, a similar reduction of the bacterial count was obtained with clinafloxacin at the dose of 10 x MIC (delta log10 CFU/ml = -2.19 +/- 0.24), with levofloxacin at 8 x MIC (delta log10 CFU/ml = -2.28 +/- 0.25), and with sparfloxacin at 4 x MIC (delta log10 CFU/ml = -2.16 +/- 0.21) after 24 h of incubation. The combination of the quinolones with trimethoprim-sulfamethoxazole or amoxicillin did not show a substantial increase in activity compared to the fluoroquinolone alone. Antagonism with rifampin was strongly suggested. No modification of the MIC was observed after 20 successive infections of HeLa cells and contact with subinhibitory concentrations of clinafloxacin, levofloxacin, and sparfloxacin for 24 h. We conclude that clinafloxacin, levofloxacin, or sparfloxacin could represent a therapeutic alternative to amoxicillin for the treatment of Listeria infections in adults, especially clinafloxacin, whose MIC is low (0.06 to 0.12 micrograms/ml), and whose best activity against intracellular L. monocytogenes was obtained at a concentration of 1.2 micrograms/ml, which is similar to clinically achievable levels. The results must be confirmed in an experimental model. PMID:8980755

  6. Bem3, a Cdc42 GTPase-activating protein, traffics to an intracellular compartment and recruits the secretory Rab GTPase Sec4 to endomembranes

    PubMed Central

    Mukherjee, Debarati; Sen, Arpita; Boettner, Douglas R.; Fairn, Gregory D.; Schlam, Daniel; Bonilla Valentin, Fernando J.; Michael McCaffery, J.; Hazbun, Tony; Staiger, Chris J.; Grinstein, Sergio; Lemmon, Sandra K.; Claudio Aguilar, R.

    2013-01-01

    Summary Cell polarity is essential for many cellular functions including division and cell-fate determination. Although RhoGTPase signaling and vesicle trafficking are both required for the establishment of cell polarity, the mechanisms by which they are coordinated are unclear. Here, we demonstrate that the yeast RhoGAP (GTPase activating protein), Bem3, is targeted to sites of polarized growth by the endocytic and recycling pathways. Specifically, deletion of SLA2 or RCY1 led to mislocalization of Bem3 to depolarized puncta and accumulation in intracellular compartments, respectively. Bem3 partitioned between the plasma membrane and an intracellular membrane-bound compartment. These Bem3-positive structures were polarized towards sites of bud emergence and were mostly observed during the pre-mitotic phase of apical growth. Cell biological and biochemical approaches demonstrated that this intracellular Bem3 compartment contained markers for both the endocytic and secretory pathways, which were reminiscent of the Spitzenkörper present in the hyphal tips of growing fungi. Importantly, Bem3 was not a passive cargo, but recruited the secretory Rab protein, Sec4, to the Bem3-containing compartments. Moreover, Bem3 deletion resulted in less efficient localization of Sec4 to bud tips during early stages of bud emergence. Surprisingly, these effects of Bem3 on Sec4 were independent of its GAP activity, but depended on its ability to efficiently bind endomembranes. This work unveils unsuspected and important details of the relationship between vesicle traffic and elements of the cell polarity machinery: (1) Bem3, a cell polarity and peripherally associated membrane protein, relies on vesicle trafficking to maintain its proper localization; and (2) in turn, Bem3 influences secretory vesicle trafficking. PMID:23943876

  7. Retinoic acid receptor-α signalling antagonizes both intracellular and extracellular amyloid-β production and prevents neuronal cell death caused by amyloid-β.

    PubMed

    Jarvis, C I; Goncalves, M B; Clarke, E; Dogruel, M; Kalindjian, S B; Thomas, S A; Maden, M; Corcoran, J P T

    2010-10-01

    Alzheimer's disease (AD) is characterized by amyloid-β (Aβ) deposition in the brain, neuronal cell loss and cognitive decline. We show here that retinoic acid receptor (RAR)α signalling in vitro can prevent both intracellular and extracellular Aβ accumulation. RARα signalling increases the expression of a disintegrin and metalloprotease 10, an α-secretase that processes the amyloid precursor protein into the non-amyloidic pathway, thus reducing Aβ production. We also show that RARα agonists are neuroprotective, as they prevent Aβ-induced neuronal cell death in cortical cultures. If RARα agonists are given to the Tg2576 mouse, the normal Aβ production in their brains is suppressed. In contrast, neither RARβ nor γ-agonists affect Aβ production or Aβ-mediated neuronal cell death. Therefore, RARα agonists have therapeutic potential for the treatment of AD.

  8. Mitofusin 2 decreases intracellular lipids in macrophages by regulating peroxisome proliferator-activated receptor-γ

    SciTech Connect

    Liu, Chun; Ge, Beihai; He, Chao; Zhang, Yi; Liu, Xiaowen; Liu, Kejian; Qian, Cuiping; Zhang, Yu; Peng, Wenzhong; Guo, Xiaomei

    2014-07-18

    Highlights: • Mfn2 decreases cellular lipid accumulation by activating cholesterol transporters. • PPARγ is involved in the Mfn2-mediated increase of cholesterol transporter expressions. • Inactivation of ERK1/2 and p38 is involved in Mfn2-induced PPARγ expression. - Abstract: Mitofusin 2 (Mfn2) inhibits atherosclerotic plaque formation, but the underlying mechanism remains elusive. This study aims to reveal how Mfn2 functions in the atherosclerosis. Mfn2 expression was found to be significantly reduced in arterial atherosclerotic lesions of both mice and human compared with healthy counterparts. Here, we observed that Mfn2 increased cellular cholesterol transporter expression in macrophages by upregulating peroxisome proliferator-activated receptor-γ, an effect achieved at least partially by inhibiting extracellular signal-regulated kinase1/2 (ERK1/2) and p38 mitogen-activated protein kinases (MAPKs) pathway. These findings provide insights into potential mechanisms of Mfn2-mediated alterations in cholesterol transporter expression, which may have significant implications for the treatment of atherosclerotic heart disease.

  9. The intracellular delivery of TAT-aequorin reveals calcium-mediated sensing of environmental and symbiotic signals by the arbuscular mycorrhizal fungus Gigaspora margarita.

    PubMed

    Moscatiello, Roberto; Sello, Simone; Novero, Mara; Negro, Alessandro; Bonfante, Paola; Navazio, Lorella

    2014-08-01

    Arbuscular mycorrhiza (AM) is an ecologically relevant symbiosis between most land plants and Glomeromycota fungi. The peculiar traits of AM fungi have so far limited traditional approaches such as genetic transformation. The aim of this work was to investigate whether the protein transduction domain of the HIV-1 transactivator of transcription (TAT) protein, previously shown to act as a potent nanocarrier for macromolecule delivery in both animal and plant cells, may translocate protein cargoes into AM fungi. We evaluated the internalization into germinated spores of Gigaspora margarita of two recombinant TAT fusion proteins consisting of either a fluorescent (GFP) or a luminescent (aequorin) reporter linked to the TAT peptide. Both TAT-fused proteins were found to enter AM fungal mycelia after a short incubation period (5-10 min). Ca2+ measurements in G. margarita mycelia pre-incubated with TAT-aequorin demonstrated the occurrence of changes in the intracellular free Ca2+ concentration in response to relevant stimuli, such as touch, cold, salinity, and strigolactones, symbiosis-related plant signals. These data indicate that the cell-penetrating properties of the TAT peptide can be used as an effective strategy for intracellularly delivering proteins of interest and shed new light on Ca2+ homeostasis and signalling in AM fungi.

  10. Biological Activities of Reactive Oxygen and Nitrogen Species: Oxidative Stress versus Signal Transduction.

    PubMed

    Weidinger, Adelheid; Kozlov, Andrey V

    2015-01-01

    In the past, reactive oxygen and nitrogen species (RONS) were shown to cause oxidative damage to biomolecules, contributing to the development of a variety of diseases. However, recent evidence has suggested that intracellular RONS are an important component of intracellular signaling cascades. The aim of this review was to consolidate old and new ideas on the chemical, physiological and pathological role of RONS for a better understanding of their properties and specific activities. Critical consideration of the literature reveals that deleterious effects do not appear if only one primary species (superoxide radical, nitric oxide) is present in a biological system, even at high concentrations. The prerequisite of deleterious effects is the formation of highly reactive secondary species (hydroxyl radical, peroxynitrite), emerging exclusively upon reaction with another primary species or a transition metal. The secondary species are toxic, not well controlled, causing irreversible damage to all classes of biomolecules. In contrast, primary RONS are well controlled (superoxide dismutase, catalase), and their reactions with biomolecules are reversible, making them ideal for physiological/pathophysiological intracellular signaling. We assume that whether RONS have a signal transducing or damaging effect is primarily defined by their quality, being primary or secondary RONS, and only secondly by their quantity. PMID:25884116

  11. Biological Activities of Reactive Oxygen and Nitrogen Species: Oxidative Stress versus Signal Transduction

    PubMed Central

    Weidinger, Adelheid; Kozlov, Andrey V.

    2015-01-01

    In the past, reactive oxygen and nitrogen species (RONS) were shown to cause oxidative damage to biomolecules, contributing to the development of a variety of diseases. However, recent evidence has suggested that intracellular RONS are an important component of intracellular signaling cascades. The aim of this review was to consolidate old and new ideas on the chemical, physiological and pathological role of RONS for a better understanding of their properties and specific activities. Critical consideration of the literature reveals that deleterious effects do not appear if only one primary species (superoxide radical, nitric oxide) is present in a biological system, even at high concentrations. The prerequisite of deleterious effects is the formation of highly reactive secondary species (hydroxyl radical, peroxynitrite), emerging exclusively upon reaction with another primary species or a transition metal. The secondary species are toxic, not well controlled, causing irreversible damage to all classes of biomolecules. In contrast, primary RONS are well controlled (superoxide dismutase, catalase), and their reactions with biomolecules are reversible, making them ideal for physiological/pathophysiological intracellular signaling. We assume that whether RONS have a signal transducing or damaging effect is primarily defined by their quality, being primary or secondary RONS, and only secondly by their quantity. PMID:25884116

  12. Isomer-nonspecific action of dichlorodiphenyltrichloroethane on aryl hydrocarbon receptor and G-protein-coupled receptor 30 intracellular signaling in apoptotic neuronal cells.

    PubMed

    Kajta, M; Litwa, E; Rzemieniec, J; Wnuk, A; Lason, W; Zelek-Molik, A; Nalepa, I; Grzegorzewska-Hiczwa, M; Tokarski, K; Golas, A; Guzik, E; Grochowalski, A; Szychowski, K A; Wojtowicz, A K

    2014-07-01

    Extended residual persistence of the pesticide dichlorodiphenyltrichloroethane (DDT) raises concerns about its long-term neurotoxic effects. Little is known, however, about DDT toxicity during the early stages of neural development. This study demonstrated that DDT-induced apoptosis of mouse embryonic neuronal cells is a caspase-9-, caspase-3-, and GSK-3β-dependent process, which involves p,p'-DDT-specific impairment of classical ERs. It also provided evidence for DDT-isomer-nonspecific alterations of AhR- and GPR30-mediated intracellular signaling, including changes in the levels of the receptor and receptor-regulated mRNAs, and also changes in the protein levels of the receptors. DDT-induced stimulation of AhR-signaling and reduction of GPR30-signaling were verified using selective ligands and specific siRNAs. Co-localization of the receptors was demonstrated with confocal microscopy, and the presence of functional GPR30 was detected by electrophysiology. This study demonstrates that stimulation of AhR-signaling and impairment of GPR30-signaling play important roles in the propagation of DDT-induced apoptosis during the early stages of neural development.

  13. Human Macrophage SCN5A Activates an Innate Immune Signaling Pathway for Antiviral Host Defense*

    PubMed Central

    Jones, Alexis; Kainz, Danielle; Khan, Faatima; Lee, Cara; Carrithers, Michael D.

    2014-01-01

    Pattern recognition receptors contain a binding domain for pathogen-associated molecular patterns coupled to a signaling domain that regulates transcription of host immune response genes. Here, a novel mechanism that links pathogen recognition to channel activation and downstream signaling is proposed. We demonstrate that an intracellular sodium channel variant, human macrophage SCN5A, initiates signaling and transcription through a calcium-dependent isoform of adenylate cyclase, ADCY8, and the transcription factor, ATF2. Pharmacological stimulation with a channel agonist or treatment with cytoplasmic poly(I:C), a mimic of viral dsRNA, activates this pathway to regulate expression of SP100-related genes and interferon β. Electrophysiological analysis reveals that the SCN5A variant mediates nonselective outward currents and a small, but detectable, inward current. Intracellular poly(I:C) markedly augments an inward voltage-sensitive sodium current and inhibits the outward nonselective current. These results suggest human macrophage SCN5A initiates signaling in an innate immune pathway relevant to antiviral host defense. It is postulated that SCN5A is a novel pathogen sensor and that this pathway represents a channel activation-dependent mechanism of transcriptional regulation. PMID:25368329

  14. Compartmentalized calcium signaling triggers subpopulation formation upon platelet activation through PAR1.

    PubMed

    Sveshnikova, Anastasia N; Ataullakhanov, Fazoil I; Panteleev, Mikhail A

    2015-04-01

    Blood platelets need to undergo activation to carry out their function of stopping bleeding. Different activation degrees lead to a stepped hierarchy of responses: ability to aggregate, granule release, and, in a fraction of platelets, phosphatidylserine (PS) exposure. This suggests the existence of decision-making mechanisms in the platelet intracellular signaling network. To identify and investigate them, we developed a computational model of PAR1-stimulated platelet signal transduction that included a minimal set of major players in the calcium signaling network. The model comprised three intracellular compartments: cytosol, dense tubular system (DTS) and mitochondria and extracellular space. Computer simulations showed that the stable resting state of platelets is maintained via a balance between calcium pumps and leaks through the DTS and plasma membranes. Stimulation of PAR1 induced oscillations in the cytosolic calcium concentrations, in good agreement with experimental observations. Further increase in the agonist level activated the mitochondrial uniporter leading to calcium uptake by mitochondria, which caused the collapse of mitochondrial membrane potential in a fraction of platelets leading to the PS exposure. The formation of this subpopulation was shown to be a stochastic process determined by the small number of activated PAR1 receptors and by heterogeneity in the number of ion pumps. These results demonstrate how a gradual increase of the activation degree can be converted into a stepped response hierarchy ultimately leading to formation of two distinct subpopulations from an initially homogeneous population. PMID:25627921

  15. Prostaglandin E2 Inhibits NLRP3 Inflammasome Activation through EP4 Receptor and Intracellular Cyclic AMP in Human Macrophages.

    PubMed

    Sokolowska, Milena; Chen, Li-Yuan; Liu, Yueqin; Martinez-Anton, Asuncion; Qi, Hai-Yan; Logun, Carolea; Alsaaty, Sara; Park, Yong Hwan; Kastner, Daniel L; Chae, Jae Jin; Shelhamer, James H

    2015-06-01

    PGE2 is a potent lipid mediator involved in maintaining homeostasis but also promotion of acute inflammation or immune suppression in chronic inflammation and cancer. Nucleotide-binding domain, leucine-rich repeat-containing protein (NLR)P3 inflammasome plays an important role in host defense. Uncontrolled activation of the NLRP3 inflammasome, owing to mutations in the NLRP3 gene, causes cryopyrin-associated periodic syndromes. In this study, we showed that NLRP3 inflammasome activation is inhibited by PGE2 in human primary monocyte-derived macrophages. This effect was mediated through PGE2 receptor subtype 4 (EP4) and an increase in intracellular cAMP, independently of protein kinase A or exchange protein directly activated by cAMP. A specific agonist of EP4 mimicked, whereas its antagonist or EP4 knockdown reversed, PGE2-mediated NLRP3 inhibition. PGE2 caused an increase in intracellular cAMP. Blockade of adenylate cyclase by its inhibitor reversed PGE2-mediated NLRP3 inhibition. Increase of intracellular cAMP by an activator of adenylate cyclase or an analog of cAMP, or a blockade of cAMP degradation by phosphodiesterase inhibitor decreased NLRP3 activation. Protein kinase A or exchange protein directly activated by cAMP agonists did not mimic, and their antagonists did not reverse, PGE2-mediated NLRP3 inhibition. Additionally, constitutive IL-1β secretion from LPS-primed PBMCs of cryopyrin-associated periodic fever syndromes patients was substantially reduced by high doses of PGE2. Moreover, blocking cytosolic phospholipase A2α by its inhibitor or small interfering RNA or inhibiting cyclooxygenase 2, resulting in inhibition of endogenous PGE2 production, caused an increase in NLRP3 inflammasome activation. Our results suggest that PGE2 might play a role in maintaining homeostasis during the resolution phase of inflammation and might serve as an autocrine and paracrine regulator.

  16. Disentangling stellar activity and planetary signals

    NASA Astrophysics Data System (ADS)

    Boisse, I.; Bouchy, F.; Hébrard, G.; Bonfils, X.; Santos, N. C.; Vauclair, S.

    2011-02-01

    Photospheric stellar activity (i.e. dark spots or bright plages) might be an important source of noise and confusion in the radial-velocity (RV) measurements. Radial-velocimetry planet search surveys as well as follow-up of photometric transit surveys require a deep understanding and precise characterization of the effects of stellar activity, in order to disentangle it from planetary signals. We simulate dark spots on a rotating stellar photosphere. The variations of the RV are characterized and analyzed according to the stellar inclination, the latitude and the number of spots. The Lomb-Scargle periodograms of the RV variations induced by activity present power at the rotational period Prot of the star and its two-first harmonics Prot/2 and Prot/3. Three adjusted sinusoids fixed at the fundamental period and its two-first harmonics allow to remove about 90% of the RV jitter amplitude. We apply and validate our approach on four known active planet-host stars: HD 189733, GJ 674, CoRoT-7 and ι Hor.

  17. Proteomes of Host Cell Membranes Modified by Intracellular Activities of Salmonella enterica*

    PubMed Central

    Vorwerk, Stephanie; Krieger, Viktoria; Deiwick, Jörg; Hensel, Michael; Hansmeier, Nicole

    2015-01-01

    Intracellular pathogens need to establish a growth-stimulating host niche for survival and replication. A unique feature of the gastrointestinal pathogen Salmonella enterica serovar Typhimurium is the creation of extensive membrane networks within its host. An understanding of the origin and function of these membranes is crucial for the development of new treatment strategies. However, the characterization of this compartment is very challenging, and only fragmentary knowledge of its composition and biogenesis exists. Here, we describe a new proteome-based approach to enrich and characterize Salmonella-modified membranes. Using a Salmonella mutant strain that does not form this unique membrane network as a reference, we identified a high-confidence set of host proteins associated with Salmonella-modified membranes. This comprehensive analysis allowed us to reconstruct the interactions of Salmonella with host membranes. For example, we noted that Salmonella redirects endoplasmic reticulum (ER) membrane trafficking to its intracellular niche, a finding that has not been described for Salmonella previously. Our system-wide approach therefore has the potential to rapidly close gaps in our knowledge of the infection process of intracellular pathogens and demonstrates a hitherto unrecognized complexity in the formation of Salmonella host niches. PMID:25348832

  18. Proteomes of host cell membranes modified by intracellular activities of Salmonella enterica.

    PubMed

    Vorwerk, Stephanie; Krieger, Viktoria; Deiwick, Jörg; Hensel, Michael; Hansmeier, Nicole

    2015-01-01

    Intracellular pathogens need to establish a growth-stimulating host niche for survival and replication. A unique feature of the gastrointestinal pathogen Salmonella enterica serovar Typhimurium is the creation of extensive membrane networks within its host. An understanding of the origin and function of these membranes is crucial for the development of new treatment strategies. However, the characterization of this compartment is very challenging, and only fragmentary knowledge of its composition and biogenesis exists. Here, we describe a new proteome-based approach to enrich and characterize Salmonella-modified membranes. Using a Salmonella mutant strain that does not form this unique membrane network as a reference, we identified a high-confidence set of host proteins associated with Salmonella-modified membranes. This comprehensive analysis allowed us to reconstruct the interactions of Salmonella with host membranes. For example, we noted that Salmonella redirects endoplasmic reticulum (ER) membrane trafficking to its intracellular niche, a finding that has not been described for Salmonella previously. Our system-wide approach therefore has the potential to rapidly close gaps in our knowledge of the infection process of intracellular pathogens and demonstrates a hitherto unrecognized complexity in the formation of Salmonella host niches.

  19. Effects of chronic sleep deprivation on autonomic activity by examining heart rate variability, plasma catecholamine, and intracellular magnesium levels.

    PubMed

    Takase, Bonpei; Akima, Takashi; Satomura, Kimio; Ohsuzu, Fumitaka; Mastui, Takemi; Ishihara, Masayuki; Kurita, Akira

    2004-10-01

    Chronic sleep deprivation is associated with cardiovascular events. In addition, autonomic activity determined from the levels of the heart rate variability (HRV), plasma catecholamine, and intracellular magnesium (Mg) are important in the pathophysiology of cardiovascular events. This study therefore aimed to determine the effects of chronic sleep deprivation on autonomic activity by examining the HRV, plasma catecholamine, and intracellular magnesium levels. Thirty (30) healthy male college students ranging in age from 20 to 24 years of age (average 22 +/- 1 years; mean +/- SD) with no coronary risk factors such as hypertension, diabetes mellitus, hyperlipidemia or a family history of premature coronary artery disease (CAD) were included in the study. Over a 4-week period, the volunteers' plasma levels of epinephrine, norepinephrine, and erythrocyte-Mg were measured. The study was made during the 4 weeks before and immediately after college finals exams. HRV, obtained from 24-hour ambulatory ECG monitoring, included time and frequency domain indices. The HRV indices and erythrocyte-Mg decreased while norepinephrine increased during chronic sleep deprivation. It is concluded that chronic sleep deprivation causes an autonomic imbalance and decreases intracellular Mg, which could be associated with chronic sleep deprivation-induced cardiovascular events. PMID:15754837

  20. Signal transduction pathways in mast cell granule-mediated endothelial cell activation.

    PubMed Central

    Chi, Luqi; Stehno-Bittel, Lisa; Smirnova, Irina; Stechschulte, Daniel J; Dileepan, Kottarappat N

    2003-01-01

    BACKGROUND: We have previously shown that incubation of human endothelial cells with mast cell granules results in potentiation of lipopolysaccharide-induced production of interleukin-6 and interleukin-8. AIMS: The objective of the present study was to identify candidate molecules and signal transduction pathways involved in the synergy between mast cell granules and lipopolysaccharide on endothelial cell activation. METHODS: Human umbilical vein endothelial cells were incubated with rat mast cell granules in the presence and absence of lipopolysaccharide, and IL-6 production was quantified. The status of c-Jun amino-terminal kinase and extracellular signal-regulated kinase 1/2 activation, nuclear factor-kappaB translocation and intracellular calcium levels were determined to identify the mechanism of synergy between mast cell granules and lipopolysaccaride. RESULTS: Mast cell granules induced low levels of interleukin-6 production by endothelial cells, and this effect was markedly enhanced by lipopolysaccharide. The results revealed that both serine proteases and histamine present in mast cell granules were involved in this activation process. Mast cell granules increased intracellular calcium, and activated c-Jun amino-terminal kinase and extracellular signal-regulated kinase 1/2. The combination of lipopolysaccharide and mast cell granules prolonged c-Jun amino-terminal kinase activity beyond the duration of induction by either stimulant alone and was entirely due to active proteases. However, both proteases and histamine contributed to calcium mobilization and extracellular signal-regulated kinase 1/2 activation. The nuclear translocation of nuclear factor-kappaB proteins was of greater magnitude in endothelial cells treated with the combination of mast cell granules and lipopolysaccharide. CONCLUSIONS:Mast cell granule serine proteases and histamine can amplify lipopolysaccharide-induced endothelial cell activation, which involves calcium mobilization, mitogen-activated

  1. Prostaglandin E2 inhibits NLRP3 inflammasome activation through EP4 receptor and intracellular cAMP in human macrophages

    PubMed Central

    Liu, Yueqin; Martinez-Anton, Asuncion; Qi, Hai-Yan; Logun, Carolea; Alsaaty, Sara; Park, Yong Hwan; Kastner, Daniel L.; Chae, Jae Jin; Shelhamer, James H.

    2015-01-01

    Prostaglandin E2 (PGE2) is a potent lipid mediator involved in maintaining homeostasis but also promotion of acute inflammation or immune suppression in chronic inflammation and cancer. NLRP3 inflammasome plays an important role in host defense. Uncontrolled activation of NLRP3 inflammasome, due to mutations in the NLRP3 gene causes cryopyrin-associated periodic syndromes (CAPS). Here, we showed that NLRP3 inflammasome activation is inhibited by PGE2 in human primary monocyte-derived macrophages. This effect was mediated through prostaglandin E receptor 4 (EP4) and an increase in intracellular cAMP, independently of protein kinase A (PKA) or exchange protein directly activated by cAMP (Epac). A specific agonist of EP4 mimicked, while its antagonist or EP4 knockdown reversed PGE2-mediated NLRP3 inhibition. PGE2 caused an increase in intracellular cAMP. Blockade of adenylate cyclase by its inhibitor reversed PGE2-mediated NLRP3 inhibition. Increase of intracellular cAMP by an activator of adenylate cyclase or an analog of cAMP, or a blockade of cAMP degradation by phosphodiesterase inhibitor decreased NLRP3 activation. PKA or Epac agonists did not mimic and their antagonists did not reverse PGE2-mediated NLRP3 inhibition. In addition, constitutive IL-1β secretion from LPS-primed PBMCs of CAPS patients was substantially reduced by high doses of PGE2. Moreover, blocking cytosolic phospholipase A2α by its inhibitor or siRNA or inhibiting cyclooxygenase 2, resulting in inhibition of endogenous PGE2 production, caused an increase in NLRP3 inflammasome activation. Our results suggest that PGE2 might play a role in maintaining homeostasis during the resolution phase of inflammation and might serve as an autocrine and paracrine regulator. PMID:25917098

  2. Microglia Activate Migration of Glioma Cells through a Pyk2 Intracellular Pathway.

    PubMed

    Rolón-Reyes, Kimberleve; Kucheryavykh, Yuriy V; Cubano, Luis A; Inyushin, Mikhail; Skatchkov, Serguei N; Eaton, Misty J; Harrison, Jeffrey K; Kucheryavykh, Lilia Y

    2015-01-01

    Glioblastoma is one of the most aggressive and fatal brain cancers due to the highly invasive nature of glioma cells. Microglia infiltrate most glioma tumors and, therefore, make up an important component of the glioma microenvironment. In the tumor environment, microglia release factors that lead to the degradation of the extracellular matrix and stimulate signaling pathways to promote glioma cell invasion. In the present study, we demonstrated that microglia can promote glioma migration through a mechanism independent of extracellular matrix degradation. Using western blot analysis, we found upregulation of proline rich tyrosine kinase 2 (Pyk2) protein phosphorylated at Tyr579/580 in glioma cells treated with microglia conditioned medium. This upregulation occurred in rodent C6 and GL261 as well as in human glioma cell lines with varying levels of invasiveness (U-87MG, A172, and HS683). siRNA knock-down of Pyk2 protein and pharmacological blockade by the Pyk2/focal-adhesion kinase (FAK) inhibitor PF-562,271 reversed the stimulatory effect of microglia on glioma migration in all cell lines. A lower concentration of PF-562,271 that selectively inhibits FAK, but not Pyk2, did not have any effect on glioma cell migration. Moreover, with the use of the CD11b-HSVTK microglia ablation mouse model we demonstrated that elimination of microglia in the implanted tumors (GL261 glioma cells were used for brain implantation) by the local in-tumor administration of Ganciclovir, significantly reduced the phosphorylation of Pyk2 at Tyr579/580 in implanted tumor cells. Taken together, these data indicate that microglial cells activate glioma cell migration/dispersal through the pro-migratory Pyk2 signaling pathway in glioma cells. PMID:26098895

  3. Microglia Activate Migration of Glioma Cells through a Pyk2 Intracellular Pathway

    PubMed Central

    Rolón-Reyes, Kimberleve; Kucheryavykh, Yuriy V.; Cubano, Luis A.; Inyushin, Mikhail; Skatchkov, Serguei N.; Eaton, Misty J.; Harrison, Jeffrey K.; Kucheryavykh, Lilia Y.

    2015-01-01

    Glioblastoma is one of the most aggressive and fatal brain cancers due to the highly invasive nature of glioma cells. Microglia infiltrate most glioma tumors and, therefore, make up an important component of the glioma microenvironment. In the tumor environment, microglia release factors that lead to the degradation of the extracellular matrix and stimulate signaling pathways to promote glioma cell invasion. In the present study, we demonstrated that microglia can promote glioma migration through a mechanism independent of extracellular matrix degradation. Using western blot analysis, we found upregulation of proline rich tyrosine kinase 2 (Pyk2) protein phosphorylated at Tyr579/580 in glioma cells treated with microglia conditioned medium. This upregulation occurred in rodent C6 and GL261 as well as in human glioma cell lines with varying levels of invasiveness (U-87MG, A172, and HS683). siRNA knock-down of Pyk2 protein and pharmacological blockade by the Pyk2/focal-adhesion kinase (FAK) inhibitor PF-562,271 reversed the stimulatory effect of microglia on glioma migration in all cell lines. A lower concentration of PF-562,271 that selectively inhibits FAK, but not Pyk2, did not have any effect on glioma cell migration. Moreover, with the use of the CD11b-HSVTK microglia ablation mouse model we demonstrated that elimination of microglia in the implanted tumors (GL261 glioma cells were used for brain implantation) by the local in-tumor administration of Ganciclovir, significantly reduced the phosphorylation of Pyk2 at Tyr579/580 in implanted tumor cells. Taken together, these data indicate that microglial cells activate glioma cell migration/dispersal through the pro-migratory Pyk2 signaling pathway in glioma cells. PMID:26098895

  4. Mechanisms of intracellular defense and activity of free radical oxidation in rat myocardium in the dynamics of chronic fluorine intoxication.

    PubMed

    Zhukova, A G; Alekhina, D A; Sazontova, T G; Prokop'ev, Yu A; Gorokhova, L G; Stryapko, N V; Mikhailova, N N

    2013-12-01

    The mechanisms of intracellular defense and activity of free radical oxidation in the myocardium were studied in the dynamics of chronic fluorine intoxication. At the early stages of fluorine intoxication (day 3-week 3), the concentrations of defense proteins HIF-1α, HSC73, and HOx-2 and activity of the main metabolic enzymes increased, which promoted maintenance of cardiomyocyte structure and function at the normal physiological level. At late stages of fluorine intoxication (weeks 6 and 9), metabolic changes in the myocardium attest to high strain of the adaptive mechanisms.

  5. Sleep Loss Activates Cellular Inflammatory Signaling

    PubMed Central

    Irwin, Michael R.; Wang, Minge; Ribeiro, Denise; Cho, Hyong Jin; Olmstead, Richard; Breen, Elizabeth Crabb; Martinez-Maza, Otoniel; Cole, Steve

    2008-01-01

    Background Accumulating evidence suggests that sleep disturbance is associated with inflammation and related disorders including cardiovascular disease, arthritis, and diabetes mellitus. This study was undertaken to test the effects of sleep loss on activation of nuclear factor (NF) -κB, a transcription factor that serves a critical role in the inflammatory signaling cascade. Methods In 14 healthy adults (7 females; 7 males), peripheral blood mononuclear cell NF-κB was repeatedly assessed, along with enumeration of lymphocyte subpopulations, in the morning after baseline sleep, partial sleep deprivation (awake from 23:00 h to 03:00 h), and recovery sleep. Results In the morning after a night of sleep loss, mononuclear cell NF-κB activation was significantly greater compared with morning levels following uninterrupted baseline or recovery sleep, in which the response was found in females but not in males. Conclusions These results identify NF-κB activation as a molecular pathway by which sleep disturbance may influence leukocyte inflammatory gene expression and the risk of inflammation-related disease. PMID:18561896

  6. Heat dissipation guides activation in signaling proteins

    PubMed Central

    Weber, Jeffrey K.; Shukla, Diwakar; Pande, Vijay S.

    2015-01-01

    Life is fundamentally a nonequilibrium phenomenon. At the expense of dissipated energy, living things perform irreversible processes that allow them to propagate and reproduce. Within cells, evolution has designed nanoscale machines to do meaningful work with energy harnessed from a continuous flux of heat and particles. As dictated by the Second Law of Thermodynamics and its fluctuation theorem corollaries, irreversibility in nonequilibrium processes can be quantified in terms of how much entropy such dynamics produce. In this work, we seek to address a fundamental question linking biology and nonequilibrium physics: can the evolved dissipative pathways that facilitate biomolecular function be identified by their extent of entropy production in general relaxation processes? We here synthesize massive molecular dynamics simulations, Markov state models (MSMs), and nonequilibrium statistical mechanical theory to probe dissipation in two key classes of signaling proteins: kinases and G-protein–coupled receptors (GPCRs). Applying machinery from large deviation theory, we use MSMs constructed from protein simulations to generate dynamics conforming to positive levels of entropy production. We note the emergence of an array of peaks in the dynamical response (transient analogs of phase transitions) that draw the proteins between distinct levels of dissipation, and we see that the binding of ATP and agonist molecules modifies the observed dissipative landscapes. Overall, we find that dissipation is tightly coupled to activation in these signaling systems: dominant entropy-producing trajectories become localized near important barriers along known biological activation pathways. We go on to classify an array of equilibrium and nonequilibrium molecular switches that harmonize to promote functional dynamics. PMID:26240354

  7. Splice cassette II of Na+,HCO3(-) cotransporter NBCn1 (slc4a7) interacts with calcineurin A: implications for transporter activity and intracellular pH control during rat artery contractions.

    PubMed

    Danielsen, Andreas A; Parker, Mark D; Lee, Soojung; Boron, Walter F; Aalkjaer, Christian; Boedtkjer, Ebbe

    2013-03-22

    Activation of Na(+),HCO3(-) cotransport in vascular smooth muscle cells (VSMCs) contributes to intracellular pH (pH(i)) control during artery contraction, but the signaling pathways involved have been unknown. We investigated whether physical and functional interactions between the Na(+),HCO3(-) cotransporter NBCn1 (slc4a7) and the Ca(2+)/calmodulin-activated serine/threonine phosphatase calcineurin exist and play a role for pHi control in VSMCs. Using a yeast two-hybrid screen, we found that splice cassette II from the N terminus of NBCn1 interacts with calcineurin Aβ. When cassette II was truncated or mutated to disrupt the putative calcineurin binding motif PTVVIH, the interaction was abolished. Native NBCn1 and calcineurin Aβ co-immunoprecipitated from A7r5 rat VSMCs. A peptide (acetyl-DDIPTVVIH-amide), which mimics the putative calcineurin binding motif, inhibited the co-immunoprecipitation whereas a mutated peptide (acetyl-DDIATAVAA-amide) did not. Na(+),HCO3(-) cotransport activity was investigated in VSMCs of mesenteric arteries after an NH4(+) prepulse. During depolarization with 50 mM extracellular K(+) to raise intracellular [Ca(2+)], Na(+),HCO3(-) cotransport activity was inhibited 20-30% by calcineurin inhibitors (FK506 and cyclosporine A). FK506 did not affect Na(+),HCO3(-) cotransport activity in VSMCs when cytosolic [Ca(2+)] was lowered by buffering, nor did it disrupt binding between NBCn1 and calcineurin Aβ. FK506 augmented the intracellular acidification of VSMCs during norepinephrine-induced artery contractions. No physical or functional interactions between calcineurin Aβ and the Na(+)/H(+) exchanger NHE1 were observed in VSMCs. In conclusion, we demonstrate a physical interaction between calcineurin Aβ and cassette II of NBCn1. Intracellular Ca(2+) activates Na(+),HCO3(-) cotransport activity in VSMCs in a calcineurin-dependent manner which is important for protection against intracellular acidification.

  8. Glucagon receptor activates extracellular signal-regulated protein kinase 1/2 via cAMP-dependent protein kinase

    PubMed Central

    Jiang, Youwei; Cypess, Aaron M.; Muse, Evan D.; Wu, Cui-Rong; Unson, Cecilia G.; Merrifield, R. B.; Sakmar, Thomas P.

    2001-01-01

    We prepared a stable cell line expressing the glucagon receptor to characterize the effect of Gs-coupled receptor stimulation on extracellular signal-regulated protein kinase 1/2 (ERK1/2) activity. Glucagon treatment of the cell line caused a dose-dependent increase in cAMP concentration, activation of cAMP-dependent protein kinase (PKA), and transient release of intracellular calcium. Glucagon treatment also caused rapid dose-dependent phosphorylation and activation of mitogen-activated protein kinase kinase/ERK kinase (MEK1/2) and ERK1/2. Inhibition of either PKA or MEK1/2 blocked ERK1/2 activation by glucagon. However, no significant activation of several upstream activators of MEK, including Ras, Rap1, and Raf, was observed in response to glucagon treatment. In addition, chelation of intracellular calcium reduced glucagon-mediated ERK1/2 activation. In transient transfection experiments, glucagon receptor mutants that bound glucagon but failed to increase intracellular cAMP and calcium concentrations showed no glucagon-stimulated ERK1/2 phosphorylation. We conclude that glucagon-induced MEK1/2 and ERK1/2 activation is mediated by PKA and that an increase in intracellular calcium concentration is required for maximal ERK activation. PMID:11517300

  9. Active voltammetric microsensors with neural signal processing.

    SciTech Connect

    Vogt, M. C.

    1998-12-11

    Many industrial and environmental processes, including bioremediation, would benefit from the feedback and control information provided by a local multi-analyte chemical sensor. For most processes, such a sensor would need to be rugged enough to be placed in situ for long-term remote monitoring, and inexpensive enough to be fielded in useful numbers. The multi-analyte capability is difficult to obtain from common passive sensors, but can be provided by an active device that produces a spectrum-type response. Such new active gas microsensor technology has been developed at Argonne National Laboratory. The technology couples an electrocatalytic ceramic-metallic (cermet) microsensor with a voltammetric measurement technique and advanced neural signal processing. It has been demonstrated to be flexible, rugged, and very economical to produce and deploy. Both narrow interest detectors and wide spectrum instruments have been developed around this technology. Much of this technology's strength lies in the active measurement technique employed. The technique involves applying voltammetry to a miniature electrocatalytic cell to produce unique chemical ''signatures'' from the analytes. These signatures are processed with neural pattern recognition algorithms to identify and quantify the components in the analyte. The neural signal processing allows for innovative sampling and analysis strategies to be employed with the microsensor. In most situations, the whole response signature from the voltammogram can be used to identify, classify, and quantify an analyte, without dissecting it into component parts. This allows an instrument to be calibrated once for a specific gas or mixture of gases by simple exposure to a multi-component standard rather than by a series of individual gases. The sampled unknown analytes can vary in composition or in concentration, the calibration, sensing, and processing methods of these active voltammetric microsensors can detect, recognize, and

  10. Signal integration by Ca(2+) regulates intestinal stem-cell activity.

    PubMed

    Deng, Hansong; Gerencser, Akos A; Jasper, Heinrich

    2015-12-10

    Somatic stem cells maintain tissue homeostasis by dynamically adjusting proliferation and differentiation in response to stress and metabolic cues. Here we identify Ca(2+) signalling as a central regulator of intestinal stem cell (ISC) activity in Drosophila. We show that dietary L-glutamate stimulates ISC division and gut growth. The metabotropic glutamate receptor (mGluR) is required in ISCs for this response, and for an associated modulation of cytosolic Ca(2+) oscillations that results in sustained high cytosolic Ca(2+) concentrations. High cytosolic Ca(2+) concentrations induce ISC proliferation by regulating Calcineurin and CREB-regulated transcriptional co-activator (Crtc). In response to a wide range of dietary and stress stimuli, ISCs reversibly transition between Ca(2+) oscillation states that represent poised or activated modes of proliferation, respectively. We propose that the dynamic regulation of intracellular Ca(2+) levels allows effective integration of diverse mitogenic signals in ISCs to adapt their proliferative activity to the needs of the tissue.

  11. Next Generation of Targeted Molecules for Non-Hodgkin Lymphomas: Small-Molecule Inhibitors of Intracellular Targets and Signaling Pathways.

    PubMed

    Choe, Hannah; Ruan, Jia

    2016-09-15

    Advances in our understanding of the molecular pathogenesis of B-cell lymphoma have guided the development of targeted therapies that disrupt aberrant signaling pathways important for communication within lymphoma cells and for their interactions with the tumor microenvironment. This has led to unprecedented therapeutic progress, with biologic agents that have begun to transform the care of patients with lymphoma and chronic lymphocytic leukemia. This review discusses the mechanisms of action, clinical development, and emerging applications of small-molecule inhibitors that target B-cell receptor signaling pathways, B-cell lymphoma-2 inhibitors, selective inhibitors of nuclear export, and epigenetic modifiers. PMID:27633417

  12. Rapid burst of H2O2 by plant growth regulators increases intracellular Ca2+ amounts and modulates CD4+ T cell activation.

    PubMed

    Ahmed, Asma; Mukherjee, Sambuddho; Deobagkar, Mukta; Naik, Tanushree; Nandi, Dipankar

    2010-11-01

    The identification of small molecules that affect T cell activation is an important area of research. Three molecules that regulate plant growth and differentiation, but not their structurally similar analogs, were identified to enhance primary mouse CD4(+) T cell activation in conjunction with soluble anti-CD3 stimulation: Indoleacetic acid (natural plant auxin), 1-Napthaleneacetic acid (synthetic plant auxin) and 2,4-Dichlorophenoxyacetic acid (synthetic plant auxin and herbicide). These effects are distinct in comparison to Curcumin, the well known phenolic immunomodulator, which lowers T cell activation. An investigation into the mechanisms of action of the three plant growth regulators revealed a rapid induction of reactive oxygen species (ROS), mainly comprising H(2)O(2). In addition, these three molecules synergize with soluble anti-CD3 signaling to enhance intracellular Ca(2+) concentrations [Ca(2+)](i), leading to greater T cell activation, e.g. induction of CD25 and IL-2. Enhanced production of TNFα and IFNγ by CD4(+) T cells is also observed upon plant growth regulator treatment with soluble anti-CD3. Interestingly, maximal IL-2 production and CD4(+) T cell cycle progression are observed upon activation with soluble anti-CD3 and phorbol 12-myristate 13-acetate (PMA), a phorbol ester. Additionally, stimulation with PMA and Ionomcyin (a Ca(2+) ionophore), which activates T cells by circumventing the TCR, and plant growth regulators also demonstrated the role of the strength of signal (SOS): T cell cycle progression is enhanced with gentle activation conditions but decreased with strong activation conditions. This study demonstrates the direct effects of three plant growth regulators on CD4(+) T cell activation and cycling.

  13. Activation of frog (Xenopus laevis) eggs by inositol trisphosphate. I. Characterization of Ca2+ release from intracellular stores

    PubMed Central

    1985-01-01

    Iontophoresis of inositol 1, 4, 5-triphosphate into frog (Xenopus laevis) eggs activated early developmental events such as membrane depolarization, cortical contraction, cortical granule exocytosis, and abortive cleavage furrow formation (pseudocleavage). Inositol 1, 4- bisphosphate also triggered these events, but only at doses approximately 100-fold higher, whereas no level of fructose-1, 6- bisphosphate tested activated eggs. Using Ca2+-selective microelectrodes, we observed that activating doses of inositol 1, 4, 5- trisphosphate triggered a Ca2+ release from intracellular stores that was indistinguishable from that previously observed at fertilization (Busa, W. B., and R. Nuccitelli, 1985, J. Cell Biol., 100:1325-1329), whereas subthreshold doses triggered only a localized Ca2+ release at the site of injection. The subthreshold IP3 response could be distinguished from the major Ca2+ release at activation with respect to their dose-response characteristics, relative timing, sensitivity to external Ca2+ levels, additivity, and behavior in the activated egg, suggesting that the Xenopus egg may possess two functionally distinct Ca2+ pools mobilized by different effectors. In light of these differences, we suggest a model for intracellular Ca2+ mobilization by sperm-egg interaction. PMID:3874873

  14. An In Vivo Selection Identifies Listeria monocytogenes Genes Required to Sense the Intracellular Environment and Activate Virulence Factor Expression.

    PubMed

    Reniere, Michelle L; Whiteley, Aaron T; Portnoy, Daniel A

    2016-07-01

    Listeria monocytogenes is an environmental saprophyte and facultative intracellular bacterial pathogen with a well-defined life-cycle that involves escape from a phagosome, rapid cytosolic growth, and ActA-dependent cell-to-cell spread, all of which are dependent on the master transcriptional regulator PrfA. The environmental cues that lead to temporal and spatial control of L. monocytogenes virulence gene expression are poorly understood. In this study, we took advantage of the robust up-regulation of ActA that occurs intracellularly and expressed Cre recombinase from the actA promoter and 5' untranslated region in a strain in which loxP sites flanked essential genes, so that activation of actA led to bacterial death. Upon screening for transposon mutants that survived intracellularly, six genes were identified as necessary for ActA expression. Strikingly, most of the genes, including gshF, spxA1, yjbH, and ohrA, are predicted to play important roles in bacterial redox regulation. The mutants identified in the genetic selection fell into three broad categories: (1) those that failed to reach the cytosolic compartment; (2) mutants that entered the cytosol, but failed to activate the master virulence regulator PrfA; and (3) mutants that entered the cytosol and activated transcription of actA, but failed to synthesize it. The identification of mutants defective in vacuolar escape suggests that up-regulation of ActA occurs in the host cytosol and not the vacuole. Moreover, these results provide evidence for two non-redundant cytosolic cues; the first results in allosteric activation of PrfA via increased glutathione levels and transcriptional activation of actA while the second results in translational activation of actA and requires yjbH. Although the precise host cues have not yet been identified, we suggest that intracellular redox stress occurs as a consequence of both host and pathogen remodeling their metabolism upon infection. PMID:27414028

  15. An In Vivo Selection Identifies Listeria monocytogenes Genes Required to Sense the Intracellular Environment and Activate Virulence Factor Expression

    PubMed Central

    Portnoy, Daniel A.

    2016-01-01

    Listeria monocytogenes is an environmental saprophyte and facultative intracellular bacterial pathogen with a well-defined life-cycle that involves escape from a phagosome, rapid cytosolic growth, and ActA-dependent cell-to-cell spread, all of which are dependent on the master transcriptional regulator PrfA. The environmental cues that lead to temporal and spatial control of L. monocytogenes virulence gene expression are poorly understood. In this study, we took advantage of the robust up-regulation of ActA that occurs intracellularly and expressed Cre recombinase from the actA promoter and 5’ untranslated region in a strain in which loxP sites flanked essential genes, so that activation of actA led to bacterial death. Upon screening for transposon mutants that survived intracellularly, six genes were identified as necessary for ActA expression. Strikingly, most of the genes, including gshF, spxA1, yjbH, and ohrA, are predicted to play important roles in bacterial redox regulation. The mutants identified in the genetic selection fell into three broad categories: (1) those that failed to reach the cytosolic compartment; (2) mutants that entered the cytosol, but failed to activate the master virulence regulator PrfA; and (3) mutants that entered the cytosol and activated transcription of actA, but failed to synthesize it. The identification of mutants defective in vacuolar escape suggests that up-regulation of ActA occurs in the host cytosol and not the vacuole. Moreover, these results provide evidence for two non-redundant cytosolic cues; the first results in allosteric activation of PrfA via increased glutathione levels and transcriptional activation of actA while the second results in translational activation of actA and requires yjbH. Although the precise host cues have not yet been identified, we suggest that intracellular redox stress occurs as a consequence of both host and pathogen remodeling their metabolism upon infection. PMID:27414028

  16. High-intensity physical exercise disrupts implicit memory in mice: involvement of the striatal glutathione antioxidant system and intracellular signaling.

    PubMed

    Aguiar, A S; Boemer, G; Rial, D; Cordova, F M; Mancini, G; Walz, R; de Bem, A F; Latini, A; Leal, R B; Pinho, R A; Prediger, R D S

    2010-12-29

    Physical exercise is a widely accepted behavioral strategy to enhance overall health, including mental function. However, there is controversial evidence showing brain mitochondrial dysfunction, oxidative damage and decreased neurotrophin levels after high-intensity exercise, which presumably worsens cognitive performance. Here we investigated learning and memory performance dependent on different brain regions, glutathione antioxidant system, and extracellular signal-regulated protein kinase 1/2 (ERK1/2), serine/threonine protein kinase (AKT), cAMP response element binding (CREB) and dopamine- and cyclic AMP-regulated phosphoprotein (DARPP)-32 signaling in adult Swiss mice submitted to 9 weeks of high-intensity exercise. The exercise did not alter the animals' performance in the reference and working memory versions of the water maze task. On the other hand, we observed a significant impairment in the procedural memory (an implicit memory that depends on basal ganglia) accompanied by a reduced antioxidant capacity and ERK1/2 and CREB signaling in this region. In addition, we found increased striatal DARPP-32-Thr-75 phosphorylation in trained mice. These findings indicate an increased vulnerability of the striatum to high-intensity exercise associated with the disruption of implicit memory in mice and accompanied by alteration of signaling proteins involved in the plasticity of this brain structure.

  17. β-Adrenergic Signaling Inhibits Gq-Dependent PKD Activation by Preventing PKD Translocation

    PubMed Central

    Nichols, C. Blake; Chang, Chia-Wei; Ferrero, Maura; Wood, Brittani M.; Stein, Matthew L.; Ferguson, Amanda J.; Ha, Derrick; Rigor, Robert R.; Bossuyt, Sven; Bossuyt, Julie

    2014-01-01

    Rationale Both β-adrenergic (β-AR) and Gq-coupled agonist (GqR) driven signaling play key roles in the events leading up to and during cardiac dysfunction. How these stimuli interact at the level of protein kinase D (PKD), a nodal point in cardiac hypertrophic signaling, remains unclear. Objective To assess the spatiotemporal dynamics of PKD activation in response to β-AR signaling alone and upon co-activation with GqR agonists. This will test our hypothesis that compartmentalized PKD signaling reconciles disparate findings of protein kinase A (PKA) facilitation and inhibition of PKD activation. Methods and Results We report on the spatial and temporal profiles of PKD activation using GFP-tagged PKD (wildtype or mutant S427E) and targeted FRET based biosensors (DKARs) in adult cardiomyocytes. We find that β-AR/PKA signaling drives local nuclear activation of PKD, without preceding sarcolemmal translocation. We also discover pronounced interference of β-AR/cAMP/PKA signaling on GqR-induced translocation and activation of PKD throughout the cardiomyocyte. We attribute these effects to direct, PKA-dependent phosphorylation of PKD-S427. We also show that phosphomimetic substitution of S427 likewise impedes GqR-induced PKD translocation and activation. In neonatal myocytes, S427E inhibits GqR-evoked cell growth and expression of hypertrophic markers. Lastly, we show altered S427 phosphorylation in TAC-induced hypertrophy. Conclusions β-AR signaling triggers local nuclear signaling and inhibits GqR-mediated PKD activation by preventing its intracellular translocation. PKA-dependent phosphorylation of PKD S427 fine-tunes the PKD responsiveness to GqR-agonists, serving as a key integration point for β-adrenergic and Gq-coupled stimuli. PMID:24643961

  18. Intracellular proteoglycans.

    PubMed Central

    Kolset, Svein Olav; Prydz, Kristian; Pejler, Gunnar

    2004-01-01

    Proteoglycans (PGs) are proteins with glycosaminoglycan chains, are ubiquitously expressed and have a wide range of functions. PGs in the extracellular matrix and on the cell surface have been the subject of extensive structural and functional studies. Less attention has so far been given to PGs located in intracellular compartments, although several reports suggest that these have biological functions in storage granules, the nucleus and other intracellular organelles. The purpose of this review is, therefore, to present some of these studies and to discuss possible functions linked to PGs located in different intracellular compartments. Reference will be made to publications relevant for the topics we present. It is beyond the scope of this review to cover all publications on PGs in intracellular locations. PMID:14759226

  19. Intracellular signaling of the Ufo/Axl receptor tyrosine kinase is mediated mainly by a multi-substrate docking-site.

    PubMed

    Braunger, J; Schleithoff, L; Schulz, A S; Kessler, H; Lammers, R; Ullrich, A; Bartram, C R; Janssen, J W

    1997-06-01

    Ufo/Axl belongs to a new family of receptor tyrosine kinases with an extracellular structure similar to that of neural cell adhesion molecules. In order to elucidate intracellular signaling, the cytoplasmic moiety of Ufo/Axl was used to screen an expression library according to the CORT (cloning of receptor targets) method. Three putative Ufo substrates were identified: phospholipase Cgamma1 (PLCgamma), as well as p85alpha and p85beta subunits of phosphatidylinositol 3'-kinase (PI3-kinase). Subsequently, chimeric EGFR/Ufo receptors consisting of the extracellular domains of the epidermal growth factor receptor (EGFR) and the transmembrane and intracellular moiety of Ufo were engineered. Using different far-Western blot analyses and coimmunoprecipitation assays, receptor binding of PLCgamma and p85 proteins as well as GRB2, c-src and lck was examined in vitro and in vivo. Competitive inhibition of substrate binding and mutagenesis experiments with EGFR/Ufo constructs revealed C-terminal tyrosine 821 (EILpYVNMDEG) as a docking site for multiple effectors, namely PLCgamma, p85 proteins, GRB2, c-src and lck. Tyrosine 779 (DGLpYALMSRC) demonstrated an additional, but lower binding affinity for the p85 proteins in vitro. In addition, binding of PLCgamma occurred through tyrosine 866 (AGRpYVLCPST). Moreover, our in vivo data indicate that further direct or indirect binding sites for PLCgamma, GRB2, c-src and lck on the human Ufo receptor may exist.

  20. C-terminal clipping of chemokine CCL1/I-309 enhances CCR8-mediated intracellular calcium release and anti-apoptotic activity.

    PubMed

    Denis, Catherine; Deiteren, Kathleen; Mortier, Anneleen; Tounsi, Amel; Fransen, Erik; Proost, Paul; Renauld, Jean-Christophe; Lambeir, Anne-Marie

    2012-01-01

    Carboxypeptidase M (CPM) targets the basic amino acids arginine and lysine present at the C-terminus of peptides or proteins. CPM is thought to be involved in inflammatory processes. This is corroborated by CPM-mediated trimming and modulation of inflammatory factors, and expression of the protease in inflammatory environments. Since the function of CPM in and beyond inflammation remains mainly undefined, the identification of natural substrates can aid in discovering the (patho)physiological role of CPM. CCL1/I-309, with its three C-terminal basic amino acids, forms a potential natural substrate for CPM. CCL1 plays a role not only in inflammation but also in apoptosis, angiogenesis and tumor biology. Enzymatic processing differently impacts the biological activity of chemokines thereby contributing to the complex regulation of the chemokine system. The aim of the present study was to investigate whether (i) CCL1/I-309 is prone to trimming by CPM, and (ii) the biological activity of CCL1 is altered after C-terminal proteolytic processing. CCL1 was identified as a novel substrate for CPM in vitro using mass spectrometry. C-terminal clipping of CCL1 augmented intracellular calcium release mediated by CCR8 but reduced the binding of CCL1 to CCR8. In line with the higher intracellular calcium release, a pronounced increase of the anti-apoptotic activity of CCL1 was observed in the BW5147 cellular model. CCR8 signaling, CCR8 binding and anti-apoptotic activity were unaffected when CPM was exposed to the carboxypeptidase inhibitor DL-2-mercaptomethyl-3-guanidino-ethylthiopropanoic acid. The results of this study suggest that CPM is a likely candidate for the regulation of biological processes relying on the CCL1-CCR8 system. PMID:22479563

  1. Intracellular Zn2+ accumulation enhances suppression of synaptic activity following spreading depolarization.

    PubMed

    Carter, Russell E; Seidel, Jessica L; Lindquist, Britta E; Sheline, Christian T; Shuttleworth, C William

    2013-06-01

    Spreading depolarization (SD) is a feed-forward wave that propagates slowly throughout brain tissue and recovery from SD involves substantial metabolic demand. Presynaptic Zn(2+) release and intracellular accumulation occurs with SD, and elevated intracellular Zn(2+) ([Zn(2+) ]i ) can impair cellular metabolism through multiple pathways. We tested here whether increased [Zn(2+) ]i could exacerbate the metabolic challenge of SD, induced by KCl, and delay recovery in acute murine hippocampal slices. [Zn(2+) ]i loading prior to SD, by transient ZnCl2 application with the Zn(2+) ionophore pyrithione (Zn/Pyr), delayed recovery of field excitatory post-synaptic potentials (fEPSPs) in a concentration-dependent manner, prolonged DC shifts, and significantly increased extracellular adenosine accumulation. These effects could be due to metabolic inhibition, occurring downstream of pyruvate utilization. Prolonged [Zn(2+) ]i accumulation prior to SD was required for effects on fEPSP recovery and consistent with this, endogenous synaptic Zn(2+) release during SD propagation did not delay recovery from SD. The effects of exogenous [Zn(2+) ]i loading were also lost in slices preconditioned with repetitive SDs, implying a rapid adaptation. Together, these results suggest that [Zn(2+) ]i loading prior to SD can provide significant additional challenge to brain tissue, and could contribute to deleterious effects of [Zn(2+) ]i accumulation in a range of brain injury models. PMID:23495967

  2. An active intracellular device to prevent lethal disease outcomes in virus-infected bacterial cells.

    PubMed

    Bagh, Sangram; Mandal, Mahuya; Ang, Jordan; McMillen, David R

    2011-03-01

    Synthetic biology includes an effort to logically control cellular behavior. One long-term goal is to implement medical interventions inside living cells, creating intracellular "disease fighters"; one may imagine a system that detects viral infection and responds to halt the spread of the virus. Here, we explore a system designed to display some of the qualitative features that such disease prevention systems should have, while not claiming that the system itself has any medical application. An intracellular disease prevention mechanism should: lie dormant in the absence of the disease state; detect the onset of a lethal disease pathway; respond to halt or mitigate the disease's effects; and be subject to external deactivation when required. We have created a device that displays these properties, in the highly simplified case of a bacterial viral disease. Our system detects the onset of the lytic phase of bacteriophage lambda in Escherichia coli, responds by preventing this lethal pathway from being followed, and is deactivated by a temperature shift. We have formulated a mathematical model of the engineered system, using parameters obtained from the literature and by local experimental measurement, and shown that the model captures the essential experimental behavior of the system in most parameter regimes.

  3. Intracellular mechanisms coupled to NPY Y2 and Y5 receptor activation and lipid accumulation in murine adipocytes.

    PubMed

    Rosmaninho-Salgado, Joana; Cortez, Vera; Estrada, Marta; Santana, Magda M; Gonçalves, Alexandra; Marques, Ana Patrícia; Cavadas, Cláudia

    2012-12-01

    The formation of adipose tissue is a process that includes the pre-adipocyte proliferation and differentiation to adipocytes that are cells specialized in lipid accumulation. The adipocyte differentiation is a process driven by the coordinated expression of various transcription factors, such as peroxisome proliferator-activated receptor (PPAR-γ). Neuropeptide Y (NPY) induces adipocyte proliferation and differentiation but the NPY receptors and the intracellular pathways involved in these processes are still not clear. In the present work we studied the role of NPY receptors and the intracellular pathways involved in the stimulatory effect of NPY on lipid accumulation. The murine pre-adipocyte cell line, 3T3-L1, was used as a cell model. Adipogenesis was evaluated by quantifying lipid accumulation by Oil red-O assay and by analyzing PPAR-γ expression using the Western blotting assay. Adipocytes were incubated with NPY (100nM) and a decrease on lipid accumulation and PPAR-γ expression was observed in the presence of NPY Y(2) receptor antagonist (BIIE0246, 1μM) or NPY Y(5) antagonist. Furthermore, NPY Y(2) (NPY(3-36), 100nM) or NPY Y(5) (NPY(19-23)(GLY(1), Ser(3), Gln(4), Thr(6), Ala(31), Aib(32), Gln(34)) PP, 100nM) receptor agonists increased lipid accumulation and PPAR-γ expression. We further investigate the intracellular pathways associated with NPY Y(2) and NPY Y(5) receptor activation. Our results show NPY induces PPAR-γ expression and lipid accumulation through NPY Y(2) and NPY Y(5) receptors activation. PKC and PLC inhibitors inhibit lipid accumulation induced by NPY Y(5) receptor agonist. Moreover, our results suggest that lipid accumulation induced by NPY Y(2) receptor activation occurs through PKA, MAPK and PI3K pathways. In conclusion, this study contributes to a step forward on the knowledge of intracellular mechanisms associated with NPY receptors activation on adipocytes and contributes to a better understanding and the development of new

  4. The PAK system links Rho GTPase signaling to thrombin-mediated platelet activation

    PubMed Central

    Baker, Sandra M.; Loren, Cassandra P.; Haley, Kristina M.; Itakura, Asako; Pang, Jiaqing; Greenberg, Daniel L.; David, Larry L.; Manser, Ed; Chernoff, Jonathan; McCarty, Owen J. T.

    2013-01-01

    Regulation of the platelet actin cytoskeleton by the Rho family of small GTPases is essential for the proper maintenance of hemostasis. However, little is known about how intracellular platelet activation from Rho GTPase family members, including Rac, Cdc42, and Rho, translate into changes in platelet actin structures. To better understand how Rho family GTPases coordinate platelet activation, we identified platelet proteins associated with Rac1, a Rho GTPase family member, and actin regulatory protein essential for platelet hemostatic function. Mass spectrometry analysis revealed that upon platelet activation with thrombin, Rac1 associates with a set of effectors of the p21-activated kinases (PAKs), including GIT1, βPIX, and guanine nucleotide exchange factor GEFH1. Platelet activation by thrombin triggered the PAK-dependent phosphorylation of GIT1, GEFH1, and other PAK effectors, including LIMK1 and Merlin. PAK was also required for the thrombin-mediated activation of the MEK/ERK pathway, Akt, calcium signaling, and phosphatidylserine (PS) exposure. Inhibition of PAK signaling prevented thrombin-induced platelet aggregation and blocked platelet focal adhesion and lamellipodia formation in response to thrombin. Together, these results demonstrate that the PAK signaling system is a key orchestrator of platelet actin dynamics, linking Rho GTPase activation downstream of thrombin stimulation to PAK effector function, MAP kinase activation, calcium signaling, and PS exposure in platelets. PMID:23784547

  5. New Constitutively Active Phytochromes Exhibit Light-Independent Signaling Activity.

    PubMed

    Jeong, A-Reum; Lee, Si-Seok; Han, Yun-Jeong; Shin, Ah-Young; Baek, Ayoung; Ahn, Taeho; Kim, Min-Gon; Kim, Young Soon; Lee, Keun Woo; Nagatani, Akira; Kim, Jeong-Il

    2016-08-01

    Plant phytochromes are photoreceptors that mediate a variety of photomorphogenic responses. There are two spectral photoisomers, the red light-absorbing Pr and far-red light-absorbing Pfr forms, and the photoreversible transformation between the two forms is important for the functioning of phytochromes. In this study, we isolated a Tyr-268-to-Val mutant of Avena sativa phytochrome A (AsYVA) that displayed little photoconversion. Interestingly, transgenic plants of AsYVA showed light-independent phytochrome signaling with a constitutive photomorphogenic (cop) phenotype that is characterized by shortened hypocotyls and open cotyledons in the dark. In addition, the corresponding Tyr-303-to-Val mutant of Arabidopsis (Arabidopsis thaliana) phytochrome B (AtYVB) exhibited nuclear localization and interaction with phytochrome-interacting factor 3 (PIF3) independently of light, conferring a constitutive photomorphogenic development to its transgenic plants, which is comparable to the first constitutively active version of phytochrome B (YHB; Tyr-276-to-His mutant). We also found that chromophore ligation was required for the light-independent interaction of AtYVB with PIF3. Moreover, we demonstrated that AtYVB did not exhibit phytochrome B activity when it was localized in the cytosol by fusion with the nuclear export signal and that AsYVA exhibited the full activity of phytochrome A when localized in the nucleus by fusion with the nuclear localization signal. Furthermore, the corresponding Tyr-269-to-Val mutant of Arabidopsis phytochrome A (AtYVA) exhibited similar cop phenotypes in transgenic plants to AsYVA. Collectively, these results suggest that the conserved Tyr residues in the chromophore-binding pocket play an important role during the Pr-to-Pfr photoconversion of phytochromes, providing new constitutively active alleles of phytochromes by the Tyr-to-Val mutation. PMID:27325667

  6. Intracellular Antioxidant Activity of Grape Skin Polyphenolic Extracts in Rat Superficial Colonocytes: In situ Detection by Confocal Fluorescence Microscopy

    PubMed Central

    Giordano, M. Elena; Ingrosso, Ilaria; Schettino, Trifone; Caricato, Roberto; Giovinazzo, Giovanna; Lionetto, M. Giulia

    2016-01-01

    Colon is exposed to a number of prooxidant conditions and several colon diseases are associated with increased levels of reactive species. Polyphenols are the most abundant antioxidants in the diet, but to date no information is available about their absorption and potential intracellular antioxidant activity on colon epithelial cells. The work was addressed to study the intracellular antioxidant activity of red grape polyphenolic extracts on rat colon epithelium experimentally exposed to prooxidant conditions. The experimental model chosen was represented by freshly isolated colon explants, which closely resemble the functional, and morphological characteristics of the epithelium in vivo. The study was carried out by in situ confocal microscopy observation on CM-H2DCFDA charged explants exposed to H2O2 (5, 10, and 15 min). The qualitative and quantitative polyphenolic composition of the extracts as well as their in vitro oxygen radical absorbing capacity (ORAC) was determined. The incubation of the explants with the polyphenolic extracts for 1 h produced a significant decrease of the H2O2 induced fluorescence. This effect was more pronounced following 15 min H2O2 exposure with respect to 5 min and it was also more evident for extracts obtained from mature grapes, which showed an increased ORAC value and qualitative peculiarities in the polyphenolic composition. The results demonstrated the ability of red grape polyphenols to cross the plasma membrane and exert a direct intracellular antioxidant activity in surface colonocytes, inducing a protection against pro-oxidant conditions. The changes in the polyphenol composition due to ripening process was reflected in a more effective antioxidant protection. PMID:27303304

  7. Intracellular Antioxidant Activity of Grape Skin Polyphenolic Extracts in Rat Superficial Colonocytes: In situ Detection by Confocal Fluorescence Microscopy.

    PubMed

    Giordano, M Elena; Ingrosso, Ilaria; Schettino, Trifone; Caricato, Roberto; Giovinazzo, Giovanna; Lionetto, M Giulia

    2016-01-01

    Colon is exposed to a number of prooxidant conditions and several colon diseases are associated with increased levels of reactive species. Polyphenols are the most abundant antioxidants in the diet, but to date no information is available about their absorption and potential intracellular antioxidant activity on colon epithelial cells. The work was addressed to study the intracellular antioxidant activity of red grape polyphenolic extracts on rat colon epithelium experimentally exposed to prooxidant conditions. The experimental model chosen was represented by freshly isolated colon explants, which closely resemble the functional, and morphological characteristics of the epithelium in vivo. The study was carried out by in situ confocal microscopy observation on CM-H2DCFDA charged explants exposed to H2O2 (5, 10, and 15 min). The qualitative and quantitative polyphenolic composition of the extracts as well as their in vitro oxygen radical absorbing capacity (ORAC) was determined. The incubation of the explants with the polyphenolic extracts for 1 h produced a significant decrease of the H2O2 induced fluorescence. This effect was more pronounced following 15 min H2O2 exposure with respect to 5 min and it was also more evident for extracts obtained from mature grapes, which showed an increased ORAC value and qualitative peculiarities in the polyphenolic composition. The results demonstrated the ability of red grape polyphenols to cross the plasma membrane and exert a direct intracellular antioxidant activity in surface colonocytes, inducing a protection against pro-oxidant conditions. The changes in the polyphenol composition due to ripening process was reflected in a more effective antioxidant protection. PMID:27303304

  8. Delineation of upstream signaling events in the salmonella pathogenicity island 2 transcriptional activation pathway.

    PubMed

    Kim, Charles C; Falkow, Stanley

    2004-07-01

    Survival and replication in the intracellular environment are critical components of the ability of Salmonella enterica serovar Typhimurium to establish systemic infection in the murine host. Intracellular survival is mediated by a number of genetic loci, including Salmonella pathogenicity island 2 (SPI2). SPI2 is a 40-kb locus encoding a type III secretion system that secretes effector molecules, which permits bacterial survival and replication in the intracellular environment of host cells. A two-component regulatory system, ssrAB, is also encoded in SPI2 and controls expression of the secretion system and effectors. While the environmental signals to which SPI2 responds in vivo are not known, activation of expression is dependent on OmpR and can be stimulated in vitro by chelation of cations or by a shift from rich to acidic minimal medium. In this work, we demonstrated that SPI2 activation is associated with OmpR in the phosphorylated form (OmpR-P). Mutations in envZ and ackA-pta, which disrupted two distinct sources of OmpR phosphorylation, indicated that SPI2 activation by chelators or a shift from rich to acidic minimal medium is largely dependent on functional EnvZ. In contrast, the PhoPQ pathway is not required for SPI2 activation in the presence of OmpR-P. As in the case of in vitro stimulation, SPI2 expression in macrophages correlates with the presence of OmpR-P. Additionally, EnvZ, but not acetyl phosphate, is required for maximal expression of SPI2 in the intracellular environment, suggesting that the in vitro SPI2 activation pathway is the same as that used in vivo.

  9. Arsenic-induced alteration in intracellular calcium homeostasis induces head kidney macrophage apoptosis involving the activation of calpain-2 and ERK in Clarias batrachus

    SciTech Connect

    Banerjee, Chaitali; Goswami, Ramansu; Datta, Soma; Rajagopal, R.; Mazumder, Shibnath

    2011-10-01

    We had earlier shown that exposure to arsenic (0.50 {mu}M) caused caspase-3 mediated head kidney macrophage (HKM) apoptosis involving the p38-JNK pathway in Clarias batrachus. Here we examined the roles of calcium (Ca{sup 2+}) and extra-cellular signal-regulated protein kinase (ERK), the other member of MAPK-pathway on arsenic-induced HKM apoptosis. Arsenic-induced HKM apoptosis involved increased expression of ERK and calpain-2. Nifedipine, verapamil and EGTA pre-treatment inhibited the activation of calpain-2, ERK and reduced arsenic-induced HKM apoptosis as evidenced from reduced caspase-3 activity, Annexin V-FITC-propidium iodide and Hoechst 33342 staining. Pre-incubation with ERK inhibitor U 0126 inhibited the activation of calpain-2 and interfered with arsenic-induced HKM apoptosis. Additionally, pre-incubation with calpain-2 inhibitor also interfered with the activation of ERK and inhibited arsenic-induced HKM apoptosis. The NADPH oxidase inhibitor apocynin and diphenyleneiodonium chloride also inhibited ERK activation indicating activation of ERK in arsenic-exposed HKM also depends on signals from NADPH oxidase pathway. Our study demonstrates the critical role of Ca{sup 2+} homeostasis on arsenic-induced HKM apoptosis. We suggest that arsenic-induced alteration in intracellular Ca{sup 2+} levels initiates pro-apoptotic ERK and calpain-2; the two pathways influence each other positively and induce caspase-3 mediated HKM apoptosis. Besides, our study also indicates the role of ROS in the activation of ERK pathway in arsenic-induced HKM apoptosis in C. batrachus. - Highlights: > Altered Ca{sup 2+} homeostasis leads to arsenic-induced HKM apoptosis. > Calpain-2 plays a critical role in the process. > ERK is pro-apoptotic in arsenic-induced HKM apoptosis. > Arsenic-induced HKM apoptosis involves cross talk between calpain-2 and ERK.

  10. Activation of Notch1 signaling is required for β-catenin–mediated human primary melanoma progression

    PubMed Central

    Balint, Klara; Xiao, Min; Pinnix, Chelsea C.; Soma, Akinobu; Veres, Imre; Juhasz, Istvan; Brown, Eric J.; Capobianco, Anthony J.; Herlyn, Meenhard; Liu, Zhao-Jun

    2005-01-01

    Notch is a highly conserved transmembrane receptor that determines cell fate. Notch signaling denotes cleavage of the Notch intracellular domain, its translocation to the nucleus, and subsequent activation of target gene transcription. Involvement of Notch signaling in several cancers is well known, but its role in melanoma remains poorly characterized. Here we show that the Notch1 pathway is activated in human melanoma. Blocking Notch signaling suppressed whereas constitutive activation of the Notch1 pathway enhanced primary melanoma cell growth both in vitro and in vivo yet had little effect on metastatic melanoma cells. Activation of Notch1 signaling enabled primary melanoma cells to gain metastatic capability. Furthermore, the oncogenic effect of Notch1 on primary melanoma cells was mediated by β-catenin, which was upregulated following Notch1 activation. Inhibiting β-catenin expression reversed Notch1-enhanced tumor growth and metastasis. Our data therefore suggest a β-catenin–dependent, stage-specific role for Notch1 signaling in promoting the progression of primary melanoma. PMID:16239965

  11. Entry of Sanfetrinem into Human Polymorphonuclear Granulocytes and Its Cell-Associated Activity against Intracellular, Penicillin-Resistant Streptococcus pneumoniae

    PubMed Central

    Cuffini, Anna Maria; Tullio, Vivian; Bonino, Alessandro; Allocco, Alessandra; Palarchio, Angela Ianni; Carlone, Nicola A.

    1998-01-01

    The entry of antibiotics into phagocytes is necessary for activity against intracellular pathogens. The ability of sanfetrinem, the first member of a new class of antibiotics, to penetrate human polymorphonuclear granulocytes and its consequences upon subsequent phagocytosis and killing of ingested penicillin-resistant Streptococcus pneumoniae have been evaluated. Sanfetrinem penetrated into human polymorphonuclear leukocytes (PMNs) at all concentrations tested, with cellular concentration/extracellular concentration ratios of 6.6 to 5.03 and 4.21 when sanfetrinem was used at 0.25 to 0.5 and 1 μg/ml, respectively, within 30 min of incubation. The uptake was complete within 5 min and was not energy dependent, since it was not affected by cell viability, environmental temperature, or the addition of a metabolic inhibitor. At a concentration of one-half the MIC, sanfetrinem significantly enhanced human PMN phagocytosis and increased intracellular bactericidal activity against penicillin-resistant S. pneumoniae. Following preexposure of PMNs to a concentration of one-half the MIC of sanfetrinem, there was a significant increase in both phagocytosis and killing compared with that for the controls, indicating the ability of sanfetrinem to interact with biological membranes and remain active within PMNs. Preexposure of streptococci to sanfetrinem made penicillin-resistant S. pneumoniae more susceptible to the bactericidal mechanisms of human PMNs than untreated organisms. PMID:9661015

  12. Subcellular optogenetic activation of Cdc42 controls local and distal signaling to drive immune cell migration

    PubMed Central

    O’Neill, Patrick R.; Kalyanaraman, Vani; Gautam, N.

    2016-01-01

    Migratory immune cells use intracellular signaling networks to generate and orient spatially polarized responses to extracellular cues. The monomeric G protein Cdc42 is believed to play an important role in controlling the polarized responses, but it has been difficult to determine directly the consequences of localized Cdc42 activation within an immune cell. Here we used subcellular optogenetics to determine how Cdc42 activation at one side of a cell affects both cell behavior and dynamic molecular responses throughout the cell. We found that localized Cdc42 activation is sufficient to generate polarized signaling and directional cell migration. The optically activated region becomes the leading edge of the cell, with Cdc42 activating Rac and generating membrane protrusions driven by the actin cytoskeleton. Cdc42 also exerts long-range effects that cause myosin accumulation at the opposite side of the cell and actomyosin-mediated retraction of the cell rear. This process requires the RhoA-activated kinase ROCK, suggesting that Cdc42 activation at one side of a cell triggers increased RhoA signaling at the opposite side. Our results demonstrate how dynamic, subcellular perturbation of an individual signaling protein can help to determine its role in controlling polarized cellular responses. PMID:26941336

  13. Ethanol Enhances TGF-β Activity by Recruiting TGF-β Receptors From Intracellular Vesicles/Lipid Rafts/Caveolae to Non-Lipid Raft Microdomains.

    PubMed

    Huang, Shuan Shian; Chen, Chun-Lin; Huang, Franklin W; Johnson, Frank E; Huang, Jung San

    2016-04-01

    Regular consumption of moderate amounts of ethanol has important health benefits on atherosclerotic cardiovascular disease (ASCVD). Overindulgence can cause many diseases, particularly alcoholic liver disease (ALD). The mechanisms by which ethanol causes both beneficial and harmful effects on human health are poorly understood. Here we demonstrate that ethanol enhances TGF-β-stimulated luciferase activity with a maximum of 0.5-1% (v/v) in Mv1Lu cells stably expressing a luciferase reporter gene containing Smad2-dependent elements. In Mv1Lu cells, 0.5% ethanol increases the level of P-Smad2, a canonical TGF-β signaling sensor, by ∼ 2-3-fold. Ethanol (0.5%) increases cell-surface expression of the type II TGF-β receptor (TβR-II) by ∼ 2-3-fold from its intracellular pool, as determined by I(125) -TGF-β-cross-linking/Western blot analysis. Sucrose density gradient ultracentrifugation and indirect immunofluorescence staining analyses reveal that ethanol (0.5% and 1%) also displaces cell-surface TβR-I and TβR-II from lipid rafts/caveolae and facilitates translocation of these receptors to non-lipid raft microdomains where canonical signaling occurs. These results suggest that ethanol enhances canonical TGF-β signaling by increasing non-lipid raft microdomain localization of the TGF-β receptors. Since TGF-β plays a protective role in ASCVD but can also cause ALD, the TGF-β enhancer activity of ethanol at low and high doses appears to be responsible for both beneficial and harmful effects. Ethanol also disrupts the location of lipid raft/caveolae of other membrane proteins (e.g., neurotransmitter, growth factor/cytokine, and G protein-coupled receptors) which utilize lipid rafts/caveolae as signaling platforms. Displacement of these membrane proteins induced by ethanol may result in a variety of pathologies in nerve, heart and other tissues.

  14. Coincident signalling between the Gi/Go-coupled delta-opioid receptor and the Gq-coupled m3 muscarinic receptor at the level of intracellular free calcium in SH-SY5Y cells.

    PubMed

    Yeo, A; Samways, D S; Fowler, C E; Gunn-Moore, F; Henderson, G

    2001-03-01

    In SH-SY5Y cells, activation of delta-opioid receptors with [D-Pen(2,5)]-enkephalin (DPDPE; 1 microM) did not alter the intracellular free Ca(2+) concentration [Ca(2+)](i). However, when DPDPE was applied during concomitant Gq-coupled m3 muscarinic receptor stimulation by carbachol or oxotremorine-M, it produced an elevation of [Ca(2+)](i). The DPDPE-evoked increase in [Ca(2+)](i) was abolished when the carbachol-sensitive intracellular Ca(2+) store was emptied. There was a marked difference between the concentration-response relationship for the elevation of [Ca(2+)](i) by carbachol (EC(50) 13 microM, Hill slope 1) and the concentration-response relationship for carbachol's permissive action in revealing the delta-opioid receptor-mediated elevation of [Ca(2+)] (EC(50) 0.7 mM; Hill slope 1.8). Sequestration of free G protein beta gamma dimers by transient transfection of cells with a beta gamma binding protein (residues 495-689 of the C terminal tail of G protein-coupled receptor kinase 2) reduced the ability of delta opioid receptor activation to elevate [Ca(2+)](i). However, DPDPE did not elevate either basal or oxotremorine-M-evoked inositol phosphate production indicating that delta-opioid receptor activation did not stimulate phospholipase C. Furthermore, delta-opioid receptor activation did not result in the reversal of muscarinic receptor desensitization, membrane hyperpolarization or stimulation of sphingosine kinase. There was no coincident signalling between the delta-opioid receptor and the lysophosphatidic acid receptor which couples to elevation of [Ca(2+)](i) in SH-SY5Y cells by a PLC-independent mechanism. In SH-SY5Y cells the coincident signalling between the endogenously expressed delta-opioid and m3 muscarinic receptors appears to occur in the receptor activation-Ca(2+) release signalling pathway at a step after the activation of phospholipase C. PMID:11259487

  15. Conjugated Bilirubin Differentially Regulates CD4+ T Effector Cells and T Regulatory Cell Function through Outside-In and Inside-Out Mechanisms: The Effects of HAV Cell Surface Receptor and Intracellular Signaling.

    PubMed

    Corral-Jara, Karla F; Trujillo-Ochoa, Jorge L; Realpe, Mauricio; Panduro, Arturo; Gómez-Leyva, Juan F; Rosenstein, Yvonne; Jose-Abrego, Alexis; Roman, Sonia; Fierro, Nora A

    2016-01-01

    We recently reported an immune-modulatory role of conjugated bilirubin (CB) in hepatitis A virus (HAV) infection. During this infection the immune response relies on CD4+ T lymphocytes (TLs) and it may be affected by the interaction of HAV with its cellular receptor (HAVCR1/TIM-1) on T cell surface. How CB might affect T cell function during HAV infection remains to be elucidated. Herein, in vitro stimulation of CD4+ TLs from healthy donors with CB resulted in a decrease in the degree of intracellular tyrosine phosphorylation and an increase in the activity of T regulatory cells (Tregs) expressing HAVCR1/TIM-1. A comparison between CD4+ TLs from healthy donors and HAV-infected patients revealed changes in the TCR signaling pathway relative to changes in CB levels. The proportion of CD4+CD25+ TLs increased in patients with low CB serum levels and an increase in the percentage of Tregs expressing HAVCR1/TIM-1 was found in HAV-infected patients relative to controls. A low frequency of 157insMTTTVP insertion in the viral receptor gene HAVCR1/TIM-1 was found in patients and controls. Our data revealed that, during HAV infection, CB differentially regulates CD4+ TLs and Tregs functions by modulating intracellular pathways and by inducing changes in the proportion of Tregs expressing HAVCR1/TIM-1. PMID:27578921

  16. Conjugated Bilirubin Differentially Regulates CD4+ T Effector Cells and T Regulatory Cell Function through Outside-In and Inside-Out Mechanisms: The Effects of HAV Cell Surface Receptor and Intracellular Signaling

    PubMed Central

    Corral-Jara, Karla F.; Gómez-Leyva, Juan F.; Rosenstein, Yvonne; Jose-Abrego, Alexis; Roman, Sonia

    2016-01-01

    We recently reported an immune-modulatory role of conjugated bilirubin (CB) in hepatitis A virus (HAV) infection. During this infection the immune response relies on CD4+ T lymphocytes (TLs) and it may be affected by the interaction of HAV with its cellular receptor (HAVCR1/TIM-1) on T cell surface. How CB might affect T cell function during HAV infection remains to be elucidated. Herein, in vitro stimulation of CD4+ TLs from healthy donors with CB resulted in a decrease in the degree of intracellular tyrosine phosphorylation and an increase in the activity of T regulatory cells (Tregs) expressing HAVCR1/TIM-1. A comparison between CD4+ TLs from healthy donors and HAV-infected patients revealed changes in the TCR signaling pathway relative to changes in CB levels. The proportion of CD4+CD25+ TLs increased in patients with low CB serum levels and an increase in the percentage of Tregs expressing HAVCR1/TIM-1 was found in HAV-infected patients relative to controls. A low frequency of 157insMTTTVP insertion in the viral receptor gene HAVCR1/TIM-1 was found in patients and controls. Our data revealed that, during HAV infection, CB differentially regulates CD4+ TLs and Tregs functions by modulating intracellular pathways and by inducing changes in the proportion of Tregs expressing HAVCR1/TIM-1. PMID:27578921

  17. Conjugated Bilirubin Differentially Regulates CD4+ T Effector Cells and T Regulatory Cell Function through Outside-In and Inside-Out Mechanisms: The Effects of HAV Cell Surface Receptor and Intracellular Signaling.

    PubMed

    Corral-Jara, Karla F; Trujillo-Ochoa, Jorge L; Realpe, Mauricio; Panduro, Arturo; Gómez-Leyva, Juan F; Rosenstein, Yvonne; Jose-Abrego, Alexis; Roman, Sonia; Fierro, Nora A

    2016-01-01

    We recently reported an immune-modulatory role of conjugated bilirubin (CB) in hepatitis A virus (HAV) infection. During this infection the immune response relies on CD4+ T lymphocytes (TLs) and it may be affected by the interaction of HAV with its cellular receptor (HAVCR1/TIM-1) on T cell surface. How CB might affect T cell function during HAV infection remains to be elucidated. Herein, in vitro stimulation of CD4+ TLs from healthy donors with CB resulted in a decrease in the degree of intracellular tyrosine phosphorylation and an increase in the activity of T regulatory cells (Tregs) expressing HAVCR1/TIM-1. A comparison between CD4+ TLs from healthy donors and HAV-infected patients revealed changes in the TCR signaling pathway relative to changes in CB levels. The proportion of CD4+CD25+ TLs increased in patients with low CB serum levels and an increase in the percentage of Tregs expressing HAVCR1/TIM-1 was found in HAV-infected patients relative to controls. A low frequency of 157insMTTTVP insertion in the viral receptor gene HAVCR1/TIM-1 was found in patients and controls. Our data revealed that, during HAV infection, CB differentially regulates CD4+ TLs and Tregs functions by modulating intracellular pathways and by inducing changes in the proportion of Tregs expressing HAVCR1/TIM-1.

  18. Bimatoprost and prostaglandin F(2 alpha) selectively stimulate intracellular calcium signaling in different cat iris sphincter cells.

    PubMed

    Spada, Clayton S; Krauss, Achim H-P; Woodward, David F; Chen, June; Protzman, Charles E; Nieves, Amelia L; Wheeler, Larry A; Scott, David F; Sachs, George

    2005-01-01

    Bimatoprost is a synthetic analog of prostaglandin F(2 alpha) ethanolamide (prostamide F(2 alpha)), and shares a pharmacological profile consistent with that of the prostamides. Like prostaglandin F(2 alpha) carboxylic acid, bimatoprost potently lowers intraocular pressure in dogs, primates and humans. In order to distinguish its mechanism of action from prostaglandin F(2 alpha), fluorescence confocal microscopy was used to examine the effects of bimatoprost, prostaglandin F(2 alpha) and 17-phenyl prostaglandin F(2 alpha) on calcium signaling in resident cells of digested cat iris sphincter, a tissue which exhibits contractile responses to both agonists. Constant superfusion conditions obviated effective conversion of bimatoprost. Serial challenge with 100 nM bimatoprost and prostaglandin F(2 alpha) consistently evoked responses in different cells within the same tissue preparation, whereas prostaglandin F(2 alpha) and 17-phenyl prostaglandin F(2 alpha) elicited signaling responses in the same cells. Bimatoprost-sensitive cells were consistently re-stimulated with bimatoprost only, and prostaglandin F(2 alpha) sensitive cells could only be re-stimulated with prostaglandin F(2 alpha). The selective stimulation of different cells in the same cat iris sphincter preparation by bimatoprost and prostaglandin F(2 alpha), along with the complete absence of observed instances in which the same cells respond to both agonists, strongly suggests the involvement of distinct receptors for prostaglandin F(2 alpha) and bimatoprost. Further, prostaglandin F(2 alpha) but not bimatoprost potently stimulated calcium signaling in isolated human embryonic kidney cells stably transfected with the feline- and human-prostaglandin F(2 alpha) FP-receptor and in human dermal fibroblast cells, and only prostaglandin F(2 alpha) competed with radioligand binding in HEK-feFP cells. These studies provide further evidence for the existence of a bimatoprost-sensitive receptor that is distinct from

  19. Interferon gamma-activated human monocytes downregulate transferrin receptors and inhibit the intracellular multiplication of Legionella pneumophila by limiting the availability of iron.

    PubMed Central

    Byrd, T F; Horwitz, M A

    1989-01-01

    We have investigated the role of iron in the intracellular biology of Legionella pneumophila in human monocytes and in the effector arm of cell-mediated immune defense against this intracellular bacterial pathogen. To determine if L. pneumophila intracellular multiplication is iron dependent, we studied the effect of the iron chelator deferoxamine on L. pneumophila infection of monocytes. Deferoxamine at 15 microM completely inhibited L. pneumophila intracellular multiplication. The inhibitory effect of deferoxamine was reversed with equimolar iron-saturated transferrin but not apotransferrin. To examine the potential role of iron in monocyte activation, we investigated the influence of iron-saturated transferrin on L. pneumophila multiplication in IFN gamma-activated monocytes. Iron transferrin, but not apotransferrin, neutralized the capacity of activated monocytes to inhibit L. pneumophila multiplication. To explore a potential mechanism by which activated monocytes might limit the availability of intracellular iron, we examined transferrin receptor expression on nonactivated and activated monocytes cultured in vitro for 5 d. By fluorescence-activated flow cytometry, activated monocytes exhibited markedly fewer transferrin receptors than nonactivated monocytes. By Scatchard analysis of 125I-transferrin binding to monocytes, nonactivated monocytes had 38,300 +/- 12,700 (mean +/- SE) transferrin binding sites, whereas activated monocytes had 10,300 +/- 1,600, a reduction of 73%. Activated and nonactivated monocytes had a similar mean Kd (1.8 +/- 0.2 nM). This study demonstrates that (a) L. pneumophila intracellular multiplication is iron dependent; (b) activated monocytes inhibit L. pneumophila multiplication by limiting the availability of intracellular iron; and (c) transferrin receptors are downregulated on IFN gamma-activated monocytes. Images PMID:2496141

  20. Proteinase activated-receptors-associated signaling in the control of gastric cancer

    PubMed Central

    Sedda, Silvia; Marafini, Irene; Caruso, Roberta; Pallone, Francesco; Monteleone, Giovanni

    2014-01-01

    Gastric cancer (GC) is the fourth most common cancer in the world and the second cause of cancer-related death. Gastric carcinogenesis is a multifactorial process, in which environmental and genetic factors interact to activate multiple intracellular signals thus leading to uncontrolled growth and survival of GC cells. One such a pathway is regulated by proteinase activated-receptors (PARs), seven transmembrane-spanning domain G protein-coupled receptors, which comprise four receptors (i.e., PAR-1, PAR-2, PAR-3, and PAR-4) activated by various proteases. Both PAR-1 and PAR-2 are over-expressed on GC cells and their activation triggers and/or amplifies intracellular pathways, which sustain gastric carcinogenesis. There is also evidence that expression of either PAR-1 or PAR-2 correlates with depth of wall invasion and metastatic dissemination and inversely with the overall survival of patients. Consistently, data emerging from experimental models of GC suggest that both these receptors can be important targets for therapeutic interventions in GC patients. In contrast, PAR-4 levels are down-regulated in GC and correlate inversely with the aggressiveness of GC, thus suggesting a negative role of this receptor in the control of GC. In this article we review the available data on the expression and role of PARs in GC and discuss whether manipulation of PAR-driven signals may be useful for interfering with GC cell behavior. PMID:25232234