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Sample records for intracellular chloride regulation

  1. Chloride Channels of Intracellular Membranes

    PubMed Central

    Edwards, John C.; Kahl, Christina R.

    2010-01-01

    Proteins implicated as intracellular chloride channels include the intracellular ClC proteins, the bestrophins, the cystic fibrosis transmembrane conductance regulator, the CLICs, and the recently described Golgi pH regulator. This paper examines current hypotheses regarding roles of intracellular chloride channels and reviews the evidence supporting a role in intracellular chloride transport for each of these proteins. PMID:20100480

  2. Human epithelial cystic fibrosis transmembrane conductance regulator without exon 5 maintains partial chloride channel function in intracellular membranes.

    PubMed Central

    Xie, J; Drumm, M L; Zhao, J; Ma, J; Davis, P B

    1996-01-01

    The cardiac isoform of the cystic fibrosis transmembrane conductance regulator (CFTR) is a splice variant of the epithelial CFTR, with lacks 30 amino acids encoded by exon 5 in the first intracellular loop. For examination of the role of exon 5 in CFTR channel function, a CFTR deletion mutant, in which exon 5 was removed from the human epithelial CFTR, was constructed. The wild type and delta exon5 CFTR were expressed in a human embryonic kidney cell line (293 HEK). Fully mature glycosylated CFTR (approximately 170 kDa) was immunoprecipitated from cells transfected with wild type CFTR cDNA, whereas cells transfected with delta exon5 CFTR express only a core-glycosylated from (approximately 140 kDa). The Western blot test performed on subcellular membrane fractions showed that delta exon5 CFTR was located in the intracellular membranes. Neither incubation at lower temperature (26 degrees C) nor stimulation of 293 HEK cells with forskolin or CPT-cAMP caused improvement in glycosylation and processing of delta exon5 CFTR proteins, indicating that the human epithelial CFTR lacking exon5 did not process properly in 293 HEK cells. On incorporation of intracellular membrane vesicles containing the delta exon5 CFTR proteins into the lipid bilayer membrane, functional phosphorylation- and ATP-dependent chloride channels were identified. CFTR channels with an 8-pS full-conductance state were observed in 14% of the experiments. The channel had an average open probability (Po) of 0.098 +/- 0.022, significantly less than that of the wild type CFTR (Po = 0.318 +/- 0.028). More frequently, the delta exon5 CFTR formed chloride channels with lower conductance states of approximately 2-3 and approximately 4-6 pS. These subconductance states were also observed with wild type CFTR but to a much lesser extent. Average Po for the 2-3-pS subconductance state, estimated from the area under the curve on an amplitude histogram, was 0.461 +/- 0.194 for delta exon5 CFTR and 0.332 +/- 0

  3. Twenty years of fluorescence imaging of intracellular chloride

    PubMed Central

    Arosio, Daniele; Ratto, Gian Michele

    2014-01-01

    Chloride homeostasis has a pivotal role in controlling neuronal excitability in the adult brain and during development. The intracellular concentration of chloride is regulated by the dynamic equilibrium between passive fluxes through membrane conductances and the active transport mediated by importers and exporters. In cortical neurons, chloride fluxes are coupled to network activity by the opening of the ionotropic GABAA receptors that provides a direct link between the activity of interneurons and chloride fluxes. These molecular mechanisms are not evenly distributed and regulated over the neuron surface and this fact can lead to a compartmentalized control of the intracellular concentration of chloride. The inhibitory drive provided by the activity of the GABAA receptors depends on the direction and strength of the associated currents, which are ultimately dictated by the gradient of chloride, the main charge carrier flowing through the GABAA channel. Thus, the intracellular distribution of chloride determines the local strength of ionotropic inhibition and influences the interaction between converging excitation and inhibition. The importance of chloride regulation is also underlined by its involvement in several brain pathologies, including epilepsy and disorders of the autistic spectra. The full comprehension of the physiological meaning of GABAergic activity on neurons requires the measurement of the spatiotemporal dynamics of chloride fluxes across the membrane. Nowadays, there are several available tools for the task, and both synthetic and genetically encoded indicators have been successfully used for chloride imaging. Here, we will review the available sensors analyzing their properties and outlining desirable future developments. PMID:25221475

  4. Chloride Regulation: A Dynamic Equilibrium Crucial for Synaptic Inhibition.

    PubMed

    Doyon, Nicolas; Vinay, Laurent; Prescott, Steven A; De Koninck, Yves

    2016-03-16

    Fast synaptic inhibition relies on tight regulation of intracellular Cl(-). Chloride dysregulation is implicated in several neurological and psychiatric disorders. Beyond mere disinhibition, the consequences of Cl(-) dysregulation are multifaceted and best understood in terms of a dynamical system involving complex interactions between multiple processes operating on many spatiotemporal scales. This dynamical perspective helps explain many unintuitive manifestations of Cl(-) dysregulation. Here we discuss how taking into account dynamical regulation of intracellular Cl(-) is important for understanding how synaptic inhibition fails, how to best detect that failure, why Cl(-) regulation is energetically so expensive, and the overall consequences for therapeutics. PMID:26985723

  5. Chloride Regulation: A Dynamic Equilibrium Crucial for Synaptic Inhibition.

    PubMed

    Doyon, Nicolas; Vinay, Laurent; Prescott, Steven A; De Koninck, Yves

    2016-03-16

    Fast synaptic inhibition relies on tight regulation of intracellular Cl(-). Chloride dysregulation is implicated in several neurological and psychiatric disorders. Beyond mere disinhibition, the consequences of Cl(-) dysregulation are multifaceted and best understood in terms of a dynamical system involving complex interactions between multiple processes operating on many spatiotemporal scales. This dynamical perspective helps explain many unintuitive manifestations of Cl(-) dysregulation. Here we discuss how taking into account dynamical regulation of intracellular Cl(-) is important for understanding how synaptic inhibition fails, how to best detect that failure, why Cl(-) regulation is energetically so expensive, and the overall consequences for therapeutics.

  6. Chloride Regulation in the Pain Pathway

    PubMed Central

    Price, Theodore J; Cervero, Fernando; Gold, Michael S; Hammond, Donna L; Prescott, Steven A

    2009-01-01

    Melzack and Wall’s Gate Control Theory of Pain laid the theoretical groundwork for a role of spinal inhibition in endogenous pain control. While the Gate Control Theory was based on the notion that spinal inhibition is dynamically regulated, mechanisms underlying the regulation of inhibition have turned out to be far more complex than Melzack and Wall could have ever imagined. Recent evidence indicates that an exquisitely sensitive form of regulation involves changes in anion equilibrium potential (Eanion), which subsequently impacts fast synaptic inhibition mediated GABAA, and to a lesser extent, glycine receptor activation, the prototypic ligand gated anion channels. The cation-chloride co-transporters (in particular NKCC1 and KCC2) have emerged as proteins that play a critical role in the dynamic regulation of Eanion which in turn appears to play a critical role in hyperalgesia and allodynia following peripheral inflammation or nerve injury. This review summarizes the current state of knowledge in this area with particular attention to how such findings relate to endogenous mechanisms of hyperalgesia and allodynia and potential applications for therapeutics based on modulation of intracellular Cl− gradients or pharmacological interventions targeting GABAA receptors PMID:19167425

  7. Transgenic mouse lines for non-invasive ratiometric monitoring of intracellular chloride

    PubMed Central

    Batti, Laura; Mukhtarov, Marat; Audero, Enrica; Ivanov, Anton; Paolicelli, Rosa Chiara; Zurborg, Sandra; Gross, Cornelius; Bregestovski, Piotr; Heppenstall, Paul A.

    2013-01-01

    Chloride is the most abundant physiological anion and participates in a variety of cellular processes including trans-epithelial transport, cell volume regulation, and regulation of electrical excitability. The development of tools to monitor intracellular chloride concentration ([Cli]) is therefore important for the evaluation of cellular function in normal and pathological conditions. Recently, several Cl-sensitive genetically encoded probes have been described which allow for non-invasive monitoring of [Cli]. Here we describe two mouse lines expressing a CFP-YFP-based Cl probe called Cl-Sensor. First, we generated transgenic mice expressing Cl-Sensor under the control of the mouse Thy1 mini promoter. Cl-Sensor exhibited good expression from postnatal day two (P2) in neurons of the hippocampus and cortex, and its level increased strongly during development. Using simultaneous whole-cell monitoring of ionic currents and Cl-dependent fluorescence, we determined that the apparent EC50 for Cli was 46 mM, indicating that this line is appropriate for measuring neuronal [Cli] in postnatal mice. We also describe a transgenic mouse reporter line for Cre-dependent conditional expression of Cl-Sensor, which was targeted to the Rosa26 locus and by incorporating a strong exogenous promoter induced robust expression upon Cre-mediated recombination. We demonstrate high levels of tissue-specific expression in two different Cre-driver lines targeting cells of the myeloid lineage and peripheral sensory neurons. Using these mice the apparent EC50 for Cli was estimated to be 61 and 54 mM in macrophages and DRG, respectively. Our data suggest that these mouse lines will be useful models for ratiometric monitoring of Cli in specific cell types in vivo. PMID:23734096

  8. Cytoskeletal Network Morphology Regulates Intracellular Transport Dynamics.

    PubMed

    Ando, David; Korabel, Nickolay; Huang, Kerwyn Casey; Gopinathan, Ajay

    2015-10-20

    Intracellular transport is essential for maintaining proper cellular function in most eukaryotic cells, with perturbations in active transport resulting in several types of disease. Efficient delivery of critical cargos to specific locations is accomplished through a combination of passive diffusion and active transport by molecular motors that ballistically move along a network of cytoskeletal filaments. Although motor-based transport is known to be necessary to overcome cytoplasmic crowding and the limited range of diffusion within reasonable timescales, the topological features of the cytoskeletal network that regulate transport efficiency and robustness have not been established. Using a continuum diffusion model, we observed that the time required for cellular transport was minimized when the network was localized near the nucleus. In simulations that explicitly incorporated network spatial architectures, total filament mass was the primary driver of network transit times. However, filament traps that redirect cargo back to the nucleus caused large variations in network transport. Filament polarity was more important than filament orientation in reducing average transit times, and transport properties were optimized in networks with intermediate motor on and off rates. Our results provide important insights into the functional constraints on intracellular transport under which cells have evolved cytoskeletal structures, and have potential applications for enhancing reactions in biomimetic systems through rational transport network design.

  9. Intracellular Na+ regulates epithelial Na+ channel maturation.

    PubMed

    Heidrich, Elisa; Carattino, Marcelo D; Hughey, Rebecca P; Pilewski, Joseph M; Kleyman, Thomas R; Myerburg, Mike M

    2015-05-01

    Epithelial Na(+) channel (ENaC) function is regulated by the intracellular Na(+) concentration ([Na(+)]i) through a process known as Na(+) feedback inhibition. Although this process is known to decrease the expression of proteolytically processed active channels on the cell surface, it is unknown how [Na(+)]i alters ENaC cleavage. We show here that [Na(+)]i regulates the posttranslational processing of ENaC subunits during channel biogenesis. At times when [Na(+)]i is low, ENaC subunits develop mature N-glycans and are processed by proteases. Conversely, glycan maturation and sensitivity to proteolysis are reduced when [Na(+)]i is relatively high. Surface channels with immature N-glycans were not processed by endogenous channel activating proteases, nor were they sensitive to cleavage by exogenous trypsin. Biotin chase experiments revealed that the immature surface channels were not converted into mature cleaved channels following a reduction in [Na(+)]i. The hypothesis that [Na(+)]i regulates ENaC maturation within the biosynthetic pathways is further supported by the finding that Brefeldin A prevented the accumulation of processed surface channels following a reduction in [Na(+)]i. Therefore, increased [Na(+)]i interferes with ENaC N-glycan maturation and prevents the channel from entering a state that allows proteolytic processing. PMID:25767115

  10. Regulation of neuronal chloride homeostasis by neuromodulators.

    PubMed

    Mahadevan, Vivek; Woodin, Melanie A

    2016-05-15

    KCC2 is the central regulator of neuronal Cl(-) homeostasis, and is critical for enabling strong hyperpolarizing synaptic inhibition in the mature brain. KCC2 hypofunction results in decreased inhibition and increased network hyperexcitability that underlies numerous disease states including epilepsy, neuropathic pain and neuropsychiatric disorders. The current holy grail of KCC2 biology is to identify how we can rescue KCC2 hypofunction in order to restore physiological levels of synaptic inhibition and neuronal network activity. It is becoming increasingly clear that diverse cellular signals regulate KCC2 surface expression and function including neurotransmitters and neuromodulators. In the present review we explore the existing evidence that G-protein-coupled receptor (GPCR) signalling can regulate KCC2 activity in numerous regions of the nervous system including the hypothalamus, hippocampus and spinal cord. We present key evidence from the literature suggesting that GPCR signalling is a conserved mechanism for regulating chloride homeostasis. This evidence includes: (1) the activation of group 1 metabotropic glutamate receptors and metabotropic Zn(2+) receptors strengthens GABAergic inhibition in CA3 pyramidal neurons through a regulation of KCC2; (2) activation of the 5-hydroxytryptamine type 2A serotonin receptors upregulates KCC2 cell surface expression and function, restores endogenous inhibition in motoneurons, and reduces spasticity in rats; and (3) activation of A3A-type adenosine receptors rescues KCC2 dysfunction and reverses allodynia in a model of neuropathic pain. We propose that GPCR-signals are novel endogenous Cl(-) extrusion enhancers that may regulate KCC2 function. PMID:26876607

  11. Regulation of neuronal chloride homeostasis by neuromodulators

    PubMed Central

    Mahadevan, Vivek; Woodin, Melanie A.

    2016-01-01

    Abstract KCC2 is the central regulator of neuronal Cl− homeostasis, and is critical for enabling strong hyperpolarizing synaptic inhibition in the mature brain. KCC2 hypofunction results in decreased inhibition and increased network hyperexcitability that underlies numerous disease states including epilepsy, neuropathic pain and neuropsychiatric disorders. The current holy grail of KCC2 biology is to identify how we can rescue KCC2 hypofunction in order to restore physiological levels of synaptic inhibition and neuronal network activity. It is becoming increasingly clear that diverse cellular signals regulate KCC2 surface expression and function including neurotransmitters and neuromodulators. In the present review we explore the existing evidence that G‐protein‐coupled receptor (GPCR) signalling can regulate KCC2 activity in numerous regions of the nervous system including the hypothalamus, hippocampus and spinal cord. We present key evidence from the literature suggesting that GPCR signalling is a conserved mechanism for regulating chloride homeostasis. This evidence includes: (1) the activation of group 1 metabotropic glutamate receptors and metabotropic Zn2+ receptors strengthens GABAergic inhibition in CA3 pyramidal neurons through a regulation of KCC2; (2) activation of the 5‐hydroxytryptamine type 2A serotonin receptors upregulates KCC2 cell surface expression and function, restores endogenous inhibition in motoneurons, and reduces spasticity in rats; and (3) activation of A3A‐type adenosine receptors rescues KCC2 dysfunction and reverses allodynia in a model of neuropathic pain. We propose that GPCR‐signals are novel endogenous Cl− extrusion enhancers that may regulate KCC2 function. PMID:26876607

  12. A role for intracellular zinc in glioma alteration of neuronal chloride equilibrium

    PubMed Central

    Di Angelantonio, S; Murana, E; Cocco, S; Scala, F; Bertollini, C; Molinari, M G; Lauro, C; Bregestovski, P; Limatola, C; Ragozzino, D

    2014-01-01

    Glioma patients commonly suffer from epileptic seizures. However, the mechanisms of glioma-associated epilepsy are far to be completely understood. Using glioma-neurons co-cultures, we found that tumor cells are able to deeply influence neuronal chloride homeostasis, by depolarizing the reversal potential of γ-aminobutyric acid (GABA)-evoked currents (EGABA). EGABA depolarizing shift is due to zinc-dependent reduction of neuronal KCC2 activity and requires glutamate release from glioma cells. Consistently, intracellular zinc loading rapidly depolarizes EGABA in mouse hippocampal neurons, through the Src/Trk pathway and this effect is promptly reverted upon zinc chelation. This study provides a possible molecular mechanism linking glioma invasion to excitation/inhibition imbalance and epileptic seizures, through the zinc–mediated disruption of neuronal chloride homeostasis. PMID:25356870

  13. Host metabolism regulates intracellular growth of Trypanosoma cruzi.

    PubMed

    Caradonna, Kacey L; Engel, Juan C; Jacobi, David; Lee, Chih-Hao; Burleigh, Barbara A

    2013-01-16

    Metabolic coupling of intracellular pathogens with host cells is essential for successful colonization of the host. Establishment of intracellular infection by the protozoan Trypanosoma cruzi leads to the development of human Chagas' disease, yet the functional contributions of the host cell toward the infection process remain poorly characterized. Here, a genome-scale functional screen identified interconnected metabolic networks centered around host energy production, nucleotide metabolism, pteridine biosynthesis, and fatty acid oxidation as key processes that fuel intracellular T. cruzi growth. Additionally, the host kinase Akt, which plays essential roles in various cellular processes, was critical for parasite replication. Targeted perturbations in these host metabolic pathways or Akt-dependent signaling pathways modulated the parasite's replicative capacity, highlighting the adaptability of this intracellular pathogen to changing conditions in the host. These findings identify key cellular process regulating intracellular T. cruzi growth and illuminate the potential to leverage host pathways to limit T. cruzi infection. PMID:23332160

  14. Host metabolism regulates intracellular growth of Trypanosoma cruzi

    PubMed Central

    Caradonna, Kacey L.; Engel, Juan C.; Jacobi, David; Lee, Chih-Hao; Burleigh, Barbara A.

    2012-01-01

    SUMMARY Metabolic coupling of intracellular pathogens with host cells is essential for successful colonization of the host. Establishment of intracellular infection by the protozoan Trypanosoma cruzi leads to the development of human Chagas disease, yet the functional contributions of the host cell toward the infection process remain poorly characterized. Here, a genome-scale functional screen identified interconnected metabolic networks centered around host energy production, nucleotide metabolism, pteridine biosynthesis, and fatty acid oxidation as key processes that fuel intracellular T. cruzi growth. Additionally, the host kinase Akt, which plays essential roles in various cellular processes, was critical for parasite replication. Targeted perturbations in these host metabolic pathways or Akt-dependent signaling pathways modulated the parasite’s replicative capacity, highlighting the adaptability of this intracellular pathogen to changing conditions in the host. These findings identify key cellular process regulating intracellular T. cruzi growth and illuminate the potential to leverage host pathways to limit T. cruzi infection. PMID:23332160

  15. Intracellular events regulating cross-presentation

    PubMed Central

    Wagner, Claudia S.; Grotzke, Jeffrey E.; Cresswell, Peter

    2012-01-01

    Cross-presentation plays a fundamental role in the induction of CD8-T cell immunity. However, although more than three decades have passed since its discovery, surprisingly little is known about the exact mechanisms involved. Here we give an overview of the components involved at different stages of this process. First, antigens must be internalized into the cross-presenting cell. The involvement of different receptors, method of antigen uptake, and nature of the antigen can influence intracellular trafficking and access to the cross-presentation pathway. Once antigens access the endocytic system, different requirements for endosomal/phagosomal processing arise, such as proteolysis and reduction of disulfide bonds. The majority of cross-presented peptides are generated by proteasomal degradation. Therefore, antigens must cross a membrane barrier in a manner analogous to the fate of misfolded proteins in the endoplasmic reticulum (ER) that are retrotranslocated into the cytosol for degradation. Indeed, some components of the ER-associated degradation machinery have been implicated in cross-presentation. Further complicating the matter, endosomal and phagosomal compartments have been suggested as alternative sites to the ER for loading of peptides on major histocompatibility complex class I molecules. Finally, the antigen presenting cells involved, particularly dendritic cell subsets and their state of maturation, influence the efficiency of cross-presentation. PMID:22675326

  16. Interaction of Human Chloride Intracellular Channel Protein 1 (CLIC1) with Lipid Bilayers: A Fluorescence Study.

    PubMed

    Hare, Joanna E; Goodchild, Sophia C; Breit, Samuel N; Curmi, Paul M G; Brown, Louise J

    2016-07-12

    Chloride intracellular channel protein 1 (CLIC1) is very unusual as it adopts a soluble glutathione S-transferase-like canonical fold but can also autoinsert into lipid bilayers to form an ion channel. The conversion between these forms involves a large, but reversible, structural rearrangement of the CLIC1 module. The only identified environmental triggers controlling the metamorphic transition of CLIC1 are pH and oxidation. Until now, there have been no high-resolution structural data available for the CLIC1 integral membrane state, and consequently, a limited understanding of how CLIC1 unfolds and refolds across the bilayer to form a membrane protein with ion channel activity exists. Here we show that fluorescence spectroscopy can be used to establish the interaction and position of CLIC1 in a lipid bilayer. Our method employs a fluorescence energy transfer (FRET) approach between CLIC1 and a dansyl-labeled lipid analogue to probe the CLIC1-lipid interface. Under oxidizing conditions, a strong FRET signal between the single tryptophan residue of CLIC1 (Trp35) and the dansyl-lipid analogue was detected. When considering the proportion of CLIC1 interacting with the lipid bilayer, as estimated by fluorescence quenching experiments, the FRET distance between Trp35 and the dansyl moiety on the membrane surface was determined to be ∼15 Å. This FRET-detected interaction provides direct structural evidence that CLIC1 associates with membranes. The results presented support the current model of an oxidation-driven interaction of CLIC1 with lipid bilayers and also propose a membrane anchoring role for Trp35. PMID:27299171

  17. A unifying mechanism for WNK kinase regulation of sodium-chloride cotransporter.

    PubMed

    Huang, Chou-Long; Cheng, Chih-Jen

    2015-11-01

    Mammalian with-no-lysine [K] (WNK) kinases are a family of four serine-threonine protein kinases, WNK1-4. Mutations of WNK1 and WNK4 in humans cause pseudohypoaldosteronism type II (PHA2), an autosomal-dominant disease characterized by hypertension and hyperkalemia. Increased Na(+) reabsorption through Na(+)-Cl(-) cotransporter (NCC) in the distal convoluted tubule plays an important role in the pathogenesis of hypertension in patients with PHA2. However, how WNK1 and WNK4 regulate NCC and how mutations of WNKs cause activation of NCC have been controversial. Here, we review current state of literature supporting a compelling model that WNK1 and WNK4 both contribute to stimulation of NCC. The precise combined effects of WNK1 and WNK4 on NCC remain unclear but likely are positive rather than antagonistic. The recent discovery that WNK kinases may function as an intracellular chloride sensor adds a new dimension to the physiological role of WNK kinases. Intracellular chloride-dependent regulation of WNK's may underlie the mechanism of regulation of NCC by extracellular K(+). Definite answer yet will require future investigation by tubular perfusion in mice with altered WNK kinase expression.

  18. Preferential intracellular pH regulation: hypotheses and perspectives.

    PubMed

    Shartau, Ryan B; Baker, Daniel W; Crossley, Dane A; Brauner, Colin J

    2016-08-01

    The regulation of vertebrate acid-base balance during acute episodes of elevated internal PCO2  is typically characterized by extracellular pH (pHe) regulation. Changes in pHe are associated with qualitatively similar changes in intracellular tissue pH (pHi) as the two are typically coupled, referred to as 'coupled pH regulation'. However, not all vertebrates rely on coupled pH regulation; instead, some preferentially regulate pHi against severe and maintained reductions in pHe Preferential pHi regulation has been identified in several adult fish species and an aquatic amphibian, but never in adult amniotes. Recently, common snapping turtles were observed to preferentially regulate pHi during development; the pattern of acid-base regulation in these species shifts from preferential pHi regulation in embryos to coupled pH regulation in adults. In this Commentary, we discuss the hypothesis that preferential pHi regulation may be a general strategy employed by vertebrate embryos in order to maintain acid-base homeostasis during severe acute acid-base disturbances. In adult vertebrates, the retention or loss of preferential pHi regulation may depend on selection pressures associated with the environment inhabited and/or the severity of acid-base regulatory challenges to which they are exposed. We also consider the idea that the retention of preferential pHi regulation into adulthood may have been a key event in vertebrate evolution, with implications for the invasion of freshwater habitats, the evolution of air breathing and the transition of vertebrates from water to land. PMID:27489212

  19. Regulation of dopamine transporter trafficking by intracellular amphetamine.

    PubMed

    Kahlig, Kristopher M; Lute, Brandon J; Wei, Yuqiang; Loland, Claus J; Gether, Ulrik; Javitch, Jonathan A; Galli, Aurelio

    2006-08-01

    The dopamine (DA) transporter (DAT) mediates the removal of released DA. DAT is the major molecular target responsible for the rewarding properties and abuse potential of the psychostimulant amphetamine (AMPH). AMPH has been shown to reduce the number of DATs at the cell surface, and this AMPH-induced cell surface DAT redistribution may result in long-lasting changes in DA homeostasis. The molecular mechanism by which AMPH induces trafficking is not clear. Because AMPH is a substrate, we do not know whether extracellular AMPH stimulates trafficking through its interaction with DAT and subsequent alteration in DAT function, thereby triggering intracellular signaling or whether AMPH must be transported and then act intracellularly. In agreement with our previous studies, extracellular AMPH caused cytosolic redistribution of the wild-type human DAT (WT-hDAT). However, AMPH did not induce cytosolic redistribution in an uptake-impaired hDAT (Y335A-hDAT) that still binds AMPH. The divalent cation zinc (Zn(2+)) inhibits WT-hDAT activity, but it restores Y335A-hDAT uptake. Coadministration of Zn(2+) and AMPH consistently reduced WT-hDAT trafficking but stimulated cytosolic redistribution of Y335A-hDAT. Furthermore, direct intracellular application of AMPH, via a whole-cell patch pipette, stimulated the trafficking of Y335A-hDAT. Taken together, these data suggest that the DAT transport cycle is not required for AMPH-induced down-regulation and that an increase of intracellular AMPH is an essential component of DAT redistribution.

  20. 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

  1. 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

  2. Intracellular Calcium Regulates Nonsense-Mediated mRNA Decay

    PubMed Central

    Nickless, Andrew; Jackson, Erin; Marasa, Jayne; Nugent, Patrick; Mercer, Robert W.; Piwnica-Worms, David; You, Zhongsheng

    2014-01-01

    The nonsense-mediated mRNA decay (NMD) pathway selectively eliminates aberrant transcripts containing premature translation termination codons (PTCs) and regulates the levels of a number of physiological mRNAs. NMD modulates the clinical outcome of a variety of human diseases, including cancer and many genetic disorders, and may represent an important target for therapeutic intervention. Here we have developed a novel multicolored, bioluminescence-based reporter system that can specifically and effectively assay NMD in live human cells. Using this reporter system, we conducted a robust high-throughput small-molecule screen in human cells and, unpredictably, identified a group of cardiac glycosides including ouabain and digoxin as potent inhibitors of NMD. Cardiac glycoside-mediated effects on NMD are dependent on binding and inhibiting the Na+/K+-ATPase on the plasma membrane and subsequent elevation of intracellular calcium levels. Induction of calcium release from endoplasmic reticulum also leads to inhibition of NMD. Thus, this study reveals intracellular calcium as a key regulator of NMD and has important implications for exploiting NMD in the treatment of disease. PMID:25064126

  3. A flow cytometric method for measurement of intracellular chloride concentration in lymphocytes using the halide-specific probe 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ).

    PubMed

    Pilas, B; Durack, G

    1997-08-01

    A flow cytometry method using the halide-specific fluorescent dye, 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ), has been developed to measure intracellular chloride concentration in single cells. Collisions with chloride quench the fluorescence of SPQ, making it possible to relate the measured fluorescence intensity to chloride concentration with a Stern-Volmer equation. To demonstrate the method, porcine lymphocytes were loaded in vitro, using a hypotonic method, with 5 mM SPQ. Fluorescence excitation was provided by a UV laser and the fluorescence emission intensity at 485 nm was recorded. Calibration was performed by using 7 microM nigericin (a K/H antiporter) and 10 microM tributyltin (a Cl/OH antiporter) to equilibrate the concentrations of intracellular and extracellular chloride. Calibration measurements were made for chloride concentrations between 0 mM and 140 mM. The calibration produced a Stern-Volmer quenching constant of 16.2 M(-1) which was used to relate measured cell fluorescence to intracellular chloride concentration. The intracellular chloride concentration for fresh porcine lymphocytes was determined to be 56.2 +/- 3.3 mM. Stable loading of cells with 5 mM SPQ was accomplished in 15 minutes, leakage of SPQ from the cells was minimal, and over 95% of the cells remained viable after loading. PMID:9266752

  4. Copper transporter 2 regulates intracellular copper and sensitivity to cisplatin.

    PubMed

    Huang, Carlos P; Fofana, Mariama; Chan, Jefferson; Chang, Christopher J; Howell, Stephen B

    2014-03-01

    Mammalian cells express two copper (Cu) influx transporters, CTR1 and CTR2. CTR1 serves as an influx transporter for both Cu and cisplatin (cDDP). In mouse embryo fibroblasts, reduction of CTR1 expression renders cells resistant to cDDP whereas reduction of CTR2 makes them hypersensitive both in vitro and in vivo. To investigate the role of CTR2 on intracellular Cu and cDDP sensitivity its expression was molecularly altered in the human epithelial 2008 cancer cell model. Intracellular exchangeable Cu(+) was measured with the fluorescent probe Coppersensor-3 (CS3). The ability of CS3 to report on changes in intracellular Cu(+) was validated by showing that Cu chelators reduced its signal, and that changes in signal accompanied alterations in expression of the major Cu influx transporter CTR1 and the two Cu efflux transporters, ATP7A and ATP7B. Constitutive knock down of CTR2 mRNA by ∼50% reduced steady-state exchangeable Cu by 22-23% and increased the sensitivity of 2008 cells by a factor of 2.6-2.9 in two separate clones. Over-expression of CTR2 increased exchangeable Cu(+) by 150% and rendered the 2008 cells 2.5-fold resistant to cDDP. The results provide evidence that CS3 can quantitatively assess changes in exchangeable Cu(+), and that CTR2 regulates both the level of exchangeable Cu(+) and sensitivity to cDDP in a model of human epithelial cancer. This study introduces CS3 and related sensors as novel tools for probing and assaying Cu-dependent sensitivity to anticancer therapeutics. PMID:24522273

  5. Copper transporter 2 regulates intracellular copper and sensitivity to cisplatin.

    PubMed

    Huang, Carlos P; Fofana, Mariama; Chan, Jefferson; Chang, Christopher J; Howell, Stephen B

    2014-03-01

    Mammalian cells express two copper (Cu) influx transporters, CTR1 and CTR2. CTR1 serves as an influx transporter for both Cu and cisplatin (cDDP). In mouse embryo fibroblasts, reduction of CTR1 expression renders cells resistant to cDDP whereas reduction of CTR2 makes them hypersensitive both in vitro and in vivo. To investigate the role of CTR2 on intracellular Cu and cDDP sensitivity its expression was molecularly altered in the human epithelial 2008 cancer cell model. Intracellular exchangeable Cu(+) was measured with the fluorescent probe Coppersensor-3 (CS3). The ability of CS3 to report on changes in intracellular Cu(+) was validated by showing that Cu chelators reduced its signal, and that changes in signal accompanied alterations in expression of the major Cu influx transporter CTR1 and the two Cu efflux transporters, ATP7A and ATP7B. Constitutive knock down of CTR2 mRNA by ∼50% reduced steady-state exchangeable Cu by 22-23% and increased the sensitivity of 2008 cells by a factor of 2.6-2.9 in two separate clones. Over-expression of CTR2 increased exchangeable Cu(+) by 150% and rendered the 2008 cells 2.5-fold resistant to cDDP. The results provide evidence that CS3 can quantitatively assess changes in exchangeable Cu(+), and that CTR2 regulates both the level of exchangeable Cu(+) and sensitivity to cDDP in a model of human epithelial cancer. This study introduces CS3 and related sensors as novel tools for probing and assaying Cu-dependent sensitivity to anticancer therapeutics.

  6. Copper Transporter 2 Regulates Intracellular Copper and Sensitivity to Cisplatin

    PubMed Central

    Huang, Carlos P.; Fofana, Mariama; Chan, Jefferson; Chang, Christopher J.; Howell, Stephen B.

    2014-01-01

    Mammalian cells express two copper (Cu) influx transporters, CTR1 and CTR2. CTR1 serves as an influx transporter for both Cu and cisplatin (cDDP). In mouse embryo fibroblasts, reduction of CTR1 expression renders cells resistant to cDDP whereas reduction of CTR2 makes them hypersensitive both in vitro and in vivo. To investigate the role of CTR2 on intracellular Cu and cDDP sensitivity its expression was molecularly altered in the human epithelial 2008 cancer cell model. Intracellular exchangeable Cu+ was measured with the fluorescent probe Coppersensor-3 (CS3). The ability of CS3 to report on changes in intracellular Cu+ was validated by showing that Cu chelators reduced its signal, and that changes in signal accompanied alterations in expression the major Cu influx transporter CTR1 and the two Cu efflux transporters, ATP7A and ATP7B. Constitutive knock down of CTR2 mRNA by ~50% reduced steady-state exchangeable Cu by 22–23% and increased the sensitivity of 2008 cells by a factor of 2.6 – 2.9 in two separate clones. Over-expression of CTR2 increased exchangeable Cu+ by 150% and rendered the 2008 cells 2.5-fold resistant to cDDP. The results provide evidence that CS3 can quantitatively assess changes in exchangeable Cu+, and that CTR2 regulates both the level of exchangeable Cu+ and sensitivity to cDDP in a model of human epithelial cancer. This study introduces CS3 and related sensors as novel tools for probing and assaying Cu-dependent sensitivity to anticancer therapeutics. PMID:24522273

  7. Role of intracellular calcium in cellular volume regulation

    SciTech Connect

    Wong, S.M.; Chase, H.S. Jr.

    1986-06-01

    We investigated the role of intracellular calcium in epithelial cell volume regulation using cells isolated from the toad urinary bladder. A suspension of cells was prepared by treatment of the bladder with collagenase followed by ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid. The cells retained their ion-transporting capabilities: ouabain (1 mM) and amiloride (10 microM) inhibited cellular uptake of /sup 86/Rb and /sup 22/Na, respectively. Using a Coulter counter to measure cellular volume, we found that we could swell cells either by reducing the extracellular osmolality or by adding the permeant solute urea (45 mM) isosmotically. Under both conditions, cells first swelled and then returned to their base-line volume, in spite of the continued presence of the stimulus to swell. Volume regulation was inhibited when cells were swelled at low extracellular (Ca) (100 nM) and was retarded in cells preloaded with the calcium buffer quin 2. Swelling increased the intracellular free calcium concentration ((Ca)i), as measured by quin 2 fluorescence: (Ca)i increased 35 +/- 9 nM (n = 6) after hypotonic swelling and 42 +/- 3 nM (n = 3) after urea swelling. Reducing extracellular (Ca) to less than 100 nM prevented the swelling-induced increase in (Ca)i, suggesting that the source of the increase in (Ca)i was extracellular. This result was confirmed in measurements of cellular uptake of 45Ca: the rate of uptake was significantly higher in swollen cells compared with control (1.1 +/- 0.2 vs. 0.4 +/- 0.1 fmol . cell-1 X 5 min-1). Our experiments provide the first demonstration that cellular swelling increases (Ca)i. This increase is likely to play a critical role in cellular volume regulation.

  8. Chloride channel-3 promotes tumor metastasis by regulating membrane ruffling and is associated with poor survival

    PubMed Central

    Li, Qin; Deng, Lulu; Wu, Hui; Lin, Guixian; Chen, Lixin; Zhang, Haifeng; Li, Chunmei; Wang, Liwei; Zhu, Jiayong; Wang, Weizhang; Chu, Fujiang; Shen, Juan; Li, Hongzhi; Mao, Jianwen

    2015-01-01

    The chloride channel-3 (ClC-3) protein is known to be a component of Cl− channels involved in cell volume regulation or acidification of intracellular vesicles. Here, we report that ClC-3 was highly expressed in the cytoplasm of metastatic carcinomatous cells and accelerated cell migration in vitro and tumor metastasis in vivo. High-grade expression of cytoplasmic ClC-3 predicted poor survival in cancer patients. We found that independent of its volume-activated Cl− channel properties, ClC-3 was able to promote cell membrane ruffling, required for tumor metastasis. ClC-3 mediated membrane ruffling by regulating keratin 18 phosphorylation to control β1 Integrin recycling. Therefore, cytoplasmic ClC-3 plays an active and key role in tumor metastasis and may be a valuable prognostic biomarker and a therapeutic target to prevent tumor spread. PMID:25537517

  9. Interdependent regulation of intracellular acidification and SHP-1 in apoptosis.

    PubMed

    Thangaraju, M; Sharma, K; Liu, D; Shen, S H; Srikant, C B

    1999-04-01

    The G protein-coupled receptor agonist somatostatin (SST)-induces apoptosis in MCF-7 human breast cancer cells. This is associated with induction of wild-type p53, Bax, and an acidic endonuclease. We have shown recently that its cytotoxic signaling is mediated via membrane-associated SHP-1 and is dependent on decrease in intracellular pH (pHi) to 6.5. Here we investigated the relationship between intracellular acidification and SHP-1 in cytotoxic signaling. Clamping of pHi at 7.25 by the proton-ionophore nigericin abolished SST-signaled apoptosis without affecting its ability to regulate SHP-1, p53, and Bax. Apoptosis could be induced by nigericin clamping of pHi to 6.5. Such acidification-induced apoptosis was not observed at pHi <6.0 or >6.7. pHi-dependent apoptosis was associated with the translocation of SHP-1 to the membrane, enhanced in cells overexpressing SHP-1, and was abolished by its inactive mutant SHP-1C455S. Acidification caused by inhibition of Na+/H+ exchanger and H+ ATPase (pHi = 6.55 and 6.65, respectively) also triggered apoptosis. The effect of concurrent inhibition of Na+/H+ exchanger and H(+)-ATPase on pHi and apoptosis was comparable with that of SST. Acidification-induced, SHP-1-dependent apoptosis occurred in breast cancer cell lines in which SST was cytotoxic (MCF-7 and T47D) or not (MDA-MB-231). We conclude that: (a) SST-induced SHP-1-dependent acidification occurs subsequent to or independent of the induction of p53 and Bax; (b) SST-induced intracellular acidification may arise due to inhibition of Na+/H+ exchanger and H(+)-ATPase; and (c) SHP-1 is necessary not only for agonist-induced acidification but also for the execution of acidification-dependent apoptosis. We suggest that combined targeting of SHP-1 and intracellular acidification may lead to a novel strategy of anticancer therapy bypassing the need for receptor-mediated signaling.

  10. Intracellular Energetic Units regulate metabolism in cardiac cells.

    PubMed

    Saks, Valdur; Kuznetsov, Andrey V; Gonzalez-Granillo, Marcela; Tepp, Kersti; Timohhina, Natalja; Karu-Varikmaa, Minna; Kaambre, Tuuli; Dos Santos, Pierre; Boucher, François; Guzun, Rita

    2012-02-01

    This review describes developments in historical perspective as well as recent results of investigations of cellular mechanisms of regulation of energy fluxes and mitochondrial respiration by cardiac work - the metabolic aspect of the Frank-Starling law of the heart. A Systems Biology solution to this problem needs the integration of physiological and biochemical mechanisms that take into account intracellular interactions of mitochondria with other cellular systems, in particular with cytoskeleton components. Recent data show that different tubulin isotypes are involved in the regular arrangement exhibited by mitochondria and ATP-consuming systems into Intracellular Energetic Units (ICEUs). Beta II tubulin association with the mitochondrial outer membrane, when co-expressed with mitochondrial creatine kinase (MtCK) specifically limits the permeability of voltage-dependent anion channel for adenine nucleotides. In the MtCK reaction this interaction changes the regulatory kinetics of respiration through a decrease in the affinity for adenine nucleotides and an increase in the affinity for creatine. Metabolic Control Analysis of the coupled MtCK-ATP Synthasome in permeabilized cardiomyocytes showed a significant increase in flux control by steps involved in ADP recycling. Mathematical modeling of compartmentalized energy transfer represented by ICEUs shows that cyclic changes in local ADP, Pi, phosphocreatine and creatine concentrations during contraction cycle represent effective metabolic feedback signals when amplified in the coupled non-equilibrium MtCK-ATP Synthasome reactions in mitochondria. This mechanism explains the regulation of respiration on beat to beat basis during workload changes under conditions of metabolic stability. This article is part of a Special Issue entitled "Local Signaling in Myocytes." PMID:21816155

  11. Ornithine decarboxylase antizyme inhibitor 2 regulates intracellular vesicle trafficking

    SciTech Connect

    Kanerva, Kristiina; Maekitie, Laura T.; Baeck, Nils; Andersson, Leif C.

    2010-07-01

    Antizyme inhibitor 1 (AZIN1) and 2 (AZIN2) are proteins that activate ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis. Both AZINs release ODC from its inactive complex with antizyme (AZ), leading to formation of the catalytically active ODC. The ubiquitously expressed AZIN1 is involved in cell proliferation and transformation whereas the role of the recently found AZIN2 in cellular functions is unknown. Here we report the intracellular localization of AZIN2 and present novel evidence indicating that it acts as a regulator of vesicle trafficking. We used immunostaining to demonstrate that both endogenous and FLAG-tagged AZIN2 localize to post-Golgi vesicles of the secretory pathway. Immuno-electron microscopy revealed that the vesicles associate mainly with the trans-Golgi network (TGN). RNAi-mediated knockdown of AZIN2 or depletion of cellular polyamines caused selective fragmentation of the TGN and retarded the exocytotic release of vesicular stomatitis virus glycoprotein. Exogenous addition of polyamines normalized the morphological changes and reversed the inhibition of protein secretion. Our findings demonstrate that AZIN2 regulates the transport of secretory vesicles by locally activating ODC and polyamine biosynthesis.

  12. Stochastic focusing: Fluctuation-enhanced sensitivity of intracellular regulation

    PubMed Central

    Paulsson, Johan; Berg, Otto G.; Ehrenberg, Måns

    2000-01-01

    Many regulatory molecules are present in low copy numbers per cell so that significant random fluctuations emerge spontaneously. Because cell viability depends on precise regulation of key events, such signal noise has been thought to impose a threat that cells must carefully eliminate. However, the precision of control is also greatly affected by the regulatory mechanisms' capacity for sensitivity amplification. Here we show that even if signal noise reduces the capacity for sensitivity amplification of threshold mechanisms, the effect on realistic regulatory kinetics can be the opposite: stochastic focusing (SF). SF particularly exploits tails of probability distributions and can be formulated as conventional multistep sensitivity amplification where signal noise provides the degrees of freedom. When signal fluctuations are sufficiently rapid, effects of time correlations in signal-dependent rates are negligible and SF works just like conventional sensitivity amplification. This means that, quite counterintuitively, signal noise can reduce the uncertainty in regulated processes. SF is exemplified by standard hyperbolic inhibition, and all probability distributions for signal noise are first derived from underlying chemical master equations. The negative binomial is suggested as a paradigmatic distribution for intracellular kinetics, applicable to stochastic gene expression as well as simple systems with Michaelis–Menten degradation or positive feedback. SF resembles stochastic resonance in that noise facilitates signal detection in nonlinear systems, but stochastic resonance is related to how noise in threshold systems allows for detection of subthreshold signals and SF describes how fluctuations can make a gradual response mechanism work more like a threshold mechanism. PMID:10852944

  13. IQGAP1: a regulator of intracellular spacetime relativity.

    PubMed

    Malarkannan, Subramaniam; Awasthi, Aradhana; Rajasekaran, Kamalakannan; Kumar, Pawan; Schuldt, Kristina M; Bartoszek, Allison; Manoharan, Niranjan; Goldner, Nicholas K; Umhoefer, Colleen M; Thakar, Monica S

    2012-03-01

    Activating and inhibiting receptors of lymphocytes collect valuable information about their mikròs kósmos. This information is essential to initiate or to turn off complex signaling pathways. Irrespective of these advances, our knowledge on how these intracellular activation cascades are coordinated in a spatiotemporal manner is far from complete. Among multiple explanations, the scaffolding proteins have emerged as a critical piece of this evolutionary tangram. Among many, IQGAP1 is one of the essential scaffolding proteins that coordinate multiple signaling pathways. IQGAP1 possesses multiple protein interaction motifs to achieve its scaffolding functions. Using these domains, IQGAP1 has been shown to regulate a number of essential cellular events. This includes actin polymerization, tubulin multimerization, microtubule organizing center formation, calcium/calmodulin signaling, Pak/Raf/Mek1/2-mediated Erk1/2 activation, formation of maestrosome, E-cadherin, and CD44-mediated signaling and glycogen synthase kinase-3/adenomatous polyposis coli-mediated β-catenin activation. In this review, we summarize the recent developments and exciting new findings of cellular functions of IQGAP1.

  14. Acute effects of mercuric chloride on intracellular GSH levels and mercury distribution in the fish Oreochromic aureus

    SciTech Connect

    Allen, P.; Min, S.Y.; Keong, W.M.

    1988-02-01

    In recent years there has been much interest in the effects of trace metals on intracellular levels of reduced glutathione (GSH). Most of the research has been performed on rats. As GSH is ubiquitous in living organisms it is of interest to establish a relationship between mercury intoxication and intracellular GSH levels in fish; especially as fish living in rivers and coastal areas are often expose to mercury as an aquatic pollutant. The role of GSH in fish trace metal toxicity has not been thoroughly investigated. The distribution of total glutathione (oxidized + reduced) in selected black sea bass organs seems to follow the established pattern for mammalian organs. Thus, it would appear that teleostian and mammalian glutathione metabolism may have many similarities. There are few reports concerning the effects of mercury during the first few hours of exposure. The aim of this investigation is to establish any changes in organ GSH and mercury levels following just 2 h exposure to mercuric chloride (HgCl/sub 2/).

  15. Neurogenic regulation of proximal bicarbonate and chloride reabsorption.

    PubMed

    Cogan, M G

    1986-01-01

    Although a change in renal nerve activity is known to alter proximal reabsorption, it is unclear whether reabsorption of NaHCO3 or NaCl or both are affected. Sprague-Dawley rats (n = 10) were studied using free-flow micropuncture techniques during euvolemia and following acute ipsilateral denervation. Glomerular filtration rate and single nephron glomerular filtration rate were stable. Absolute proximal bicarbonate reabsorption fell following denervation (933 +/- 40 to 817 +/- 30 pmol/min) with a parallel reduction in chloride reabsorption (1,643 +/- 116 to 1,341 +/- 129 peq/min). Urinary sodium, potassium, bicarbonate, and chloride excretion all increased significantly. To further assess the physiological significance of neurogenic modulation of proximal transport, other rats (n = 6) were subjected to acute unilateral nephrectomy (AUN). There is evidence that AUN induces a contralateral natriuresis (renorenal reflex) at least partially by causing inhibition of efferent renal nerve traffic. AUN caused significant changes in proximal NaHCO3 and NaCl reabsorption as well as in whole kidney electrolyte excretion in the same pattern as had denervation. Prior denervation of the remaining kidney prevented the proximal and whole kidney response to AUN (n = 6). In conclusion, depression of renal nerve activity inhibits both NaHCO3 and NaCl reabsorption in the rat superficial proximal convoluted tubule. The data are consistent with the hypothesis that changes in renal nerve activity modify whole kidney electrolyte excretion under physiological conditions at least partially by regulating proximal transport.

  16. Regulating Intracellular Calcium in Plants: From Molecular Genetics to Physiology

    SciTech Connect

    Heven Sze

    2008-06-22

    To grow, develop, adapt, and reproduce, plants have evolved mechanisms to regulate the uptake, translocation and sorting of calcium ions into different cells and subcellular compartments. Yet how plants accomplish this remarkable feat is still poorly understood. The spatial and temporal changes in intracellular [Ca2+] during growth and during responses to hormonal and environmental stimuli indicate that Ca2+ influx and efflux transporters are diverse and tightly regulated in plants. The specific goals were to determine the biological roles of multiple Ca pumps (ECAs) in the model plant Arabidopsis thaliana. We had pioneered the use of K616 yeast strain to functionally express plant Ca pumps, and demonstrated two distinct types of Ca pumps in plants (Sze et al., 2000. Annu Rev Plant Biol. 51,433). ACA2 represented one type that was auto-inhibited by the N-terminal region and stimulated by calmodulin. ECA1 represented another type that was not sensitive to calmodulin and phylogenetically distinct from ACAs. The goal to determine the biological roles of multiple ECA-type Ca pumps in Arabidopsis has been accomplished. Although we demonstrated ECA1 was a Ca pump by functional expression in yeast, the in vivo roles of ECAs was unclear. A few highlights are described. ECA1 and/or ECA4 are Ca/Mn pumps localized to the ER and are highly expressed in all cell types. Using homozygous T-DNA insertional mutants of eca1, we demonstrated that the ER-bound ECA1 supports growth and confers tolerance of plants growing on medium low in Ca or containing toxic levels of Mn. This is the first genetic study to determine the in vivo function of a Ca pump in plants. A phylogenetically distinct ECA3 is also a Ca/Mn pump that is localized to endosome, such as post-Golgi compartments. Although it is expressed at lower levels than ECA1, eca3 mutants are impaired in Ca-dependent root growth and in pollen tube elongation. Increased secretion of wall proteins in mutants suggests that Ca and Mn

  17. Intracellular calcium levels can regulate Importin-dependent nuclear import

    SciTech Connect

    Kaur, Gurpreet; Ly-Huynh, Jennifer D.; Jans, David A.

    2014-07-18

    Highlights: • High intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import. • The effect of Ca{sup 2+} on nuclear import does not relate to changes in the nuclear pore. • High intracellular calcium can result in mislocalisation of Impβ1, Ran and RCC1. - Abstract: We previously showed that increased intracellular calcium can modulate Importin (Imp)β1-dependent nuclear import of SRY-related chromatin remodeling proteins. Here we extend this work to show for the first time that high intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import generally. The basis of this relates to the mislocalisation of the transport factors Impβ1 and Ran, which show significantly higher nuclear localization in contrast to various other factors, and RCC1, which shows altered subnuclear localisation. The results here establish for the first time that intracellular calcium modulates conventional nuclear import through direct effects on the nuclear transport machinery.

  18. Regulation of the epithelial Na(+) channel by intracellular Na(+).

    PubMed

    Awayda, M S

    1999-08-01

    The hypothesis that the intracellular Na(+) concentration ([Na(+)](i)) is a regulator of the epithelial Na(+) channel (ENaC) was tested with the Xenopus oocyte expression system by utilizing a dual-electrode voltage clamp. [Na(+)](i) averaged 48.1 +/- 2.2 meq (n = 27) and was estimated from the amiloride-sensitive reversal potential. [Na(+)](i) was increased by direct injection of 27.6 nl of 0.25 or 0.5 M Na(2)SO(4). Within minutes of injection, [Na(+)](i) stabilized and remained elevated at 97.8 +/- 6.5 meq (n = 9) and 64. 9 +/- 4.4 (n = 5) meq 30 min after the initial injection of 0.5 and 0.25 M Na(2)SO(4), respectively. This increase of [Na(+)](i) caused a biphasic inhibition of ENaC currents. In oocytes injected with 0.5 M Na(2)SO(4) (n = 9), a rapid decrease of inward amiloride-sensitive slope conductance (g(Na)) to 0.681 +/- 0.030 of control within the first 3 min and a secondary, slower decrease to 0.304 +/- 0.043 of control at 30 min were observed. Similar but smaller inhibitions were also observed with the injection of 0.25 M Na(2)SO(4). Injection of isotonic K(2)SO(4) (70 mM) or isotonic K(2)SO(4) made hypertonic with sucrose (70 mM K(2)SO(4)-1.2 M sucrose) was without effect. Injection of a 0.5 M concentration of either K(2)SO(4), N-methyl-D-glucamine (NMDG) sulfate, or 0.75 M NMDG gluconate resulted in a much smaller initial inhibition (<14%) and little or no secondary decrease. Thus increases of [Na(+)](i) have multiple specific inhibitory effects on ENaC that can be temporally separated into a rapid phase that was complete within 2-3 min and a delayed slow phase that was observed between 5 and 30 min. PMID:10444397

  19. Metabolic regulation of neutrophil spreading, membrane tubulovesicular extensions (cytonemes) formation and intracellular pH upon adhesion to fibronectin

    SciTech Connect

    Galkina, Svetlana I. . E-mail: galkina@genebee.msu.su; Sud'ina, Galina F.; Klein, Thomas

    2006-08-01

    Circulating leukocytes have a round cell shape and roll along vessel walls. However, metabolic disorders can lead them to adhere to the endothelium and spread (flatten). We studied the metabolic regulation of adhesion, spreading and intracellular pH (pHi) of neutrophils (polymorphonuclear leukocytes) upon adhesion to fibronectin-coated substrata. Resting neutrophils adhered and spread on fibronectin. An increase in pHi accompanied neutrophil spreading. Inhibition of oxidative phosphorylation or inhibition of P- and F-type ATPases affected neither neutrophil spreading nor pHi. Inhibition of glucose metabolism or V-ATPase impaired neutrophil spreading, blocked the increase in the pHi and induced extrusion of membrane tubulovesicular extensions (cytonemes), anchoring cells to substrata. Omission of extracellular Na{sup +} and inhibition of chloride channels caused a similar effect. We propose that these tubulovesicular extensions represent protrusions of exocytotic trafficking, supplying the plasma membrane of neutrophils with ion exchange mechanisms and additional membrane for spreading. Glucose metabolism and V-type ATPase could affect fusion of exocytotic trafficking with the plasma membrane, thus controlling neutrophil adhesive state and pHi. Cl{sup -} efflux through chloride channels and Na{sup +} influx seem to be involved in the regulation of the V-ATPase by carrying out charge compensation for the proton-pumping activity and through V-ATPase in regulation of neutrophil spreading and pHi.

  20. Structural dynamics of soluble chloride intracellular channel protein CLIC1 examined by amide hydrogen-deuterium exchange mass spectrometry.

    PubMed

    Stoychev, Stoyan H; Nathaniel, Christos; Fanucchi, Sylvia; Brock, Melissa; Li, Sheng; Asmus, Kyle; Woods, Virgil L; Dirr, Heini W

    2009-09-01

    Chloride intracellular channel protein 1 (CLIC1) functions as an anion channel in plasma and nuclear membranes when its soluble monomeric form converts to an integral-membrane form. The transmembrane region of CLIC1 is located in its thioredoxin-like domain 1, but the mechanism whereby the protein converts to its membrane conformation has yet to be determined. Since channel formation in membranes is enhanced at low pH (5 to 5.5), a condition that is found at the surface of membranes, the structural dynamics of soluble CLIC1 was studied at pH 7 and at pH 5.5 in the absence of membranes by amide hydrogen-deuterium exchange mass spectrometry (DXMS). Rapid hydrogen exchange data indicate that CLIC1 displays a similar core structure at these pH values. Domain 1 is less stable than the all-helical domain 2, and, while the structure of domain 1 remains intact, its conformational flexibility is further increased in an acidic environment (pH 5.5). In the absence of membrane, an acidic environment appears to prime the solution structure of CLIC1 by destabilizing domain 1 in order to lower the activation energy barrier for its conversion to the membrane-insertion conformation. The significantly enhanced H/D-exchange rates at pH 5.5 displayed by two segments (peptides 11-31 and 68-82) could be due to the protonation of acidic residues in salt bridges. One of these segments (peptide 11-31) includes part of the transmembrane region which, in the solution structure, consists of helix alpha1. This helix is intrinsically stable and is most likely retained in the membrane conformation. Strand beta2, another element of the transmembrane region, displays a propensity to form a helical structure and has putative N- and C-capping motifs, suggesting that it too most likely forms a helix in a lipid bilayer.

  1. The regulation of chloride homeostasis in the small nonspiking visual interneurons of the fly compound eye.

    PubMed

    Uusitalo, R O; Weckström, M

    1994-04-01

    1. We have used intracellular recordings and ionophoretic injections in vivo to investigate the ion exchange mechanisms responsible for the maintenance of the ion gradients in the large monopolar cells (LMCs) of the first optic ganglion of the blowfly, Calliphora vicinia. 2. Ionophoretic chloride injections caused a rapid approximately 20-mV depolarization of the resting potential (Erp) and abolished or even reversed the light-ON response (OR), which is caused by histamine-gated chloride conductance, as the chloride equilibrium potential (ECl) was increased beyond the Erp, i.e., 50 mV upward. Ionophoretic sodium injections were found to mimic the action of the ionophoretic chloride injections and thus also to cause chloride accumulation inside the cell. 3. Ionophoretic injections of bicarbonate only had the effect of hyperpolarizing the Erp by 5-15 mV for 1-25 s, but chloride gradient, i.e., ECl remained unchanged. Intracellular proton load caused depolarization of the Erp by 15 +/- 5 mV (mean +/- SE) for 20-25 s and a slight 15 +/- 5-mV decrease of the peak OR. Ionophoretic injections of potassium, acetate, and furosemide failed to cause any physiological effect. 4. The time constant for the recovery of the peak OR after sodium load increased linearly as a function of injected charge whereby the time constant for the recovery after chloride accumulation increased slowly up to 50 nC of injected charge, after which it increased rapidly, possibly indicating substrate inhibition. The time constant for the recovery of peak OR after sodium load was from 5 to 65 nC greater than that of chloride.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Novel Roles for Chloride Channels, Exchangers, and Regulators in Chronic Inflammatory Airway Diseases

    PubMed Central

    Sala-Rabanal, Monica; Yurtsever, Zeynep; Berry, Kayla N.; Brett, Tom J.

    2015-01-01

    Chloride transport proteins play critical roles in inflammatory airway diseases, contributing to the detrimental aspects of mucus overproduction, mucus secretion, and airway constriction. However, they also play crucial roles in contributing to the innate immune properties of mucus and mucociliary clearance. In this review, we focus on the emerging novel roles for a chloride channel regulator (CLCA1), a calcium-activated chloride channel (TMEM16A), and two chloride exchangers (SLC26A4/pendrin and SLC26A9) in chronic inflammatory airway diseases. PMID:26612971

  3. Regulation of lung surfactant secretion by intracellular pH.

    PubMed

    Chander, A

    1989-12-01

    We investigated secretion of lung surfactant phosphatidylcholine (PC) using isolated perfused rat lung preparation after labeling the lung lipids in vitro with [methyl-3H]choline. The perfusion medium was Krebs-Ringer bicarbonate buffer (pH 7.4) containing 10 mM glucose and 3% fatty acid-poor bovine serum albumin. After ventilation of lungs with air containing 5% CO2 (control) for 1 h, 0.91% +/- 0.04 (mean +/- SE, n = 6) of total lung lipid radioactivity (greater than 95% in PC) was recovered in the cell-free lavage fluid. The secretion of PC was increased with terbutaline (50 microM), 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP, 100 microM), phorbol L2-myristate 13-acetate (30 ng/ml), and ATP (1 mM), in each case by approximately 150%. Secretion of PC was also increased by 160% if the lungs were ventilated with air containing 0% CO2. The low CO2-mediated PC secretion was time and concentration dependent. The dose-response curve for 0-10% CO2 was S-shaped. The low CO2-induced increase in PC secretion could be largely reversed with diffusible weak acids (25 mM, acetate or butyrate) in the perfusion medium. An increase (70%) in secretion was also induced with 10 mM NH4Cl, suggesting a role for intracellular alkalosis. These observations suggest that intracellular alkalosis stimulates lung surfactant secretion. Alkalosis-stimulated secretion of PC was additive with that with terbutaline (5 X 10(-7) to 5 X 10(-4) M) or 10(-4) M 8-BrcAMP, suggesting that alkalosis effect was not mediated through the beta-adrenergic pathway of surfactant secretion.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2514603

  4. Regulation of Chloride Channels by Protein Kinase C in Normal and Cystic Fibrosis Airway Epithelia

    NASA Astrophysics Data System (ADS)

    Li, Ming; McCann, John D.; Anderson, Matthew P.; Clancy, John P.; Liedtke, Carole M.; Nairn, Angus C.; Greengard, Paul; Welsh, Michael J.

    1989-06-01

    Apical membrane chloride channels control chloride secretion by airway epithelial cells. Defective regulation of these channels is a prominent characteristic of cystic fibrosis. In normal intact cells, activation of protein kinase C (PKC) by phorbol ester either stimulated or inhibited chloride secretion, depending on the physiological status of the cell. In cell-free membrane patches, PKC also had a dual effect: at a high calcium concentration, PKC inactivated chloride channels; at a low calcium concentration, PKC activated chloride channels. In cystic fibrosis cells, PKC-dependent channel inactivation was normal, but activation was defective. Thus it appears that PKC phosphorylates and regulates two different sites on the channel or on an associated membrane protein, one of which is defective in cystic fibrosis.

  5. Possible regulation of caffeine-induced intracellular Ca2+ mobilization by intracellular free Na+.

    PubMed

    Sorimachi, M; Yamagami, K; Nishimura, S; Kuramoto, K

    1992-12-01

    To gain some understanding of the regulatory mechanism involved in caffeine-induced Ca2+ release in adrenal chromaffin cells, we took advantage of the paradoxical observation that removal of divalent cations potentiated the secretory response to caffeine. We measured the concentration of cytosolic free Ca2+ ([Ca]in) in isolated cat chromaffin cells, by fura-2 microfluorometry, to see whether there was any correlation between the secretory response and the rise in [Ca]in. The caffeine-induced [Ca]in rise and catecholamine secretion were increased by treatment of cells with a divalent cation-deficient solution. These potentiated responses were strongly inhibited either by pretreatment with ryanodine, by the reduction of the external Na+ concentration, or by the addition of Ca2+ channel blockers. Removal of divalent cations caused a large rise in the cytosolic free Na+ concentration ([Na]in), which was measured using SBFI microfluorometry. This rise in [Na]in was reduced either by adding Ca2+ channel blockers or by reducing the external Na+ concentration. These results show a good correlation between caffeine-induced Ca2+ release and [Na]in at the time of stimulation, suggesting that caffeine-induced Ca2+ release is regulated by [Na]in. PMID:1431907

  6. Rab proteins: The key regulators of intracellular vesicle transport

    SciTech Connect

    Bhuin, Tanmay; Roy, Jagat Kumar

    2014-10-15

    Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes. - Highlights: • Rab proteins regulate different signalling pathways. • Deregulation of Rabs is the fundamental causes of a variety of human diseases. • This paper gives potential directions in developing therapeutic targets. • This paper also gives ample directions for modulating pathways central to normal physiology. • These are the huge challenges for drug discovery and delivery in near future.

  7. A cAMP-Regulated Chloride Channel in Lymphocytes that is Affected in Cystic Fibrosis

    NASA Astrophysics Data System (ADS)

    Chen, Jennifer H.; Schulman, Howard; Gardner, Phyllis

    1989-02-01

    A defect in regulation of a chloride channel appears to be the molecular basis for cystic fibrosis (CF), a common lethal genetic disease. It is shown here that a chloride channel with kinetic and regulatory properties similar to those described for secretory epithelial cells is present in both T and B lymphocyte cell lines. The regulation of the channels by adenosine 3',5'-monophosphate (cAMP)--dependent protein kinase in transformed B cells from CF patients is defective. Thus, lymphocytes may be an accessible source of CF tissue for study of this defect, for cloning of the chloride channel complex, and for diagnosis of the disease.

  8. Regulation of intracellular heme trafficking revealed by subcellular reporters.

    PubMed

    Yuan, Xiaojing; Rietzschel, Nicole; Kwon, Hanna; Walter Nuno, Ana Beatriz; Hanna, David A; Phillips, John D; Raven, Emma L; Reddi, Amit R; Hamza, Iqbal

    2016-08-30

    Heme is an essential prosthetic group in proteins that reside in virtually every subcellular compartment performing diverse biological functions. Irrespective of whether heme is synthesized in the mitochondria or imported from the environment, this hydrophobic and potentially toxic metalloporphyrin has to be trafficked across membrane barriers, a concept heretofore poorly understood. Here we show, using subcellular-targeted, genetically encoded hemoprotein peroxidase reporters, that both extracellular and endogenous heme contribute to cellular labile heme and that extracellular heme can be transported and used in toto by hemoproteins in all six subcellular compartments examined. The reporters are robust, show large signal-to-background ratio, and provide sufficient range to detect changes in intracellular labile heme. Restoration of reporter activity by heme is organelle-specific, with the Golgi and endoplasmic reticulum being important sites for both exogenous and endogenous heme trafficking. Expression of peroxidase reporters in Caenorhabditis elegans shows that environmental heme influences labile heme in a tissue-dependent manner; reporter activity in the intestine shows a linear increase compared with muscle or hypodermis, with the lowest heme threshold in neurons. Our results demonstrate that the trafficking pathways for exogenous and endogenous heme are distinct, with intrinsic preference for specific subcellular compartments. We anticipate our results will serve as a heuristic paradigm for more sophisticated studies on heme trafficking in cellular and whole-animal models.

  9. Regulation of intracellular heme trafficking revealed by subcellular reporters.

    PubMed

    Yuan, Xiaojing; Rietzschel, Nicole; Kwon, Hanna; Walter Nuno, Ana Beatriz; Hanna, David A; Phillips, John D; Raven, Emma L; Reddi, Amit R; Hamza, Iqbal

    2016-08-30

    Heme is an essential prosthetic group in proteins that reside in virtually every subcellular compartment performing diverse biological functions. Irrespective of whether heme is synthesized in the mitochondria or imported from the environment, this hydrophobic and potentially toxic metalloporphyrin has to be trafficked across membrane barriers, a concept heretofore poorly understood. Here we show, using subcellular-targeted, genetically encoded hemoprotein peroxidase reporters, that both extracellular and endogenous heme contribute to cellular labile heme and that extracellular heme can be transported and used in toto by hemoproteins in all six subcellular compartments examined. The reporters are robust, show large signal-to-background ratio, and provide sufficient range to detect changes in intracellular labile heme. Restoration of reporter activity by heme is organelle-specific, with the Golgi and endoplasmic reticulum being important sites for both exogenous and endogenous heme trafficking. Expression of peroxidase reporters in Caenorhabditis elegans shows that environmental heme influences labile heme in a tissue-dependent manner; reporter activity in the intestine shows a linear increase compared with muscle or hypodermis, with the lowest heme threshold in neurons. Our results demonstrate that the trafficking pathways for exogenous and endogenous heme are distinct, with intrinsic preference for specific subcellular compartments. We anticipate our results will serve as a heuristic paradigm for more sophisticated studies on heme trafficking in cellular and whole-animal models. PMID:27528661

  10. Chloride channels as drug targets

    PubMed Central

    Verkman, Alan S.; Galietta, Luis J. V.

    2013-01-01

    Chloride channels represent a relatively under-explored target class for drug discovery as elucidation of their identity and physiological roles has lagged behind that of many other drug targets. Chloride channels are involved in a wide range of biological functions, including epithelial fluid secretion, cell-volume regulation, neuroexcitation, smooth-muscle contraction and acidification of intracellular organelles. Mutations in several chloride channels cause human diseases, including cystic fibrosis, macular degeneration, myotonia, kidney stones, renal salt wasting and hyperekplexia. Chloride-channel modulators have potential applications in the treatment of some of these disorders, as well as in secretory diarrhoeas, polycystic kidney disease, osteoporosis and hypertension. Modulators of GABAA (γ-aminobutyric acid A) receptor chloride channels are in clinical use and several small-molecule chloride-channel modulators are in preclinical development and clinical trials. Here, we discuss the broad opportunities that remain in chloride-channel-based drug discovery. PMID:19153558

  11. Modulation of intracellular calcium homeostasis by trimethyltin chloride in human tumour cells: neuroblastoma SY5Y and cervix adenocarcinoma HeLa S3.

    PubMed

    Florea, Ana-Maria; Splettstoesser, Frank; Dopp, Elke; Rettenmeier, Albert W; Büsselberg, Dietrich

    2005-12-01

    Physiological modifications of intracellular Ca2+ ([Ca2+]i) levels trigger and/or regulate a diversity of cellular activities (e.g. neurotransmitter release, synaptic plasticity, muscular contraction, cell proliferation), while calcium overloads could result in cytotoxicity. Previously, we have shown that trimethyltin chloride (Me3SnCl; TMT) modulates calcium homeostasis in cervix adenocarcinoma (HeLa S3) cells [Florea, A.-M., Dopp, E., Büsselberg, D., 2005. TMT induces elevated calcium transients in HeLa cells: types and levels of response. Cell Calcium 37, 252-258]. Here we compare [Ca2+]i-changes induced by trimethyltin chloride in neuroblastoma SY5Y and HeLa S3 cells using calcium-sensitive dyes (fluo-4/AM (fluo-4) and rhod-2/AM (rhod-2)) and laser scanning microscopy (LSM). TMT-induced calcium elevations in neuroblastoma SY5Y as well as in HeLa S3 cells. [Ca2+]i rose to a sustained plateau or to transient spikes. Overall, the detected averaged increase of the maximum calcium elevation were: 0.5 microM approximately 125.6%; 5 microM approximately 130.1%; 500 microM approximately 145% in HeLa S3 cells and 0.5 microM approximately 133.3%; 5 microM approximately 136.1%; 500 microM approximately 147.1% in neuroblastoma SY5Y cells. The calcium rise derived from internal stores did not significantly depend on the presence of calcium in the external solution: approximately 109% (no calcium added) versus approximately 117% (2 mM calcium; 5 microM TMT) in HeLa cells. This difference was similar in neuroblastoma SY5Y cells, were approximately 127% versus approximately 136% increase (5 microM TMT) were measured. Staining of calcium stores with rhod-2 showed a TMT-induced [Ca2+]i-decrease in the stores followed by an increase of the calcium concentration in the nuclei of the two cell lines tested. Our results suggest that toxic effects in human tumour cells after exposure to trimethyltin compounds might be due to an elevation of [Ca2+]i.

  12. Ectdomain shedding and regulated intracellular proteolysis in the central nervous system.

    PubMed

    Montes de Oca-B, Pavel

    2010-12-01

    The term Ectodomain Shedding (ES) refers to extracellular domain proteolytic release from cell membrane molecules. This proteolysis is mediated mainly by matrix metalloproteases (MMP) or disintegrin and metalloproteases (ADAM), although some other proteases may mediate it. Virtually, all functional categories of cell membrane molecules are subject of this kind of proteolysis, for this reason ES is involved in different cellular processes such as proliferation, apoptosis, migration, differentiation or pathologies such as inflammation, cancer and degeneration among others. ES releases membrane molecule's extracellular domain (or ectodomain) to the extracellular milieu where it can play different biological functions. ES of transmembrane molecules also generates membrane attached terminal fragments comprising transmembrane and intracellular domains that enable their additional processing by intracellular proteases known as Regulated Intracellular Proteolysis (RIP). This second proteolytic cleavage delivers molecule's intracellular domain (ICD) that carry out intracellular functions. RIP is mediated by the group of intracellular cleaving proteases (i-CLiPs) that include presenilin from the γ-secretase complex. In the CNS the best well known ES is that of the Amyloid Precursor Protein, although many other membrane molecules expressed by cells of the CNS are also subject to ES and RIP. In this review, these molecules are summarized, and some meaningful examples are highlighted and described. In addition, ES and RIP implications in the context of cell biology are discussed. Finally, some considerations that rise from the study of ES and RIP are formulated in view of the unexpected roles of intracellular fragments.

  13. Chloride flux in phagocytes.

    PubMed

    Wang, Guoshun

    2016-09-01

    Phagocytes, such as neutrophils and macrophages, engulf microbes into phagosomes and launch chemical attacks to kill and degrade them. Such a critical innate immune function necessitates ion participation. Chloride, the most abundant anion in the human body, is an indispensable constituent of the myeloperoxidase (MPO)-H2 O2 -halide system that produces the potent microbicide hypochlorous acid (HOCl). It also serves as a balancing ion to set membrane potentials, optimize cytosolic and phagosomal pH, and regulate phagosomal enzymatic activities. Deficient supply of this anion to or defective attainment of this anion by phagocytes is linked to innate immune defects. However, how phagocytes acquire chloride from their residing environment especially when they are deployed to epithelium-lined lumens, and how chloride is intracellularly transported to phagosomes remain largely unknown. This review article will provide an overview of chloride protein carriers, potential mechanisms for phagocytic chloride preservation and acquisition, intracellular chloride supply to phagosomes for oxidant production, and methods to measure chloride levels in phagocytes and their phagosomes. PMID:27558337

  14. Ontogeny of intracellular isosmotic regulation in the european lobster Homarus gammarus (L.).

    PubMed

    Haond, C; Bonnal, L; Sandeaux, R; Charmantier, G; Trilles, J P

    1999-01-01

    Intracellular free amino acids were measured in the abdominal muscle of the three larval instars, postlarvae, and juveniles of the lobster Homarus gammarus, acclimated to seawater (35 per thousand) and to a dilute medium (22 per thousand), to study intracellular isosmotic regulation throughout the development of this species. Transfer to low salinity was followed by a highly significant drop of free amino acids level in all developmental stages. The main regulated amino acids were glycine, proline, and alanine. The level of regulation of total free amino acids changed at metamorphosis: the decrease in total free amino acids at low salinity was 46% in the three larval instars, but it was only 29% in postlarvae and 20% in juveniles. These results suggest that free amino acids, mainly glycine, proline, and alanine, are involved in intracellular isosmotic regulation in the lobster, with different levels of involvement in pre- and postmetamorphic stages. The ontogenetic changes in intracellular isosmotic regulation are discussed in relation to the changes in extracellular regulation (osmoregulation) in the lobster. PMID:10521321

  15. 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

  16. Intracellular Regulation of Cross-Presentation during Dendritic Cell Maturation

    PubMed Central

    Wagner, Claudia S.; Grotzke, Jeff; Cresswell, Peter

    2013-01-01

    We have investigated the effect of different maturation stimuli on the ability of mature dendritic cells (DCs) to cross-present newly acquired particulate antigens. Cross-presentation was impaired in DCs matured by treatment with TNF-α, CpG and LPS, but was less affected upon CD40L-induced maturation. The difference could not be explained by decreased antigen uptake or translocation into the cytosol, but decreased cross-presentation ability did correlate with increased phagosomal/lysosomal acidification. Nevertheless, intra-phagosomal degradation of OVA was not increased in matured samples, suggesting that decreasing phagosomal pH may also regulate cross-presentation by a mechanism other than enhancing degradation. PMID:24098562

  17. Synphilin-1 binds ATP and regulates intracellular energy status.

    PubMed

    Li, Tianxia; Liu, Jingnan; Smith, Wanli W

    2014-01-01

    Recent studies have suggested that synphilin-1, a cytoplasmic protein, is involved in energy homeostasis. Overexpression of synphilin-1 in neurons results in hyperphagia and obesity in animal models. However, the mechanism by which synphilin-1 alters energy homeostasis is unknown. Here, we used cell models and biochemical approaches to investigate the cellular functions of synphilin-1 that may affect energy balance. Synphilin-1 was pulled down by ATP-agarose beads, and the addition of ATP and ADP reduced this binding, indicating that synphilin-1 bound ADP and ATP. Synphilin-1 also bound GMP, GDP, and GTP but with a lower affinity than it bound ATP. In contrast, synphilin-1 did not bind with CTP. Overexpression of synphilin-1 in HEK293T cells significantly increased cellular ATP levels. Genetic alteration to abolish predicted ATP binding motifs of synphilin-1 or knockdown of synphilin-1 by siRNA reduced cellular ATP levels. Together, these data demonstrate that synphilin-1 binds and regulates the cellular energy molecule, ATP. These findings provide a molecular basis for understanding the actions of synphilin-1 in energy homeostasis.

  18. Regulation and control of intracellular algae (= zooxanthellae) in hard corals

    PubMed Central

    Jones, R. J.; Yellowlees, D.

    1997-01-01

    To examine algal (= zooxanthellae) regulation and control, and the factors determining algal densities in hard corals, the zooxanthellae mitotic index and release rates were regularly determined in branch tips from a colony of a staghorn coral, Acropora formosa, recovering from a coral 'bleaching' event (the stress-related dissociation of the coral–algal symbiosis). Mathematical models based upon density-dependent decreases in the algal division frequency and increases in algal release rates during the post-bleaching recovery period accurately predict the observed recovery period (ca. 20 weeks). The models suggest that (i) the colony recovered its algal population from the division of the remaining zooxanthellae, and (ii) the continual loss of zooxanthellae significantly slowed the recovery of the coral. Possible reasons for the 'paradoxical' loss of healthy zooxanthellae from the bleached coral are discussed in terms of endodermal processes occurring in the recovering coral and the redistribution of newly formed zooxanthellae to aposymbiotic host cells. At a steady-state algal density of 2.1 x 106 zooxanthellae cm-2 at the end of the recovery period, the zooxanthellae would have to form a double layer of cells in the coral tissues, consistent with microscopic observations. Neighbouring colonies of A. formosa with inherently higher algal densities possess proportionately smaller zooxanthellae. Results suggest that space availability and the size of the algal symbionts determines the algal densities in the coral colonies. The large increases in the algal densities reported in corals exposed to elevated nutrient concentrations (i.e between a two- and five-fold increase in the algal standing stock) are not consistent with this theory. We suggest that increases of this magnitude are a product of the experimental conditions: reasons for this statement are discussed. We propose that the stability of the coral–algal symbiosis under non-stress conditions, and the

  19. Intracellular pathways regulating ciliary beating of rat brain ependymal cells

    PubMed Central

    Nguyen, Thien; Chin, Wei-Chun; O’Brien, Jennifer A; Verdugo, Pedro; Berger, Albert J

    2001-01-01

    The mammalian brain ventricles are lined with ciliated ependymal cells. As yet little is known about the mechanisms by which neurotransmitters regulate cilia beat frequency (CBF). Application of 5-HT to ependymal cells in cultured rat brainstem slices caused CBF to increase. 5-HT had an EC50 of 30 μM and at 100 μM attained a near-maximal CBF increase of 52.7 ± 4.1 % (mean ± s.d.) (n= 8). Bathing slices in Ca2+-free solution markedly reduced the 5-HT-mediated increase in CBF. Fluorescence measurements revealed that 5-HT caused a marked transient elevation in cytosolic Ca2+ ([Ca2+]c) that then slowly decreased to a plateau level. Analysis showed that the [Ca2+]c transient was due to release of Ca2+ from inositol 1,4,5-trisphosphate (IP3)-sensitive stores; the plateau was probably due to extracellular Ca2+ influx through Ca2+ release-activated Ca2+ (CRAC) channels. Application of ATP caused a sustained decrease in CBF. ATP had an EC50 of about 50 μM and 100 μM ATP resulted in a maximal 57.5 ± 6.5 % (n= 12) decrease in CBF. The ATP-induced decrease in CBF was unaffected by lowering extracellular [Ca2+], and no changes in [Ca2+]c were observed. Exposure of ependymal cells to forskolin caused a decrease in CBF. Ciliated ependymal cells loaded with caged cAMP exhibited a 54.3 ± 7.5 % (n= 9) decrease in CBF following uncaging. These results suggest that ATP reduces CBF by a Ca2+-independent cAMP-mediated pathway. Application of 5-HT and adenosine-5′-O-3-thiotriphosphate (ATP-γ-S) to acutely isolated ciliated ependymal cells resulted in CBF responses similar to those of ependymal cells in cultured slices suggesting that these neurotransmitters act directly on these cells. The opposite response of ciliated ependymal cells to 5-HT and ATP provides a novel mechanism for their active involvement in central nervous system signalling. PMID:11179397

  20. Regulation of acetylcholine release by intracellular acidification of developing motoneurons in Xenopus cell cultures

    PubMed Central

    Chen, Yu-Hwa; Wu, Mei-Lin; Fu, Wen-Mei

    1998-01-01

    The effects of intracellular pH changes on the acetylcholine (ACh) release and cytoplasmic Ca2+ concentration at developing neuromuscular synapses were studied in Xenopus nerve-muscle co-cultures. Spontaneous and evoked ACh release of motoneurons was monitored by using whole-cell voltage-clamped myocytes. Intracellular alkalinization with 15 mm NH4Cl slightly reduced the frequency of spontaneous synaptic currents (SSCs). However, cytosolic acidification following withdrawal of extracellular NH4Cl caused a marked and transient increase in spontaneous ACh release. Another method of cytosolic acidification was used in which NaCl in Ringer solution was replaced with weak organic acids. The increase in spontaneous ACh release paralleled the level of intracellular acidification resulting from addition of these organic acids. Acetate and propionate but not isethionate, methylsulphate and glucuronate, caused an increase in intracellular pH and a marked increase in spontaneous ACh release. Impulse-evoked ACh release was slightly augmented by intracellular alkalinization and inhibited by cytosolic acidification. Cytosolic acidification was accompanied by an elevation in the cytoplasmic Ca2+ concentration ([Ca2+]i), resulting from both external Ca2+ influx and intracellular Ca2+ mobilization. In contrast, the increase in [Ca2+]i induced by high K+ was inhibited by cytosolic acidification. We conclude that cytosolic acidification regulates spontaneous and evoked ACh release differentially in Xenopus motoneurons, increasing spontaneous ACh release but inhibiting evoked ACh release. PMID:9490814

  1. Identification of metastasis-associated proteins involved in gallbladder carcinoma metastasis by proteomic analysis and functional exploration of chloride intracellular channel 1.

    PubMed

    Wang, Jian-Wei; Peng, Shu-You; Li, Jiang-Tao; Wang, Yong; Zhang, Zhi-Ping; Cheng, Yan; Cheng, De-Qing; Weng, Wei-Hong; Wu, Xiang-Song; Fei, Xiao-Zhou; Quan, Zhi-Wei; Li, Ji-Yu; Li, Song-Gang; Liu, Ying-Bin

    2009-08-18

    Advanced gallbladder cancer has an extremely poor prognosis because of metastasis. Identification of metastasis-related biomarkers is essential to improve patient survival. In the present study, metastasis-associated proteins were identified by comparative proteomic analysis and the metastasis-related function of the candidate protein, chloride intracellular channel 1 (CLIC1), was further elucidated. Two cell lines with high or low metastatic potential (termed GBC-SD18H and GBC-SD18L, respectively), originating from the same parental gallbladder carcinoma GBC-SD cell line, were identified by spontaneous metastasis in vivo and characterized by metastatic phenotypes analysis in vitro. Subsequently, a proteomic approach comprised of two-dimensional gel electrophoresis analysis and mass spectroscopy was used to identify and compare the protein expression patterns between GBC-SD18L and GBC-SD18H. Twenty-six proteins were identified and further verified by one-dimensional Western blotting and semiquantitative reverse transcriptase polymerase chain reaction analysis. It was determined that CLIC1, ezrin, vimentin, annexin A3, WD repeat domain 1, triosephosphate isomerase, C1-tetrahydrofolate synthase, Rho GDP-dissociation inhibitor 1, T-complex protein 1, heterogeneous nuclear ribonucleoprotein K, glutamate dehydrogenase 1, proteasome activator complex subunit 3 and Rab GDP-dissociation inhibitor beta were significantly up-regulated in the highly metastatic GBC-SD18H cell line compared to the poorly metastatic GBC-SD18L cell line. However, phosphoglycerate kinase 1 and programmed cell death protein 8 were significantly down-regulated in the highly metastatic GBC-SD18H cell line compared to GBC-SD18L. Considering that CLIC1 was profuse in highly metastatic GBC-SD18H but scarce in poorly metastatic GBC-SD18L, the association of CLIC1 with metastasis was further elucidated by the overexpression and RNA interference of CLIC1 in GBC-SD18L cells and GBC-SD18H cells, respectively

  2. Stress-induced inhibition of nonsense mediated RNA decay regulates intracellular cystine transport and intracellular glutathione through regulation of the cystine/glutamate exchanger SLC7A11

    PubMed Central

    Martin, Leenus; Gardner, Lawrence B.

    2014-01-01

    SLC7A11 encodes a subunit of the xCT cystine/glutamate amino acid transport system and plays a critical role in the generation of glutathione and the protection of cells from oxidative stress. Expression of SLC7A11 promotes tumorigenesis and chemotherapy resistance, but while SLC7A11 has been previously noted to be upregulated in hypoxic cells its regulation has not been fully delineated. We have recently shown that nonsense mediated RNA decay (NMD) is inhibited by cellular stresses generated by the tumor microenvironment, including hypoxia, and augments tumorigenesis. Here we demonstrate that the inhibition of NMD by various cellular stresses leads to the stabilization and upregulation of SLC7A11 mRNA and protein. The inhibition of NMD and upregulation of SLC7A11 augments intracellular cystine transport, and increases intracellular levels of cysteine and glutathione. Accordinglyy, the inhibition of NMD protects cells against oxidative stress via SLC7A11 upregulation. Together our studies identify a mechanism for the dynamic regulation of SLC7A11, through the stress-inhibited regulation of NMD, and add to the growing evidence that the inhibition of NMD is an adaptive response. PMID:25399695

  3. 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

  4. Transcriptional regulation of the Chlamydia heat shock stress response in an intracellular infection

    PubMed Central

    Hanson, Brett R.; Tan, Ming

    2015-01-01

    Summary Bacteria encode heat shock proteins that aid in survival during stressful growth conditions. In addition, the major heat shock proteins of the intracellular bacterium Chlamydia trachomatis have been associated with immune pathology and disease. We developed a ChIP-qPCR method to study the regulation of chlamydial heat shock gene regulation during an intracellular infection. This approach allowed us to show that chlamydial heat shock genes are regulated by the transcription factor HrcA within an infected cell, providing validation for previous in vitro findings. Induction of chlamydial heat shock gene expression by elevated temperature was due to loss of HrcA binding to heat shock promoters, supporting a mechanism of derepression. This heat shock response was rapid, while recovery of HrcA binding and return to non-stress transcript levels occurred more slowly. We also found that control of heat shock gene expression was differentially regulated over the course of the intracellular Chlamydia infection. There was evidence of HrcA-mediated regulation of heat shock genes throughout the chlamydial developmental cycle but the level of repression was lower at early times. This is the first study of Chlamydia-infected cells showing the effect of an environmental signal on transcription factor-DNA binding and target gene expression in the bacterium. PMID:26075961

  5. Regulation of intracellular pH in cnidarians: response to acidosis in Anemonia viridis.

    PubMed

    Laurent, Julien; Venn, Alexander; Tambutté, Éric; Ganot, Philippe; Allemand, Denis; Tambutté, Sylvie

    2014-02-01

    The regulation of intracellular pH (pHi) is a fundamental aspect of cell physiology that has received little attention in studies of the phylum Cnidaria, which includes ecologically important sea anemones and reef-building corals. Like all organisms, cnidarians must maintain pH homeostasis to counterbalance reductions in pHi, which can arise because of changes in either intrinsic or extrinsic parameters. Corals and sea anemones face natural daily changes in internal fluids, where the extracellular pH can range from 8.9 during the day to 7.4 at night. Furthermore, cnidarians are likely to experience future CO₂-driven declines in seawater pH, a process known as ocean acidification. Here, we carried out the first mechanistic investigation to determine how cnidarian pHi regulation responds to decreases in extracellular and intracellular pH. Using the anemone Anemonia viridis, we employed confocal live cell imaging and a pH-sensitive dye to track the dynamics of pHi after intracellular acidosis induced by acute exposure to decreases in seawater pH and NH₄Cl prepulses. The investigation was conducted on cells that contained intracellular symbiotic algae (Symbiodinium sp.) and on symbiont-free endoderm cells. Experiments using inhibitors and Na⁺-free seawater indicate a potential role of Na⁺/H⁺ plasma membrane exchangers (NHEs) in mediating pHi recovery following intracellular acidosis in both cell types. We also measured the buffering capacity of cells, and obtained values between 20.8 and 43.8 mM per pH unit, which are comparable to those in other invertebrates. Our findings provide the first steps towards a better understanding of acid-base regulation in these basal metazoans, for which information on cell physiology is extremely limited. PMID:24256552

  6. Regulation of intracellular pH in cnidarians: response to acidosis in Anemonia viridis.

    PubMed

    Laurent, Julien; Venn, Alexander; Tambutté, Éric; Ganot, Philippe; Allemand, Denis; Tambutté, Sylvie

    2014-02-01

    The regulation of intracellular pH (pHi) is a fundamental aspect of cell physiology that has received little attention in studies of the phylum Cnidaria, which includes ecologically important sea anemones and reef-building corals. Like all organisms, cnidarians must maintain pH homeostasis to counterbalance reductions in pHi, which can arise because of changes in either intrinsic or extrinsic parameters. Corals and sea anemones face natural daily changes in internal fluids, where the extracellular pH can range from 8.9 during the day to 7.4 at night. Furthermore, cnidarians are likely to experience future CO₂-driven declines in seawater pH, a process known as ocean acidification. Here, we carried out the first mechanistic investigation to determine how cnidarian pHi regulation responds to decreases in extracellular and intracellular pH. Using the anemone Anemonia viridis, we employed confocal live cell imaging and a pH-sensitive dye to track the dynamics of pHi after intracellular acidosis induced by acute exposure to decreases in seawater pH and NH₄Cl prepulses. The investigation was conducted on cells that contained intracellular symbiotic algae (Symbiodinium sp.) and on symbiont-free endoderm cells. Experiments using inhibitors and Na⁺-free seawater indicate a potential role of Na⁺/H⁺ plasma membrane exchangers (NHEs) in mediating pHi recovery following intracellular acidosis in both cell types. We also measured the buffering capacity of cells, and obtained values between 20.8 and 43.8 mM per pH unit, which are comparable to those in other invertebrates. Our findings provide the first steps towards a better understanding of acid-base regulation in these basal metazoans, for which information on cell physiology is extremely limited.

  7. Intracellular pH regulation in isolated hepatopancreas cells from the Roman snail (Helix pomatia).

    PubMed

    Manzl, Claudia; Krumschnabel, Gerhard; Schwarzbaum, Pablo J; Chabicovsky, Monika; Dallinger, Reinhard

    2004-01-01

    The mechanisms of intracellular pH (pHi) regulation were studied in isolated hepatopancreas cells from the Roman snail, Helix pomatia. The relationship between intracellular and extracellular pH indicated that pHi is actively regulated in these cells. At least three pHi-regulatory ion transporters were found to be present in these cells and to be responsible for the maintenance of pHi: an amiloride-sensitive Na+/H+ exchanger, a 4-acetamido-4'-isothiocyanostilbene-2,2'disulfonic acid (SITS)-sensitive, presumably Na(+)-dependent, Cl-/HCO3-exchanger, and a bafilomycin-sensitive H(+)-pump. Inhibition of one of these transporters alone did not affect steady state pHi, whereas incubation with amiloride and SITS in combination resulted in a significant intracellular acidification. Following the induction of intracellular acidosis by addition of the weak acid Na+propionate, the Na+/H+ exchanger was immediately activated leading to a rapid recovery of pHi towards the baseline level. Both the SITS-sensitive mechanism and the H(+)-pump responded more slowly, but were of similar importance for pHi recovery. Measurement of pHi recovery from acidification in the three discernible types of hepatopancreas cells with a video fluorescence image system revealed slightly differing response patterns, the physiological significance of which remains to be determined. PMID:14695690

  8. Identification of a transcriptional regulator that controls intracellular gene expression in Salmonella Typhi.

    PubMed

    Haghjoo, Erik; Galán, Jorge E

    2007-06-01

    Salmonella enterica serovar Typhi (S. Typhi), the aetiological agent of typhoid fever, is an exclusively human pathogen. Little is known about specific factors that may confer to this bacterium its unique pathogenic features. One of these determinants is CdtB, a homologue of the active subunit of the cytolethal distending toxin, which causes DNA damage leading to cell cycle arrest and distension of intoxicated cells. A unique property of S. Typhi CdtB is that it is only synthesized when this bacterium is within an intracellular compartment. Through a genetic screen, we have identified a transcriptional regulatory protein that controls the intracellular expression of cdtB. This regulator, which we have named IgeR, is a member of the DeoR family of transcriptional regulatory proteins and is highly conserved in all S. enterica serovars. IgeR directly binds the cdtB promoter and represses its expression in the extracellular environment. Microarray analysis identified additional IgeR-regulated genes that are involved in virulence. Constitutive expression of igeR resulted in the reduction of intracellular expression of cdtB by S. Typhi and in significant impairment of the virulence of Salmonella enterica serovar Typhimurium (S. Typhimurium) in mice. We propose that IgeR may co-ordinate gene expression during Salmonella's transition from an extracellular to an intracellular environment. PMID:17555437

  9. TMEM203 Is a Novel Regulator of Intracellular Calcium Homeostasis and Is Required for Spermatogenesis

    PubMed Central

    Shambharkar, Prashant B.; Bittinger, Mark; Latario, Brian; Xiong, ZhaoHui; Bandyopadhyay, Somnath; Davis, Vanessa; Lin, Victor; Yang, Yi; Valdez, Reginald; Labow, Mark A.

    2015-01-01

    Intracellular calcium signaling is critical for initiating and sustaining diverse cellular functions including transcription, synaptic signaling, muscle contraction, apoptosis and fertilization. Trans-membrane 203 (TMEM203) was identified here in cDNA overexpression screens for proteins capable of modulating intracellular calcium levels using activation of a calcium/calcineurin regulated transcription factor as an indicator. Overexpression of TMEM203 resulted in a reduction of Endoplasmic Reticulum (ER) calcium stores and elevation in basal cytoplasmic calcium levels. TMEM203 protein was localized to the ER and found associated with a number of ER proteins which regulate ER calcium entry and efflux. Mouse Embryonic Fibroblasts (MEFs) derived from Tmem203 deficient mice had reduced ER calcium stores and altered calcium homeostasis. Tmem203 deficient mice were viable though male knockout mice were infertile and exhibited a severe block in spermiogenesis and spermiation. Expression profiling studies showed significant alternations in expression of calcium channels and pumps in testes and concurrently Tmem203 deficient spermatocytes demonstrated significantly altered calcium handling. Thus Tmem203 is an evolutionarily conserved regulator of cellular calcium homeostasis, is required for spermatogenesis and provides a causal link between intracellular calcium regulation and spermiogenesis. PMID:25996873

  10. Intracellular oxygen determined by respiration regulates localization of Ras and prenylated proteins

    PubMed Central

    Kim, A; Davis, R; Higuchi, M

    2015-01-01

    Reduction of mitochondrial DNA (mtDNA) content induces the reduction of oxidative phosphorylation and dependence on fermentative glycolysis, that is, the Warburg effect. In aggressive prostate cancer (PCa), the reduction of mtDNA reduces oxygen consumption, increases intracellular oxygen concentration, and induces constitutive activation of Ras. Many essential proteins for cell death, growth, differentiation, and development, such as Ras, require prenylation for subcellular localization and activation. Prenylation of a protein is defined as the attachment of isoprenoids to a cysteine residue at or near the C-terminus. 3-Hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR) produces isoprenoids, and is posttranslationally regulated by oxygen. We investigated a critical role of intracellular oxygen in membrane localization of prenylated proteins. Localization of prenylated proteins (H-Ras, prelamin A/C, and Rab5a) was observed in poorly differentiated PCa (PC-3) and well-differentiated PCa (LNCaP) cells. PC-3 cells exhibited high intracellular oxygen concentration, and H-Ras, prelamin A/C, and Rab5a were localized to various membranes (Golgi and plasma membrane, nuclear membrane, and early endosomes, respectively). Remarkably, exogenous hypoxia (0.2% O2) in PC-3 cells induced intracellular hypoxia and changed the localization of the prenylated proteins. H-Ras and Rab5a were translocated to cytosol, and prelamin A/C was in the nucleus forming an abnormal nuclear envelope. The localization was reversed by mevalonate indicating the involvement of mevalonate pathway. In contrast, in LNCaP cells, exhibiting low intracellular oxygen concentration, H-Ras and Rab5a were localized in the cytosol, and prelamin A/C was inside the nucleus forming an inadequate nuclear envelope. Exogenous hyperoxia (40% O2) increased the intracellular oxygen concentration and induced Ras translocation from cytosol to the membrane. Prelamin A/C was translocated to the nuclear membrane and formed a

  11. Intracellular oxygen determined by respiration regulates localization of Ras and prenylated proteins.

    PubMed

    Kim, A; Davis, R; Higuchi, M

    2015-07-16

    Reduction of mitochondrial DNA (mtDNA) content induces the reduction of oxidative phosphorylation and dependence on fermentative glycolysis, that is, the Warburg effect. In aggressive prostate cancer (PCa), the reduction of mtDNA reduces oxygen consumption, increases intracellular oxygen concentration, and induces constitutive activation of Ras. Many essential proteins for cell death, growth, differentiation, and development, such as Ras, require prenylation for subcellular localization and activation. Prenylation of a protein is defined as the attachment of isoprenoids to a cysteine residue at or near the C-terminus. 3-Hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR) produces isoprenoids, and is posttranslationally regulated by oxygen. We investigated a critical role of intracellular oxygen in membrane localization of prenylated proteins. Localization of prenylated proteins (H-Ras, prelamin A/C, and Rab5a) was observed in poorly differentiated PCa (PC-3) and well-differentiated PCa (LNCaP) cells. PC-3 cells exhibited high intracellular oxygen concentration, and H-Ras, prelamin A/C, and Rab5a were localized to various membranes (Golgi and plasma membrane, nuclear membrane, and early endosomes, respectively). Remarkably, exogenous hypoxia (0.2% O2) in PC-3 cells induced intracellular hypoxia and changed the localization of the prenylated proteins. H-Ras and Rab5a were translocated to cytosol, and prelamin A/C was in the nucleus forming an abnormal nuclear envelope. The localization was reversed by mevalonate indicating the involvement of mevalonate pathway. In contrast, in LNCaP cells, exhibiting low intracellular oxygen concentration, H-Ras and Rab5a were localized in the cytosol, and prelamin A/C was inside the nucleus forming an inadequate nuclear envelope. Exogenous hyperoxia (40% O2) increased the intracellular oxygen concentration and induced Ras translocation from cytosol to the membrane. Prelamin A/C was translocated to the nuclear membrane and formed a

  12. A novel putative auxin carrier family regulates intracellular auxin homeostasis in plants.

    PubMed

    Barbez, Elke; Kubeš, Martin; Rolčík, Jakub; Béziat, Chloé; Pěnčík, Aleš; Wang, Bangjun; Rosquete, Michel Ruiz; Zhu, Jinsheng; Dobrev, Petre I; Lee, Yuree; Zažímalovà, Eva; Petrášek, Jan; Geisler, Markus; Friml, Jiří; Kleine-Vehn, Jürgen

    2012-05-01

    The phytohormone auxin acts as a prominent signal, providing, by its local accumulation or depletion in selected cells, a spatial and temporal reference for changes in the developmental program. The distribution of auxin depends on both auxin metabolism (biosynthesis, conjugation and degradation) and cellular auxin transport. We identified in silico a novel putative auxin transport facilitator family, called PIN-LIKES (PILS). Here we illustrate that PILS proteins are required for auxin-dependent regulation of plant growth by determining the cellular sensitivity to auxin. PILS proteins regulate intracellular auxin accumulation at the endoplasmic reticulum and thus auxin availability for nuclear auxin signalling. PILS activity affects the level of endogenous auxin indole-3-acetic acid (IAA), presumably via intracellular accumulation and metabolism. Our findings reveal that the transport machinery to compartmentalize auxin within the cell is of an unexpected molecular complexity and demonstrate this compartmentalization to be functionally important for a number of developmental processes. PMID:22504182

  13. 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

  14. 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

  15. Regulation of the collagenase-3 receptor and its role in intracellular ligand processing in rat osteoblastic cells

    NASA Technical Reports Server (NTRS)

    Walling, H. W.; Chan, P. T.; Omura, T. H.; Barmina, O. Y.; Fiacco, G. J.; Jeffrey, J. J.; Partridge, N. C.

    1998-01-01

    We have previously described a specific, saturable receptor for rat collagenase-3 in the rat osteosarcoma cell line, UMR 106-01. Binding of rat collagenase-3 to this receptor is coupled to the internalization and eventual degradation of the enzyme and correlates with observed extracellular levels of the enzyme. In this study we have shown that decreased binding, internalization, and degradation of 125I-rat collagenase-3 were observed in cells after 24 h of parathyroid hormone treatment; these activities returned to control values after 48 h and were increased substantially (twice control levels) after 96 h of treatment with the hormone. Subcellular fractionation studies to identify the route of uptake and degradation of collagenase-3 localized intracellular accumulation of 125I-rat collagenase-3 initially in Golgi-associated lysosomes and later in secondary lysosomes. Maximal lysosomal accumulation of the radiolabel and stimulation of general lysosomal activity occurred after 72 h of parathyroid hormone treatment. Preventing fusion of endosomes with lysosomes (by temperature shift, colchicine, or monensin) resulted in no internalized 125I-collagenase-3 in either lysosomal fraction. Treatment of UMR cells with the above agents or ammonium chloride decreased excretion of 125I-labeled degradation products of collagenase-3. These experiments demonstrated that degradation of collagenase-3 required receptor-mediated endocytosis and sequential processing by endosomes and lysosomes. Thus, parathyroid hormone regulates the expression and synthesis of collagenase-3 as well as the abundance and functioning of the collagenase-3 receptor and the intracellular degradation of its ligand. The coordinate changes in the secretion of collagenase-3 and expression of the receptor determine the net abundance of the enzyme in the extracellular space.

  16. The Arabidopsis Thylakoid Chloride Channel AtCLCe Functions in Chloride Homeostasis and Regulation of Photosynthetic Electron Transport.

    PubMed

    Herdean, Andrei; Nziengui, Hugues; Zsiros, Ottó; Solymosi, Katalin; Garab, Győző; Lundin, Björn; Spetea, Cornelia

    2016-01-01

    Chloride ions can be translocated across cell membranes through Cl(-) channels or Cl(-)/H(+) exchangers. The thylakoid-located member of the Cl(-) channel CLC family in Arabidopsis thaliana (AtCLCe) was hypothesized to play a role in photosynthetic regulation based on the initial photosynthetic characterization of clce mutant lines. The reduced nitrate content of Arabidopsis clce mutants suggested a role in regulation of plant nitrate homeostasis. In this study, we aimed to further investigate the role of AtCLCe in the regulation of ion homeostasis and photosynthetic processes in the thylakoid membrane. We report that the size and composition of proton motive force were mildly altered in two independent Arabidopsis clce mutant lines. Most pronounced effects in the clce mutants were observed on the photosynthetic electron transport of dark-adapted plants, based on the altered shape and associated parameters of the polyphasic OJIP kinetics of chlorophyll a fluorescence induction. Other alterations were found in the kinetics of state transition and in the macro-organization of photosystem II supercomplexes, as indicated by circular dichroism measurements. Pre-treatment with KCl but not with KNO3 restored the wild-type photosynthetic phenotype. Analyses by transmission electron microscopy revealed a bow-like arrangement of the thylakoid network and a large thylakoid-free stromal region in chloroplast sections from the dark-adapted clce plants. Based on these data, we propose that AtCLCe functions in Cl(-) homeostasis after transition from light to dark, which affects chloroplast ultrastructure and regulation of photosynthetic electron transport.

  17. The Arabidopsis Thylakoid Chloride Channel AtCLCe Functions in Chloride Homeostasis and Regulation of Photosynthetic Electron Transport

    PubMed Central

    Herdean, Andrei; Nziengui, Hugues; Zsiros, Ottó; Solymosi, Katalin; Garab, Győző; Lundin, Björn; Spetea, Cornelia

    2016-01-01

    Chloride ions can be translocated across cell membranes through Cl− channels or Cl−/H+ exchangers. The thylakoid-located member of the Cl− channel CLC family in Arabidopsis thaliana (AtCLCe) was hypothesized to play a role in photosynthetic regulation based on the initial photosynthetic characterization of clce mutant lines. The reduced nitrate content of Arabidopsis clce mutants suggested a role in regulation of plant nitrate homeostasis. In this study, we aimed to further investigate the role of AtCLCe in the regulation of ion homeostasis and photosynthetic processes in the thylakoid membrane. We report that the size and composition of proton motive force were mildly altered in two independent Arabidopsis clce mutant lines. Most pronounced effects in the clce mutants were observed on the photosynthetic electron transport of dark-adapted plants, based on the altered shape and associated parameters of the polyphasic OJIP kinetics of chlorophyll a fluorescence induction. Other alterations were found in the kinetics of state transition and in the macro-organization of photosystem II supercomplexes, as indicated by circular dichroism measurements. Pre-treatment with KCl but not with KNO3 restored the wild-type photosynthetic phenotype. Analyses by transmission electron microscopy revealed a bow-like arrangement of the thylakoid network and a large thylakoid-free stromal region in chloroplast sections from the dark-adapted clce plants. Based on these data, we propose that AtCLCe functions in Cl− homeostasis after transition from light to dark, which affects chloroplast ultrastructure and regulation of photosynthetic electron transport. PMID:26904077

  18. 5,5'-Dithio-bis(2-nitrobenzoic acid) modification of cysteine improves the crystal quality of human chloride intracellular channel protein 2

    SciTech Connect

    Mi Wei; Li Lanfen; Su Xiaodong

    2008-04-18

    Structural studies of human chloride intracellular channel protein 2 (CLIC2) had been hampered by the problem of generating suitable crystals primarily due to the protein containing exposed cysteines. Several chemical reagents were used to react with the cysteines on CLIC2 in order to modify the redox state of the protein. We have obtained high quality crystals that diffracted to better than 2.5 A at a home X-ray source by treating the protein with 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB). After solving the crystal structure of CLIC2, we found that the DTNB had reacted with the Cys{sup 114}, and made CLIC2 in a homogenous oxidized state. This study demonstrated that the DTNB modification drastically improved the crystallization of CLIC2, and it implied that this method may be useful for other proteins containing exposed cysteines in general.

  19. [The activation effect of nobiletin on cystic fibrosis transmembrane conductance regulator chloride channel].

    PubMed

    Yang, Shuang; Yu, Bo; Zhang, Yao-Fang; Wang, Xue; Yang, Hong

    2013-06-01

    Aim of the present study is to investigate activation effect of nobiletin on cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel activity. CFTR-mediated iodide influx assay and patch-clamp tests were done on FRT cells stably co-transfected with human CFTR and EYFP/H148Q. Nobiletin potently activated CFTR chloride channel activity in a dose- and time-dependent manner. The CFTR blocker CFTR(inh)-172 could completely reverse the effect. Preliminary mechanism study indicated that nobiletin activated CFTR chloride channel through a direct binding way. In addition, ex vivo tests done on mice trachea showed that nobiletin time-dependently stimulated submucosal gland fluid secretion. Nobiletin may be a therapeutic lead compound in treating CFTR-related diseases including disseminated bronchiectasis.

  20. The calcium-activated chloride channel Anoctamin 1 contributes to the regulation of renal function.

    PubMed

    Faria, Diana; Rock, Jason R; Romao, Ana M; Schweda, Frank; Bandulik, Sascha; Witzgall, Ralph; Schlatter, Eberhard; Heitzmann, Dirk; Pavenstädt, Hermann; Herrmann, Edwin; Kunzelmann, Karl; Schreiber, Rainer

    2014-06-01

    The role of calcium-activated chloride channels for renal function is unknown. By immunohistochemistry we demonstrate dominant expression of the recently identified calcium-activated chloride channels, Anoctamin 1 (Ano1, TMEM16A) in human and mouse proximal tubular epithelial (PTE) cells, with some expression in podocytes and other tubular segments. Ano1-null mice had proteinuria and numerous large reabsorption vesicles in PTE cells. Selective knockout of Ano1 in podocytes (Ano1-/-/Nphs2-Cre) did not impair renal function, whereas tubular knockout in Ano1-/-/Ksp-Cre mice increased urine protein excretion and decreased urine electrolyte concentrations. Purinergic stimulation activated calcium-dependent chloride currents in isolated proximal tubule epithelial cells from wild-type but not from Ano1-/-/Ksp-Cre mice. Ano1 currents were activated by acidic pH, suggesting parallel stimulation of Ano1 chloride secretion with activation of the proton-ATPase. Lack of calcium-dependent chloride secretion in cells from Ano1-/-/Ksp-Cre mice was paralleled by attenuated proton secretion and reduced endosomal acidification, which compromised proximal tubular albumin uptake. Tubular knockout of Ano1 enhanced serum renin and aldosterone concentrations, probably leading to enhanced compensatory distal tubular reabsorption, thus maintaining normal blood pressure levels. Thus, Ano1 has a role in proximal tubular proton secretion and protein reabsorption. The results correspond to regulation of the proton-ATPase by the Ano1-homolog Ist2 in yeast.

  1. 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

  2. Intracellular role of adenylyl cyclase in regulation of lateral pseudopod formation during Dictyostelium chemotaxis.

    PubMed

    Stepanovic, Vesna; Wessels, Deborah; Daniels, Karla; Loomis, William F; Soll, David R

    2005-04-01

    Cyclic AMP (cAMP) functions as the extracellular chemoattractant in the aggregation phase of Dictyostelium development. There is some question, however, concerning what role, if any, it plays intracellularly in motility and chemotaxis. To test for such a role, the behavior of null mutants of acaA, the adenylyl cyclase gene that encodes the enzyme responsible for cAMP synthesis during aggregation, was analyzed in buffer and in response to experimentally generated spatial and temporal gradients of extracellular cAMP. acaA- cells were defective in suppressing lateral pseudopods in response to a spatial gradient of cAMP and to an increasing temporal gradient of cAMP. acaA- cells were incapable of chemotaxis in natural waves of cAMP generated by majority control cells in mixed cultures. These results indicate that intracellular cAMP and, hence, adenylyl cyclase play an intracellular role in the chemotactic response. The behavioral defects of acaA- cells were surprisingly similar to those of cells of null mutants of regA, which encodes the intracellular phosphodiesterase that hydrolyzes cAMP and, hence, functions opposite adenylyl cyclase A (ACA). This result is consistent with the hypothesis that ACA and RegA are components of a receptor-regulated intracellular circuit that controls protein kinase A activity. In this model, the suppression of lateral pseudopods in the front of a natural wave depends on a complete circuit. Hence, deletion of any component of the circuit (i.e., RegA or ACA) would result in the same chemotactic defect.

  3. 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.

  4. Phosphate-Regulated Induction of Intracellular Ribonucleases in Cultured Tomato (Lycopersicon esculentum) Cells 1

    PubMed Central

    Löffler, Andreas; Abel, Steffen; Jost, Wolfgang; Beintema, Jaap J.; Glund, Konrad

    1992-01-01

    Four intracellular RNases were found to be induced in cultured tomato (Lycopersicon esculentum) cells upon phosphate starvation. Localization studies revealed three (RNases LV 1-3) in the vacuoles and one (RNase LX) outside these organelles. All of these RNases were purified to homogeneity and were shown to be type I RNases on the basis of type of splitting, substrate, and base specificity at the cleavage site, molecular weight, isoelectric point, and pH optimum. Moreover, RNase LV 3 was shown by fingerprinting of tryptic digests on reversed-phase high-performance liquid chromatography and sequencing the N terminus and two tryptic peptides to be structurally very similar to a recently characterized extracellular RNase LE which is also phosphate regulated (Nürnberger et al. [1990] Plant Physiol 92: 970-976; Jost et al. [1991] Eur J Biochem 198: 1-6). Expression of the four intracellular RNases is induced by depleting the cells of phosphate and repressed by adding phosphate. Our studies indicate that higher plants, in addition to secreting enzymes for scavanging phosphate under starvation conditions, also induce intracellularly emergency rescue systems. ImagesFigure 1Figure 2Figure 3Figure 4 PMID:16668816

  5. Galectin-3 regulates intracellular trafficking of epidermal growth factor receptor through Alix and promotes keratinocyte migration

    PubMed Central

    Liu, Wei; Hsu, Daniel K.; Chen, Huan-Yuan; Yang, Ri-Yao; Carraway, Kermit L.; Isseroff, Roslyn R.; Liu, Fu-Tong

    2012-01-01

    The epidermal growth factor receptor (EGFR)-mediated signaling pathways are important in a variety of cellular processes, including cell migration and wound re-epithelialization. Intracellular trafficking of EGFR is critical for maintaining EGFR surface expression. Galectin-3, a member of an animal lectin family, has been implicated in a number of physiological and pathological processes. Through studies of galectin-3-deficient mice and cells isolated from these mice, we demonstrated that absence of galectin-3 impairs keratinocyte migration and skin wound re-epithelialization. We have linked this pro-migratory function to a crucial role of cytosolic galectin-3 in controlling intracellular trafficking and cell surface expression of EGFR after EGF stimulation. Without galectin-3, the surface levels of EGFR are dramatically reduced and the receptor accumulates diffusely in the cytoplasm. This is associated with reduced rates of both endocytosis and recycling of the receptor. We have provided evidence that this novel function of galectin-3 may be mediated through interaction with its binding partner Alix, which is a protein component of the endosomal sorting complex required for transport (ESCRT) machinery. Our results suggest that galectin-3 is potentially a critical regulator of a number of important cellular responses through its intracellular control of trafficking of cell surface receptors. PMID:22785133

  6. 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.

  7. Apoplastic and intracellular plant sugars regulate developmental transitions in witches’ broom disease of cacao

    PubMed Central

    Barau, Joan; Grandis, Adriana; Carvalho, Vinicius Miessler de Andrade; Teixeira, Gleidson Silva; Zaparoli, Gustavo Henrique Alcalá; do Rio, Maria Carolina Scatolin; Rincones, Johana; Buckeridge, Marcos Silveira; Pereira, Gonçalo Amarante Guimarães

    2015-01-01

    Witches’ broom disease (WBD) of cacao differs from other typical hemibiotrophic plant diseases by its unusually long biotrophic phase. Plant carbon sources have been proposed to regulate WBD developmental transitions; however, nothing is known about their availability at the plant–fungus interface, the apoplastic fluid of cacao. Data are provided supporting a role for the dynamics of soluble carbon in the apoplastic fluid in prompting the end of the biotrophic phase of infection. Carbon depletion and the consequent fungal sensing of starvation were identified as key signalling factors at the apoplast. MpNEP2, a fungal effector of host necrosis, was found to be up-regulated in an autophagic-like response to carbon starvation in vitro. In addition, the in vivo artificial manipulation of carbon availability in the apoplastic fluid considerably modulated both its expression and plant necrosis rate. Strikingly, infected cacao tissues accumulated intracellular hexoses, and showed stunted photosynthesis and the up-regulation of senescence markers immediately prior to the transition to the necrotrophic phase. These opposite findings of carbon depletion and accumulation in different host cell compartments are discussed within the frame of WBD development. A model is suggested to explain phase transition as a synergic outcome of fungal-related factors released upon sensing of extracellular carbon starvation, and an early senescence of infected tissues probably triggered by intracellular sugar accumulation. PMID:25540440

  8. Apoplastic and intracellular plant sugars regulate developmental transitions in witches' broom disease of cacao.

    PubMed

    Barau, Joan; Grandis, Adriana; Carvalho, Vinicius Miessler de Andrade; Teixeira, Gleidson Silva; Zaparoli, Gustavo Henrique Alcalá; do Rio, Maria Carolina Scatolin; Rincones, Johana; Buckeridge, Marcos Silveira; Pereira, Gonçalo Amarante Guimarães

    2015-03-01

    Witches' broom disease (WBD) of cacao differs from other typical hemibiotrophic plant diseases by its unusually long biotrophic phase. Plant carbon sources have been proposed to regulate WBD developmental transitions; however, nothing is known about their availability at the plant-fungus interface, the apoplastic fluid of cacao. Data are provided supporting a role for the dynamics of soluble carbon in the apoplastic fluid in prompting the end of the biotrophic phase of infection. Carbon depletion and the consequent fungal sensing of starvation were identified as key signalling factors at the apoplast. MpNEP2, a fungal effector of host necrosis, was found to be up-regulated in an autophagic-like response to carbon starvation in vitro. In addition, the in vivo artificial manipulation of carbon availability in the apoplastic fluid considerably modulated both its expression and plant necrosis rate. Strikingly, infected cacao tissues accumulated intracellular hexoses, and showed stunted photosynthesis and the up-regulation of senescence markers immediately prior to the transition to the necrotrophic phase. These opposite findings of carbon depletion and accumulation in different host cell compartments are discussed within the frame of WBD development. A model is suggested to explain phase transition as a synergic outcome of fungal-related factors released upon sensing of extracellular carbon starvation, and an early senescence of infected tissues probably triggered by intracellular sugar accumulation.

  9. Intracellular trafficking by Star regulates cleavage of the Drosophila EGF receptor ligand Spitz

    PubMed Central

    Tsruya, Rachel; Schlesinger, Ayelet; Reich, Aderet; Gabay, Limor; Sapir, Amir; Shilo, Ben-Zion

    2002-01-01

    Spitz (Spi) is a TGFα homolog that is a cardinal ligand for the Drosophila EGF receptor throughout development. Cleavage of the ubiquitously expressed transmembrane form of Spi (mSpi) precedes EGF receptor activation. We show that the Star and Rhomboid (Rho) proteins are necessary for Spi cleavage in Drosophila cells. Complexes between the Spi and Star proteins, as well as between the Star and Rho proteins were identified, but no Spi–Star–Rho triple complex was detected. This observation suggests a sequential activity of Star and Rho in mSpi processing. The interactions between Spi and Star regulate the intracellular trafficking of Spi. The Spi precursor is retained in the periphery of the nucleus. Coexpression of Star promotes translocation of Spi to a compartment where Rho is present both in cells and in embryos. A Star deletion construct that maintains binding to Spi and Rho, but is unable to facilitate Spi translocation, lost biological activity. These results underscore the importance of regulated intracellular trafficking in processing of a TGFα family ligand. PMID:11799065

  10. 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

  11. Regulation of Intracellular Structural Tension by Talin in the Axon Growth and Regeneration.

    PubMed

    Dingyu, Wang; Fanjie, Meng; Zhengzheng, Ding; Baosheng, Huang; Chao, Yang; Yi, Pan; Huiwen, Wu; Jun, Guo; Gang, Hu

    2016-09-01

    Intracellular tension is the most important characteristic of neuron polarization as well as the growth and regeneration of axons, which can be generated by motor proteins and conducted along the cytoskeleton. To better understand this process, we created Förster resonance energy transfer (FRET)-based tension probes that can be incorporated into microfilaments to provide a real-time measurement of forces in neuron cytoskeletons. We found that our probe could be used to assess the structural tension of neuron polarity. Nerve growth factor (NGF) upregulated structural forces, whereas the glial-scar inhibitors chondroitin sulfate proteoglycan (CSPG) and aggrecan weakened such forces. Notably, the tension across axons was distributed uniformly and remarkably stronger than that in the cell body in NGF-stimulated neurons. The mechanosensors talin/vinculin could antagonize the effect of glial-scar inhibitors via structural forces. However, E-cadherin was closely associated with glial-scar inhibitor-induced downregulation of structural forces. Talin/vinculin was involved in the negative regulation of E-cadherin transcription through the nuclear factor-kappa B pathway. Collectively, this study clarified the mechanism underlying intracellular tension in the growth and regeneration of axons which, conversely, can be regulated by talin and E-cadherin.

  12. Intracellular pH regulation in chicken enterocytes: the importance of extracellular pH.

    PubMed

    Peral, M J; Calonge, M L; Ilundáin, A A

    1995-11-01

    The present work reports the effect of pHo on pHi and Na(+)-H+ exchanger activity. Intracellular pH tended to follow pHo, but the proton distribution across the cell membrane is not at electrochemical equilibrium. Removal of external Na+ acidified the cells by both reversing the direction of the Na(+)-H+ exchanger and hyperpolarizing the cell membrane potential. The relationship between pHo and the rate of Na(+)-dependent proton efflux following an acid load suggests that external protons interact with the Na(+)-H+ exchanger at a single site with an apparent pK (-log of the dissociation constant) of 7.22. The results demonstrate that maintenance of pHo in the physiological range is essential for maintenance of normal cell pH and that the activity of the Na(+)-H+ exchanger involved in pHi regulation is affected by external protons. The results also suggest that, at least at low pHo, some intracellular mechanism is involved in pHi regulation. PMID:8962700

  13. CFTR chloride channels are regulated by a SNAP-23/syntaxin 1A complex

    PubMed Central

    Cormet-Boyaka, Estelle; Di, Anke; Chang, Steven Y.; Naren, Anjaparavanda P.; Tousson, Albert; Nelson, Deborah J.; Kirk, Kevin L.

    2002-01-01

    Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate membrane fusion reactions in eukaryotic cells by assembling into complexes that link vesicle-associated SNAREs with SNAREs on target membranes (t-SNAREs). Many SNARE complexes contain two t-SNAREs that form a heterodimer, a putative intermediate in SNARE assembly. Individual t-SNAREs (e.g., syntaxin 1A) also regulate synaptic calcium channels and cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial chloride channel that is defective in cystic fibrosis. Whether the regulation of ion channels by individual t-SNAREs is related to SNARE complex assembly and membrane fusion is unknown. Here we show that CFTR channels are coordinately regulated by two cognate t-SNAREs, SNAP-23 (synaptosome-associated protein of 23 kDa) and syntaxin 1A. SNAP-23 physically associates with CFTR by binding to its amino-terminal tail, a region that modulates channel gating. CFTR-mediated chloride currents are inhibited by introducing excess SNAP-23 into HT29-Cl.19A epithelial cells. Conversely, CFTR activity is stimulated by a SNAP-23 antibody that blocks the binding of this t-SNARE to the CFTR amino-terminal tail. The physical and functional interactions between SNAP-23 and CFTR depend on syntaxin 1A, which binds to both proteins. We conclude that CFTR channels are regulated by a t-SNARE complex that may tune CFTR activity to rates of membrane traffic in epithelial cells. PMID:12209004

  14. Intracellular osteopontin stabilizes TRAF3 to positively regulate innate antiviral response

    PubMed Central

    Zhao, Kai; Zhang, Meng; Zhang, Lei; Wang, Peng; Song, Guanhua; Liu, Bingyu; Wu, Haifeng; Yin, Zhinan; Gao, Chengjiang

    2016-01-01

    Osteopontin (OPN) is a multifunctional protein involved in both innate immunity and adaptive immunity. However, the function of OPN, especially the intracellular form OPN (iOPN) on innate antiviral immune response remains elusive. Here, we demonstrated that iOPN is an essential positive regulator to protect the host from virus infection. OPN deficiency or knockdown significantly attenuated virus-induced IRF3 activation, IFN-β production and antiviral response. Consistently, OPN-deficient mice were more susceptible to VSV infection than WT mice. Mechanistically, iOPN was found to interact with tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3) and inhibit Triad3A-mediated K48-linked polyubiquitination and degradation of TRAF3 through the C-terminal fragment of iOPN. Therefore, our findings delineated a new function for iOPN to act as a positive regulator in innate antiviral immunity through stabilization of TRAF3. PMID:27026194

  15. Substrate Regulation of Single Potassium and Chloride Ion Channels in Arabidopsis Plasma Membrane

    PubMed Central

    Lew, Roger R.

    1991-01-01

    Patch clamp measurements of excised inside-out patches of Arabidopsis thaliana plasma membrane reveal at least two ion channels which conduct either potassium or chloride. The conductance of the potassium channel ranged from 5 to 70 picosiemens depending on KCl concentration. The conductance increased linearly with increasing cytoplasmic-side [KCl]; the extent of this dependence declined as extracytoplasmic-side [KCl] was increased. This indicates that substrate regulation of the potassium channel is a consequence of the molecular architecture of the channel: in particular, multi-ion binding sites within the channel pore. The chloride channel conductance (ranging from 5-40 picosiemens) was independent of cytoplasmic-side [KCl] until a threshold concentration of about 300 millimolar was reached. Such behavior is expected only if the channel is allosterically regulated by cytoplasmic-side K+ and/or Cl−. The median open times of either channel (about 200 milliseconds for the potassium channel and 20 milliseconds for the chloride channel) were unaffected by substrate concentrations. PMID:16668031

  16. Intracellular localization of differentially regulated RNA-specific adenosine deaminase isoforms in inflammation.

    PubMed

    Yang, Jing-Hua; Nie, Yongzhan; Zhao, Qingchuan; Su, Yingjun; Pypaert, Marc; Su, Haili; Rabinovici, Reuven

    2003-11-14

    Adenosine-to-inosine (A-to-I) RNA editing is a post-transcriptional process that amplifies the repertoire of protein production. Recently, the induction of this process through up-regulation of the editing enzyme RNA-specific adenosine deaminase 1 (ADAR1) was documented during acute inflammation. Here we report that the inflammation-induced up-regulation of ADAR1 involves differential production and intracellular localization of several isoforms with distinct RNA-binding domains and localization signals. These include the full-length ADAR1 (p150) and two functionally active short isoforms (p80 and p110). ADAR1 p80 starts at a methionine 519 (M519) due to alternative splicing in exon 2, which deletes the putative nuclear localization signal, the Z-DNA binding domain, and the entire RNA binding domain I. ADAR1 p110 is the mouse homologue of the human ADAR1 110-kDa variant (M246), which retains the second half of the Z-DNA binding domain, all RNA binding domains, and the deaminase domain. Additional variations are found in the third RNA binding domain of ADAR1; they are differentially regulated during inflammation, generating isoforms with different levels of activities. Studies in several cell types transfected with ADAR1-EGFP chimeras demonstrated that the p150 and p80 variants are localized in the cytoplasm and nucleolus, respectively. In agreement with this observation, endogenous ADAR1 was identified in the cytoplasm and nucleolus of mouse splenocytes and HeLa cells. Since the ADAR1 variants are differentially regulated during acute inflammation, it suggests that the localization of these variants and of A-to-I RNA editing in the cytoplasm, nucleus, and nucleolus is intracellularly reorganized in response to inflammatory stimulation. PMID:12954622

  17. Chloride Anions Regulate Kinetics but Not Voltage-Sensor Qmax of the Solute Carrier SLC26a5.

    PubMed

    Santos-Sacchi, Joseph; Song, Lei

    2016-06-01

    In general, SLC26 solute carriers serve to transport a variety of anions across biological membranes. However, prestin (SLC26a5) has evolved, now serving as a motor protein in outer hair cells (OHCs) of the mammalian inner ear and is required for cochlear amplification, a mechanical feedback mechanism to boost auditory performance. The mechanical activity of the OHC imparted by prestin is driven by voltage and controlled by anions, chiefly intracellular chloride. Current opinion is that chloride anions control the Boltzmann characteristics of the voltage sensor responsible for prestin activity, including Qmax, the total sensor charge moved within the membrane, and Vh, a measure of prestin's operating voltage range. Here, we show that standard narrow-band, high-frequency admittance measures of nonlinear capacitance (NLC), an alternate representation of the sensor's charge-voltage (Q-V) relationship, is inadequate for assessment of Qmax, an estimate of the sum of unitary charges contributed by all voltage sensors within the membrane. Prestin's slow transition rates and chloride-binding kinetics adversely influence these estimates, contributing to the prevalent concept that intracellular chloride level controls the quantity of sensor charge moved. By monitoring charge movement across frequency, using measures of multifrequency admittance, expanded displacement current integration, and OHC electromotility, we find that chloride influences prestin kinetics, thereby controlling charge magnitude at any particular frequency of interrogation. Importantly, however, this chloride dependence vanishes as frequency decreases, with Qmax asymptoting at a level irrespective of the chloride level. These data indicate that prestin activity is significantly low-pass in the frequency domain, with important implications for cochlear amplification. We also note that the occurrence of voltage-dependent charge movements in other SLC26 family members may be hidden by inadequate

  18. [Roles of intracellular calcium and monomeric G-proteins in regulating exocytosis of human neutrophils].

    PubMed

    Zhu, Ying; Wang, Jun-Han; Wu, Jian-Min; Xu, Tao; Zhang, Chun-Guang

    2003-12-25

    Neutrophils play a major role in host defense against microbial infection. There are some clues indicate that neutrophils may also play a role in the pathophysiology of the airway obstruction in chronic asthma. We studied the roles of intracellular calcium and GTP gamma S in the regulation of neutrophils exocytosis using pipette perfusion and membrane capacitance measurement technique in whole cell patch clamp configuration. The results showed that the membrane capacitance increase induced by calcium revealed a biphasic process. The first phase occurred when the calcium level was between 0.2-14 micromol/L with a plateau amplitude of 1.23 pF and a calcium EC50 of 1.1 micromol/L. This phase might correspond to the release of the tertiary granules. The second phase occurred when the calcium concentration was between 20-70 micromol/L with a plateau increment of 6.36 pF, the calcium EC50 being about 33 micromol/L. This phase might represent the release of the primary and secondary granules. Intracellular calcium also simultaneously increased the exocytotic rate and the eventual extent in neutrophils. On the other hand, GTP gamma S can increase the exocytotic rate in a dose-dependent manner but had no effect on the eventual extent of membrane capacitance increment (>6 pF) if the cell was stimulated for a long period (>20 min). GTP gamma S (ranging from 20 to 100 micromol/L) induced the neutrophils to release all four types of the granules at very low intracellular calcium level. PMID:14695488

  19. Vitamin E and Phosphoinositides Regulate the Intracellular Localization of the Hepatic α-Tocopherol Transfer Protein.

    PubMed

    Chung, Stacey; Ghelfi, Mikel; Atkinson, Jeffrey; Parker, Robert; Qian, Jinghui; Carlin, Cathleen; Manor, Danny

    2016-08-12

    α-Tocopherol (vitamin E) is an essential nutrient for all vertebrates. From the eight naturally occurring members of the vitamin E family, α-tocopherol is the most biologically active species and is selectively retained in tissues. The hepatic α-tocopherol transfer protein (TTP) preferentially selects dietary α-tocopherol and facilitates its transport through the hepatocyte and its secretion to the circulation. In doing so, TTP regulates body-wide levels of α-tocopherol. The mechanisms by which TTP facilitates α-tocopherol trafficking in hepatocytes are poorly understood. We found that the intracellular localization of TTP in hepatocytes is dynamic and responds to the presence of α-tocopherol. In the absence of the vitamin, TTP is localized to perinuclear vesicles that harbor CD71, transferrin, and Rab8, markers of the recycling endosomes. Upon treatment with α-tocopherol, TTP- and α-tocopherol-containing vesicles translocate to the plasma membrane, prior to secretion of the vitamin to the exterior of the cells. The change in TTP localization is specific to α-tocopherol and is time- and dose-dependent. The aberrant intracellular localization patterns of lipid binding-defective TTP mutants highlight the importance of protein-lipid interaction in the transport of α-tocopherol. These findings provide the basis for a proposed mechanistic model that describes TTP-facilitated trafficking of α-tocopherol through hepatocytes. PMID:27307040

  20. Intracellular univalent cations and the regulation of the BALB/c-3T3 cell cycle

    PubMed Central

    1981-01-01

    Addition of serum to density-arrested BALB/c-3T3 cells causes a rapid increase in uptake of Na+ and K+, followed 12 h later by the onset of DNA synthesis. We explored the role of intracellular univalent cation concentrations in the regulation of BALB/c-3T3 cell growth by serum growth factors. As cells grew to confluence, intracellular Na+ and K+ concentrations ([Na+]i and [K+]i) fell from 40 and 180 to 15 and 90 mmol/liter, respectively. Stimulation of growth of density-inhibited cells by the addition of serum growth factors increased [Na]i by 30% and [K+]i by 13-25% in early G0/G1, resulting in an increase in total univalent cation concentration. Addition of ouabain to stimulated cells resulted in a concentration-dependent steady decrease in [K+]i and increase in [Na+]i. Ouabain (100 microM) decreased [K+]i to approximately 60 mmol/liter by 12 h, and also prevented the serum- stimulated increase in 86Rb+ uptake. However, 100 microM ouabain did not inhibit DNA synthesis. A time-course experiment was done to determine the effect of 100 microM ouabain on [K+]i throughout G0/G1 and S phase. The addition of serum growth factors to density-inhibited cells stimulated equal rates of entry into the S phase in the presence or absence of 100 microM ouabain. However, in the presence of ouabain, there was a decrease in [K+]i. Therefore, an increase in [K+]i is not required for entry into S phase; serum growth factors do not regulate cell growth by altering [K+]i. The significance of increased total univalent cation concentration is discussed. PMID:7204489

  1. 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.

  2. Reversible intracellular translocation of KRas but not HRas in hippocampal neurons regulated by Ca2+/calmodulin.

    PubMed

    Fivaz, Marc; Meyer, Tobias

    2005-08-01

    The Ras/MAPK pathway regulates synaptic plasticity and cell survival in neurons of the central nervous system. Here, we show that KRas, but not HRas, acutely translocates from the plasma membrane (PM) to the Golgi complex and early/recycling endosomes in response to neuronal activity. Translocation is reversible and mediated by the polybasic-prenyl membrane targeting motif of KRas. We provide evidence that KRas translocation occurs through sequestration of the polybasic-prenyl motif by Ca2+/calmodulin (Ca2+/CaM) and subsequent release of KRas from the PM, in a process reminiscent of GDP dissociation inhibitor-mediated membrane recycling of Rab and Rho GTPases. KRas translocation was accompanied by partial intracellular redistribution of its activity. We conclude that the polybasic-prenyl motif acts as a Ca2+/CaM-regulated molecular switch that controls PM concentration of KRas and redistributes its activity to internal sites. Our data thus define a novel signaling mechanism that differentially regulates KRas and HRas localization and activity in neurons.

  3. β-PIX controls intracellular viscoelasticity to regulate lung cancer cell migration

    PubMed Central

    Yu, Helen Wenshin; Chen, Yin-Quan; Huang, Chi-Ming; Liu, Ching-Yi; Chiou, Arthur; Wang, Yang-Kao; Tang, Ming-Jer; Kuo, Jean-Cheng

    2015-01-01

    Cancer metastasis occurs via a progress involving abnormal cell migration. Cell migration, a dynamic physical process, is controlled by the cytoskeletal system, which includes the dynamics of actin organization and cellular adhesive organelles, focal adhesions (FAs). However, it is not known whether the organization of actin cytoskeletal system has a regulatory role in the physiologically relevant aspects of cancer metastasis. In the present studies, it was found that lung adenocarcinoma cells isolated from the secondary lung cancer of the lymph nodes, H1299 cells, show specific dynamics in terms of the actin cytoskeleton and FAs. This results in a higher level of mobility and this is regulated by an immature FA component, β-PIX (PAK-interacting exchange factor-β). In H1299 cells, β-PIX's activity was found not to be down-regulated by sequestration onto stress fibres, as the cells did not bundle actin filaments into stress fibres. Thus, β-PIX mainly remained localized at FAs, which allowed maturation of nascent adhesions into focal complexes; this resulted in actin polymerization, increased actin network integrity, changes in the intracellular microrheology at the peripheral of the cell, and cell polarity, which in turn regulated cell migration. Perturbation of β-PIX caused an inhibition of cell migration, including migration velocity, accumulated distance and directional persistence. Our results demonstrate the importance of β-PIX to the regulation of high mobility of lung adenocarcinoma cell line H1299 and that this occurs via regulation of FA dynamics, changes in actin cytoskeleton organization and cell polarity. PMID:25683605

  4. Chloride channels activated by swell can regulate the NADPH oxidase generated membrane depolarisation in activated human neutrophils

    SciTech Connect

    Ahluwalia, Jatinder

    2008-01-11

    Chloride channels activated by swell have important functions in many physiological processes. The phagocyte NADPH oxidase is essential for host defence and it generates superoxide by transferring electrons from the donor NADPH to the acceptor O{sub 2}. This electron current, induces a depolarisation of the plasma membrane. In this study, I report that chloride channels activated by swell can counteract the depolarisation induced by the NADPH oxidase. When a chloride conductance was activated by swelling, its inhibition by either 50 {mu}M NPPB or removing external chloride, depolarised the plasma membrane potential to +26 mV {+-} 3.1 (n = 4) and +40 {+-} 1 mV (n = 4), respectively. These channels were partially inhibited by the NADPH oxidase inhibitor AEBSF (1 mM) and potently inhibited by ZnCl{sub 2} (3 mM). These currents were not activated by a phosphorylation step and elevations in intracellular calcium did not appear to activate chloride currents similar to those activated by swell.

  5. Coxiella burnetii Transcriptional Analysis Reveals Serendipity Clusters of Regulation in Intracellular Bacteria

    PubMed Central

    Leroy, Quentin; Lebrigand, Kevin; Armougom, Fabrice; Barbry, Pascal; Thiéry, Richard; Raoult, Didier

    2010-01-01

    Coxiella burnetii, the causative agent of the zoonotic disease Q fever, is mainly transmitted to humans through an aerosol route. A spore-like form allows C. burnetii to resist different environmental conditions. Because of this, analysis of the survival strategies used by this bacterium to adapt to new environmental conditions is critical for our understanding of C. burnetii pathogenicity. Here, we report the early transcriptional response of C. burnetii under temperature stresses. Our data show that C. burnetii exhibited minor changes in gene regulation under short exposure to heat or cold shock. While small differences were observed, C. burnetii seemed to respond similarly to cold and heat shock. The expression profiles obtained using microarrays produced in-house were confirmed by quantitative RT-PCR. Under temperature stresses, 190 genes were differentially expressed in at least one condition, with a fold change of up to 4. Globally, the differentially expressed genes in C. burnetii were associated with bacterial division, (p)ppGpp synthesis, wall and membrane biogenesis and, especially, lipopolysaccharide and peptidoglycan synthesis. These findings could be associated with growth arrest and witnessed transformation of the bacteria to a spore-like form. Unexpectedly, clusters of neighboring genes were differentially expressed. These clusters do not belong to operons or genetic networks; they have no evident associated functions and are not under the control of the same promoters. We also found undescribed but comparable clusters of regulation in previously reported transcriptomic analyses of intracellular bacteria, including Rickettsia sp. and Listeria monocytogenes. The transcriptomic patterns of C. burnetii observed under temperature stresses permits the recognition of unpredicted clusters of regulation for which the trigger mechanism remains unidentified but which may be the result of a new mechanism of epigenetic regulation. PMID:21203564

  6. 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.

  7. Circadian Clock in a Mouse Colon Tumor Regulates Intracellular Iron Levels to Promote Tumor Progression.

    PubMed

    Okazaki, Fumiyasu; Matsunaga, Naoya; Okazaki, Hiroyuki; Azuma, Hiroki; Hamamura, Kengo; Tsuruta, Akito; Tsurudome, Yuya; Ogino, Takashi; Hara, Yukinori; Suzuki, Takuya; Hyodo, Kenji; Ishihara, Hiroshi; Kikuchi, Hiroshi; To, Hideto; Aramaki, Hironori; Koyanagi, Satoru; Ohdo, Shigehiro

    2016-03-25

    Iron is an important biological catalyst and is critical for DNA synthesis during cell proliferation. Cellular iron uptake is enhanced in tumor cells to support increased DNA synthesis. Circadian variations in DNA synthesis and proliferation have been identified in tumor cells, but their relationship with intracellular iron levels is unclear. In this study, we identified a 24-h rhythm in iron regulatory protein 2 (IRP2) levels in colon-26 tumors implanted in mice. Our findings suggest that IRP2 regulates the 24-h rhythm of transferrin receptor 1 (Tfr1) mRNA expression post-transcriptionally, by binding to RNA stem-loop structures known as iron-response elements. We also found thatIrp2mRNA transcription is promoted by circadian clock genes, including brain and muscle Arnt-like 1 (BMAL1) and the circadian locomotor output cycles kaput (CLOCK) heterodimer. Moreover, growth in colon-26(Δ19) tumors expressing the clock-mutant protein (CLOCK(Δ19)) was low compared with that in wild-type colon-26 tumor. The time-dependent variation of cellular iron levels, and the proliferation rate in wild-type colon-26 tumor was decreased by CLOCK(Δ19)expression. Our findings suggest that circadian organization contributes to tumor cell proliferation by regulating iron metabolism in the tumor.

  8. Design and engineering of intracellular-metabolite-sensing/regulation gene circuits in Saccharomyces cerevisiae.

    PubMed

    Wang, Meng; Li, Sijin; Zhao, Huimin

    2016-01-01

    The development of high-throughput phenotyping tools is lagging far behind the rapid advances of genotype generation methods. To bridge this gap, we report a new strategy for design, construction, and fine-tuning of intracellular-metabolite-sensing/regulation gene circuits by repurposing bacterial transcription factors and eukaryotic promoters. As proof of concept, we systematically investigated the design and engineering of bacterial repressor-based xylose-sensing/regulation gene circuits in Saccharomyces cerevisiae. We demonstrated that numerous properties, such as induction ratio and dose-response curve, can be fine-tuned at three different nodes, including repressor expression level, operator position, and operator sequence. By applying these gene circuits, we developed a cell sorting based, rapid and robust high-throughput screening method for xylose transporter engineering and obtained a sugar transporter HXT14 mutant with 6.5-fold improvement in xylose transportation capacity. This strategy should be generally applicable and highly useful for evolutionary engineering of proteins, pathways, and genomes in S. cerevisiae.

  9. 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

  10. 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

  11. Calmodulin regulation of TMEM16A and 16B Ca(2+)-activated chloride channels.

    PubMed

    Yang, Tingting; Colecraft, Henry M

    2016-01-01

    Ca(2+)-activated chloride channels encoded by TMEM16A and 16B are important for regulating epithelial mucus secretion, cardiac and neuronal excitability, smooth muscle contraction, olfactory transduction, and cell proliferation. Whether and how the ubiquitous Ca(2+) sensor calmodulin (CaM) regulates the activity of TMEM16A and 16B channels has been controversial and the subject of an ongoing debate. Recently, using a bioengineering approach termed ChIMP (Channel Inactivation induced by Membrane-tethering of an associated Protein) we argued that Ca(2+)-free CaM (apoCaM) is pre-associated with functioning TMEM16A and 16B channel complexes in live cells. Further, the pre-associated apoCaM mediates Ca(2+)-dependent sensitization of activation (CDSA) and Ca(2+)-dependent inactivation (CDI) of some TMEM16A splice variants. In this review, we discuss these findings in the context of previous and recent results relating to Ca(2+)-dependent regulation of TMEM16A/16B channels and the putative role of CaM. We further discuss potential future directions for these nascent ideas on apoCaM regulation of TMEM16A/16B channels, noting that such future efforts will benefit greatly from the pioneering work of Dr. David T. Yue and colleagues on CaM regulation of voltage-dependent calcium channels.

  12. Aluminum-dependent regulation of intracellular silicon in the aquatic invertebrate Lymnaea stagnalis

    PubMed Central

    Desouky, Mahmoud; Jugdaohsingh, Ravin; McCrohan, Catherine R.; White, Keith N.; Powell, Jonathan J.

    2002-01-01

    Silicon is essential for some plants, diatoms, and sponges but, in higher animals, its endogenous regulation has not been demonstrated. Silicate ions may be natural ligands for aluminum and here we show that, in the freshwater snail (Lymnaea stagnalis), intracellular silicon seems specifically up-regulated in response to sublethal aluminum exposure. X-ray microanalysis showed that exposure of snails to low levels of aluminum led to its accumulation in lysosomal granules, accompanied by marked up-regulation of silicon. Increased lysosomal levels of silicon were a specific response to aluminum because cadmium and zinc had no such effect. Furthermore, intra-lysosomal sulfur from metallothionein and other sulfur-containing ligands was increased after exposure to cadmium and zinc but not aluminum. To ensure that these findings indicated a specific in vivo response, and not ex vivo formation of hydroxy-aluminosilicates (HAS) from added aluminum (555 μg/liter) and water-borne silicon (43 μg/liter), two further studies were undertaken. In a ligand competition assay the lability of aluminum (527 μg/liter) was completely unaffected by the presence of silicon (46 μg/liter), suggesting the absence of HAS. In addition, exogenous silicon (6.5 mg/liter), added to the water column to promote formation of HAS, caused a decrease in lysosomal aluminum accumulation, showing that uptake of HAS would not explain the loading of aluminum into lysosomal granules. These findings, and arguments on the stability, lability, and kinetics of aluminum–silicate interactions, suggest that a silicon-specific mechanism exists for the in vivo detoxification of aluminum, which provides regulatory evidence of silicon in a multicellular organism. PMID:11891333

  13. Altered intracellular pH regulation in neutrophils from patients with cystic fibrosis.

    PubMed

    Coakley, R J; Taggart, C; Canny, G; Greally, P; O'Neill, S J; McElvaney, N G

    2000-07-01

    Cystic fibrosis (CF) is a condition characterized by neutrophil-mediated lung damage and bacterial colonization. The physiological basis for reported functional alterations in CF neutrophils, including increased release of neutrophil elastase, myeloperoxidase, and oxidants, is unknown. These processes are, however, regulated by intracellular pH (pH(i)). We demonstrate here that pH(i) regulation is altered in neutrophils from CF patients. Although resting pH(i) is similar, pH(i) after acid loading and activation (N-formyl-methionyl-leucyl-phenylalanine and phorbol 12-myristate 13-acetate) is more acidic in CF cells than in normal cells. Furthermore, patients with non-CF-related bronchiectasis handle acid loading and activation in a fashion similar to subjects with normal neutrophils, suggesting that chronic pulmonary inflammation alone does not explain the difference in pH(i). This is further supported by data showing that normal neutrophils exposed to the CF pulmonary milieu respond by increasing pH(i) as opposed to decreasing pH(i) as seen in activated CF neutrophils. These pH(i) differences in activated or acid-loaded CF neutrophils are abrogated by ZnCl(2) but not by amiloride and bafilomycin A(1), suggesting that passive proton conductance is abnormal in CF. In addition, DIDS, which inhibits HCO(3)(-)/Cl(-) exchange, causes alkalinization of control but not of CF neutrophils, suggesting that anion transport is also abnormal in CF neutrophils. In summary, we have shown that pH(i) regulation in CF neutrophils is intrinsically abnormal, potentially contributing to the pulmonary manifestations of the condition.

  14. Biodegradable DNA-Brush Block Copolymer Spherical Nucleic Acids Enable Transfection Agent-Free Intracellular Gene Regulation.

    PubMed

    Zhang, Chuan; Hao, Liangliang; Calabrese, Colin M; Zhou, Yu; Choi, Chung Hang J; Xing, Hang; Mirkin, Chad A

    2015-10-28

    By grafting multiple DNA strands onto one terminus of a polyester chain, a DNA-brush block copolymer that can assemble into micelle structure is constructed. These micelle spherical nucleic acids have a density of nucleic acids that is substantively higher than linear DNA block copolymer structures, which makes them effective cellular transfection and intracellular gene regulation agents.

  15. Regulation of the cell surface expression of chloride transporters during epileptogenesis.

    PubMed

    González, Marco I

    2016-08-15

    The process is commonly known as epileptogenesis refers to the cascade of molecular and cellular changes that transform the brain to make it hyperexcitable and capable of generate recurrent spontaneous seizures. Unfortunately, our understanding of the molecular changes that affect the brain during epileptogenesis remains incomplete. Recent evidence suggests that dysfunction of cation-chloride transporters (CCCs) might be one of the factors that contribute to the deficits in inhibitory neurotransmission observed during epileptogenesis. This study analyzed the cell surface expression of CCCs during epileptogenesis and during chronic epilepsy to evaluate if a loss of CCCs from the plasma membrane might contribute to hyperexcitability. Alterations in the plasma membrane expression of CCCs were mostly detected during the early phase of the epileptogenic period, suggesting that dysfunction of CCCs might contribute to the alterations in the chloride gradient previously detected. Together, the findings presented here suggest that aberrant regulation of the plasma membrane levels of CCCs might contribute to the impartment of GABAergic neurotransmission and that CCCs dysfunction might be relevant for the initial appearance of spontaneous seizures. PMID:27345384

  16. Regulation of the sodium-potassium pump in cultured rat skeletal myotubes by intracellular sodium ions

    SciTech Connect

    Brodie, C.; Sampson, S.R.

    1989-07-01

    The properties of the Na-K pump and some of the factors controlling its amount and function were studied in rat myotubes in culture. The number of Na-K pump sites was quantified by measuring the amount of (/sup 3/H)ouabain bound to whole-cell preparations. Activity of the pump was determined by measurement of ouabain-sensitive /sup 86/Rb-uptake and component of membrane potential. Chronic treatment of myotubes with tetrodotoxin (TTX), which lowers (Na)i, decreased the number of Na-K pumps, the ouabain-sensitive 86Rb uptake, and the size of the electrogenic pump component of Em. In contrast, chronic treatment with either ouabain or veratridine, which increases (Na+)i, resulted in an elevated level of Na-K pump sites. This effect was blocked by inhibitors of protein synthesis. Neither rates of degradation nor affinity of pump sites in cells treated with TTX, veratridine, or ouabain differred from those in control cells. The number and activity of Na-K pump sites were unaffected by chronic elevation in (Ca)i or chronic depolarization. We conclude that alterations in the level in intracellular Na ions play the major role in regulation of Na-K pump synthesis in cultured mammalian skeletal muscle.

  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. 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.

  19. Intracellular pH regulation by acid-base transporters in mammalian neurons

    PubMed Central

    Ruffin, Vernon A.; Salameh, Ahlam I.; Boron, Walter F.; Parker, Mark D.

    2014-01-01

    Intracellular pH (pHi) regulation in the brain is important in both physiological and physiopathological conditions because changes in pHi generally result in altered neuronal excitability. In this review, we will cover 4 major areas: (1) The effect of pHi on cellular processes in the brain, including channel activity and neuronal excitability. (2) pHi homeostasis and how it is determined by the balance between rates of acid loading (JL) and extrusion (JE). The balance between JE and JL determine steady-state pHi, as well as the ability of the cell to defend pHi in the face of extracellular acid-base disturbances (e.g., metabolic acidosis). (3) The properties and importance of members of the SLC4 and SLC9 families of acid-base transporters expressed in the brain that contribute to JL (namely the Cl-HCO3 exchanger AE3) and JE (the Na-H exchangers NHE1, NHE3, and NHE5 as well as the Na+- coupled HCO3− transporters NBCe1, NBCn1, NDCBE, and NBCn2). (4) The effect of acid-base disturbances on neuronal function and the roles of acid-base transporters in defending neuronal pHi under physiopathologic conditions. PMID:24592239

  20. Strain-specific regulation of intracellular Wolbachia density in multiply infected insects.

    PubMed

    Mouton, L; Henri, H; Bouletreau, M; Vavre, F

    2003-12-01

    Vertically transmitted symbionts suffer a severe reduction in numbers when they pass through host generations, resulting in genetic homogeneity or even clonality of their populations. Wolbachia endosymbionts that induce cytoplasmic incompatibility in their hosts depart from this rule, because cytoplasmic incompatibility actively maintains multiple infection within hosts. Hosts and symbionts are thus probably under peculiar selective pressures that must shape the way intracellular bacterial populations are regulated. We studied the density and location of Wolbachia within adult Leptopilina heterotoma, a haplodiploid wasp that is parasitic on Drosophila and that is naturally infected with three Wolbachia strains, but for which we also obtained one simply infected and two doubly infected lines. Comparison of these four lines by quantitative polymerase chain reaction using a real-time detection system showed that total Wolbachia density varies according to the infection status of individuals, while the specific density of each Wolbachia strain remains constant regardless of the presence of other strains. This suggests that Wolbachia strains do not compete with one another within the same host individual, and that a strain-specific regulatory mechanism is operating. We discuss the regulatory mechanisms that are involved, and how this process might have evolved as a response to selective pressures acting on both partners. PMID:14629360

  1. Monitoring of Intracellular Tau Aggregation Regulated by OGA/OGT Inhibitors

    PubMed Central

    Lim, Sungsu; Haque, Md. Mamunul; Nam, Ghilsoo; Ryoo, Nayeon; Rhim, Hyewhon; Kim, Yun Kyung

    2015-01-01

    Abnormal phosphorylation of tau has been considered as a key pathogenic mechanism inducing tau aggregation in multiple neurodegenerative disorders, collectively called tauopathies. Recent evidence showed that tau phosphorylation sites are protected with O-linked β-N-acetylglucosamine (O-GlcNAc) in normal brain. In pathological condition, tau is de-glycosylated and becomes a substrate for kinases. Despite the importance of O-GlcNAcylation in tau pathology, O-GlcNAc transferase (OGT), and an enzyme catalyzing O-GlcNAc to tau, has not been carefully investigated in the context of tau aggregation. Here, we investigated intracellular tau aggregation regulated by BZX2, an inhibitor of OGT. Upon the inhibition of OGT, tau phosphorylation increased 2.0-fold at Ser199 and 1.5-fold at Ser396, resulting in increased tau aggregation. Moreover, the BZX2 induced tau aggregation was efficiently reduced by the treatment of Thiamet G, an inhibitor of O-GlcNAcase (OGA). Our results demonstrated the protective role of OGT in tau aggregation and also suggest the counter-regulatory mechanism of OGA and OGT in tau pathology. PMID:26343633

  2. Apical Na+/H+ antiporter and glycolysis-dependent H+-ATPase regulate intracellular pH in the rabbit S3 proximal tubule.

    PubMed Central

    Kurtz, I

    1987-01-01

    The apical transport processes responsible for proton secretion were studied in the isolated perfused rabbit S3 proximal tubule. Intracellular pH (pHi) was measured with the pH dye, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. Steady state pHi in S3 tubules in nominally HCO3(-)-free solutions was 7.08 +/- 0.03. Removal of Na+ (lumen) caused a decrease in pHi of 0.34 +/- 0.06 pH/min. The decrease in pHi was inhibited 62% by 1 mM amiloride (lumen) and was unaffected by 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (lumen) and Cl- removal (lumen, bath). After a brief exposure to 20 mM NH4Cl, pHi fell by approximately 0.7 and recovered at a rate of 0.89 +/- 0.15 pH/min in the nominal absence of Na+, HCO3-, organic anions, and SO4(2-) (lumen, bath). 1 mM N,N'-dicyclohexylcarbodiimide (lumen), 1 mM N-ethylmaleimide (lumen), 0.5 mM colchicine (bath), and 0.5 mM iodoacetic acid (lumen, bath) inhibited the Na+-independent pHi recovery rate by 73%, 55%, 77%, and 86%, respectively, whereas 1 mM KCN (lumen, bath) did not inhibit pHi recovery. Reduction of intracellular, but not extracellular chloride, also decreased the Na+-independent pHi recovery rate. In conclusion, the S3 proximal tubule has an apical Na+/H+ antiporter with a Michaelis constant for Na+ of 29 mM and a maximum velocity of 0.47 pH/min. S3 tubules also possess a plasma membrane H+-ATPase that can regulate pHi, has a requirement for intracellular chloride, and utilizes ATP derived primarily from glycolysis. PMID:2888787

  3. Emerging role of cystic fibrosis transmembrane conductance regulator - an epithelial chloride channel in gastrointestinal cancers.

    PubMed

    Hou, Yuning; Guan, Xiaoqing; Yang, Zhe; Li, Chunying

    2016-03-15

    Cystic fibrosis transmembrane conductance regulator (CFTR), a glycoprotein with 1480 amino acids, has been well established as a chloride channel mainly expressed in the epithelial cells of various tissues and organs such as lungs, sweat glands, gastrointestinal system, and reproductive organs. Although defective CFTR leads to cystic fibrosis, a common genetic disorder in the Caucasian population, there is accumulating evidence that suggests a novel role of CFTR in various cancers, especially in gastroenterological cancers, such as pancreatic cancer and colon cancer. In this review, we summarize the emerging findings that link CFTR with various cancers, with focus on the association between CFTR defects and gastrointestinal cancers as well as the underlying mechanisms. Further study of CFTR in cancer biology may help pave a new way for the diagnosis and treatment of gastrointestinal cancers. PMID:26989463

  4. Emerging role of cystic fibrosis transmembrane conductance regulator - an epithelial chloride channel in gastrointestinal cancers

    PubMed Central

    Hou, Yuning; Guan, Xiaoqing; Yang, Zhe; Li, Chunying

    2016-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR), a glycoprotein with 1480 amino acids, has been well established as a chloride channel mainly expressed in the epithelial cells of various tissues and organs such as lungs, sweat glands, gastrointestinal system, and reproductive organs. Although defective CFTR leads to cystic fibrosis, a common genetic disorder in the Caucasian population, there is accumulating evidence that suggests a novel role of CFTR in various cancers, especially in gastroenterological cancers, such as pancreatic cancer and colon cancer. In this review, we summarize the emerging findings that link CFTR with various cancers, with focus on the association between CFTR defects and gastrointestinal cancers as well as the underlying mechanisms. Further study of CFTR in cancer biology may help pave a new way for the diagnosis and treatment of gastrointestinal cancers. PMID:26989463

  5. Intracellular calcium regulation among subpopulations of rat dorsal root ganglion neurons

    PubMed Central

    Lu, Shao-Gang; Zhang, Xiulin; Gold, Michael S

    2006-01-01

    Primary afferent neurons are functionally heterogeneous. To determine whether this functional heterogeneity reflects, in part, heterogeneity in the regulation of the concentration of intracellular Ca2+ ([Ca2+]i), the magnitude and decay of evoked Ca2+ transients were assessed in subpopulations of dorsal root ganglion (DRG) neurons with voltage clamp and fura-2 ratiometric imaging. To determine whether differences in evoked Ca2+ transients among subpopulations of DRG neurons reflected differences in the contribution of Ca2+ regulatory mechanisms, pharmacological techniques were employed to assess the contribution of influx, efflux, release and uptake pathways. Subpopulations of DRG neurons were defined by cell body size, binding of the plant lectin IB4 and responsiveness to the algogenic compound capsaicin (CAP). Ca2+ transients were evoked with 30 mm K+ or voltage steps to 0 mV. There were marked differences between subpopulations of neurons with respect to both the magnitude and decay of the Ca2+ transient, with the largest and most slowly decaying Ca2+ transients in small-diameter, IB4-positive, CAP-responsive neurons. The smallest and most rapidly decaying transients were in large-diameter, IB4-negative and CAP-unresponsive DRG neurons. These differences were not due to a differential distribution of voltage-gated Ca2+ currents. However, these differences did appear to reflect a differential contribution of other influx, efflux, release and uptake mechanisms between subpopulations of neurons. These results suggest that electrical activity in subpopulations of DRG neurons will have a differential influence on Ca2+-regulated phenomena such as spike adaptation, transmitter release and gene transcription. Significantly more activity should be required in large-diameter non-nociceptive afferents than in small-diameter nociceptive afferents to have a comparable influence on these processes. PMID:16945973

  6. Functional Impact of Ryanodine Receptor Oxidation on Intracellular Calcium Regulation in the Heart

    PubMed Central

    Mazurek, Stefan R.

    2016-01-01

    Type 2 ryanodine receptor (RyR2) serves as the major intracellular Ca2+ release channel that drives heart contraction. RyR2 is activated by cytosolic Ca2+ via the process of Ca2+-induced Ca2+ release (CICR). To ensure stability of Ca2+ dynamics, the self-reinforcing CICR must be tightly controlled. Defects in this control cause sarcoplasmic reticulum (SR) Ca2+ mishandling, which manifests in a variety of cardiac pathologies that include myocardial infarction and heart failure. These pathologies are also associated with oxidative stress. Given that RyR2 contains a large number of cysteine residues, it is no surprise that RyR2 plays a key role in the cellular response to oxidative stress. RyR’s many cysteine residues pose an experimental limitation in defining a specific target or mechanism of action for oxidative stress. As a result, the current understanding of redox-mediated RyR2 dysfunction remains incomplete. Several oxidative modifications, including S-glutathionylation and S-nitrosylation, have been suggested playing an important role in the regulation of RyR2 activity. Moreover, oxidative stress can increase RyR2 activity by forming disulfide bonds between two neighboring subunits (intersubunit cross-linking). Since intersubunit interactions within the RyR2 homotetramer complex dictate the channel gating, such posttranslational modification of RyR2 would have a significant impact on RyR2 function and Ca2+ regulation. This review summarizes recent findings on oxidative modifications of RyR2 and discusses contributions of these RyR2 modifications to SR Ca2+ mishandling during cardiac pathologies. PMID:27251471

  7. Modular TRAPP complexes regulate intracellular protein trafficking through multiple Ypt/Rab GTPases in Saccharomyces cerevisiae.

    PubMed

    Zou, Shenshen; Liu, Yutao; Zhang, Xiu Qi; Chen, Yong; Ye, Min; Zhu, Xiaoping; Yang, Shu; Lipatova, Zhanna; Liang, Yongheng; Segev, Nava

    2012-06-01

    Ypt/Rab are key regulators of intracellular trafficking in all eukaryotic cells. In yeast, Ypt1 is essential for endoplasmic reticulum (ER)-to-Golgi transport, whereas Ypt31/32 regulate Golgi-to-plasma membrane and endosome-to-Golgi transport. TRAPP is a multisubunit complex that acts as an activator of Ypt/Rab GTPases. Trs85 and Trs130 are two subunits specific for TRAPP III and TRAPP II, respectively. Whereas TRAPP III was shown to acts as a Ypt1 activator, it is still controversial whether TRAPP II acts as a Ypt1 or Ypt31/32 activator. Here, we use GFP-Snc1 as a tool to study transport in Ypt and TRAPP mutant cells. First, we show that expression of GFP-Snc1 in trs85Δ mutant cells results in temperature sensitivity. Second, we suggest that in ypt1ts and trs85Δ, but not in ypt31Δ/32ts and trs130ts mutant cells, GFP-Snc1 accumulates in the ER. Third, we show that overexpression of Ypt1, but not Ypt31/32, can suppress both the growth and GFP-Snc1 accumulation phenotypes of trs85Δ mutant cells. In contrast, overexpression of Ypt31, but not Ypt1, suppresses the growth and GFP-Snc1 transport phenotypes of trs130ts mutant cells. These results provide genetic support for functional grouping of Ypt1 with Trs85-containing TRAPP III and Ypt31/32 with Trs130-containing TRAPP II.

  8. Structural Dynamics of Soluble Chloride Intracellular Channel Protein CLIC1 Examined by Amide Hydrogen-Deuterium Exchange Mass Spectrometry (DXMS)†

    PubMed Central

    Stoychev, Stoyan H.; Nathaniel, Christos; Fanucchi, Sylvia; Brock, Melissa; Li, Sheng; Asmus, Kyle; Woods, Virgil L.; Dirr, Heini W.

    2009-01-01

    Chloride intracellular channel protein 1 (CLIC1) functions as an anion channel in plasma and nuclear membranes when its soluble monomeric form converts to an integral-membrane form. The transmembrane region of CLIC1 is located in its thioredoxin-like domain 1 but the mechanism whereby the protein converts to its membrane conformation has yet to be determined. Since channel formation in membranes is enhanced at low pH (5 to 5.5), a condition that is found at the surface of membranes, the structural dynamics of soluble CLIC1 was studied at pH 7 and at pH 5.5 in the absence of membranes by amide hydrogen-deuterium exchange mass spectrometry (DXMS). Rapid hydrogen exchange data indicate that CLIC1 displays a similar core structure at these pH values. Domain 1 is less stable than the all-helical domain 2 and, while the structure of domain 1 remains intact, its conformational flexibility is further increased in an acidic environment (pH 5.5). In the absence of membrane, an acidic environment appears to prime the solution structure of CLIC1 by destabilising domain 1 in order to lower the activation energy barrier for its conversion to the membrane-insertion conformation. The significantly enhanced H/D-exchange rates at pH 5.5 displayed by two segments (peptides 11-31 and 68-82) could be due to the protonation of acidic residues in salt bridges. One of these segments (peptide 11-31) includes part of the transmembrane region which, in the solution structure, consists of helix α1. This helix is intrinsically stable and is most likely retained in the membrane conformation. Strand β2, another element of the transmembrane region, displays a propensity to form a helical structure and has putative N- and C-capping motifs, suggesting that it too most likely forms a helix in a lipid bilayer. PMID:19650640

  9. Candida albicans erythroascorbate peroxidase regulates intracellular methylglyoxal and reactive oxygen species independently of D-erythroascorbic acid.

    PubMed

    Kwak, Min-Kyu; Song, Sung-Hyun; Ku, MyungHee; Kang, Sa-Ouk

    2015-07-01

    Candida albicans D-erythroascorbate peroxidase (EAPX1), which can catalyze the oxidation of D-erythroascorbic acid (EASC) to water, was observed to be inducible in EAPX1-deficient and EAPX1-overexpressing cells via activity staining. EAPX1-deficient cells have remarkably increased intracellular reactive oxygen species and methylglyoxal independent of the intracellular EASC content. The increased methylglyoxal caused EAPX1-deficient cells to activate catalase-peroxidase and cytochrome c peroxidase, which led to defects in cell growth, viability, mitochondrial respiration, filamentation and virulence. These findings indicate that EAPX1 mediates cell differentiation and virulence by regulating intracellular methylglyoxal along with oxidative stresses, regardless of endogenous EASC biosynthesis or alternative oxidase expression. PMID:25957768

  10. Developmental expression of chicken antithrombin III is regulated by increased RNA abundance and intracellular processing.

    PubMed

    Amrani, D L; Rosenberg, J; Samad, F; Bergtrom, G; Banfield, D K

    1993-01-23

    We isolated and sequenced a 432 bp cDNA to cAT-III, that encoded 115 nucleotides of 5' untranslated sequence, a 17 amino acid long signal peptide and residues 1-88 of the mature protein, and used it to prepare a probe for measuring and correlating the developmental changes of steady-state cAT-III mRNA levels with known changes in antigen levels. Densitometric analysis of nuclease protection (n = 2), Northern blot (n = 4), and slot blots (n = 3) of total RNA from chick livers of 16-day-old embryos to 6-day-old chicks showed a 2.6 +/- 0.5-fold increase in steady-state cAT-III mRNA levels. Assay of functional mRNA levels by in vitro translation of poly(A)+ RNA and specific immunoprecipitation of 35S-Met-labelled cAT-III was comparable to RNA analysis (16-day-old embryos vs. 10-day-old hatchlings). We evaluated whether there were developmental differences in post-translational secretion which may also contribute to the regulation of the circulating level of this protein. Pulse-chase studies of freshly-isolated hepatocytes from 16-day-old embryos and 10-day-old hatchlings maintained in suspension demonstrated a approx. 5.0-5.5-fold increase in cAT-III levels at steady-state secretion. The above findings indicate that changes in circulating cAT-III levels during late embryonic development are primarily due to increased abundance of cAT-III mRNA. In addition, we postulate that post-translational intracellular processing may account for further differences in circulating protein levels. PMID:8424948

  11. 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.

  12. Intra-ChIP: studying gene regulation in an intracellular pathogen.

    PubMed

    Hanson, Brett R; Tan, Ming

    2016-08-01

    Intracellular bacteria that reside within a host cell use a variety of strategies to exploit this unique niche. While these organisms are technically challenging to study in the context of an infected host cell, recent advances have led to an improved understanding of how the intracellular environment impacts bacterial gene expression. We recently demonstrated that chromatin immunoprecipitation (ChIP) can be used to quantify transcription factor binding in the obligate intracellular pathogen Chlamydia trachomatis within infected cells. Furthermore, we showed it was possible to experimentally modulate transcription factor binding while simultaneously measuring changes in transcription. Here we discuss these findings as well as other recent work that has used ChIP to study intracellular pathogens within infected cells. We also discuss technical considerations associated with this approach and its possible future applications.

  13. Sulfate-chloride exchange by lobster hepatopancreas is regulated by pH-sensitive modifier sites

    SciTech Connect

    Cattey, M.A.; Ahearn, G.A.; Gerencser, G.A. Univ. of Florida, Gainesville )

    1991-03-15

    {sup 35}SO{sub 4}{sup 2{minus}} uptake by Atlantic lobster (Homarus americanus) hepatopancreatic epithelial brush border membrane vesicles (BBMV) was stimulated by internal Cl{sup {minus}}, but not internal HCO{sub 3}{sup {minus}}, or external Na{sup +}. Sulfate-chloride exchange was stimulated by inside positive, and inhibited by inside negative, trans-membrane K diffusion potentials. {sup 35}SO{sub 4}{sup 2{minus}}-Cl{sup {minus}} exchange was strongly inhibited by 4,4{prime} diisothiocyanostilbene-2,2{prime}-disulfonic acid (DIDS), 4-acetamido-4{prime}-isotheocynaostilbene-2,2{prime}-disulfonic acid, (SITS), and thiosulfate. Chloride, bicarbonate, furosamide, and bumetanide slightly, yet significantly, cis-inhibited {sup 35}SO{sub 4}{sup 2{minus}}-Cl{sup {minus}} exchange. Altering bilateral pH from 8.0 to 5.4 stimulated {sup 35}SO{sub 4}{sup 2{minus}}-Cl{sup {minus}} exchange when vesicles were loaded with Cl{sup {minus}}, but reduced bilateral pH alone or the presence of pH gradients did not affect {sup 35}SO{sub 4}{sup 2{minus}} transport in the absence of internal Cl{sup {minus}}. {sup 36}Cl uptake into SO{sub 4}{sup 2{minus}}-loaded BBMV was stimulated by an internal negative membrane potential and inhibited when the interior was electrically positive. A model is proposed which suggests that SO{sub 4}{sup 2{minus}}-Cl{sup {minus}} exchange is regulated by internal and external pH-sensitive modifier sites on the anion antiporter and by coupling to the electrogenic 2 Na{sup +}/1 H{sup +} antiporter and by coupling to the electrogenic 2 Na{sup +}/1 H{sup +} antiporter on the same membrane.

  14. 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.

  15. Function and regulation of TRPM7, as well as intracellular magnesium content, are altered in cells expressing ΔF508-CFTR and G551D-CFTR.

    PubMed

    Huguet, F; Calvez, M L; Benz, N; Le Hir, S; Mignen, O; Buscaglia, P; Horgen, F D; Férec, C; Kerbiriou, M; Trouvé, P

    2016-09-01

    Cystic fibrosis (CF), one of the most common fatal hereditary disorders, is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The CFTR gene product is a multidomain adenosine triphosphate-binding cassette (ABC) protein that functions as a chloride (Cl(-)) channel that is regulated by intracellular magnesium [Mg(2+)]i. The most common mutations in CFTR are a deletion of a phenylalanine residue at position 508 (ΔF508-CFTR, 70-80 % of CF phenotypes) and a Gly551Asp substitution (G551D-CFTR, 4-5 % of alleles), which lead to decreased or almost abolished Cl(-) channel function, respectively. Magnesium ions have to be finely regulated within cells for optimal expression and function of CFTR. Therefore, the melastatin-like transient receptor potential cation channel, subfamily M, member 7 (TRPM7), which is responsible for Mg(2+) entry, was studies and [Mg(2+)]i measured in cells stably expressing wildtype CFTR, and two mutant proteins (ΔF508-CFTR and G551D-CFTR). This study shows for the first time that [Mg(2+)]i is decreased in cells expressing ΔF508-CFTR and G551D-CFTR mutated proteins. It was also observed that the expression of the TRPM7 protein is increased; however, membrane localization was altered for both ΔF508del-CFTR and G551D-CFTR. Furthermore, both the function and regulation of the TRPM7 channel regarding Mg(2+) is decreased in the cells expressing the mutated CFTR. Ca(2+) influx via TRPM7 were also modified in cells expressing a mutated CFTR. Therefore, there appears to be a direct involvement of TRPM7 in CF physiopathology. Finally, we propose that the TRPM7 activator Naltriben is a new potentiator for G551D-CFTR as the function of this mutant increases upon activation of TRPM7 by Naltriben.

  16. Regulation of biofilm formation and cellular buoyancy through modulating intracellular cyclic di-GMP levels in engineered cyanobacteria.

    PubMed

    Agostoni, Marco; Waters, Christopher M; Montgomery, Beronda L

    2016-02-01

    The second messenger cyclic dimeric (3'→5') GMP (cyclic di-GMP or c-di-GMP) has been implicated in the transition between motile and sessile lifestyles in bacteria. In this study, we demonstrate that biofilm formation, cellular aggregation or flocculation, and cellular buoyancy are under the control of c-di-GMP in Synechocystis sp. PCC 6803 (Synechocystis) and Fremyella diplosiphon. Synechocystis is a unicellular cyanobacterium and displays lower levels of c-di-GMP; F. diplosiphon is filamentous and displays higher intracellular c-di-GMP levels. We transformed Synechocystis and F. diplosiphon with a plasmid for constitutive expression of genes encoding diguanylate cylase (DGC) and phosphodiesterase (PDE) proteins from Vibrio cholerae or Escherichia coli, respectively. These engineered strains allowed us to modulate intracellular c-di-GMP levels. Biofilm formation and cellular deposition were induced in the DGC-expressing Synechocystis strain which exhibited high intracellular levels of c-di-GMP; whereas strains expressing PDE in Synechocystis and F. diplosiphon to drive low intracellular levels of c-di-GMP exhibited enhanced cellular buoyancy. In addition, the PDE-expressing F. diplosiphon strain showed elevated chlorophyll levels. These results imply roles for coordinating c-di-GMP homeostasis in regulating native cyanobacterial phenotypes. Engineering exogenous DGC or PDE proteins to regulate intracellular c-di-GMP levels represents an effective tool for uncovering cryptic phenotypes or modulating phenotypes in cyanobacteria for practical applications in biotechnology applicable in photobioreactors and in green biotechnologies, such as energy-efficient harvesting of cellular biomass or the treatment of metal-containing wastewaters.

  17. 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

  18. Development and regulation of chloride homeostasis in the central nervous system.

    PubMed

    Watanabe, Miho; Fukuda, Atsuo

    2015-01-01

    γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter of the mature central nervous system (CNS). The developmental switch of GABAergic transmission from excitation to inhibition is induced by changes in Cl(-) gradients, which are generated by cation-Cl(-) co-transporters. An accumulation of Cl(-) by the Na(+)-K(+)-2Cl(-) co-transporter (NKCC1) increases the intracellular Cl(-) concentration ([Cl(-)]i) such that GABA depolarizes neuronal precursors and immature neurons. The subsequent ontogenetic switch, i.e., upregulation of the Cl(-)-extruder KCC2, which is a neuron-specific K(+)-Cl(-) co-transporter, with or without downregulation of NKCC1, results in low [Cl(-)]i levels and the hyperpolarizing action of GABA in mature neurons. Development of Cl(-) homeostasis depends on developmental changes in NKCC1 and KCC2 expression. Generally, developmental shifts (decreases) in [Cl(-)]i parallel the maturation of the nervous system, e.g., early in the spinal cord, hypothalamus and thalamus, followed by the limbic system, and last in the neocortex. There are several regulators of KCC2 and/or NKCC1 expression, including brain-derived neurotrophic factor (BDNF), insulin-like growth factor (IGF), and cystic fibrosis transmembrane conductance regulator (CFTR). Therefore, regionally different expression of these regulators may also contribute to the regional developmental shifts of Cl(-) homeostasis. KCC2 and NKCC1 functions are also regulated by phosphorylation by enzymes such as PKC, Src-family tyrosine kinases, and WNK1-4 and their downstream effectors STE20/SPS1-related proline/alanine-rich kinase (SPAK)-oxidative stress responsive kinase-1 (OSR1). In addition, activation of these kinases is modulated by humoral factors such as estrogen and taurine. Because these transporters use the electrochemical driving force of Na(+) and K(+) ions, topographical interaction with the Na(+)-K(+) ATPase and its modulators such as creatine kinase (CK) should modulate

  19. Development and regulation of chloride homeostasis in the central nervous system

    PubMed Central

    Watanabe, Miho; Fukuda, Atsuo

    2015-01-01

    γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter of the mature central nervous system (CNS). The developmental switch of GABAergic transmission from excitation to inhibition is induced by changes in Cl− gradients, which are generated by cation-Cl− co-transporters. An accumulation of Cl− by the Na+-K+-2Cl− co-transporter (NKCC1) increases the intracellular Cl− concentration ([Cl−]i) such that GABA depolarizes neuronal precursors and immature neurons. The subsequent ontogenetic switch, i.e., upregulation of the Cl−-extruder KCC2, which is a neuron-specific K+-Cl− co-transporter, with or without downregulation of NKCC1, results in low [Cl−]i levels and the hyperpolarizing action of GABA in mature neurons. Development of Cl− homeostasis depends on developmental changes in NKCC1 and KCC2 expression. Generally, developmental shifts (decreases) in [Cl−]i parallel the maturation of the nervous system, e.g., early in the spinal cord, hypothalamus and thalamus, followed by the limbic system, and last in the neocortex. There are several regulators of KCC2 and/or NKCC1 expression, including brain-derived neurotrophic factor (BDNF), insulin-like growth factor (IGF), and cystic fibrosis transmembrane conductance regulator (CFTR). Therefore, regionally different expression of these regulators may also contribute to the regional developmental shifts of Cl− homeostasis. KCC2 and NKCC1 functions are also regulated by phosphorylation by enzymes such as PKC, Src-family tyrosine kinases, and WNK1–4 and their downstream effectors STE20/SPS1-related proline/alanine-rich kinase (SPAK)-oxidative stress responsive kinase-1 (OSR1). In addition, activation of these kinases is modulated by humoral factors such as estrogen and taurine. Because these transporters use the electrochemical driving force of Na+ and K+ ions, topographical interaction with the Na+-K+ ATPase and its modulators such as creatine kinase (CK) should modulate functions of Cl

  20. Chloride current in mammalian cardiac myocytes. Novel mechanism for autonomic regulation of action potential duration and resting membrane potential

    PubMed Central

    1990-01-01

    The properties of the autonomically regulated chloride current (ICl) were studied in isolated guinea pig ventricular myocytes. This current was elicited upon exposure to isoproterenol (ISO) and reversed upon concurrent exposure to acetylcholine (ACh). ICl was time independent and exhibited outward rectification. The responses to ISO and ACh could be blocked by propranolol and atropine, respectively, and ICl was also elicited by forskolin, 8-bromoadenosine 3',5'-cyclic monophosphate, and 3-isobutyl-l-methylxanthine, indicating that the current is regulated through a cAMP-dependent pathway. The reversal potential of the ISO- induced current followed the predicted chloride equilibrium potential, consistent with it being carried predominantly by Cl-. Activation of ICl produced changes in the resting membrane potential and action potential duration, which were Cl- gradient dependent. These results indicate that under physiological conditions ICl may play an important role in regulating action potential duration and resting membrane potential in mammalian cardiac myocytes. PMID:2165130

  1. Vitamin C controls the cystic fibrosis transmembrane conductance regulator chloride channel.

    PubMed

    Fischer, Horst; Schwarzer, Christian; Illek, Beate

    2004-03-01

    Vitamin C (l-ascorbate) is present in the respiratory lining fluid of human lungs, and local deficits occur during oxidative stress. Here we report a unique function of vitamin C on the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), a cAMP-dependent Cl channel that regulates epithelial surface fluid secretion. Vitamin C (100 microM) induced the openings of CFTR Cl channels by increasing its average open probability from 0 to 0.21 +/- 0.08, without a detectable increase in intracellular cAMP levels. Exposure of the apical airway surface to vitamin C stimulated the transepithelial Cl secretion to 68% of forskolin-stimulated currents. The average half-maximal stimulatory constant was 36.5 +/- 2.9 microM, which corresponds to physiological concentrations. When vitamin C was instilled into the nasal epithelium of human subjects, it effectively activated Cl transport in vivo. In CF epithelia, previous treatment of the underlying trafficking defect with trimethylamine oxide or expression of WT CFTR restored the activation of Cl transport by vitamin C. Sodium dependency and phloretin sensitivity, as well as the expression of transcripts for sodium-dependent vitamin C transporter (SVCT)-1 and SVCT2, support a model in which an apical vitamin C transporter is central for relaying the effect of vitamin C to CFTR. We conclude that cellular vitamin C is a biological regulator of CFTR-mediated Cl secretion in epithelia. The pool of vitamin C in the respiratory tract represents a potential nutraceutical and pharmaceutical target for the complementary treatment of sticky airway secretions by enhancing epithelial fluid secretion.

  2. Chloride regulates leaf cell size and water relations in tobacco plants.

    PubMed

    Franco-Navarro, Juan D; Brumós, Javier; Rosales, Miguel A; Cubero-Font, Paloma; Talón, Manuel; Colmenero-Flores, José M

    2016-02-01

    Chloride (Cl(-)) is a micronutrient that accumulates to macronutrient levels since it is normally available in nature and actively taken up by higher plants. Besides a role as an unspecific cell osmoticum, no clear biological roles have been explicitly associated with Cl(-) when accumulated to macronutrient concentrations. To address this question, the glycophyte tobacco (Nicotiana tabacum L. var. Habana) has been treated with a basal nutrient solution supplemented with one of three salt combinations containing the same cationic balance: Cl(-)-based (CL), nitrate-based (N), and sulphate+phosphate-based (SP) treatments. Under non-saline conditions (up to 5 mM Cl(-)) and no water limitation, Cl(-) specifically stimulated higher leaf cell size and led to a moderate increase of plant fresh and dry biomass mainly due to higher shoot expansion. When applied in the 1-5 mM range, Cl(-) played specific roles in regulating leaf osmotic potential and turgor, allowing plants to improve leaf water balance parameters. In addition, Cl(-) also altered water relations at the whole-plant level through reduction of plant transpiration. This was a consequence of a lower stomatal conductance, which resulted in lower water loss and greater photosynthetic and integrated water-use efficiency. In contrast to Cl(-), these effects were not observed for essential anionic macronutrients such as nitrate, sulphate, and phosphate. We propose that the abundant uptake and accumulation of Cl(-) responds to adaptive functions improving water homeostasis in higher plants.

  3. Chloride regulates leaf cell size and water relations in tobacco plants

    PubMed Central

    Franco-Navarro, Juan D.; Brumós, Javier; Rosales, Miguel A.; Cubero-Font, Paloma; Talón, Manuel; Colmenero-Flores, José M.

    2016-01-01

    Chloride (Cl–) is a micronutrient that accumulates to macronutrient levels since it is normally available in nature and actively taken up by higher plants. Besides a role as an unspecific cell osmoticum, no clear biological roles have been explicitly associated with Cl– when accumulated to macronutrient concentrations. To address this question, the glycophyte tobacco (Nicotiana tabacum L. var. Habana) has been treated with a basal nutrient solution supplemented with one of three salt combinations containing the same cationic balance: Cl–-based (CL), nitrate-based (N), and sulphate+phosphate-based (SP) treatments. Under non-saline conditions (up to 5mM Cl–) and no water limitation, Cl– specifically stimulated higher leaf cell size and led to a moderate increase of plant fresh and dry biomass mainly due to higher shoot expansion. When applied in the 1–5mM range, Cl– played specific roles in regulating leaf osmotic potential and turgor, allowing plants to improve leaf water balance parameters. In addition, Cl– also altered water relations at the whole-plant level through reduction of plant transpiration. This was a consequence of a lower stomatal conductance, which resulted in lower water loss and greater photosynthetic and integrated water-use efficiency. In contrast to Cl–, these effects were not observed for essential anionic macronutrients such as nitrate, sulphate, and phosphate. We propose that the abundant uptake and accumulation of Cl– responds to adaptive functions improving water homeostasis in higher plants. PMID:26602947

  4. Reactive Neurogenesis and Down-Regulation of the Potassium-Chloride Cotransporter KCC2 in the Cochlear Nuclei after Cochlear Deafferentation.

    PubMed

    Tighilet, Brahim; Dutheil, Sophie; Siponen, Marina I; Noreña, Arnaud J

    2016-01-01

    While many studies have been devoted to investigating the homeostatic plasticity triggered by cochlear hearing loss, the cellular and molecular mechanisms involved in these central changes remain elusive. In the present study, we investigated the possibility of reactive neurogenesis after unilateral cochlear nerve section in the cochlear nucleus (CN) of cats. We found a strong cell proliferation in all the CN sub-divisions ipsilateral to the lesion. Most of the newly generated cells survive up to 1 month after cochlear deafferentation in all cochlear nuclei (except the dorsal CN) and give rise to a variety of cell types, i.e., microglial cells, astrocytes, and neurons. Interestingly, many of the newborn neurons had an inhibitory (GABAergic) phenotype. This result is intriguing since sensory deafferentation is usually accompanied by enhanced excitation, consistent with a reduction in central inhibition. The membrane potential effect of GABA depends, however, on the intra-cellular chloride concentration, which is maintained at low levels in adults by the potassium chloride co-transporter KCC2. The KCC2 density on the plasma membrane of neurons was then assessed after cochlear deafferentation in the cochlear nuclei ipsilateral and contralateral to the lesion. Cochlear deafferentation is accompanied by a strong down-regulation of KCC2 ipsilateral to the lesion at 3 and 30 days post-lesion. This study suggests that reactive neurogenesis and down-regulation of KCC2 is part of the vast repertoire involved in homeostatic plasticity triggered by hearing loss. These central changes may also play a role in the generation of tinnitus and hyperacusis. PMID:27630564

  5. Reactive Neurogenesis and Down-Regulation of the Potassium-Chloride Cotransporter KCC2 in the Cochlear Nuclei after Cochlear Deafferentation

    PubMed Central

    Tighilet, Brahim; Dutheil, Sophie; Siponen, Marina I.; Noreña, Arnaud J.

    2016-01-01

    While many studies have been devoted to investigating the homeostatic plasticity triggered by cochlear hearing loss, the cellular and molecular mechanisms involved in these central changes remain elusive. In the present study, we investigated the possibility of reactive neurogenesis after unilateral cochlear nerve section in the cochlear nucleus (CN) of cats. We found a strong cell proliferation in all the CN sub-divisions ipsilateral to the lesion. Most of the newly generated cells survive up to 1 month after cochlear deafferentation in all cochlear nuclei (except the dorsal CN) and give rise to a variety of cell types, i.e., microglial cells, astrocytes, and neurons. Interestingly, many of the newborn neurons had an inhibitory (GABAergic) phenotype. This result is intriguing since sensory deafferentation is usually accompanied by enhanced excitation, consistent with a reduction in central inhibition. The membrane potential effect of GABA depends, however, on the intra-cellular chloride concentration, which is maintained at low levels in adults by the potassium chloride co-transporter KCC2. The KCC2 density on the plasma membrane of neurons was then assessed after cochlear deafferentation in the cochlear nuclei ipsilateral and contralateral to the lesion. Cochlear deafferentation is accompanied by a strong down-regulation of KCC2 ipsilateral to the lesion at 3 and 30 days post-lesion. This study suggests that reactive neurogenesis and down-regulation of KCC2 is part of the vast repertoire involved in homeostatic plasticity triggered by hearing loss. These central changes may also play a role in the generation of tinnitus and hyperacusis. PMID:27630564

  6. Reactive Neurogenesis and Down-Regulation of the Potassium-Chloride Cotransporter KCC2 in the Cochlear Nuclei after Cochlear Deafferentation

    PubMed Central

    Tighilet, Brahim; Dutheil, Sophie; Siponen, Marina I.; Noreña, Arnaud J.

    2016-01-01

    While many studies have been devoted to investigating the homeostatic plasticity triggered by cochlear hearing loss, the cellular and molecular mechanisms involved in these central changes remain elusive. In the present study, we investigated the possibility of reactive neurogenesis after unilateral cochlear nerve section in the cochlear nucleus (CN) of cats. We found a strong cell proliferation in all the CN sub-divisions ipsilateral to the lesion. Most of the newly generated cells survive up to 1 month after cochlear deafferentation in all cochlear nuclei (except the dorsal CN) and give rise to a variety of cell types, i.e., microglial cells, astrocytes, and neurons. Interestingly, many of the newborn neurons had an inhibitory (GABAergic) phenotype. This result is intriguing since sensory deafferentation is usually accompanied by enhanced excitation, consistent with a reduction in central inhibition. The membrane potential effect of GABA depends, however, on the intra-cellular chloride concentration, which is maintained at low levels in adults by the potassium chloride co-transporter KCC2. The KCC2 density on the plasma membrane of neurons was then assessed after cochlear deafferentation in the cochlear nuclei ipsilateral and contralateral to the lesion. Cochlear deafferentation is accompanied by a strong down-regulation of KCC2 ipsilateral to the lesion at 3 and 30 days post-lesion. This study suggests that reactive neurogenesis and down-regulation of KCC2 is part of the vast repertoire involved in homeostatic plasticity triggered by hearing loss. These central changes may also play a role in the generation of tinnitus and hyperacusis.

  7. DNA Damage–Induced Bcl-xL Deamidation Is Mediated by NHE-1 Antiport Regulated Intracellular pH

    PubMed Central

    Zhao, Rui; Oxley, David; Smith, Trevor S; Follows, George A; Green, Anthony R; Alexander, Denis R

    2007-01-01

    The pro-survival protein Bcl-xL is critical for the resistance of tumour cells to DNA damage. We have previously demonstrated, using a mouse cancer model, that oncogenic tyrosine kinase inhibition of DNA damage–induced Bcl-xL deamidation tightly correlates with T cell transformation in vivo, although the pathway to Bcl-xL deamidation remains unknown and its functional consequences unclear. We show here that rBcl-xL deamidation generates an iso-Asp52/iso-Asp66 species that is unable to sequester pro-apoptotic BH3-only proteins such as Bim and Puma. DNA damage in thymocytes results in increased expression of the NHE-1 Na/H antiport, an event both necessary and sufficient for subsequent intracellular alkalinisation, Bcl-xL deamidation, and apoptosis. In murine thymocytes and tumour cells expressing an oncogenic tyrosine kinase, this DNA damage–induced cascade is blocked. Enforced intracellular alkalinisation mimics the effects of DNA damage in murine tumour cells and human B-lineage chronic lymphocytic leukaemia cells, thereby causing Bcl-xL deamidation and increased apoptosis. Our results define a signalling pathway leading from DNA damage to up-regulation of the NHE-1 antiport, to intracellular alkalanisation to Bcl-xL deamidation, to apoptosis, representing the first example, to our knowledge, of how deamidation of internal asparagine residues can be regulated in a protein in vivo. Our findings also suggest novel approaches to cancer therapy. PMID:17177603

  8. The C-terminal tail of tetraspanin proteins regulates their intracellular distribution in the parasite Trichomonas vaginalis.

    PubMed

    Coceres, V M; Alonso, A M; Nievas, Y R; Midlej, V; Frontera, L; Benchimol, M; Johnson, P J; de Miguel, N

    2015-08-01

    The parasite Trichomonas vaginalis is the causative agent of trichomoniasis, a prevalent sexually transmitted infection. Here, we report the cellular analysis of T.vaginalis tetraspanin family (TvTSPs). This family of membrane proteins has been implicated in cell adhesion, migration and proliferation in vertebrates. We found that the expression of several members of the family is up-regulated upon contact with vaginal ectocervical cells. We demonstrate that most TvTSPs are localized on the surface and intracellular vesicles and that the C-terminal intracellular tails of surface TvTSPs are necessary for proper localization. Analyses of full-length TvTSP8 and a mutant that lacks the C-terminal tail indicates that surface-localized TvTSP8 is involved in parasite aggregation, suggesting a role for this protein in parasite : parasite interaction.

  9. The C-terminal tail of tetraspanin proteins regulates their intracellular distribution in the parasite Trichomonas vaginalis.

    PubMed

    Coceres, V M; Alonso, A M; Nievas, Y R; Midlej, V; Frontera, L; Benchimol, M; Johnson, P J; de Miguel, N

    2015-08-01

    The parasite Trichomonas vaginalis is the causative agent of trichomoniasis, a prevalent sexually transmitted infection. Here, we report the cellular analysis of T.vaginalis tetraspanin family (TvTSPs). This family of membrane proteins has been implicated in cell adhesion, migration and proliferation in vertebrates. We found that the expression of several members of the family is up-regulated upon contact with vaginal ectocervical cells. We demonstrate that most TvTSPs are localized on the surface and intracellular vesicles and that the C-terminal intracellular tails of surface TvTSPs are necessary for proper localization. Analyses of full-length TvTSP8 and a mutant that lacks the C-terminal tail indicates that surface-localized TvTSP8 is involved in parasite aggregation, suggesting a role for this protein in parasite : parasite interaction. PMID:25703821

  10. 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

  11. A pH-independent DNA nanodevice for quantifying chloride transport in organelles of living cells

    NASA Astrophysics Data System (ADS)

    Saha, Sonali; Prakash, Ved; Halder, Saheli; Chakraborty, Kasturi; Krishnan, Yamuna

    2015-07-01

    The concentration of chloride ions in the cytoplasm and subcellular organelles of living cells spans a wide range (5-130 mM), and is tightly regulated by intracellular chloride channels or transporters. Chloride-sensitive protein reporters have been used to study the role of these chloride regulators, but they are limited to a small range of chloride concentrations and are pH-sensitive. Here, we show that a DNA nanodevice can precisely measure the activity and location of subcellular chloride channels and transporters in living cells in a pH-independent manner. The DNA nanodevice, called Clensor, is composed of sensing, normalizing and targeting modules, and is designed to localize within organelles along the endolysosomal pathway. It allows fluorescent, ratiometric sensing of chloride ions across the entire physiological regime. We used Clensor to quantitate the resting chloride concentration in the lumen of acidic organelles in Drosophila melanogaster. We showed that lumenal lysosomal chloride, which is implicated in various lysosomal storage diseases, is regulated by the intracellular chloride transporter DmClC-b.

  12. 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

  13. Modulating intracellular acidification by regulating the incubation time of proton caged compounds.

    PubMed

    Carbone, Marilena; Sabbatella, Gianfranco; Antonaroli, Simonetta; Orlando, Viviana; Biagioni, Stefano; Nucara, Alessandro

    2016-09-01

    A proton caged compound, the 1-(2-nitrophenyl)- ethylhexadecyl sulfonate (HDNS), was dosed into HEK-293 at different incubation times. Samples were irradiated with filtered UV light for inducing photolysis of the HDNS and then probed by infrared spectroscopy. The intracellular acidification reaction can be followed by monitoring the consequent CO2 peak intensity variation. The total CO2 produced is similar for all the samples, hence it is only a function of the initial HDNS concentration. The way it is achieved, though, is different for the different incubation times and follows kinetics, which results in a combination of a linear CO2 increase and a steep CO2 increase followed by a decay. This is interpreted in terms of confinement of the HDNS into intracellular vesicles of variable average size and sensitive to UV light when they reach critical dimensions. PMID:27017356

  14. Subcellular Dynamics of Multifunctional Protein Regulation: Mechanisms of GAPDH Intracellular Translocation

    PubMed Central

    Sirover, Michael A.

    2012-01-01

    Multidimensional proteins such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) exhibit distinct activities unrelated to their originally identified functions. Apart from glycolysis, GAPDH participates in iron metabolism, membrane trafficking, histone biosynthesis, the maintenance of DNA integrity and receptor mediated cell signaling. Further, multifunctional proteins exhibit distinct changes in their subcellular localization reflecting their new activities. As such, GAPDH is not only a cytosolic protein but is localized in the membrane, the nucleus, polysomes, the ER and the Golgi. In addition, although the initial subcellular localizations of multifunctional proteins may be of significance, dynamic changes in intracellular distribution may occur as a consequence of those new activities. As such, regulatory mechanisms may exist through which cells control multifunctional protein expression as a function of their subcellular localization. The temporal sequence through which subcellular translocation and the acquisition of new GAPDH functions is considered as well as post-translational modification as a basis for its intracellular transport. PMID:22388977

  15. Modulating intracellular acidification by regulating the incubation time of proton caged compounds.

    PubMed

    Carbone, Marilena; Sabbatella, Gianfranco; Antonaroli, Simonetta; Orlando, Viviana; Biagioni, Stefano; Nucara, Alessandro

    2016-09-01

    A proton caged compound, the 1-(2-nitrophenyl)- ethylhexadecyl sulfonate (HDNS), was dosed into HEK-293 at different incubation times. Samples were irradiated with filtered UV light for inducing photolysis of the HDNS and then probed by infrared spectroscopy. The intracellular acidification reaction can be followed by monitoring the consequent CO2 peak intensity variation. The total CO2 produced is similar for all the samples, hence it is only a function of the initial HDNS concentration. The way it is achieved, though, is different for the different incubation times and follows kinetics, which results in a combination of a linear CO2 increase and a steep CO2 increase followed by a decay. This is interpreted in terms of confinement of the HDNS into intracellular vesicles of variable average size and sensitive to UV light when they reach critical dimensions.

  16. Agouti regulation of intracellular calcium: Role in the insulin resistance of viable yellow mice

    SciTech Connect

    Zemel, M.B.; Kim, J.H.; Woychik, R.P.; Michaud, E.J.; Hadwell, S.H.; Patel, I.R.; Wilkison, W.O.

    1995-05-23

    Several dominant mutations at the agouti locus in the mouse cause a syndrome of marked obesity, hyperinsulinemia, and insulin resistance. Although it is known that the agouti gene is expressed in an ectopic manner in these mutants, the precise mechanism by which the agouti gene product mediates these effects is unclear. Since intracellular Ca{sup 2+} is believed to play a role in mediating insulin action and dysregulation of Ca{sup 2+} flux is observed in diabetic animals and humans, we examined the status of intracellular Ca{sup 2+} in mice carrying the dominant agouti allele, viable yellow (A{sup vy}). We show here that in mice carrying this mutation, the intracellular free calcium concentration ([Ca{sup 2+}]{sub i}) is elevated in skeletal muscle, and the degree of elevation is closely correlated with the degree to which the mutant traits are expressed in individual animals. Moreover, we demonstrate that the agouti gene product is capable of inducing increased [Ca{sup 2+}]{sub i} in cultured and freshly isolated skeletal muscle myocytes from wild-type mice. Based on these findings, we present a model in which we propose that the agouti polypeptide promotes insulin resistance in mutant animals through its ability to increase [Ca{sup 2+}]{sub i}. 36 refs., 3 figs., 2 tabs.

  17. Controlled intracellular proteolysis during postpartal involution of the uterus: characterization and regulation of an alkaline proteinase.

    PubMed

    Roth, M; Hoechst, M; Afting, E G

    1981-01-01

    The postpartal involution of the uterus is predominantly due to cellular hypotrophy. This implies an intracellular proteolytic system which must be carefully controlled pre and post partum. We have characterized and partially purified a proteinase with an alkaline pH-optimum of activity and a proteinase inhibitor protein which inhibits this proteinase very strongly. The alkaline proteinase copurifies with the actomyosin complex of the uterine myometrium and degrades the actomyosin complex with a concomitant loss of its myosin-ATPase activity. The alkaline proteinase is a very labile enzyme, markedly sensitive to SH-group modifying agents and has very high molecular weight at the present state of purification. This proteolytic enzyme could specifically be separated from the main components of the actomyosin complex by extraction with low ionic strength phosphate buffers. The proteinase inhibitor protein may control the activity of this alkaline proteinase during pregnancy and involution. The inhibitor protein raises 15-fold during pregnancy, possibly blocks important steps of intracellular proteolysis and permits organ growth. The dramatic fall of the inhibitor protein activity after parturition, which precedes the loss of weight, could release the proteolytic system, including the alkaline proteinase, and permits controlled intracellular degradation.

  18. Burkholderia pseudomallei Differentially Regulates Host Innate Immune Response Genes for Intracellular Survival in Lung Epithelial Cells

    PubMed Central

    Vellasamy, Kumutha Malar; Mariappan, Vanitha; Shankar, Esaki M.; Vadivelu, Jamuna

    2016-01-01

    Background Burkholderia pseudomallei, the causative agent of melioidosis poses a serious threat to humankind. B. pseudomallei secretes numerous virulence proteins that alter host cell functions to escape from intracellular immune sensors. However, the events underlying disease pathogenesis are poorly understood. Methods We determined the ability of B. pseudomallei to invade and survive intracellularly in A549 human lung epithelial cells, and also investigated the early transcriptional responses using an Illumina HumanHT-12 v4 microarray platform, after three hours of exposure to live B. pseudomallei (BCMS) and its secreted proteins (CCMS). Results We found that the ability of B. pseudomallei to invade and survive intracellularly correlated with increase of multiplicity of infection and duration of contact. Activation of host carbohydrate metabolism and apoptosis as well as suppression of amino acid metabolism and innate immune responses both by live bacteria and its secreted proteins were evident. These early events might be linked to initial activation of host genes directed towards bacterial dissemination from lungs to target organs (via proposed in vivo mechanisms) or to escape potential sensing by macrophages. Conclusion Understanding the early responses of A549 cells toward B. pseudomallei infection provide preliminary insights into the likely pathogenesis mechanisms underlying melioidosis, and could contribute to development of novel intervention strategies to combat B. pseudomallei infections. PMID:27367858

  19. Stabilization of the Serum Lithium Concentration by Regulation of Sodium Chloride Intake: Case Report.

    PubMed

    Tomita, Takashi; Goto, Hidekazu; Sumiya, Kenji; Yoshida, Tadashi; Tanaka, Katsuya; Kohda, Yukinao

    2016-01-01

    To avoid fluctuation of the serum lithium concentration (CLi), sodium chloride (NaCl) intake was regulated in oral alimentation. A 62-year-old woman was hospitalized and orally administered 400 mg of lithium carbonate a day to treat her mania. Her CLi was found to be 0.75-0.81 mEq/L. Vomiting made it difficult for the patient to ingest meals orally, and therefore parenteral nutrition with additional oral intake of protein-fortified food was initiated. On day 22, parenteral nutrition was switched to oral alimentation to enable oral intake of food. The total NaCl equivalent amount was decreased to 1.2 g/d, and the CLi increased to 1.15 mEq/L on day 26. Oral alimentation with semi-solid food blended in a mixer was immediately initiated. Although the total NaCl equivalent amount was increased to 4.5-5.0 g/d, her CLi remained high at 1.14-1.17 mEq/L on days 33 and 49, respectively. We investigated oral administration of NaCl (1.8 g/d) on day 52. The total NaCl equivalent amount was increased to 6.3-6.8 g/d, and the CLi decreased to 1.08-0.97 mEq/L on days 63 and 104, respectively. After the start of the orally administered NaCl, her diet was changed to a completely blended diet on day 125. The total NaCl equivalent amount was increased to 9.0-14.5 g/d, and the CLi decreased to 0.53 mEq/L on day 152; therefore, the oral administration of NaCl was discontinued on day 166. The CLi was found to be 0.70-0.85 mEq/L on days 176 and 220.

  20. Stabilization of the Serum Lithium Concentration by Regulation of Sodium Chloride Intake: Case Report.

    PubMed

    Tomita, Takashi; Goto, Hidekazu; Sumiya, Kenji; Yoshida, Tadashi; Tanaka, Katsuya; Kohda, Yukinao

    2016-01-01

    To avoid fluctuation of the serum lithium concentration (CLi), sodium chloride (NaCl) intake was regulated in oral alimentation. A 62-year-old woman was hospitalized and orally administered 400 mg of lithium carbonate a day to treat her mania. Her CLi was found to be 0.75-0.81 mEq/L. Vomiting made it difficult for the patient to ingest meals orally, and therefore parenteral nutrition with additional oral intake of protein-fortified food was initiated. On day 22, parenteral nutrition was switched to oral alimentation to enable oral intake of food. The total NaCl equivalent amount was decreased to 1.2 g/d, and the CLi increased to 1.15 mEq/L on day 26. Oral alimentation with semi-solid food blended in a mixer was immediately initiated. Although the total NaCl equivalent amount was increased to 4.5-5.0 g/d, her CLi remained high at 1.14-1.17 mEq/L on days 33 and 49, respectively. We investigated oral administration of NaCl (1.8 g/d) on day 52. The total NaCl equivalent amount was increased to 6.3-6.8 g/d, and the CLi decreased to 1.08-0.97 mEq/L on days 63 and 104, respectively. After the start of the orally administered NaCl, her diet was changed to a completely blended diet on day 125. The total NaCl equivalent amount was increased to 9.0-14.5 g/d, and the CLi decreased to 0.53 mEq/L on day 152; therefore, the oral administration of NaCl was discontinued on day 166. The CLi was found to be 0.70-0.85 mEq/L on days 176 and 220. PMID:26935095

  1. Changes in intracellular copper concentration and copper-regulating gene expression after PC12 differentiation into neurons.

    PubMed

    Ogra, Yasumitsu; Tejima, Aya; Hatakeyama, Naohiro; Shiraiwa, Moeko; Wu, Siyuan; Ishikawa, Tsutomu; Yawata, Ayako; Anan, Yasumi; Suzuki, Noriyuki

    2016-01-01

    It is suspected that some neurodegenerative diseases are a result of the disturbance of copper (Cu) homeostasis, although it remains unclear whether the disturbance of Cu homeostasis has aberrant effects on neurons. Herein, we investigated Cu metabolism specifically in neurons in terms of changes in the intracellular Cu concentration and the expression of Cu-regulating genes, such as Cu transporters and metallothioneins (MTs), before and after the differentiation of rat pheochromocytoma cells (PC12 cells) into neurons. After the differentiation, Cu and Zn imaging with fluorescent probes revealed an increase in intracellular Cu concentration. The concentrations of other essential metals, which were determined by an inductively coupled plasma mass spectrometer, were not altered. The mRNA expression of the Cu influx transporter, Ctr1, was decreased after the differentiation, and the differentiated cells acquired tolerance to Cu and cisplatin, another substrate of Ctr1. In addition, the expression of MT-3, a brain-specific isoform, was increased, contrary to the decreased expression of MT-1 and MT-2. Taken together, the differentiation of PC12 cells into neurons induced MT-3 expression, thereby resulting in intracellular Cu accumulation. The decrease in Ctr1 expression was assumed to be a response aimed at abolishing the physiological accumulation of Cu after the differentiation. PMID:27623342

  2. Changes in intracellular copper concentration and copper-regulating gene expression after PC12 differentiation into neurons

    PubMed Central

    Ogra, Yasumitsu; Tejima, Aya; Hatakeyama, Naohiro; Shiraiwa, Moeko; Wu, Siyuan; Ishikawa, Tsutomu; Yawata, Ayako; Anan, Yasumi; Suzuki, Noriyuki

    2016-01-01

    It is suspected that some neurodegenerative diseases are a result of the disturbance of copper (Cu) homeostasis, although it remains unclear whether the disturbance of Cu homeostasis has aberrant effects on neurons. Herein, we investigated Cu metabolism specifically in neurons in terms of changes in the intracellular Cu concentration and the expression of Cu-regulating genes, such as Cu transporters and metallothioneins (MTs), before and after the differentiation of rat pheochromocytoma cells (PC12 cells) into neurons. After the differentiation, Cu and Zn imaging with fluorescent probes revealed an increase in intracellular Cu concentration. The concentrations of other essential metals, which were determined by an inductively coupled plasma mass spectrometer, were not altered. The mRNA expression of the Cu influx transporter, Ctr1, was decreased after the differentiation, and the differentiated cells acquired tolerance to Cu and cisplatin, another substrate of Ctr1. In addition, the expression of MT-3, a brain-specific isoform, was increased, contrary to the decreased expression of MT-1 and MT-2. Taken together, the differentiation of PC12 cells into neurons induced MT-3 expression, thereby resulting in intracellular Cu accumulation. The decrease in Ctr1 expression was assumed to be a response aimed at abolishing the physiological accumulation of Cu after the differentiation. PMID:27623342

  3. Protein-protein interactions involving voltage-gated sodium channels: Post-translational regulation, intracellular trafficking and functional expression.

    PubMed

    Shao, Dongmin; Okuse, Kenji; Djamgoz, Mustafa B A

    2009-07-01

    Voltage-gated sodium channels (VGSCs), classically known to play a central role in excitability and signalling in nerves and muscles, have also been found to be expressed in a range of 'non-excitable' cells, including lymphocytes, fibroblasts and endothelia. VGSC abnormalities are associated with various diseases including epilepsy, long-QT syndrome 3, Brugada syndrome, sudden infant death syndrome and, more recently, various human cancers. Given their pivotal role in a wide range of physiological and pathophysiological processes, regulation of functional VGSC expression has been the subject of intense study. An emerging theme is post-translational regulation and macro-molecular complexing by protein-protein interactions and intracellular trafficking, leading to changes in functional VGSC expression in plasma membrane. This partially involves endoplasmic reticulum associated degradation and ubiquitin-proteasome system. Several proteins have been shown to associate with VGSCs. Here, we review the interactions involving VGSCs and the following proteins: p11, ankyrin, syntrophin, beta-subunit of VGSC, papin, ERM and Nedd4 proteins. Protein kinases A and C, as well as Ca(2+)-calmodulin dependent kinase II that have also been shown to regulate intracellular trafficking of VGSCs by changing the balance of externalization vs. internalization, and an effort is made to separate these effects from the short-term phosphorylation of mature proteins in plasma membrane. Two further modulatory mechanisms are reciprocal interactions with the cytoskeleton and, late-stage, activity-dependent regulation. Thus, the review gives an updated account of the range of post-translational molecular mechanisms regulating functional VGSC expression. However, many details of VGSC subtype-specific regulation and pathophysiological aspects remain unknown and these are highlighted throughout for completeness. PMID:19401147

  4. 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.

  5. Functional Architecture of the Cytoplasmic Entrance to the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore.

    PubMed

    El Hiani, Yassine; Linsdell, Paul

    2015-06-19

    As an ion channel, the cystic fibrosis transmembrane conductance regulator must form a continuous pathway for the movement of Cl(-) and other anions between the cytoplasm and the extracellular solution. Both the structure and the function of the membrane-spanning part of this pathway are well defined. In contrast, the structure of the pathway that connects the cytoplasm to the membrane-spanning regions is unknown, and functional roles for different parts of the protein forming this pathway have not been described. We used patch clamp recording and substituted cysteine accessibility mutagenesis to identify positively charged amino acid side chains that attract cytoplasmic Cl(-) ions to the inner mouth of the pore. Our results indicate that the side chains of Lys-190, Arg-248, Arg-303, Lys-370, Lys-1041, and Arg-1048, located in different intracellular loops of the protein, play important roles in the electrostatic attraction of Cl(-) ions. Mutation and covalent modification of these residues have charge-dependent effects on the rate of Cl(-) permeation, demonstrating their functional role in maximization of Cl(-) flux. Other nearby positively charged side chains were not involved in electrostatic interactions with Cl(-). The location of these Cl(-)-attractive residues suggests that cytoplasmic Cl(-) ions enter the pore via a lateral portal located between the cytoplasmic extensions to the fourth and sixth transmembrane helices; a secondary, functionally less relevant portal might exist between the extensions to the 10th and 12th transmembrane helices. These results define the cytoplasmic mouth of the pore and show how it attracts Cl(-) ions from the cytoplasm. PMID:25944907

  6. Polysaccharide capsule and sialic acid-mediated regulation promote biofilm-like intracellular bacterial communities during cystitis.

    PubMed

    Anderson, Gregory G; Goller, Carlos C; Justice, Sheryl; Hultgren, Scott J; Seed, Patrick C

    2010-03-01

    Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs). A murine UTI model has revealed an infection cascade whereby UPEC undergoes cycles of invasion of the bladder epithelium, intracellular proliferation in polysaccharide-containing biofilm-like masses called intracellular bacterial communities (IBC), and then dispersal into the bladder lumen to initiate further rounds of epithelial colonization and invasion. We predicted that the UPEC K1 polysaccharide capsule is a key constituent of the IBC matrix. Compared to prototypic E. coli K1 strain UTI89, a capsule assembly mutant had a fitness defect in functionally TLR4(+) and TLR4(-) mice, suggesting a protective role of capsule in inflamed and noninflamed hosts. K1 capsule assembly and synthesis mutants had dramatically reduced IBC formation, demonstrating the common requirement for K1 polysaccharide in IBC development. The capsule assembly mutant appeared dispersed in the cytoplasm of the bladder epithelial cells and failed to undergo high-density intracellular replication during later stages of infection, when the wild-type strain continued to form serial generations of IBC. Deletion of the sialic acid regulator gene nanR partially restored IBC formation in the capsule assembly mutant. These data suggest that capsule is necessary for efficient IBC formation and that aberrant sialic acid accumulation, resulting from disruption of K1 capsule assembly, produces a NanR-mediated defect in intracellular proliferation and IBC development. Together, these data demonstrate the complex but important roles of UPEC polysaccharide encapsulation and sialic acid signaling in multiple stages of UTI pathogenesis.

  7. Chlorotoxin does not inhibit volume-regulated, calcium-activated and cyclic AMP-activated chloride channels.

    PubMed

    Maertens, C; Wei, L; Tytgat, J; Droogmans, G; Nilius, B

    2000-02-01

    It was the aim of this study to look for a high-affinity and selective polypeptide toxin, which could serve as a probe for the volume-regulated anion channel (VRAC) or the calcium-activated chloride channel (CaCC). We have partially purified chlorotoxin, including new and homologous short chain insectotoxins, from the crude venom of Leiurus quinquestriatus quinquestriatus (Lqq) by means of gel filtration chromatography. Material eluting between 280 and 420 min, corresponding to fractions 15-21, was lyophilized and tested on VRAC and CaCC, using the whole-cell patch-clamp technique. We have also tested the commercially available chlorotoxin on VRAC, CaCC, the cystic fibrosis transmembrane conductance regulator (CFTR) and on the glioma specific chloride channel (GCC). VRAC and the correspondent current, I(Cl,swell), was activated in Cultured Pulmonary Artery Endothelial (CPAE) cells by a 25% hypotonic solution. Neither of the fractions 16-21 significantly inhibited I(Cl,swell) (n=4-5). Ca(2+)-activated Cl(-) currents, I(Cl,Ca), activated by loading T84 cells via the patch pipette with 1 microM free Ca(2+), were not inhibited by any of the tested fractions (15-21), (n=2-5). Chlorotoxin (625 nM) did neither effect I(Cl,swell) nor I(Cl,Ca) (n=4-5). The CFTR channel, transiently transfected in COS cells and activated by a cocktail containing IBMX and forskolin, was not affected by 1.2 microM chlorotoxin (n=5). In addition, it did not affect currents through GCC. We conclude that submicromolar concentrations of chlorotoxin do not block volume-regulated, Ca(2+)-activated and CFTR chloride channels and that it can not be classified as a general chloride channel toxin.

  8. Epigenetics: A New Model for Intracellular Parasite-Host Cell Regulation.

    PubMed

    Robert McMaster, W; Morrison, Charlotte J; Kobor, Michael S

    2016-07-01

    Intracellular protozoan parasites are an extremely important class of pathogens that cause a spectrum of diseases in human and animal hosts. There is a growing body of evidence suggesting that protozoan parasites, like other prokaryotic and viral pathogens, manipulate host cells via epigenetic modifications of the host genome that alter transcription and corresponding signaling pathways. In light of these data, we examine the role of epigenetics in downregulation of host macrophages by Leishmania that could potentially lead to a permanent state of inactivation, thus favoring pathogen survival and disease progression. PMID:27142564

  9. GmCLC1 Confers Enhanced Salt Tolerance through Regulating Chloride Accumulation in Soybean

    PubMed Central

    Wei, Peipei; Wang, Longchao; Liu, Ailin; Yu, Bingjun; Lam, Hon-Ming

    2016-01-01

    The family of chloride channel proteins that mediate Cl- transportation play vital roles in plant nutrient supply, cellular action potential and turgor pressure adjustment, stomatal movement, hormone signal recognition and transduction, Cl- homeostasis, and abiotic and biotic stress tolerance. The anionic toxicity, mainly caused by chloride ions (Cl-), on plants under salt stress remains poorly understood. In this work, we investigated the function of soybean Cl-/H+ antiporter GmCLC1 under salt stress in transgenic Arabidopsis thaliana, soybean, and yeast. We found that GmCLC1 enhanced salt tolerance in transgenic A. thaliana by reducing the Cl- accumulation in shoots and hence released the negative impact of salt stress on plant growth. Overexpression of GmCLC1 in the hairy roots of soybean sequestered more Cl- in their roots and transferred less Cl- to their shoots, leading to lower relative electrolyte leakage values in the roots and leaves. When either the soybean GmCLC1 or the yeast chloride transporter gene, GEF1, was transformed into the yeast gef1 mutant, and then treated with different chloride salts (MnCl2, KCl, NaCl), enhanced survival rate was observed. The result indicates that GmCLC1 and GEF1 exerted similar effects on alleviating the stress of diverse chloride salts on the yeast gef1 mutant. Together, this work suggests a protective function of GmCLC1 under Cl- stress. PMID:27504114

  10. GmCLC1 Confers Enhanced Salt Tolerance through Regulating Chloride Accumulation in Soybean.

    PubMed

    Wei, Peipei; Wang, Longchao; Liu, Ailin; Yu, Bingjun; Lam, Hon-Ming

    2016-01-01

    The family of chloride channel proteins that mediate Cl(-) transportation play vital roles in plant nutrient supply, cellular action potential and turgor pressure adjustment, stomatal movement, hormone signal recognition and transduction, Cl(-) homeostasis, and abiotic and biotic stress tolerance. The anionic toxicity, mainly caused by chloride ions (Cl(-)), on plants under salt stress remains poorly understood. In this work, we investigated the function of soybean Cl(-)/H(+) antiporter GmCLC1 under salt stress in transgenic Arabidopsis thaliana, soybean, and yeast. We found that GmCLC1 enhanced salt tolerance in transgenic A. thaliana by reducing the Cl(-) accumulation in shoots and hence released the negative impact of salt stress on plant growth. Overexpression of GmCLC1 in the hairy roots of soybean sequestered more Cl(-) in their roots and transferred less Cl(-) to their shoots, leading to lower relative electrolyte leakage values in the roots and leaves. When either the soybean GmCLC1 or the yeast chloride transporter gene, GEF1, was transformed into the yeast gef1 mutant, and then treated with different chloride salts (MnCl2, KCl, NaCl), enhanced survival rate was observed. The result indicates that GmCLC1 and GEF1 exerted similar effects on alleviating the stress of diverse chloride salts on the yeast gef1 mutant. Together, this work suggests a protective function of GmCLC1 under Cl(-) stress. PMID:27504114

  11. Intracellular Na+ regulation of Na+ pump sites in cultured vascular smooth muscle cells

    SciTech Connect

    Allen, J.C.; Navran, S.S.; Seidel, C.L.; Dennison, D.K.; Amann, J.M.; Jemelka, S.K.

    1989-04-01

    Enzymatically dispersed cells from canine saphenous vein and femoral artery were grown in fetal calf serum and studied at day 0 (freshly dispersed) through confluence in primary culture. Intracellular Na levels (Nai), but not intracellular K (Ki), were increased after 24 h in culture and then decreased to a steady state by 4 days. Na+ pump site number (( /sup 3/H) ouabain binding) increased through day 3 and remained elevated. Nai was still elevated at 2 days when the Na+ pump site number began to increase. Total pump turnover (maximum ouabain-inhibited /sup 86/Rb uptake) reflected the increase in Na+ pump site number. These key events precede the observed increases in both protein production and cellular proliferation. If the same cells are maintained in defined medium, without fetal calf serum, Nai, Ki, and the number of (/sup 3/H)ouabain binding sites do not change with time. These data are consistent with the suggestion that the initial mitogenic response of vascular smooth muscle cells to fetal calf serum involves an increased Na+ influx, and a Nai accumulation, caused by low Na+ pump density. The synthesis of new pump sites effects a decrease in the accumulated Nai, which may be related to cell proliferation.

  12. LacI(Ts)-regulated expression as an in situ intracellular biomolecular thermometer.

    PubMed

    McCabe, K M; Lacherndo, E J; Albino-Flores, I; Sheehan, E; Hernandez, M

    2011-05-01

    In response to needs for in situ thermometry, a temperature-sensitive vector was adapted to report changes in the intracellular heat content of Escherichia coli in near-real time. This model system utilized vectors expressing increasing quantities of β-galactosidase in response to stepwise temperature increases through a biologically relevant range (22 to 45°C). As judged by calibrated fluorometric and colorimetric reporters, both whole E. coli cells and lysates expressed significant repeatable changes in β-galactosidase activity that were sensitive to temperature changes of less than 1°C (35 to 45°C). This model system suggests that changes in cellular heat content can be detected independently of the medium in which cells are maintained, a feature of particular importance where the medium is heterogeneous or nonaqueous, or otherwise has a low heat transfer capacity. We report here that the intracellular temperature can be reliably obtained in near-real time using reliable fluorescent reporting systems from cellular scales, with a 20°C range of detection and at least 0.7°C sensitivity between 35 and 45°C. PMID:21378059

  13. Regulation of osmoadaptation in the moderate halophile Halobacillus halophilus: chloride, glutamate and switching osmolyte strategies

    PubMed Central

    Saum, Stephan H; Müller, Volker

    2008-01-01

    The moderate halophile Halobacillus halophilus is the paradigm for chloride dependent growth in prokaryotes. Recent experiments shed light on the molecular basis of the chloride dependence that is reviewed here. In the presence of moderate salinities Halobacillus halophilus mainly accumulates glutamine and glutamate to adjust turgor. The transcription of glnA2 (encoding a glutamine synthetase) as well as the glutamine synthetase activity were identified as chloride dependent steps. Halobacillus halophilus switches its osmolyte strategy and produces proline as the main compatible solute at high salinities. Furthermore, Halobacillus halophilus also shifts its osmolyte strategy at the transition from the exponential to the stationary phase where proline is exchanged by ectoine. Glutamate was found as a “second messenger” essential for proline production. This observation leads to a new model of sensing salinity by sensing the physico-chemical properties of different anions. PMID:18442383

  14. Regulation of osmoadaptation in the moderate halophile Halobacillus halophilus: chloride, glutamate and switching osmolyte strategies.

    PubMed

    Saum, Stephan H; Müller, Volker

    2008-04-28

    The moderate halophile Halobacillus halophilus is the paradigm for chloride dependent growth in prokaryotes. Recent experiments shed light on the molecular basis of the chloride dependence that is reviewed here. In the presence of moderate salinities Halobacillus halophilus mainly accumulates glutamine and glutamate to adjust turgor. The transcription of glnA2 (encoding a glutamine synthetase) as well as the glutamine synthetase activity were identified as chloride dependent steps. Halobacillus halophilus switches its osmolyte strategy and produces proline as the main compatible solute at high salinities. Furthermore, Halobacillus halophilus also shifts its osmolyte strategy at the transition from the exponential to the stationary phase where proline is exchanged by ectoine. Glutamate was found as a "second messenger" essential for proline production. This observation leads to a new model of sensing salinity by sensing the physico-chemical properties of different anions.

  15. Regulation of intracellular pH in neuronal and glial tumour cells, studied by multinuclear NMR spectroscopy.

    PubMed

    Flögel, U; Willker, W; Leibfritz, D

    1994-06-01

    The effect of extracellular pH (pHe) on intracellular pH (pHi) and cellular metabolism was examined by multinuclear NMR spectroscopy of cells in vivo and in vitro. A decrease in pHe from 7.4 to 6.4 led to a significant drop in pHi, in both neuronal and glial tumour cells, as detected by in vivo 31P NMR of cells embedded in basement membrane gel threads. A more than 50% decrease in both the phosphocreatine (PCr) level and derivatives of glycolysis (i.e., glycerol 3-phosphate) was observed, concomitantly to the fall in pHi. A 50% decrease in intracellular lactate levels was seen in in vivo 1H NMR spectra under these conditions. Reperfusion with fresh medium (pHe 7.4) resulted in the full recovery of pHi, simultaneously with an increase in both PCr and intracellular lactate back to their control levels. Perchloric acid and lipid extract measurements confirmed the observations made by in vivo 31P and 1H NMR spectroscopy and further showed a decrease both in tricarboxylic acid cycle activity and phospholipid synthesis. The data revealed no significant differences between the neuronal and glial tumour cells investigated. pHi measurements in the presence of inhibitors of the various pH regulatory mechanisms showed that the Na+/H+ exchanger, the carbonic anhydrase and at least one of the bicarbonate-transport systems are involved in pH regulation of both cell types. The results suggest that Na+/H+ exchange is the preferred mechanism by which both neuronal and glial cells regulate their pHi after extracellular acidification.

  16. Intracellular calcium and its sodium-independent regulation in voltage-clamped snail neurones.

    PubMed Central

    Kennedy, H J; Thomas, R C

    1995-01-01

    1. We have used both Ca(2+)-sensitive microelectrodes and fura-2 to measure the intracellular free calcium ion concentration ([Ca2+]i or its negative log, pCai) of snail neurones voltage clamped to -50 or -60 mV. Using Ca(2+)-sensitive microelectrodes, [Ca2+]i was found to be approximately 174 nM and pCai, 6.76 +/- 0.09 (mean +/- S.E.M.; n = 11); using fura-2, [Ca2+]i was approximately 40 nM and pCai, 7.44 +/- 0.06 (mean +/- S.E.M., n = 10). 2. Depolarizations (1-20 s) caused an increase in [Ca2+]i which was abolished by removal of extracellular Ca2+, indicating that the rise in [Ca2+]i was due to Ca2+ influx through voltage-activated Ca2+ channels. 3. Caffeine (10-20 mM) caused an increase in [Ca2+]i in the presence or absence of extracellular Ca2+. The effects of caffeine on [Ca2+]i could be prevented by ryanodine. 4. Thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a small increase in resting [Ca2+]i and slowed the rate of recovery from Ca2+ loads following 20 s depolarizations. 5. Neither replacement of extracellular sodium with N-methyl-D-glucamine (NMDG), nor loading the cells with intracellular sodium, had any effect on resting [Ca2+]i or the rate of recovery of [Ca2+]i following depolarizations. 6. The mitochondrial uncoupling agent carbonyl cyanide m-chlorophenylhydrazone (CCmP) caused a small gradual rise in resting [Ca2+]i. Removal of extracellular sodium during exposure to CCmP had no further effect on [Ca2+]i. 7. Intracellular orthovanadate caused an increase in resting [Ca2+]i and prevented the full recovery of [Ca2+]i following small Ca2+ loads, but removal of extracellular sodium did not cause a rise in [Ca2+]i. We conclude that there is no Na(+)-Ca2+ exchanger present in the cell body of these neurones and that [Ca2+]i is maintained by an ATP-dependent Ca2+ pump. Images Figure 1 PMID:7623274

  17. Tri-modal regulation of cardiac muscle relaxation; intracellular calcium decline, thin filament deactivation, and cross-bridge cycling kinetics

    PubMed Central

    Biesiadecki, Brandon J.; Davis, Jonathan P.; Ziolo, Mark T.; Janssen, Paul M.L.

    2014-01-01

    Cardiac muscle relaxation is an essential step in the cardiac cycle. Even when the contraction of the heart is normal and forceful, a relaxation phase that is too slow will limit proper filling of the ventricles. Relaxation is too often thought of as a mere passive process that follows contraction. However, many decades of advancements in our understanding of cardiac muscle relaxation have shown it is a highly complex and well-regulated process. In this review, we will discuss three distinct events that can limit the rate of cardiac muscle relaxation: the rate of intracellular calcium decline, the rate of thin-filament de-activation, and the rate of cross-bridge cycling. Each of these processes are directly impacted by a plethora of molecular events. In addition, these three processes interact with each other, further complicating our understanding of relaxation. Each of these processes is continuously modulated by the need to couple bodily oxygen demand to cardiac output by the major cardiac physiological regulators. Length-dependent activation, frequency-dependent activation, and β-adrenergic regulation all directly and indirectly modulate calcium decline, thin-filament deactivation, and cross-bridge kinetics. We hope to convey our conclusion that cardiac muscle relaxation is a process of intricate checks and balances, and should not be thought of as a single rate-limiting step that is regulated at a single protein level. Cardiac muscle relaxation is a system level property that requires fundamental integration of three governing systems: intracellular calcium decline, thin filament deactivation, and cross-bridge cycling kinetics. PMID:25484996

  18. Intracellular pH-regulating mechanism of the squid axon. Relation between the external Na+ and HCO-3 dependences

    PubMed Central

    1985-01-01

    The intracellular pH-regulating mechanism of the squid axon was examined for its dependence on the concentrations of external Na+ and HCO3-, always at an external pH (pHo) of 8.0. Axons having an initial intracellular pH (pHi) of approximately 7.4 were internally dialyzed with a solution of pH 6.5 that contained 400 mM Cl- and no Na+. After pHi had fallen to approximately 6.6, dialysis was halted, thereby returning control of pHi to the axon. With external Na+ and HCO-3 present, intracellular pH (pHi) increased because of the activity of the pHi-regulating system. The acid extrusion rate (i.e., equivalent efflux of H+, JH) is the product of the pHi recovery rate, intracellular buffering power, and the volume-to-surface ratio. The [HCO3-]o dependence of JH was examined at three fixed levels of [Na+]o: 425, 212, and 106 mM. In all three cases, the apparent Jmax was approximately 19 pmol X cm-2 X s-1. However, the apparent Km (HCO3-) was approximately inversely proportional to [Na+]o, rising from 2.6 to 5.4 to 9.7 mM as [Na+]o was lowered from 425 to 212 to 106 mM, respectively. The [Na+]o dependence of JH was similarly examined at three fixed levels of [HCO3-]o: 12, 6, and 3 mM. The Jmax values did not vary significantly from those in the first series of experiments. The apparent Km (Na+), however, was approximately inversely related to [HCO3-]o, rising from 71 to 174 to 261 mM as [HCO3-]o was lowered from 12 to 6 to 3 mM, respectively. These results agree with the predictions of the ion-pair model of acid extrusion, which has external Na+ and CO3= combining to form the ion pair NaCO3-, which then exchanges for internal Cl-. When the JH data are replotted as a function of [NaCO3- ]o, data from all six groups of experiments fall along the same Michaelis-Menten curve, with an apparent Km (NaCO3-) of 80 microM. The ordered and random binding of Na+ and CO3= cannot be ruled out as possible models, but are restricted in allowable combinations of rate constants. PMID

  19. Influence of changes in external potassium and chloride ions on membrane potential and intracellular potassium ion activity in rabbit ventricular muscle.

    PubMed

    Fozzard, H A; Lee, C O

    1976-04-01

    1. The membrane responses of rabbit papillary muscles to rapid changes in [K](o) and [Cl](o) were measured with open-tipped micropipettes and with closed micropipettes made from K-selective glass.2. The muscle cells behaved primarily as a K electrode, and responses to changes in [K](o) with constant [Cl](o) or with constant [K](o) x [Cl](o) were substantially the same.3. When [Cl](o) was changed at a constant [K](o) the membrane potentials changed rapidly and symmetrically by a small value and remained constant for 30 min.4. Measurement of potential with K(+)-selective micro-electrodes in these experiments showed no change in intracellular K activity. In addition to permitting calculation of K permeability, these measurements reassured us that the K(+)-selective electrodes were well insulated and not influenced by electrical shunts at the impalement site.5. Although the membrane response to changes in [Cl](o) was small, it was possible to calculate that the permeability ratio (P(Cl)/P(K)), was 0.11. The Cl and K conductances were about 0.015 mmho/cm(2) and 0.09 mmho/cm(2) respectively, resulting in a conductance ratio (g(Cl)/g(K)) of about 0.17.6. The time course of depolarization by increase in [K](o) was rapid (half-time 5 sec), but repolarization on return to lower [K](o) was much slower (half-time 50 sec). The depolarization time course was easily fitted by the potential change calculated by assuming the need for K diffusion into the extracellular spaces and taking account of the logarithmic relation between membrane potential and [K](o). These calculations did not fit the time course of repolarization, which was slowed in the fashion expected from an inward-rectifying membrane.7. The influence of [K](i) on membrane potential was investigated by changes in tonicity of the external solution. Hypotonic solution produced a change in intracellular K activity close to that produced by ideal water movement. However, in hypertonic solution, intracellular K activity

  20. Regulation of the system x(C)- cystine/glutamate exchanger by intracellular glutathione levels in rat astrocyte primary cultures.

    PubMed

    Seib, Todd Michael; Patel, Sarjubhai Amratbhai; Bridges, Richard James

    2011-10-01

    The system x(C)- (Sx(C)-) transporter functions to mediate the exchange of extracellular cystine (L-Cys(2)) and intracellular glutamate (L-Glu). Internalized L-Cys(2) serves as a rate-limiting precursor for the biosynthesis of glutathione (GSH), while the externalized L-Glu can contribute to either excitatory signaling or excitotoxicity. In the present study the influence of culture conditions (with and without dibutyryl-cAMP) and GSH levels on the expression of Sx(C)- were investigated in primary rat astrocyte cultures. Sx(C)- activity in dbcAMP-treated cells was nearly sevenfold greater than in untreated astrocytes and increased further (∼threefold) following the depletion of intracellular GSH with buthionine sulfoximine. This increase in Sx(C)- triggered by GSH depletion was only observed in the dbcAMP-treated phenotype and was distinct from the Nrf2-mediated response initiated by exposure to electrophiles. Changes in Sx(C)- activity correlated with increases in both protein and mRNA levels of the xCT subunit of the Sx(C)- heterodimer, an increase in the V(max) for L-Glu uptake and was linked temporally to GSH levels. This induction of Sx(C)- was not mimicked by hydrogen peroxide nor attenuated by nonspecific antioxidants but was partially prevented by the co-administration of the cell-permeant thiols GSH-ethyl ester and N-acetylcysteine. These findings demonstrate that the expression of Sx(C)- on astrocytes is dynamically regulated by intracellular GSH levels in a cell- and phenotype-dependent manner. The presence of this pathway likely reflects the inherent vulnerability of the CNS to oxidative damage and raises interesting questions as to the functional consequences of changes in Sx(C)- activity in CNS injury and disease.

  1. Ionic osmolytes and intracellular calcium regulate tissue production in chondrocytes cultured in a 3D charged hydrogel.

    PubMed

    Farnsworth, Nikki L; Mead, Benjamin E; Antunez, Lorena R; Palmer, Amy E; Bryant, Stephanie J

    2014-11-01

    The goal of this study was to investigate the role of fixed negative charges in regulating cartilage-like tissue production by chondrocytes under static and dynamic three-dimensional culture, and to determine whether intracellular calcium ([Ca(2+)]i) is involved in mediating this response. Initial experiments using the 3D neutral hydrogel were conducted in static isotonic culture with ionic and non-ionic osmolytes added to the culture medium. Tissue production by bovine chondrocytes with non-ionic osmolytes was 1.9-fold greater than with ionic osmolytes, suggesting that the ionic nature of the osmolyte is an important regulator of tissue production. To investigate fixed negative charges, a 3D culture system containing encapsulated chondrocytes was employed based on a synthetic and neutral hydrogel platform within which negatively charged chondroitin sulfate was incorporated in a controlled manner. Incorporation of negative charges did not affect the mechanical properties of the hydrogel; however, intracellular ion concentration was elevated from the culture medium (330 mOsm) and estimated to be similar to that in ~400 mOsm culture medium. With dynamic loading, GAG synthesis decreased by 26% in neutral hydrogels cultured in 400mOsm medium, and increased by 26% in charged gels cultured in 330 mOsm. Treatment of chondrocyte-seeded hydrogels with the Ca(2+) chelator BAPTA-AM decreased GAG synthesis by 32-46% and was similar among all conditions, suggesting multiple roles for Ca(2+) mediated tissue production including with ionic osmolytes. In conclusion, findings from this study suggest that a dynamic ionic environment regulates tissue synthesis and points to [Ca(2+)]i signaling as a potential mediator. PMID:25128592

  2. Arsenic promotes ubiquitinylation and lysosomal degradation of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels in human airway epithelial cells.

    PubMed

    Bomberger, Jennifer M; Coutermarsh, Bonita A; Barnaby, Roxanna L; Stanton, Bruce A

    2012-05-18

    Arsenic exposure significantly increases respiratory bacterial infections and reduces the ability of the innate immune system to eliminate bacterial infections. Recently, we observed in the gill of killifish, an environmental model organism, that arsenic exposure induced the ubiquitinylation and degradation of cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel that is essential for the mucociliary clearance of respiratory pathogens in humans. Accordingly, in this study, we tested the hypothesis that low dose arsenic exposure reduces the abundance and function of CFTR in human airway epithelial cells. Arsenic induced a time- and dose-dependent increase in multiubiquitinylated CFTR, which led to its lysosomal degradation, and a decrease in CFTR-mediated chloride secretion. Although arsenic had no effect on the abundance or activity of USP10, a deubiquitinylating enzyme, siRNA-mediated knockdown of c-Cbl, an E3 ubiquitin ligase, abolished the arsenic-stimulated degradation of CFTR. Arsenic enhanced the degradation of CFTR by increasing phosphorylated c-Cbl, which increased its interaction with CFTR, and subsequent ubiquitinylation of CFTR. Because epidemiological studies have shown that arsenic increases the incidence of respiratory infections, this study suggests that one potential mechanism of this effect involves arsenic-induced ubiquitinylation and degradation of CFTR, which decreases chloride secretion and airway surface liquid volume, effects that would be proposed to reduce mucociliary clearance of respiratory pathogens.

  3. An intracellular anion channel critical for pigmentation.

    PubMed

    Bellono, Nicholas W; Escobar, Iliana E; Lefkovith, Ariel J; Marks, Michael S; Oancea, Elena

    2014-12-16

    Intracellular ion channels are essential regulators of organellar and cellular function, yet the molecular identity and physiological role of many of these channels remains elusive. In particular, no ion channel has been characterized in melanosomes, organelles that produce and store the major mammalian pigment melanin. Defects in melanosome function cause albinism, characterized by vision and pigmentation deficits, impaired retinal development, and increased susceptibility to skin and eye cancers. The most common form of albinism is caused by mutations in oculocutaneous albinism II (OCA2), a melanosome-specific transmembrane protein with unknown function. Here we used direct patch-clamp of skin and eye melanosomes to identify a novel chloride-selective anion conductance mediated by OCA2 and required for melanin production. Expression of OCA2 increases organelle pH, suggesting that the chloride channel might regulate melanin synthesis by modulating melanosome pH. Thus, a melanosomal anion channel that requires OCA2 is essential for skin and eye pigmentation.

  4. The space of enzyme regulation in HeLa cells can be inferred from its intracellular metabolome.

    PubMed

    Diener, Christian; Muñoz-Gonzalez, Felipe; Encarnación, Sergio; Resendis-Antonio, Osbaldo

    2016-06-23

    During the transition from a healthy state to a cancerous one, cells alter their metabolism to increase proliferation. The underlying metabolic alterations may be caused by a variety of different regulatory events on the transcriptional or post-transcriptional level whose identification contributes to the rational design of therapeutic targets. We present a mechanistic strategy capable of inferring enzymatic regulation from intracellular metabolome measurements that is independent of the actual mechanism of regulation. Here, enzyme activities are expressed by the space of all feasible kinetic constants (k-cone) such that the alteration between two phenotypes is given by their corresponding kinetic spaces. Deriving an expression for the transformation of the healthy to the cancer k-cone we identified putative regulated enzymes between the HeLa and HaCaT cell lines. We show that only a few enzymatic activities change between those two cell lines and that this regulation does not depend on gene transcription but is instead post-transcriptional. Here, we identify phosphofructokinase as the major driver of proliferation in HeLa cells and suggest an optional regulatory program, associated with oxidative stress, that affects the activity of the pentose phosphate pathway.

  5. The space of enzyme regulation in HeLa cells can be inferred from its intracellular metabolome

    PubMed Central

    Diener, Christian; Muñoz-Gonzalez, Felipe; Encarnación, Sergio; Resendis-Antonio, Osbaldo

    2016-01-01

    During the transition from a healthy state to a cancerous one, cells alter their metabolism to increase proliferation. The underlying metabolic alterations may be caused by a variety of different regulatory events on the transcriptional or post-transcriptional level whose identification contributes to the rational design of therapeutic targets. We present a mechanistic strategy capable of inferring enzymatic regulation from intracellular metabolome measurements that is independent of the actual mechanism of regulation. Here, enzyme activities are expressed by the space of all feasible kinetic constants (k-cone) such that the alteration between two phenotypes is given by their corresponding kinetic spaces. Deriving an expression for the transformation of the healthy to the cancer k-cone we identified putative regulated enzymes between the HeLa and HaCaT cell lines. We show that only a few enzymatic activities change between those two cell lines and that this regulation does not depend on gene transcription but is instead post-transcriptional. Here, we identify phosphofructokinase as the major driver of proliferation in HeLa cells and suggest an optional regulatory program, associated with oxidative stress, that affects the activity of the pentose phosphate pathway. PMID:27335086

  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. Programmed Nanococktail for Intracellular Cascade Reaction Regulating Self-Synergistic Tumor Targeting Therapy.

    PubMed

    Chen, Wei-Hai; Luo, Guo-Feng; Qiu, Wen-Xiu; Lei, Qi; Hong, Sheng; Wang, Shi-Bo; Zheng, Di-Wei; Zhu, Cheng-Hui; Zeng, Xuan; Feng, Jun; Cheng, Si-Xue; Zhang, Xian-Zheng

    2016-02-10

    In this work, a ZnO based nanococktail with programmed functions is designed and synthesized for self-synergistic tumor targeting therapy. The nanococktail can actively target tumors via specific interaction of hyaluronic acid (HA) with CD44 receptors and respond to HAase-rich tumor microenvironment to induce intracellular cascade reaction for controlled therapy. The exposed cell-penetrating peptide (R8) potentiates the cellular uptake of therapeutic nanoparticles into targeted tumor cells. Then ZnO cocktail will readily degrade in acidic endo/lysosomes and induce the production of desired reactive oxygen species (ROS) in situ. The destructive ROS not only leads to serious cell damage but also triggers the on-demand drug release for precise chemotherapy, thus achieving enhanced antitumor efficiency synergistically. After tail vein injection of ZnO cocktail, a favorable tumor apoptosis rate (71.2 ± 8.2%) is detected, which is significantly superior to that of free drug, doxorubicin (12.9 ± 5.2%). Both in vitro and in vivo studies demonstrate that the tailor-made ZnO cocktail with favorable biocompatibility, promising tumor specificity, and self-synergistically therapeutic capacity opens new avenues for cancer therapy.

  8. Use of ion-sensitive microelectrodes to study intracellular free magnesium concentration and its regulation in mammalian cardiac muscle.

    PubMed

    Hall, S K; Fry, C H; Buri, A; McGuigan, J A

    Ion-sensitive microelectrodes (ISEs) have been used to measure intracellular [Mg2+] ([Mg2+]i) in cardiac muscle, although most measurements have tended to overestimate the value due to the poor selectivity of the Mg2+ ionophore in the sarcoplasm and to inaccurate collation of individual ISE measurements. This paper highlights the correct method for analysis of data from multiple ISE experiments. Since [Mg2+]i is constrained at a lower concentration than would be expected by passive distribution of the ion, some of the possible mechanisms underlying Mg2+ extrusion from ferret ventricular myocardium were investigated. During elevation of the extracellular [Mg], mean [Mg2+]i rose from 1.61 to 1.91 mM. The same intervention had no significant effect on membrane potential, intracellular [Na+] or pH measured with ISEs, and there was no change in resting [Ca2+], as assessed from fura-2 fluorescence. The data are not consistent with a simple mechanism for Na(+)-Mg2+ exchange as the primary mode of Mg2+ regulation in cardiac muscle or with an Mg2+ extrusion mechanism involving steady-state ion exchange.

  9. Regulated phosphorylation and dephosphorylation of tau protein: effects on microtubule interaction, intracellular trafficking and neurodegeneration.

    PubMed Central

    Billingsley, M L; Kincaid, R L

    1997-01-01

    This review attempts to summarize what is known about tau phosphorylation in the context of both normal cellular function and dysfunction. However, conceptions of tau function continue to evolve, and it is likely that the regulation of tau distribution and metabolism is complex. The roles of microtubule-associated kinases and phosphatases have yet to be fully described, but may afford insight into how tau phosphorylation at the distal end of the axon regulates cytoskeletal-membrane interactions. Finally, lipid and glycosaminoglycan modification of tau structure affords yet more complexity for regulation and aggregation. Continued work will help to determine what is causal and what is coincidental in Alzheimer's disease, and may lead to identification of therapeutic targets for halting the progression of paired helical filament formation. PMID:9169588

  10. NK Cell-Mediated Regulation of Protective Memory Responses against Intracellular Ehrlichial Pathogens

    PubMed Central

    Habib, Samar; El Andaloussi, Abdeljabar; Hisham, Ahmed; Ismail, Nahed

    2016-01-01

    Ehrlichiae are gram-negative obligate intracellular bacteria that cause potentially fatal human monocytic ehrlichiosis. We previously showed that natural killer (NK) cells play a critical role in host defense against Ehrlichia during primary infection. However, the contribution of NK cells to the memory response against Ehrlichia remains elusive. Primary infection of C57BL/6 mice with Ehrlichia muris provides long-term protection against a second challenge with the highly virulent Ixodes ovatus Ehrlichia (IOE), which ordinarily causes fatal disease in naïve mice. Here, we show that the depletion of NK cells in E. muris-primed mice abrogates the protective memory response against IOE. Approximately, 80% of NK cell-depleted E. muris-primed mice succumbed to lethal IOE infection on days 8–10 after IOE infection, similar to naïve mice infected with the same dose of IOE. The lack of a recall response in NK cell-depleted mice correlated with an increased bacterial burden, extensive liver injury, decreased frequency of Ehrlichia-specific IFN-γ-producing memory CD4+ and CD8+ T-cells, and a low titer of Ehrlichia-specific antibodies. Intraperitoneal infection of mice with E. muris resulted in the production of IL-15, IL-12, and IFN-γ as well as an expansion of activated NKG2D+ NK cells. The adoptive transfer of purified E. muris-primed hepatic and splenic NK cells into Rag2-/-Il2rg-/- recipient mice provided protective immunity against challenge with E. muris. Together, these data suggest that E. muris-induced memory-like NK cells, which contribute to the protective, recall response against Ehrlichia. PMID:27092553

  11. Factors Regulating Cell Wall Thickening and Intracellular Iodophilic Polysaccharide Storage in Streptococcus mutans

    PubMed Central

    Mattingly, S. J.; Daneo-Moore, L.; Shockman, G. D.

    1977-01-01

    The effects of a series of different antibiotics on the synthesis and accumulation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), protein, cell wall peptidoglycan (PG), and intracellular iodophilic polysaccharide (IPS) in Streptococcus mutans FA-1 were examined. d-Cycloserine, penicillin G, or vancomycin treatment resulted in rapid inhibitions of PG synthesis and a consequent decrease in the relative amount of lysine found in PG fractions. Decreases in culture turbidity, an indicator of gross cellular lysis, were not observed. Secondary inhibitions of the rates and extent of syntheses of DNA, RNA, and protein were observed. With all three inhibitors of PG synthesis, IPS synthesis continued for varying time intervals but, at most, resulted in only relatively small and transient increases in cellular IPS content. Chloramphenicol inhibited protein synthesis but permitted continued synthesis of RNA and PG. After 6 h, the cells contained 42% of their [3H] lysine in the PG fraction compared with 25% in exponential-phase cells, a good indication of thickened cell walls. In the presence of chloramphenicol, cellular IPS content increased about 2.5-fold during the first 45 min and then decreased to a level (13%) at 6 h very similar to that of exponential-phase cells (about 10%). Rifampin inhibition of RNA (and, consequently, also protein) synthesis resulted in accumulation of cellular PG and IPS. After 6 h, IPS accounted for 38% of the cellular dry weight, and the cells contained 43% of their lysine in PG. Thus, rifampin-inhibited cells appear to have both thickened walls and a high IPS content. The correlation between inhibition of RNA synthesis and IPS accumulation was confirmed by exposing cultures to rifampin for 60 min and then removing the drug, thus permitting the cells to regrow. Upon removal of rifampin and resumption of RNA synthesis, cellular IPS content rapidly decreased to the level expected for exponentialphase cells. PMID:892902

  12. Phosphorylation Regulating the Ratio of Intracellular CRY1 Protein Determines the Circadian Period

    PubMed Central

    Liu, Na; Zhang, Eric Erquan

    2016-01-01

    The core circadian oscillator in mammals is composed of transcription/translation feedback loop, in which cryptochrome (CRY) proteins play critical roles as repressors of their own gene expression. Although post-translational modifications, such as phosphorylation of CRY1, are crucial for circadian rhythm, little is known about how phosphorylated CRY1 contributes to the molecular clockwork. To address this, we created a series of CRY1 mutants with single amino acid substitutions at potential phosphorylation sites and performed a cell-based, phenotype-rescuing screen to identify mutants with aberrant rhythmicity in CRY-deficient cells. We report 10 mutants with an abnormal circadian period length, including long period (S280D and S588D), short period (S158D, S247D, T249D, Y266D, Y273D, and Y432D), and arrhythmicity (S71D and S404D). When expressing mutated CRY1 in HEK293 cells, we show that most of the mutants (S71D, S247D, T249D, Y266D, Y273D, and Y432D) exhibited reduction in repression activity compared with wild-type (WT) CRY1, whereas other mutants had no obvious change. Correspondingly, these mutants also showed differences in protein stability and cellular localization. We show that most of mutants are more stable than WT, except S158D, T249D, and S280D. Although the characteristics of the 10 mutants are various, they all impair the ratio balance of intracellular CRY1 protein. Thus, we conclude that the mutations caused distinct phenotypes most likely through the ratio of functional CRY1 protein in cells. PMID:27721804

  13. Intracellular pH regulation in isolated rat bile duct epithelial cells.

    PubMed Central

    Strazzabosco, M; Mennone, A; Boyer, J L

    1991-01-01

    To evaluate ion transport mechanisms in bile duct epithelium (BDE), BDE cells were isolated from bile duct-ligated rats. After short-term culture pHi was measured with a single cell microfluorimetric set-up using the fluorescent pHi indicator BCECF, and calibrated with nigericin in high K+ concentration buffer. Major contaminants were identified using vital markers. In HCO3(-)-free media, baseline pHi (7.03 +/- 0.12) decreased by 0.45 +/- 0.18 pH units after Na+ removal and by 0.12 +/- .04 after amiloride administration (1 mM). After acid loading (20 mM NH4Cl) pHi recovery was inhibited by both Na+ removal and amiloride (JH+ = 0.74 +/- 1.1, and JH+ = 2.28 +/- 0.8, respectively, vs. 5.47 +/- 1.97 and 5.97 +/- 1.76 mM/min, in controls, respectively). In HCO3- containing media baseline pHi was higher (7.16 +/- 0.1, n = 36, P less than 0.05) and was decreased by Na+ substitution but not by amiloride. Na+ removal inhibited pHi recovery after an intracellular acid load (0.27 +/- 0.26, vs. 7.7 +/- 4.1 mM/min, in controls), whereas amiloride reduced JH+ only by 27%. pH recovery was inhibited by DIDS (0.5-1 mM), but not by Cl- depletion. Finally, acute Cl- removal increased pHi by 0.18 pH units in the absence but not presence of DIDS. These data indicate that BDE cells possess mechanisms for Na+/H+ exchange, Na+:HCO3- symport and Cl-/HCO3 exchange. Therefore BDE may be capable of transepithelial H+/HCO3- transport. Images PMID:2022723

  14. By Regulating Mitochondrial Ca2+-Uptake UCP2 Modulates Intracellular Ca2+

    PubMed Central

    Gebing, Tina; Reda, Sara; Schwaiger, Astrid; Leitner, Johannes; Wolny, Martin; Eckardt, Lars; Hoppe, Uta C.

    2016-01-01

    Introduction The possible role of UCP2 in modulating mitochondrial Ca2+-uptake (mCa2+-uptake) via the mitochondrial calcium uniporter (MCU) is highly controversial. Methods Thus, we analyzed mCa2+-uptake in isolated cardiac mitochondria, MCU single-channel activity in cardiac mitoplasts, dual Ca2+-transients from mitochondrial ((Ca2+)m) and intracellular compartment ((Ca2+)c) in the whole-cell configuration in cardiomyocytes of wild-type (WT) and UCP2-/- mice. Results Isolated mitochondria showed a Ru360 sensitive mCa2+-uptake, which was significantly decreased in UCP2-/- (229.4±30.8 FU vs. 146.3±23.4 FU, P<0.05). Single-channel registrations confirmed a Ru360 sensitive voltage-gated Ca2+-channel in mitoplasts, i.e. mCa1, showing a reduced single-channel activity in UCP2-/- (Po,total: 0.34±0.05% vs. 0.07±0.01%, P<0.05). In UCP2-/- cardiomyocytes (Ca2+)m was decreased (0.050±0.009 FU vs. 0.021±0.005 FU, P<0.05) while (Ca2+)c was unchanged (0.032±0.002 FU vs. 0.028±0.004 FU, P>0.05) and transsarcolemmal Ca2+-influx was inhibited suggesting a possible compensatory mechanism. Additionally, we observed an inhibitory effect of ATP on mCa2+-uptake in WT mitoplasts and (Ca2+)m of cardiomyocytes leading to an increase of (Ca2+)c while no ATP dependent effect was observed in UCP2-/-. Conclusion Our results indicate regulatory effects of UCP2 on mCa2+-uptake. Furthermore, we propose, that previously described inhibitory effects on MCU by ATP may be mediated via UCP2 resulting in changes of excitation contraction coupling. PMID:26849136

  15. 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

  16. Critical role for NAD glycohydrolase in regulation of erythropoiesis by hematopoietic stem cells through control of intracellular NAD content.

    PubMed

    Nam, Tae-Sik; Park, Kwang-Hyun; Shawl, Asif Iqbal; Kim, Byung-Ju; Han, Myung-Kwan; Kim, Youngho; Moss, Joel; Kim, Uh-Hyun

    2014-06-01

    NAD glycohydrolases (NADases) catalyze the hydrolysis of NAD to ADP-ribose and nicotinamide. Although many members of the NADase family, including ADP-ribosyltransferases, have been cloned and characterized, the structure and function of NADases with pure hydrolytic activity remain to be elucidated. Here, we report the structural and functional characterization of a novel NADase from rabbit reticulocytes. The novel NADase is a glycosylated, glycosylphosphatidylinositol-anchored cell surface protein exclusively expressed in reticulocytes. shRNA-mediated knockdown of the NADase in bone marrow cells resulted in a reduction of erythroid colony formation and an increase in NAD level. Furthermore, treatment of bone marrow cells with NAD, nicotinamide, or nicotinamide riboside, which induce an increase in NAD content, resulted in a significant decrease in erythroid progenitors. These results indicate that the novel NADase may play a critical role in regulating erythropoiesis of hematopoietic stem cells by modulating intracellular NAD. PMID:24759100

  17. Aging is a primary risk factor for cardiac arrhythmias: disruption of intracellular Ca2+ regulation as a key suspect.

    PubMed

    Hatch, Fiona; Lancaster, Matthew K; Jones, Sandra A

    2011-08-01

    Aging is an inevitable time-dependent progression associated with a functional decline of the cardiovascular system even in 'healthy' individuals. Age positively correlates with an increasing risk of cardiac problems including arrhythmias. Not only the prevalence but also the severity of arrhythmias escalates with age. The reasons for this are multifactorial but dysregulation of intracellular calcium within the heart is likely to play a key role in initiating and perpetuating these life-threatening events. We now know that several aspects of cardiac calcium regulation significantly change with advancing age - changes that could produce electrical instability. Further development of knowledge of the mechanisms underlying these changes will allow us to reduce what currently is an inevitable increase in the incidence of arrhythmias in the elderly.

  18. The CovS/CovR Acid Response Regulator Is Required for Intracellular Survival of Group B Streptococcus in Macrophages

    PubMed Central

    Cumley, Nicola J.; Smith, Leanne M.; Anthony, Mark

    2012-01-01

    Group B Streptococcus (GBS) is a leading cause of neonatal meningitis and septicemia. The ability of this organism to survive inside phagocytic cells is poorly understood but thought to be an important step for the establishment of disease in the host. Here, we demonstrate that GBS shows prolonged survival within J774 macrophages and that the capacity to survive is not significantly changed across a diverse range of strains representing different serotypes, multilocus sequence types (MLST), and sites of clinical isolation. Using staining for the lysosome-associated membrane protein (LAMP) and by pharmacological inhibition of phagosome acidification, we demonstrate that streptococci reside in a phagosome and that acidification of the phagosome is required for GBS to survive intracellularly. Moreover, we show that the GBS two-component system CovS/CovR, which is the major acid response regulator in this organism, is required for survival inside the phagosome. PMID:22331428

  19. 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.

  20. 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

  1. New mechanisms that regulate Saccharomyces cerevisiae short peptide transporter achieve balanced intracellular amino acid concentrations.

    PubMed

    Melnykov, Artem V

    2016-01-01

    The budding yeast Saccharomyces cerevisiae is able to take up large quantities of amino acids in the form of di- and tripeptides via a short peptide transporter, Ptr2p. It is known that PTR2 can be induced by certain peptides and amino acids, and the mechanisms governing this upregulation are understood at the molecular level. We describe two new opposing mechanisms of regulation that emphasize potential toxicity of amino acids: the first is upregulation of PTR2 in a population of cells, caused by amino acid secretion that accompanies peptide uptake; the second is loss of Ptr2p activity, due to transporter internalization following peptide uptake. Our findings emphasize the importance of proper amino acid balance in the cell and extend understanding of peptide import regulation in yeast.

  2. 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

  3. N-methylhemeanthidine chloride, a novel Amaryllidaceae alkaloid, inhibits pancreatic cancer cell proliferation via down-regulating AKT activation

    SciTech Connect

    Guo, Guoli; Yao, Guangmin; Zhan, Guanqun; Hu, Yufeng; Yue, Ming; Cheng, Ling; Liu, Yaping; Ye, Qi; Qing, Guoliang; Zhang, Yonghui; Liu, Hudan

    2014-11-01

    We previously reported the isolation of a novel Amaryllidaceae alkaloid, N-methylhemeanthidine chloride (NMHC), from Zephyranthes candida, which exhibits potent cytotoxicity in a spectrum of tumor cells. However, the mechanism of action remains unclear. Using multiple cell lines derived from human pancreatic cancer, one of the most mortal and refractory human malignancies, we further studied the NMHC-mediated cytotoxicity and found that it induced drastic cytotoxicity in pancreatic cancer cells whereas an insignificant effect on a noncancerous cell line. The NMHC-mediated growth inhibition was more severe than the first-line chemotherapeutic agent gemcitabine, leading to cell cycle arrest, apoptotic death and decreased glycolysis. NMHC exerted its function through down-regulating AKT activation, and the ectopic expression of activated AKT rescued the growth inhibition. Consistently, NMHC injections in a pancreatic cancer xenograft model manifested the anti-tumor effect in vivo. Engrafted tumor cells underwent AKT attenuation and apoptotic death upon treatments. As such, we here demonstrate the AKT inhibition may be one of the mechanisms by which NMHC decreases tumor cell survival rate in vitro and in vivo. Our data thereby suggest that NMHC holds great promise as a potent chemotherapeutic agent against pancreatic cancer and sheds new light on obtaining such agents from natural products toward therapeutic purposes. - Highlights: • N-methylhemeanthidine chloride (NMHC) is a novel Amaryllidaceae alkaloid. • NMHC exhibits potent anti-neoplastic activity. • NMHC leads to cell cycle arrest, apoptotic death and decreased metabolism. • NMHC down-regulates the AKT signaling pathway.

  4. Direct Sensing of Intracellular pH by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Cl− Channel*♦

    PubMed Central

    Chen, Jeng-Haur; Cai, Zhiwei; Sheppard, David N.

    2009-01-01

    In cystic fibrosis (CF), dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel disrupts epithelial ion transport and perturbs the regulation of intracellular pH (pHi). CFTR modulates pHi through its role as an ion channel and by regulating transport proteins. However, it is unknown how CFTR senses pHi. Here, we investigate the direct effects of pHi on recombinant CFTR using excised membrane patches. By altering channel gating, acidic pHi increased the open probability (Po) of wild-type CFTR, whereas alkaline pHi decreased Po and inhibited Cl− flow through the channel. Acidic pHi potentiated the MgATP dependence of wild-type CFTR by increasing MgATP affinity and enhancing channel activity, whereas alkaline pHi inhibited the MgATP dependence of wild-type CFTR by decreasing channel activity. Because these data suggest that pHi modulates the interaction of MgATP with the nucleotide-binding domains (NBDs) of CFTR, we examined the pHi dependence of site-directed mutations in the two ATP-binding sites of CFTR that are located at the NBD1:NBD2 dimer interface (site 1: K464A-, D572N-, and G1349D-CFTR; site 2: G551D-, K1250M-, and D1370N-CFTR). Site 2 mutants, but not site 1 mutants, perturbed both potentiation by acidic pHi and inhibition by alkaline pHi, suggesting that site 2 is a critical determinant of the pHi sensitivity of CFTR. The effects of pHi also suggest that site 2 might employ substrate-assisted catalysis to ensure that ATP hydrolysis follows NBD dimerization. We conclude that the CFTR Cl− channel senses directly pHi. The direct regulation of CFTR by pHi has important implications for the regulation of epithelial ion transport. PMID:19837660

  5. Embryonic common snapping turtles (Chelydra serpentina) preferentially regulate intracellular tissue pH during acid-base challenges.

    PubMed

    Shartau, Ryan B; Crossley, Dane A; Kohl, Zachary F; Brauner, Colin J

    2016-07-01

    The nests of embryonic turtles naturally experience elevated CO2 (hypercarbia), which leads to increased blood PCO2  and a respiratory acidosis, resulting in reduced blood pH [extracellular pH (pHe)]. Some fishes preferentially regulate tissue pH [intracellular pH (pHi)] against changes in pHe; this has been proposed to be associated with exceptional CO2 tolerance and has never been identified in amniotes. As embryonic turtles may be CO2 tolerant based on nesting strategy, we hypothesized that they preferentially regulate pHi, conferring tolerance to severe acute acid-base challenges. This hypothesis was tested by investigating pH regulation in common snapping turtles (Chelydra serpentina) reared in normoxia then exposed to hypercarbia (13 kPa PCO2 ) for 1 h at three developmental ages: 70% and 90% of incubation, and yearlings. Hypercarbia reduced pHe but not pHi, at all developmental ages. At 70% of incubation, pHe was depressed by 0.324 pH units while pHi of brain, white muscle and lung increased; heart, liver and kidney pHi remained unchanged. At 90% of incubation, pHe was depressed by 0.352 pH units but heart pHi increased with no change in pHi of other tissues. Yearlings exhibited a pHe reduction of 0.235 pH units but had no changes in pHi of any tissues. The results indicate common snapping turtles preferentially regulate pHi during development, but the degree of response is reduced throughout development. This is the first time preferential pHi regulation has been identified in an amniote. These findings may provide insight into the evolution of acid-base homeostasis during development of amniotes, and vertebrates in general. PMID:27091863

  6. Embryonic common snapping turtles (Chelydra serpentina) preferentially regulate intracellular tissue pH during acid-base challenges.

    PubMed

    Shartau, Ryan B; Crossley, Dane A; Kohl, Zachary F; Brauner, Colin J

    2016-07-01

    The nests of embryonic turtles naturally experience elevated CO2 (hypercarbia), which leads to increased blood PCO2  and a respiratory acidosis, resulting in reduced blood pH [extracellular pH (pHe)]. Some fishes preferentially regulate tissue pH [intracellular pH (pHi)] against changes in pHe; this has been proposed to be associated with exceptional CO2 tolerance and has never been identified in amniotes. As embryonic turtles may be CO2 tolerant based on nesting strategy, we hypothesized that they preferentially regulate pHi, conferring tolerance to severe acute acid-base challenges. This hypothesis was tested by investigating pH regulation in common snapping turtles (Chelydra serpentina) reared in normoxia then exposed to hypercarbia (13 kPa PCO2 ) for 1 h at three developmental ages: 70% and 90% of incubation, and yearlings. Hypercarbia reduced pHe but not pHi, at all developmental ages. At 70% of incubation, pHe was depressed by 0.324 pH units while pHi of brain, white muscle and lung increased; heart, liver and kidney pHi remained unchanged. At 90% of incubation, pHe was depressed by 0.352 pH units but heart pHi increased with no change in pHi of other tissues. Yearlings exhibited a pHe reduction of 0.235 pH units but had no changes in pHi of any tissues. The results indicate common snapping turtles preferentially regulate pHi during development, but the degree of response is reduced throughout development. This is the first time preferential pHi regulation has been identified in an amniote. These findings may provide insight into the evolution of acid-base homeostasis during development of amniotes, and vertebrates in general.

  7. Core Amino Acid Residues in the Morphology-Regulating Protein, Mms6, for Intracellular Magnetite Biomineralization

    PubMed Central

    Yamagishi, Ayana; Narumiya, Kaori; Tanaka, Masayoshi; Matsunaga, Tadashi; Arakaki, Atsushi

    2016-01-01

    Living organisms produce finely tuned biomineral architectures with the aid of biomineral-associated proteins. The functional amino acid residues in these proteins have been previously identified using in vitro and in silico experimentation in different biomineralization systems. However, the investigation in living organisms is limited owing to the difficulty in establishing appropriate genetic techniques. Mms6 protein, isolated from the surface of magnetite crystals synthesized in magnetotactic bacteria, was shown to play a key role in the regulation of crystal morphology. In this study, we have demonstrated a defect in the specific region or substituted acidic amino acid residues in the Mms6 protein for observing their effect on magnetite biomineralization in vivo. Analysis of the gene deletion mutants and transformants of Magnetospirillum magneticum AMB-1 expressing partially truncated Mms6 protein revealed that deletions in the N-terminal or C-terminal regions disrupted proper protein localization to the magnetite surface, resulting in a change in the crystal morphology. Moreover, single amino acid substitutions at Asp123, Glu124, or Glu125 in the C-terminal region of Mms6 clearly indicated that these amino acid residues had a direct impact on magnetite crystal morphology. Thus, these consecutive acidic amino acid residues were found to be core residues regulating magnetite crystal morphology. PMID:27759096

  8. Intra-cellular mechanism of Anti-Müllerian hormone (AMH) in regulation of follicular development.

    PubMed

    Hayes, Emily; Kushnir, Vitaly; Ma, Xiaoting; Biswas, Anindita; Prizant, Hen; Gleicher, Norbert; Sen, Aritro

    2016-09-15

    Anti-Müllerian hormone (AMH) is a member of the transforming growth factor-β superfamily and plays a crucial role in testicular and ovarian functions. In clinical practice, AMH is used as a diagnostic and/or prognostic marker in women in association with ovulation induction and in various pathophysiological conditions. Despite widespread clinical use of AMH, our mechanistic understanding of AMH actions in regulating follicular development is limited. Using a mouse model, we in this study report that in vivo AMH treatment while stalls follicular development and inhibits ovulation, also prevents follicular atresia. We further show that these AMH actions are mediated through induction of two miRNAs, miR-181a and miR-181b, which regulate various aspects of FSH signaling and follicular growth, ultimately affecting downstream gene expression and folliculogenesis. We also report that in this mouse model AMH pre-treatment prior to superovulation improves oocyte yield. These studies, therefore, offer new mechanistic insight into AMH actions in folliculogenesis and point toward potential utilization of AMH as a therapeutic agent. PMID:27235859

  9. Neprilysin and Aβ Clearance: Impact of the APP Intracellular Domain in NEP Regulation and Implications in Alzheimer’s Disease

    PubMed Central

    Grimm, Marcus O. W.; Mett, Janine; Stahlmann, Christoph P.; Haupenthal, Viola J.; Zimmer, Valerie C.; Hartmann, Tobias

    2013-01-01

    One of the characteristic hallmarks of Alzheimer’s disease (AD) is an accumulation of amyloid β (Aβ) leading to plaque formation and toxic oligomeric Aβ complexes. Besides the de novo synthesis of Aβ caused by amyloidogenic processing of the amyloid precursor protein (APP), Aβ levels are also highly dependent on Aβ degradation. Several enzymes are described to cleave Aβ. In this review we focus on one of the most prominent Aβ degrading enzymes, the zinc-metalloprotease Neprilysin (NEP). In the first part of the review we discuss beside the general role of NEP in Aβ degradation the alterations of the enzyme observed during normal aging and the progression of AD. In vivo and cell culture experiments reveal that a decreased NEP level results in an increased Aβ level and vice versa. In a pathological situation like AD, it has been reported that NEP levels and activity are decreased and it has been suggested that certain polymorphisms in the NEP gene result in an increased risk for AD. Conversely, increasing NEP activity in AD mouse models revealed an improvement in some behavioral tests. Therefore it has been suggested that increasing NEP might be an interesting potential target to treat or to be protective for AD making it indispensable to understand the regulation of NEP. Interestingly, it is discussed that the APP intracellular domain (AICD), one of the cleavage products of APP processing, which has high similarities to Notch receptor processing, might be involved in the transcriptional regulation of NEP. However, the mechanisms of NEP regulation by AICD, which might be helpful to develop new therapeutic strategies, are up to now controversially discussed and summarized in the second part of this review. In addition, we review the impact of AICD not only in the transcriptional regulation of NEP but also of further genes. PMID:24391587

  10. Role of Sodium Bicarbonate Cotransporters in Intracellular pH Regulation and Their Regulatory Mechanisms in Human Submandibular Glands.

    PubMed

    Namkoong, Eun; Shin, Yong-Hwan; Bae, Jun-Seok; Choi, Seulki; Kim, Minkyoung; Kim, Nahyun; Hwang, Sung-Min; Park, Kyungpyo

    2015-01-01

    Sodium bicarbonate cotransporters (NBCs) are involved in the pH regulation of salivary glands. However, the roles and regulatory mechanisms among different NBC isotypes have not been rigorously evaluated. We investigated the roles of two different types of NBCs, electroneutral (NBCn1) and electrogenic NBC (NBCe1), with respect to pH regulation and regulatory mechanisms using human submandibular glands (hSMGs) and HSG cells. Intracellular pH (pHi) was measured and the pHi recovery rate from cell acidification induced by an NH4Cl pulse was recorded. Subcellular localization and protein phosphorylation were determined using immunohistochemistry and co-immunoprecipitation techniques. We determined that NBCn1 is expressed on the basolateral side of acinar cells and the apical side of duct cells, while NBCe1 is exclusively expressed on the apical membrane of duct cells. The pHi recovery rate in hSMG acinar cells, which only express NBCn1, was not affected by pre-incubation with 5 μM PP2, an Src tyrosine kinase inhibitor. However, in HSG cells, which express both NBCe1 and NBCn1, the pHi recovery rate was inhibited by PP2. The apparent difference in regulatory mechanisms for NBCn1 and NBCe1 was evaluated by artificial overexpression of NBCn1 or NBCe1 in HSG cells, which revealed that the pHi recovery rate was only inhibited by PP2 in cells overexpressing NBCe1. Furthermore, only NBCe1 was significantly phosphorylated and translocated by NH4Cl, which was inhibited by PP2. Our results suggest that both NBCn1 and NBCe1 play a role in pHi regulation in hSMG acinar cells, and also that Src kinase does not regulate the activity of NBCn1.

  11. Murine MicroRNA-214 regulates intracellular adhesion molecule (ICAM1) gene expression in genital Chlamydia muridarum infection

    PubMed Central

    Arkatkar, Tanvi; Gupta, Rishein; Li, Weidang; Yu, Jieh-Juen; Wali, Shradha; Neal Guentzel, M; Chambers, James P; Christenson, Lane K; Arulanandam, Bernard P

    2015-01-01

    The hallmark of chlamydial infection is the development of upper genital pathology in the form of hydrosalpinx and oviduct and/or tubal dilatation. Although molecular events leading to genital tissue presentation and cellular architectural remodelling are unclear, early-stage host immune responses are believed to contribute to these long-term sequelae. Recently, we reported the contribution of selected infection-associated microRNAs (miRs) in the generation of host immunity at early-stage infection (day 6 after intravaginal Chlamydia muridarum challenge in C57BL/6 mice). In this report, we describe the contribution of an infection-associated microRNA, i.e. miR-214, to host immunity. Chlamydia muridarum infection in the C57BL/6 mouse genital tract significantly down-regulated miR-214 while up-regulating intracellular adhesion molecule 1 (ICAM1) gene expression. These in vivo observations were confirmed by establishing direct regulation of ICAM-1 by miR-214 in ex vivo genital cell cultures in the presence of miR-214 mimic and inhibitor. Because, ICAM-1 contributes to recruitment of neutrophils following infection, we also demonstrated that alteration of ICAM1 by miR-214 in interleukin-17A-deficient (IL-17A−/−) mice correlated with reduction of neutrophils infiltrating genital tissue at day 6 after challenge. Additionally, these early-stage events resulted in significantly decreased genital pathology in IL-17A−/− mice compared with C57BL/6 mice. This report provides evidence for early-stage regulation of ICAM1 by microRNAs, resulting in reduction of genital pathology associated with chlamydial infection. PMID:25865776

  12. Aphid amino acid transporter regulates glutamine supply to intracellular bacterial symbionts.

    PubMed

    Price, Daniel R G; Feng, Honglin; Baker, James D; Bavan, Selvan; Luetje, Charles W; Wilson, Alex C C

    2014-01-01

    Endosymbiotic associations have played a major role in evolution. However, the molecular basis for the biochemical interdependence of these associations remains poorly understood. The aphid-Buchnera endosymbiosis provides a powerful system to elucidate how these symbioses are regulated. In aphids, the supply of essential amino acids depends on an ancient nutritional symbiotic association with the gamma-proteobacterium Buchnera aphidicola. Buchnera cells are densely packed in specialized aphid bacteriocyte cells. Here we confirm that five putative amino acid transporters are highly expressed and/or highly enriched in Acyrthosiphon pisum bacteriocyte tissues. When expressed in Xenopus laevis oocytes, two bacteriocyte amino acid transporters displayed significant levels of glutamine uptake, with transporter ACYPI001018, LOC100159667 (named here as Acyrthosiphon pisum glutamine transporter 1, ApGLNT1) functioning as the most active glutamine transporter. Transporter ApGLNT1 has narrow substrate selectivity, with high glutamine and low arginine transport capacity. Notably, ApGLNT1 has high binding affinity for arginine, and arginine acts as a competitive inhibitor for glutamine transport. Using immunocytochemistry, we show that ApGLNT1 is localized predominantly to the bacteriocyte plasma membrane, a location consistent with the transport of glutamine from A. pisum hemolymph to the bacteriocyte cytoplasm. On the basis of functional transport data and localization, we propose a substrate feedback inhibition model in which the accumulation of the essential amino acid arginine in A. pisum hemolymph reduces the transport of the precursor glutamine into bacteriocytes, thereby regulating amino acid biosynthesis in the bacteriocyte. Structural similarities in the arrangement of hosts and symbionts across endosymbiotic systems suggest that substrate feedback inhibition may be mechanistically important in other endosymbioses.

  13. Differential regulation of Sciaenops ocellatus viperin expression by intracellular and extracellular bacterial pathogens.

    PubMed

    Dang, Wei; Zhang, Min; Hu, Yong-hua; Sun, Li

    2010-08-01

    Viperin is an antiviral protein that has been found to exist in diverse vertebrate organisms and is involved in innate immunity against the infection of a wide range of viruses. However, it is largely unclear as to the potential role played by viperin in bacterial infection. In this study, we identified the red drum Sciaenops ocellatus viperin gene (SoVip) and analyzed its expression in relation to bacterial challenge. The complete gene of SoVip is 2570 bp in length and contains six exons and five introns. The open reading frame of SoVip is 1065 bp, which is flanked by a 5'-untranslated region (UTR) of 34 bp and a 3'-UTR of 350 bp. The deduced amino acid sequence of SoVip shares extensive identities with the viperins of several fish species and possesses the conserved domain of the radical S-adenosylmethionine superfamily proteins. Expressional analysis showed that constitutive expression of SoVip was relatively high in blood, muscle, brain, spleen, and liver, and low in kidney, gill, and heart. Experimental challenges with poly(I:C) and bacterial pathogens indicated that SoVip expression in liver was significantly upregulated by poly(I:C) and the fish pathogen Edwardsiella tarda but down-regulated by the fish pathogens Listonella anguillarum and Streptococcus iniae. Similar differential induction patterns were also observed at cellular level with primary hepatocytes challenged with E. tarda, L. anguillarum, and S. iniae. Infection study showed that all three bacterial pathogens could attach to cultured primary hepatocytes but only E. tarda was able to invade into and survive in hepatocytes. Together these results indicate that SoVip is involved in host immune response during bacterial infection and is differentially regulated at transcription level by different bacterial pathogens. PMID:20420911

  14. Intracellular distribution of differentially phosphorylated dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A).

    PubMed

    Kaczmarski, Wojciech; Barua, Madhabi; Mazur-Kolecka, Bozena; Frackowiak, Janusz; Dowjat, Wieslaw; Mehta, Pankaj; Bolton, David; Hwang, Yu-Wen; Rabe, Ausma; Albertini, Giorgio; Wegiel, Jerzy

    2014-02-01

    The gene encoding dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is located within the Down syndrome (DS) critical region of chromosome 21. DYRK1A interacts with a plethora of substrates in the cytosol, cytoskeleton, and nucleus. Its overexpression is a contributing factor to the developmental alterations and age-associated pathology observed in DS. We hypothesized that the intracellular distribution of DYRK1A and cell-compartment-specific functions are associated with DYRK1A posttranslational modifications. Fractionation showed that, in both human and mouse brain, almost 80% of DYRK1A was associated with the cytoskeleton, and the remaining DYRK1A was present in the cytosolic and nuclear fractions. Coimmunoprecipitation revealed that DYRK1A in the brain cytoskeleton fraction forms complexes with filamentous actin, neurofilaments, and tubulin. Two-dimensional gel analysis of the fractions revealed DYRK1A with distinct isoelectric points: 5.5-6.5 in the nucleus, 7.2-8.2 in the cytoskeleton, and 8.7 in the cytosol. Phosphate-affinity gel electrophoresis demonstrated several bands of DYRK1A with different mobility shifts for nuclear, cytoskeletal, and cytosolic DYRK1A, indicating modification by phosphorylation. Mass spectrometry analysis disclosed one phosphorylated site in the cytosolic DYRK1A and multiple phosphorylated residues in the cytoskeletal DYRK1A, including two not previously described. This study supports the hypothesis that intracellular distribution and compartment-specific functions of DYRK1A may depend on its phosphorylation pattern. PMID:24327345

  15. The GEF1 proton-chloride exchanger affects tombusvirus replication via regulation of copper metabolism in yeast.

    PubMed

    Sasvari, Zsuzsanna; Kovalev, Nikolay; Nagy, Peter D

    2013-02-01

    Replication of plus-strand RNA viruses [(+)RNA viruses] is performed by viral replicases, whose function is affected by many cellular factors in infected cells. In this paper, we demonstrate a surprising role for Gef1p proton-chloride exchanger in replication of Tomato bushy stunt virus (TBSV) model (+)RNA virus. A genetic approach revealed that Gef1p, which is the only proton-chloride exchanger in Saccharomyces cerevisiae, is required for TBSV replication in the yeast model host. We also show that the in vitro activity of the purified tombusvirus replicase from gef1Δ yeast was low and that the in vitro assembly of the viral replicase in a cell extract was inhibited by the cytosolic fraction obtained from gef1Δ yeast. Altogether, our data reveal that Gef1p modulates TBSV replication via regulating Cu(2+) metabolism in the cell. This conclusion is supported by several lines of evidence, including the direct inhibitory effect of Cu(2+) ions on the in vitro assembly of the viral replicase, on the activity of the viral RNA-dependent RNA polymerase, and an inhibitory effect of deletion of CCC2 copper pump on TBSV replication in yeast, while altered iron metabolism did not reduce TBSV replication. In addition, applying a chloride channel blocker impeded TBSV replication in Nicotiana benthamiana protoplasts or in whole plants. Overall, blocking Gef1p function seems to inhibit TBSV replication through altering Cu(2+) ion metabolism in the cytosol, which then inhibits the normal functions of the viral replicase.

  16. Regulating toxin-antitoxin expression: controlled detonation of intracellular molecular timebombs.

    PubMed

    Hayes, Finbarr; Kędzierska, Barbara

    2014-01-15

    Genes for toxin-antitoxin (TA) complexes are widely disseminated in bacteria, including in pathogenic and antibiotic resistant species. The toxins are liberated from association with the cognate antitoxins by certain physiological triggers to impair vital cellular functions. TAs also are implicated in antibiotic persistence, biofilm formation, and bacteriophage resistance. Among the ever increasing number of TA modules that have been identified, the most numerous are complexes in which both toxin and antitoxin are proteins. Transcriptional autoregulation of the operons encoding these complexes is key to ensuring balanced TA production and to prevent inadvertent toxin release. Control typically is exerted by binding of the antitoxin to regulatory sequences upstream of the operons. The toxin protein commonly works as a transcriptional corepressor that remodels and stabilizes the antitoxin. However, there are notable exceptions to this paradigm. Moreover, it is becoming clear that TA complexes often form one strand in an interconnected web of stress responses suggesting that their transcriptional regulation may prove to be more intricate than currently understood. Furthermore, interference with TA gene transcriptional autoregulation holds considerable promise as a novel antibacterial strategy: artificial release of the toxin factor using designer drugs is a potential approach to induce bacterial suicide from within.

  17. Induction of DKK1 by ox-LDL negatively regulates intracellular lipid accumulation in macrophages.

    PubMed

    Zhang, Yu; Ge, Cheng; Wang, Lin; Liu, Xinxin; Chen, Yifei; Li, Mengmeng; Zhang, Mei

    2015-01-01

    Dickkopf1 (DKK1), a canonical Wnt/β-catenin pathway antagonist, is closely associated with cardiovascular disease and adipogenesis. We performed an in vitro study to determine whether oxidized low-density lipoprotein (ox-LDL) increased the expression of DKK1 in macrophages and whether β-catenin and liver X receptor α (LXRα) were involved in this regulation. Induction of DKK1 expression by ox-LDL decreased the level of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) via a Wnt/β-catenin pathway and increased ATP-binding cassette transporter A/G1 (ABCA/G1) levels via a signal transducer and activator of transcription 3 (STAT3) pathway. Lower LOX-1 and higher ABCA/G1 levels inhibited cholesterol loading in macrophages. In conclusion, ox-LDL may induce DKK1 expression in macrophages to inhibit the accumulation of lipids through a mechanism that involves downregulation of LOX-1-mediated lipid uptake and upregulation of ABCA/G1-dependent cholesterol efflux.

  18. 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.

  19. Induction of DKK1 by ox-LDL negatively regulates intracellular lipid accumulation in macrophages.

    PubMed

    Zhang, Yu; Ge, Cheng; Wang, Lin; Liu, Xinxin; Chen, Yifei; Li, Mengmeng; Zhang, Mei

    2015-01-01

    Dickkopf1 (DKK1), a canonical Wnt/β-catenin pathway antagonist, is closely associated with cardiovascular disease and adipogenesis. We performed an in vitro study to determine whether oxidized low-density lipoprotein (ox-LDL) increased the expression of DKK1 in macrophages and whether β-catenin and liver X receptor α (LXRα) were involved in this regulation. Induction of DKK1 expression by ox-LDL decreased the level of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) via a Wnt/β-catenin pathway and increased ATP-binding cassette transporter A/G1 (ABCA/G1) levels via a signal transducer and activator of transcription 3 (STAT3) pathway. Lower LOX-1 and higher ABCA/G1 levels inhibited cholesterol loading in macrophages. In conclusion, ox-LDL may induce DKK1 expression in macrophages to inhibit the accumulation of lipids through a mechanism that involves downregulation of LOX-1-mediated lipid uptake and upregulation of ABCA/G1-dependent cholesterol efflux. PMID:25436422

  20. Intracellular protein O-GlcNAc modification integrates nutrient status with transcriptional and metabolic regulation.

    PubMed

    Nagel, Alexis K; Ball, Lauren E

    2015-01-01

    The inducible, nutrient-sensitive posttranslational modification of protein Ser/Thr residues with O-linked β-N-acetylglucosamine (O-GlcNAc) occurs on histones, transcriptional regulators, metabolic enzymes, oncogenes, tumor suppressors, and many critical intermediates of growth factor signaling. Cycling of O-GlcNAc modification on and off of protein substrates is catalyzed by the actions of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. To date, there are less than 150 publications addressing the role of O-GlcNAc modification in cancer and over half were published in the last 2 years. These studies have clearly established that increased expression of OGT and hyper-O-GlcNAcylation is common to human cancers of breast, prostate, colon, lung, and pancreas. Furthermore, attenuating OGT activity reduces tumor growth in vitro and metastasis in vivo. This chapter discusses the structure and function of the O-GlcNAc cycling enzymes, mechanisms by which protein O-GlcNAc modification sense changes in nutrient status, the influence of O-GlcNAc cycling enzymes on glucose metabolism, and provides an overview of recent observations regarding the role of O-GlcNAcylation in cancer.

  1. Chemotaxis behavior toward an odor is regulated by constant sodium chloride stimulus in Caenorhabditis elegans.

    PubMed

    Shingai, Ryuzo; Ichijo, Hiroshi; Wakabayashi, Tokumitsu; Tanaka, Hidetoshi; Ogurusu, Tarou

    2014-01-01

    We studied the chemotaxis behavior of Caenorhabditis elegans toward a chemoattractant in the presence of background sensory stimulus. Chemotaxis toward an odor butanone was greater in the presence of sodium chloride (NaCl) than that without NaCl. By contrast, chemotaxis toward NaCl was not affected by a butanone background. The salt-sensing ASE neuron-deficient che-1(p674) mutants and worms with ASE genetically ablated showed high chemotaxis toward butanone, regardless of the presence of a NaCl background. Therefore, in wild-type worms, information from ASE in the absence of NaCl suppresses butanone chemotaxis, while the suppression is removed in the presence of NaCl.

  2. Effects of energy deficit, dietary protein, and feeding on intracellular regulators of skeletal muscle proteolysis.

    PubMed

    Carbone, John W; Margolis, Lee M; McClung, James P; Cao, Jay J; Murphy, Nancy E; Sauter, Edward R; Combs, Gerald F; Young, Andrew J; Pasiakos, Stefan M

    2013-12-01

    This study was undertaken to characterize the ubiquitin proteasome system (UPS) response to varied dietary protein intake, energy deficit (ED), and consumption of a mixed meal. A randomized, controlled trial of 39 adults consuming protein at 0.8 (recommended dietary allowance [RDA]), 1.6 (2×-RDA), or 2.4 (3×-RDA) g · kg(-1) · d(-1) for 31 d. A 10-d weight maintenance (WM) period was followed by 21 d of 40% ED. Ubiquitin (Ub)-mediated proteolysis and associated gene expression were assessed in the postabsorptive (fasted) and postprandial (fed; 480 kcal, 20 g protein) states after WM and ED by using muscle biopsies, fluorescence-based assays, immunoblot analysis, and real-time qRT-PCR. In the assessment of UPS responses to varied protein intakes, ED, and feeding, the RDA, WM, and fasted measures served as appropriate controls. ED resulted in the up-regulation of UPS-associated gene expression, as mRNA expression of the atrogenes muscle RING finger-1 (MuRF1) and atrogin-1 were 1.2- and 1.3-fold higher (P<0.05) for ED than for WM. However, mixed-meal consumption attenuated UPS-mediated proteolysis, independent of energy status or dietary protein, as the activities of the 26S proteasome subunits β1, β2, and β5 were lower (P<0.05) for fed than for fasted. Muscle protein ubiquitylation was also 45% lower (P<0.05) for fed than for fasted, regardless of dietary protein and energy manipulations. Independent of habitual protein intake and despite increased MuRF1 and atrogin-1 mRNA expression during ED, consuming a protein-containing mixed meal attenuates Ub-mediated proteolysis.

  3. Effects of energy deficit, dietary protein, and feeding on intracellular regulators of skeletal muscle proteolysis.

    PubMed

    Carbone, John W; Margolis, Lee M; McClung, James P; Cao, Jay J; Murphy, Nancy E; Sauter, Edward R; Combs, Gerald F; Young, Andrew J; Pasiakos, Stefan M

    2013-12-01

    This study was undertaken to characterize the ubiquitin proteasome system (UPS) response to varied dietary protein intake, energy deficit (ED), and consumption of a mixed meal. A randomized, controlled trial of 39 adults consuming protein at 0.8 (recommended dietary allowance [RDA]), 1.6 (2×-RDA), or 2.4 (3×-RDA) g · kg(-1) · d(-1) for 31 d. A 10-d weight maintenance (WM) period was followed by 21 d of 40% ED. Ubiquitin (Ub)-mediated proteolysis and associated gene expression were assessed in the postabsorptive (fasted) and postprandial (fed; 480 kcal, 20 g protein) states after WM and ED by using muscle biopsies, fluorescence-based assays, immunoblot analysis, and real-time qRT-PCR. In the assessment of UPS responses to varied protein intakes, ED, and feeding, the RDA, WM, and fasted measures served as appropriate controls. ED resulted in the up-regulation of UPS-associated gene expression, as mRNA expression of the atrogenes muscle RING finger-1 (MuRF1) and atrogin-1 were 1.2- and 1.3-fold higher (P<0.05) for ED than for WM. However, mixed-meal consumption attenuated UPS-mediated proteolysis, independent of energy status or dietary protein, as the activities of the 26S proteasome subunits β1, β2, and β5 were lower (P<0.05) for fed than for fasted. Muscle protein ubiquitylation was also 45% lower (P<0.05) for fed than for fasted, regardless of dietary protein and energy manipulations. Independent of habitual protein intake and despite increased MuRF1 and atrogin-1 mRNA expression during ED, consuming a protein-containing mixed meal attenuates Ub-mediated proteolysis. PMID:23965841

  4. Apical membrane sodium and chloride entry during osmotic swelling of renal (A6) epithelial cells.

    PubMed

    Crowe, W E; Ehrenfeld, J; Brochiero, E; Wills, N K

    1995-03-01

    To assess the role of chloride in cell volume and sodium transport regulation, we measured cell height changes (CH), transepithelial chloride and sodium fluxes, and intracellular chloride content during challenge with hyposmotic solutions under open circuit (OC) conditions. CH maximally increased following hyposmotic challenge within approximately 5 minutes. The change in CH was smaller under short circuit (SC) conditions or following replacement of chloride in the mucosal solution by gluconate or cyclamate (Cl(-)-freem). When corrected for the osmotically inactive cell volume (30 +/- 2%), delta CH for controls (OC) were greater than predicted for an ideal osmometer. In contrast, delta CH for Cl(-)-freem or SC conditions were similar to that predicted for an ideal osmometer. Na+ and Cl- mucosa-to-serosa fluxes increased following hyposmotic challenge. Chloride fluxes increased maximally within 5 min, then decreased. In contrast, the Na+ flux increased slowly and reached a steady state after approximately 25 min. Under isosmotic conditions, exposure to Cl(-)-freem solutions led to decreases in the transepithelial conductance, Na+ flux, and CH. Chloride permeabilities in the apical and basolateral membranes were detected using the fluorescent intracellular chloride indicator MQAE. The results indicate that during osmotic swelling, the entry of both sodium and chloride is increased. The time courses of these increases differ, suggesting distinct mechanisms for the osmotic regulation of these apical membrane transport processes. PMID:7541082

  5. Fasting and postprandial regulation of the intracellular localization of adiponectin and of adipokines secretion by dietary fat in rats

    PubMed Central

    Olivares-García, V; Torre-Villalvazo, I; Velázquez-Villegas, L; Alemán, G; Lara, N; López-Romero, P; Torres, N; Tovar, A R; Díaz-Villaseñor, A

    2015-01-01

    Background/Objective: Dietary fat sources modulate fasting serum concentration of adipokines, particularly adiponectin. However, previous studies utilized obese animals in which adipose tissue function is severely altered. Thus, the present study aimed to assess the postprandial regulation of adipokine secretion in nonobese rats that consumed high-fat diet (HFD) composed of different types of fat for a short time. Methods: The rats were fed a control diet or a HFD containing coconut, safflower or soybean oil (rich in saturated fatty acid, monounsaturated fatty acid or polyunsaturated fatty acid, respectively) for 21 days. The serum concentrations of adiponectin, leptin, retinol, retinol-binding protein-4 (RBP-4), visfatin and resistin were determined at fasting and after refeeding. Adiponectin multimerization and intracellular localization, as well as the expression of endoplasmic reticulum (ER) chaperones and transcriptional regulators, were evaluated in epididymal white adipose tissue. Results: In HFD-fed rats, serum adiponectin was significantly decreased 30 min after refeeding. With coconut oil, all three multimeric forms were reduced; with safflower oil, only the high-molecular-weight (HMW) and medium-molecular-weight (MMW) forms were decreased; and with soybean oil, only the HMW form was diminished. These reductions were due not to modifications in mRNA abundance or adiponectin multimerization but rather to an increment in intracellular localization at the ER and plasma membrane. Thus, when rats consumed a HFD, the type of dietary fat differentially affected the abundance of endoplasmic reticulum resident protein 44 kDa (ERp44), sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor-γ (PPARγ) mRNAs, all of which are involved in the post-translational processing of adiponectin required for its secretion. Leptin, RBP-4, resistin and visfatin serum concentrations did not change during fasting, whereas modest alterations were observed after

  6. Volatile profiling reveals intracellular metabolic changes in Aspergillus parasiticus: veA regulates branched chain amino acid and ethanol metabolism

    PubMed Central

    2010-01-01

    Background Filamentous fungi in the genus Aspergillus produce a variety of natural products, including aflatoxin, the most potent naturally occurring carcinogen known. Aflatoxin biosynthesis, one of the most highly characterized secondary metabolic pathways, offers a model system to study secondary metabolism in eukaryotes. To control or customize biosynthesis of natural products we must understand how secondary metabolism integrates into the overall cellular metabolic network. By applying a metabolomics approach we analyzed volatile compounds synthesized by Aspergillus parasiticus in an attempt to define the association of secondary metabolism with other metabolic and cellular processes. Results Volatile compounds were examined using solid phase microextraction - gas chromatography/mass spectrometry. In the wild type strain Aspergillus parasiticus SU-1, the largest group of volatiles included compounds derived from catabolism of branched chain amino acids (leucine, isoleucine, and valine); we also identified alcohols, esters, aldehydes, and lipid-derived volatiles. The number and quantity of the volatiles produced depended on media composition, time of incubation, and light-dark status. A block in aflatoxin biosynthesis or disruption of the global regulator veA affected the volatile profile. In addition to its multiple functions in secondary metabolism and development, VeA negatively regulated catabolism of branched chain amino acids and synthesis of ethanol at the transcriptional level thus playing a role in controlling carbon flow within the cell. Finally, we demonstrated that volatiles generated by a veA disruption mutant are part of the complex regulatory machinery that mediates the effects of VeA on asexual conidiation and sclerotia formation. Conclusions 1) Volatile profiling provides a rapid, effective, and powerful approach to identify changes in intracellular metabolic networks in filamentous fungi. 2) VeA coordinates the biosynthesis of secondary

  7. Intracellular pH-regulating mechanism of the squid axon. Interaction between DNDS and extracellular Na+ and HCO3-

    PubMed Central

    1989-01-01

    Intracellular pH (pHi) of the squid axon is regulated by a stilbenesensitive transporter that couples the influx of Na+ and HCO3- (or the equivalent) to the efflux of Cl-. According to one model, the extracellular ion pair NaCO3- exchanges for intracellular Cl-. In the present study, the ion-pair model was tested by examining the interaction of the reversible stilbene derivative 4,4'-dinitrostilbene- 2,2'-disulfonate (DNDS) with extracellular Na+ and HCO3-. Axons (initial pHi approximately 7.4) were internally dialyzed with a pH 6.5 solution containing 400 mM Cl- but no Na+. After pHi, as measured with a glass microelectrode, had fallen to approximately 6.6, dialysis was halted. In the presence of both external Na+ and HCO3- (pHo = 8.0, 22 degrees C), pHi increased due to the pHi-regulating mechanism. At a fixed [Na+]o of 425 mM and [HCO3-]o of 12 mM, DNDS reversibly reduced the equivalent acid-extrusion rate (JH) calculated from the rate of pHi recovery. The best-fit value for maximal inhibition was 104%, and for the [DNDS]o at half-maximal inhibition, 0.3 mM. At a [Na+]o of 425 mM, the [HCO3-]o dependence of JH was examined at 0, 0.1, and 0.25 mM DNDS. Although Jmax was always approximately 20 pmol cm-2 s-1, Km(HCO3-) was 2.6, 5.7, and 12.7 mM, respectively. Thus, DNDS is competitive with HCO3-. At a [HCO3-]o of 12 mM, the [Na+]o dependence of JH was examined at 0 and 0.1 mM DNDS. Although Jmax was approximately 20 pmol cm-2 s-1 in both cases, Km(Na+) was 71 and 179 mM, respectively. At a [HCO3-]o of 48 mM, Jmax was approximately 20 pmol cm-2 s-1 at [DNDS]o levels of 0, 0.1, and 0.25 mM. However, Km(Na+) was 22, 45, and 90 mM, respectively. Thus, DNDS (an anion) is also competitive with Na+. The results are consistent with simple competition between DNDS and NaCO3-, and place severe restrictions on other kinetic models. PMID:2915212

  8. Regulation of L-type calcium current by intracellular magnesium in rat cardiac myocytes

    PubMed Central

    Wang, Min; Tashiro, Michiko; Berlin, Joshua R

    2004-01-01

    calcineurin. Thus, physiologically relevant [Mg2+]i modulates ICa by counteracting the effects of Ca2+ channel phosphorylation and by an unknown [Ca2+]i-dependent mechanism. The magnitude of these effects suggests that changes in [Mg2+]i could be critical in regulating l-type channel gating. PMID:14617671

  9. Role of H(+)-pyrophosphatase activity in the regulation of intracellular pH in a scuticociliate parasite of turbot: Physiological effects.

    PubMed

    Mallo, Natalia; Lamas, Jesús; de Felipe, Ana-Paula; Sueiro, Rosa-Ana; Fontenla, Francisco; Leiro, José-Manuel

    2016-10-01

    The scuticociliatosis is a very serious disease that affects the cultured turbot, and whose causal agent is the anphizoic and marine euryhaline ciliate Philasterides dicentrarchi. Several protozoans possess acidic organelles that contain high concentrations of pyrophosphate (PPi), Ca(2+) and other elements with essential roles in vesicular trafficking, pH homeostasis and osmoregulation. P. dicentrarchi possesses a pyrophosphatase (H(+)-PPase) that pumps H(+) through the membranes of vacuolar and alveolar sacs. These compartments share common features with the acidocalcisomes described in other parasitic protozoa (e.g. acid content and Ca(2+) storage). We evaluated the effects of Ca(2+) and ATP on H (+)-PPase activity in this ciliate and analyzed their role in maintaining intracellular pH homeostasis and osmoregulation, by the addition of PPi and inorganic molecules that affect osmolarity. Addition of PPi led to acidification of the intracellular compartments, while the addition of ATP, CaCl2 and bisphosphonates analogous of PPi and Ca(2+) metabolism regulators led to alkalinization and a decrease in H(+)-PPase expression in trophozoites. Addition of NaCl led to proton release, intracellular Ca(2+) accumulation and downregulation of H(+)-PPase expression. We conclude that the regulation of the acidification of intracellular compartments may be essential for maintaining the intracellular pH homeostasis necessary for survival of ciliates and their adaptation to salt stress, which they will presumably face during the endoparasitic phase, in which the salinity levels are lower than in their natural environment.

  10. Role of H(+)-pyrophosphatase activity in the regulation of intracellular pH in a scuticociliate parasite of turbot: Physiological effects.

    PubMed

    Mallo, Natalia; Lamas, Jesús; de Felipe, Ana-Paula; Sueiro, Rosa-Ana; Fontenla, Francisco; Leiro, José-Manuel

    2016-10-01

    The scuticociliatosis is a very serious disease that affects the cultured turbot, and whose causal agent is the anphizoic and marine euryhaline ciliate Philasterides dicentrarchi. Several protozoans possess acidic organelles that contain high concentrations of pyrophosphate (PPi), Ca(2+) and other elements with essential roles in vesicular trafficking, pH homeostasis and osmoregulation. P. dicentrarchi possesses a pyrophosphatase (H(+)-PPase) that pumps H(+) through the membranes of vacuolar and alveolar sacs. These compartments share common features with the acidocalcisomes described in other parasitic protozoa (e.g. acid content and Ca(2+) storage). We evaluated the effects of Ca(2+) and ATP on H (+)-PPase activity in this ciliate and analyzed their role in maintaining intracellular pH homeostasis and osmoregulation, by the addition of PPi and inorganic molecules that affect osmolarity. Addition of PPi led to acidification of the intracellular compartments, while the addition of ATP, CaCl2 and bisphosphonates analogous of PPi and Ca(2+) metabolism regulators led to alkalinization and a decrease in H(+)-PPase expression in trophozoites. Addition of NaCl led to proton release, intracellular Ca(2+) accumulation and downregulation of H(+)-PPase expression. We conclude that the regulation of the acidification of intracellular compartments may be essential for maintaining the intracellular pH homeostasis necessary for survival of ciliates and their adaptation to salt stress, which they will presumably face during the endoparasitic phase, in which the salinity levels are lower than in their natural environment. PMID:27480055

  11. Idh1 protects murine hepatocytes from endotoxin-induced oxidative stress by regulating the intracellular NADP(+)/NADPH ratio.

    PubMed

    Itsumi, M; Inoue, S; Elia, A J; Murakami, K; Sasaki, M; Lind, E F; Brenner, D; Harris, I S; Chio, I I C; Afzal, S; Cairns, R A; Cescon, D W; Elford, A R; Ye, J; Lang, P A; Li, W Y; Wakeham, A; Duncan, G S; Haight, J; You-Ten, A; Snow, B; Yamamoto, K; Ohashi, P S; Mak, T W

    2015-11-01

    Isocitrate dehydrogenase-1 (Idh1) is an important metabolic enzyme that produces NADPH by converting isocitrate to α-ketoglutarate. Idh1 is known to reduce reactive oxygen species (ROS) induced in cells by treatment with lipopolysaccharide (LPS) in vitro. Here, we used Idh1-deficient knockout (Idh1 KO) mice to investigate the role of Idh1 in antioxidant defense in vivo. Idh1 KO mice showed heightened susceptibility to death induced by LPS and exhibited increased serum levels of inflammatory cytokines such as tumor necrosis factor-α and interleukin-6. The serum of LPS-injected Idh1 KO mice also contained elevated levels of AST, a marker of inflammatory liver damage. Furthermore, after LPS injection, livers of Idh1 KO mice showed histological evidence of elevated oxidative DNA damage compared with livers of wild-type (WT) mice. Idh1 KO livers showed a faster and more pronounced oxidative stress than WT livers. In line with that, Idh1 KO hepatocytes showed higher ROS levels and an increase in the NADP(+)/NADPH ratio when compared with hepatocytes isolated from WT mice. These results suggest that Idh1 has a physiological function in protecting cells from oxidative stress by regulating the intracellular NADP(+)/NADPH ratio. Our findings suggest that stimulation of Idh1 activity may be an effective therapeutic strategy for reducing oxidative stress during inflammatory responses, including the early stages of septic shock.

  12. Lysosome-associated membrane proteins (LAMPs) regulate intracellular positioning of mitochondria in MC3T3-E1 cells.

    PubMed

    Rajapakshe, Anupama R; Podyma-Inoue, Katarzyna A; Terasawa, Kazue; Hasegawa, Katsuya; Namba, Toshimitsu; Kumei, Yasuhiro; Yanagishita, Masaki; Hara-Yokoyama, Miki

    2015-02-01

    The intracellular positioning of both lysosomes and mitochondria meets the requirements of degradation and energy supply, which are respectively the two major functions for cellular maintenance. The positioning of both lysosomes and mitochondria is apparently affected by the nutrient status of the cells. However, the mechanism coordinating the positioning of the organelles has not been sufficiently elucidated. Lysosome-associated membrane proteins-1 and -2 (LAMP-1 and LAMP-2) are highly glycosylated proteins that are abundant in lysosomal membranes. In the present study, we demonstrated that the siRNA-mediated downregulation of LAMP-1, LAMP-2 or their combination enhanced the perinuclear localization of mitochondria, in the pre-osteoblastic cell line MC3T3-E1. On the other hand, in the osteocytic cell line MLO-Y4, in which both the lysosomes and mitochondria originally accumulate in the perinuclear region and mitochondria also fill dendrites, the effect of siRNA of LAMP-1 or LAMP-2 was barely observed. LAMPs are not directly associated with mitochondria, and there do not seem to be any accessory molecules commonly required to recruit the motor proteins to lysosomes and mitochondria. Our results suggest that LAMPs may regulate the positioning of lysosomes and mitochondria. A possible mechanism involving the indirect and context-dependent action of LAMPs is discussed.

  13. 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.

  14. Arv1 regulates PM and ER membrane structure and homeostasis but is dispensable for intracellular sterol transport

    PubMed Central

    Georgiev, Alexander G.; Johansen, Jesper; Ramanathan, Vidhya D.; Sere, Yves Y.; Beh, Christopher T.; Menon, Anant K.

    2013-01-01

    The pan-eukaryotic endoplasmic reticulum (ER) membrane protein Arv1 has been suggested to play a role in intracellular sterol transport. We tested this proposal by comparing sterol traffic in wild-type and Arv1-deficient Saccharomyces cerevisiae. We used fluorescence microscopy to track the retrograde movement of exogenously supplied dehydroergosterol (DHE) from the plasma membrane (PM) to the ER and lipid droplets and high performance liquid chromatography to quantify, in parallel, the transport-coupled formation of DHE esters. Metabolic labeling and subcellular fractionation were used to assay anterograde transport of ergosterol from the ER to the PM. We report that sterol transport between the ER and PM is unaffected by Arv1 deficiency. Instead, our results indicate differences in ER morphology and the organization of the PM lipid bilayer between wild-type and arv1Δ cells suggesting a distinct role for Arv1 in membrane homeostasis. In arv1Δ cells, specific defects affecting single C-terminal transmembrane domain proteins suggest that Arv1 might regulate membrane insertion of tail-anchored proteins involved in membrane homoeostasis. PMID:23668914

  15. Arv1 regulates PM and ER membrane structure and homeostasis but is dispensable for intracellular sterol transport.

    PubMed

    Georgiev, Alexander G; Johansen, Jesper; Ramanathan, Vidhya D; Sere, Yves Y; Beh, Christopher T; Menon, Anant K

    2013-08-01

    The pan-eukaryotic endoplasmic reticulum (ER) membrane protein Arv1 has been suggested to play a role in intracellular sterol transport. We tested this proposal by comparing sterol traffic in wild-type and Arv1-deficient Saccharomyces cerevisiae. We used fluorescence microscopy to track the retrograde movement of exogenously supplied dehydroergosterol (DHE) from the plasma membrane (PM) to the ER and lipid droplets and high performance liquid chromatography to quantify, in parallel, the transport-coupled formation of DHE esters. Metabolic labeling and subcellular fractionation were used to assay anterograde transport of ergosterol from the ER to the PM. We report that sterol transport between the ER and PM is unaffected by Arv1 deficiency. Instead, our results indicate differences in ER morphology and the organization of the PM lipid bilayer between wild-type and arv1Δ cells suggesting a distinct role for Arv1 in membrane homeostasis. In arv1Δ cells, specific defects affecting single C-terminal transmembrane domain proteins suggest that Arv1 might regulate membrane insertion of tail-anchored proteins involved in membrane homoeostasis.

  16. Regulation of CLC-1 chloride channel biosynthesis by FKBP8 and Hsp90β

    PubMed Central

    Peng, Yi-Jheng; Huang, Jing-Jia; Wu, Hao-Han; Hsieh, Hsin-Ying; Wu, Chia-Ying; Chen, Shu-Ching; Chen, Tsung-Yu; Tang, Chih-Yung

    2016-01-01

    Mutations in human CLC-1 chloride channel are associated with the skeletal muscle disorder myotonia congenita. The disease-causing mutant A531V manifests enhanced proteasomal degradation of CLC-1. We recently found that CLC-1 degradation is mediated by cullin 4 ubiquitin ligase complex. It is currently unclear how quality control and protein degradation systems coordinate with each other to process the biosynthesis of CLC-1. Herein we aim to ascertain the molecular nature of the protein quality control system for CLC-1. We identified three CLC-1-interacting proteins that are well-known heat shock protein 90 (Hsp90)-associated co-chaperones: FK506-binding protein 8 (FKBP8), activator of Hsp90 ATPase homolog 1 (Aha1), and Hsp70/Hsp90 organizing protein (HOP). These co-chaperones promote both the protein level and the functional expression of CLC-1 wild-type and A531V mutant. CLC-1 biosynthesis is also facilitated by the molecular chaperones Hsc70 and Hsp90β. The protein stability of CLC-1 is notably increased by FKBP8 and the Hsp90β inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) that substantially suppresses cullin 4 expression. We further confirmed that cullin 4 may interact with Hsp90β and FKBP8. Our data are consistent with the idea that FKBP8 and Hsp90β play an essential role in the late phase of CLC-1 quality control by dynamically coordinating protein folding and degradation. PMID:27580824

  17. Regulation of CLC-1 chloride channel biosynthesis by FKBP8 and Hsp90β.

    PubMed

    Peng, Yi-Jheng; Huang, Jing-Jia; Wu, Hao-Han; Hsieh, Hsin-Ying; Wu, Chia-Ying; Chen, Shu-Ching; Chen, Tsung-Yu; Tang, Chih-Yung

    2016-01-01

    Mutations in human CLC-1 chloride channel are associated with the skeletal muscle disorder myotonia congenita. The disease-causing mutant A531V manifests enhanced proteasomal degradation of CLC-1. We recently found that CLC-1 degradation is mediated by cullin 4 ubiquitin ligase complex. It is currently unclear how quality control and protein degradation systems coordinate with each other to process the biosynthesis of CLC-1. Herein we aim to ascertain the molecular nature of the protein quality control system for CLC-1. We identified three CLC-1-interacting proteins that are well-known heat shock protein 90 (Hsp90)-associated co-chaperones: FK506-binding protein 8 (FKBP8), activator of Hsp90 ATPase homolog 1 (Aha1), and Hsp70/Hsp90 organizing protein (HOP). These co-chaperones promote both the protein level and the functional expression of CLC-1 wild-type and A531V mutant. CLC-1 biosynthesis is also facilitated by the molecular chaperones Hsc70 and Hsp90β. The protein stability of CLC-1 is notably increased by FKBP8 and the Hsp90β inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) that substantially suppresses cullin 4 expression. We further confirmed that cullin 4 may interact with Hsp90β and FKBP8. Our data are consistent with the idea that FKBP8 and Hsp90β play an essential role in the late phase of CLC-1 quality control by dynamically coordinating protein folding and degradation. PMID:27580824

  18. Regulation of intracellular pH during H+-coupled oligopeptide absorption in enterocytes from guinea-pig ileum

    PubMed Central

    Hayashi, Hisayoshi; Suzuki, Yuichi

    1998-01-01

    The mechanisms for regulating the intracellular pH (pHi) level during oligopeptide absorption were investigated in the enterocytes from guinea-pig ileum by identifying the acid-base transporters responsible for extruding H+ that enters the cell through the H+-oligopeptide cotransporter. The pHi level was measured by microfluorometry in an isolated villus tip loaded with the pH-sensitive fluoroprobe 2′7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The oligopeptide-induced increment in the short-circuit current (Isc) was determined in a mucosal sheet in Ussing chambers. A CO2/HCO3−-buffered solution was used. The superfusion of glycylglycine (Gly-Gly, l0 mM) caused a decrease in pHi level, which returned to the basal level after removing Gly-Gly. This pHi recovery was strongly dependent on extracellular Na+. Amiloride partially inhibited the pHi recovery rate with an IC50 value of 41 μM, the maximum inhibition being approximately 70%. In the presence of amiloride at its maximum concentration (0.3 mM), the addition of 0.6 mM DIDS caused a further decrease, but did not abolish the pHi recovery rate. In the absence of CO2 and HCO3−, the pHi recovery was almost completely abolished by 0.3 mM amiloride. The intracellular H+ accumulation induced by 0.3 mM amiloride or by 0.6 mM DIDS, as estimated from the pHi decrease and buffer capacity, was significantly greater during Gly-Gly superfusion than under resting conditions. The increase in Isc induced by luminal glycylproline was attenuated by either removing serosal Na+ or by adding 0.5 mM amiloride or 0.6 mM DIDS to the serosal side. We conclude that both Na+-dependent, amiloride-sensitive acid extrusion, probably by the Na+-H+ exchanger, and Na+- and HCO3−-dependent, DIDS-sensitive acid extrusion, possibly by the Na+-HCO3− cotransporter, are involved in extruding H+ that enters cells by the H+-oligopeptide cotransport. It is proposed that these acid extrusion (or base loading) mechanisms are present

  19. Chloride channel ClC-5 binds to aspartyl aminopeptidase to regulate renal albumin endocytosis.

    PubMed

    Lee, Aven; Slattery, Craig; Nikolic-Paterson, David J; Hryciw, Deanne H; Wilk, Sherwin; Wilk, Elizabeth; Zhang, Yuan; Valova, Valentina A; Robinson, Phillip J; Kelly, Darren J; Poronnik, Philip

    2015-04-01

    ClC-5 is a chloride/proton exchanger that plays an obligate role in albumin uptake by the renal proximal tubule. ClC-5 forms an endocytic complex with the albumin receptor megalin/cubilin. We have identified a novel ClC-5 binding partner, cytosolic aspartyl aminopeptidase (DNPEP; EC 3.4.11.21), that catalyzes the release of N-terminal aspartate/glutamate residues. The physiological role of DNPEP remains largely unresolved. Mass spectrometric analysis of proteins binding to the glutathione-S-transferase (GST)-ClC-5 C terminus identified DNPEP as an interacting partner. Coimmunoprecipitation confirmed that DNPEP and ClC-5 also associated in cells. Further experiments using purified GST-ClC-5 and His-DNPEP proteins demonstrated that the two proteins bound directly to each other. In opossum kidney (OK) cells, confocal immunofluorescence studies revealed that DNPEP colocalized with albumin-containing endocytic vesicles. Overexpression of wild-type DNPEP increased cell-surface levels of ClC-5 and albumin uptake. Analysis of DNPEP-immunoprecipitated products from rat kidney lysate identified β-actin and tubulin, suggesting a role for DNPEP in cytoskeletal maintenance. A DNase I inhibition assay showed a significant decrease in the amount of G actin when DNPEP was overexpressed in OK cells, suggesting a role for DNPEP in stabilizing the cytoskeleton. DNPEP was not present in the urine of healthy rats; however, it was readily detected in the urine in rat models of mild and heavy proteinuria (diabetic nephropathy and anti-glomerular basement membrane disease, respectively). Urinary levels of DNPEP were found to correlate with the severity of proteinuria. Therefore, we have identified another key molecular component of the albumin endocytic machinery in the renal proximal tubule and describe a new role for DNPEP in stabilizing the actin cytoskeleton.

  20. TRPC1 regulates calcium-activated chloride channels in salivary gland cells.

    PubMed

    Sun, Yuyang; Birnbaumer, Lutz; Singh, Brij B

    2015-11-01

    Calcium-activated chloride channel (CaCC) plays an important role in modulating epithelial secretion. It has been suggested that in salivary tissues, sustained fluid secretion is dependent on Ca(2+) influx that activates ion channels such as CaCC to initiate Cl(-) efflux. However direct evidence as well as the molecular identity of the Ca(2+) channel responsible for activating CaCC in salivary tissues is not yet identified. Here we provide evidence that in human salivary cells, an outward rectifying Cl(-) current was activated by increasing [Ca(2+)]i, which was inhibited by the addition of pharmacological agents niflumic acid (NFA), an antagonist of CaCC, or T16Ainh-A01, a specific TMEM16a inhibitor. Addition of thapsigargin (Tg), that induces store-depletion and activates TRPC1-mediated Ca(2+) entry, potentiated the Cl(-) current, which was inhibited by the addition of a non-specific TRPC channel blocker SKF96365 or removal of external Ca(2+). Stimulation with Tg also increased plasma membrane expression of TMEM16a protein, which was also dependent on Ca(2+) entry. Importantly, in salivary cells, TRPC1 silencing, but not that of TRPC3, inhibited CaCC especially upon store depletion. Moreover, primary acinar cells isolated from submandibular gland also showed outward rectifying Cl(-) currents upon increasing [Ca(2+)]i. These Cl(-) currents were again potentiated with the addition of Tg, but inhibited in the presence of T16Ainh-A01. Finally, acinar cells isolated from the submandibular glands of TRPC1 knockout mice showed significant inhibition of the outward Cl(-) currents without decreasing TMEM16a expression. Together the data suggests that Ca(2+) entry via the TRPC1 channels is essential for the activation of CaCC.

  1. Dietary salt regulates the phosphorylation of OSR1/SPAK kinases and the sodium chloride cotransporter through aldosterone.

    PubMed

    Chiga, Motoko; Rai, Tatemitsu; Yang, Sung-Sen; Ohta, Akihito; Takizawa, Toichiro; Sasaki, Sei; Uchida, Shinichi

    2008-12-01

    Pseudohypoaldosteronism type II (PHAII) is caused by mutations in the WNK1 and WNK4 genes (WNK with-no-lysine kinase). In a mouse model of this disease where a mutant of Wnk4 D561A was knocked in, increased phosphorylation of the sodium chloride cotransporter (NCC) was found and the transporter was concentrated on the apical membrane of the distal tubules. In addition, we recently found that other kinases, such as the oxidative stress response kinase-1/STE20/SPS1-related proline alanine-rich kinase (OSR1/SPAK), also showed increased phosphorylation in these mice. Here we determined whether this kinase cascade is regulated by dietary salt intake. We found that the phosphorylation states of NCC and OSR1/SPAK were increased by low-salt diets and decreased by high-salt diets; a regulation completely lost in the knock-in mice. Increased phosphorylation was reversed by spironolactone and this decreased phosphorylation was reversed by administration of exogenous aldosterone. These studies suggest that that the WNK-OSR1/SPAK-NCC cascade may be a novel effector system of aldosterone action in the kidney.

  2. The interaction between AMPKβ2 and the PP1-targeting subunit R6 is dynamically regulated by intracellular glycogen content.

    PubMed

    Oligschlaeger, Yvonne; Miglianico, Marie; Dahlmans, Vivian; Rubio-Villena, Carla; Chanda, Dipanjan; Garcia-Gimeno, Maria Adelaida; Coumans, Will A; Liu, Yilin; Voncken, J Willem; Luiken, Joost J F P; Glatz, Jan F C; Sanz, Pascual; Neumann, Dietbert

    2016-04-01

    AMP-activated protein kinase (AMPK) is a metabolic stress-sensing kinase. We previously showed that glucose deprivation induces autophosphorylation of AMPKβ at Thr-148, which prevents the binding of AMPK to glycogen. Furthermore, in MIN6 cells, AMPKβ1 binds to R6 (PPP1R3D), a glycogen-targeting subunit of protein phosphatase type 1 (PP1), thereby regulating the glucose-induced inactivation of AMPK. In the present study, we further investigated the interaction of R6 with AMPKβ and the possible dependency on Thr-148 phosphorylation status. Yeast two-hybrid (Y2H) analyses and co-immunoprecipitation (IP) of the overexpressed proteins in human embryonic kidney (HEK) 293T) cells revealed that both AMPKβ1 and AMPK-β2 wild-type (WT) isoforms bind to R6. The AMPKβ-R6 interaction was stronger with the muscle-specific AMPKβ2-WT and required association with the substrate-binding motif of R6. When HEK293T cells or C2C12 myotubes were cultured in high-glucose medium, AMPKβ2-WT and R6 weakly interacted. In contrast, glycogen depletion significantly enhanced this protein interaction. Mutation of AMPKβ2 Thr-148 prevented the interaction with R6 irrespective of the intracellular glycogen content. Treatment with the AMPK activator oligomycin enhanced the AMPKβ2-R6 interaction in conjunction with increased Thr-148 phosphorylation in cells grown in low-glucose medium. These data are in accordance with R6 binding directly to AMPKβ2 when both proteins detach from the diminishing glycogen particle, which is simultaneous with increased AMPKβ2 Thr-148 autophosphorylation. Such a model points to a possible control of AMPK by PP1-R6 upon glycogen depletion in muscle.

  3. 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

  4. Cortisol regulates sodium homeostasis by stimulating the transcription of sodium-chloride transporter (NCC) in zebrafish (Danio rerio).

    PubMed

    Lin, Chia-Hao; Hu, Huei-Jyun; Hwang, Pung-Pung

    2016-02-15

    In mammals, sodium/hydrogen exchanger (NHE) and sodium-chloride cotransporter (NCC) are expressed in renal tubules, and exhibit functional redundancy and mutual compensation in Na(+) uptake. In teleosts, the gills of the adult and skin of the embryonic stage function as external kidneys, and ionocytes are responsible for ionoregulation in these tissues. NHE- and NCC-expressing ionocytes mutually cooperate to adjust Na(+) uptake, which is analogous to the activity of the mammalian kidney. Cortisol is a hormone that controls Na(+) uptake through regulating NCC expression and activity in mammals; however, cortisol-mediated control of NCC expression is little understood in non-mammalian vertebrates, such as teleosts. It is essential for our understanding of the evolution of such regulation to determine whether cortisol has a conserved effect on NCC in vertebrates. In the present study, we treated zebrafish embryos with low Na(+) medium (LNa, 0.04 mM Na(+)) for 3 d to stimulate the mRNA expression of nhe3b, ncc, and cyp11b1 (a cortisol-synthesis enzyme) and whole body cortisol level. Exogenous cortisol treatment (20 mg/l, 3 d) resulted in an elevation of whole-body Na(+) content, ncc expression, and the density of ncc-expressing cells in zebrafish larvae. In loss-of-function experiments, microinjection of glucocorticoid receptor (gr) morpholino (MO) suppressed sodium content, ncc expression, and the density of ncc-expressing cells, but injection of mr MO had no such effects. In addition, exogenous cortisol treatment and gr MO injection also altered ncc expression and the density of ncc-expressing cells in gcm2 morphant larvae. Taken together, cortisol and GR appear to regulate Na(+) absorption through stimulating ncc expression and the differentiation of ncc-expressing ionocytes, providing new insights into the actions of cortisol on Na(+) uptake.

  5. Intracellular pH of giant salivary gland cells of the leech Haementeria ghilianii: regulation and effects on secretion.

    PubMed

    Wuttke, W A; Munsch, T; Berry, M S

    1994-04-01

    1. Intracellular pH (pHi) and membrane potential (Em) of giant salivary gland cells of the leech, Haementeria ghilianii, were measured with double-barrelled, neutral-carrier, pH-sensitive microelectrodes. 2. Em was -51 +/- 11.2 mV and pHi was 6.98 +/- 0.1 (mean +/- S.D., N = 41) in Hepes-buffered saline (nominally HCO3(-)-free; extracellular pH, pHe = 7.4). pHi was independent of Em. 3. Amiloride (2 mmol l-1) had no effect on resting pHi or on pHi recovery from an acid load (induced by the NH4+ pre-pulse technique). Removal of external Na+ produced a progressive acidification which was blocked by amiloride, and the drug also slowed the recovery of pHi on reintroduction of Na+. The results indicate the presence of an electroneutral Na+/H+ exchanger whose access to amiloride is competitively blocked by Na+. 4. In certain smaller cells of the gland, which probably form a separate population, removal of external Na+ did not affect pHi, and recovery from an acid load was blocked by amiloride. There may, therefore, be two types of Na+/H+ exchanger, differing in reversibility and sensitivity to amiloride. 5. Recovery of pHi from NH4(+)-induced acid loading was not affected by bicarbonate-buffered saline (2% CO2; 11 mmol l-1 HCO3-) or by addition of the anion-exchange blocker SITS (10(-4) mol l-1). This suggests that there is no significant contribution of a HCO3(-)-dependent transport mechanism to pHi regulation in the gland cells. 6. Removal of external Cl- slowly reduced pHi and there was a transient increase (overshoot) in pHi when Cl- was reintroduced. These effects of Cl- are probably explained by changes in the Na+ gradient. Intracellular Na+ and Cl- activities were measured with ion-selective microelectrodes. 7. Acidification with NH4+ was difficult, probably because of the cells' poor permeability to this ion. Attempts to introduce NH4+ via the Na+ pump or Na+/Cl- transporter were not successful. The H+/K+ ionophore nigericin (1 microgram ml-1), however, produced

  6. Regulation of chloride secretion in dog tracheal epithelium by protein kinase C

    SciTech Connect

    Barthelson, R.A.; Jacoby, D.B.; Widdicombe, J.H. )

    1987-12-01

    The effects of stimulating protein kinase C on Cl{sup {minus}} secretion across dog tracheal epithelium were investigated. The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), and the synthetic diacylglycerol, 1-oleolyl-2-acetylglycerol (OAG), which stimulate protein kinase C (PKC), both stimulated short-circuit current (I{sub sc}) with K{sub d} of 10 nM and 1 {mu}M, respectively. In Cl{sup {minus}}-free solution, the increases in I{sub sc} were virtually abolished, suggesting that these compounds stimulate Cl{sup {minus}} secretion, a hypothesis confirmed for TPA by measurement of {sup 36}Cl{sup {minus}} fluxes. The stimulations of Cl{sup {minus}} secretion were not sensitive to indomethacin, nor were cAMP levels elevated during stimulation. In addition to its transient stimulatory effect, TPA at high doses caused the eventual lowering of the base-line I{sub sc} and a block of subsequent stimulation by cAMP-mediated agonists. This was probably not the result of toxicity or an effect on adenylate cyclase or on cAMP-dependent protein kinase. Cell extracts from both cultured and native dog tracheal epithelial cells showed strong PKC activities. These results suggest that PKC may play a role in regulating Cl{sup {minus}} secretion across dog tracheal epithelium.

  7. The sodium chloride cotransporter SLC12A3: new roles in sodium, potassium, and blood pressure regulation.

    PubMed

    Moes, Arthur D; van der Lubbe, Nils; Zietse, Robert; Loffing, Johannes; Hoorn, Ewout J

    2014-01-01

    SLC12A3 encodes the thiazide-sensitive sodium chloride cotransporter (NCC), which is primarily expressed in the kidney, but also in intestine and bone. In the kidney, NCC is located in the apical plasma membrane of epithelial cells in the distal convoluted tubule. Although NCC reabsorbs only 5 to 10% of filtered sodium, it is important for the fine-tuning of renal sodium excretion in response to various hormonal and non-hormonal stimuli. Several new roles for NCC in the regulation of sodium, potassium, and blood pressure have been unraveled recently. For example, the recent discoveries that NCC is activated by angiotensin II but inhibited by dietary potassium shed light on how the kidney handles sodium during hypovolemia (high angiotensin II) and hyperkalemia. The additive effect of angiotensin II and aldosterone maximizes sodium reabsorption during hypovolemia, whereas the inhibitory effect of potassium on NCC increases delivery of sodium to the potassium-secreting portion of the nephron. In addition, great steps have been made in unraveling the molecular machinery that controls NCC. This complex network consists of kinases and ubiquitinases, including WNKs, SGK1, SPAK, Nedd4-2, Cullin-3, and Kelch-like 3. The pathophysiological significance of this network is illustrated by the fact that modification of each individual protein in the network changes NCC activity and results in salt-dependent hypotension or hypertension. This review aims to summarize these new insights in an integrated manner while identifying unanswered questions.

  8. Regulation of chloride transport in parotid secretory granules by membrane fluidity.

    PubMed

    Gasser, K W; Goldsmith, A; Hopfer, U

    1990-08-01

    Zymogen granule membranes contain Cl- conductance and Cl/anion exchange activities that become important for primary fluid production after fusion with the apical plasma membrane of the acinar cell. We have used steady-state fluorescence anisotropy of diphenylhexatriene derivatives and measurements of Cl- transport in isolated secretory granules to determine the contribution of membrane fluidity to the regulation of transport across the granule membrane. Secretory granules from several unstimulated glands (rat pancreas and parotid, rabbit gastric glands) were shown to have low membrane fluidity compared to plasma membranes. In addition, Cl- transport activity in different granule preparations showed a strong correlation to the membrane fluidity when measured with 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH), but not with 3-[p-(6-phenyl)-1,3,5-hexatrienyl)-phenyl]propionic acid (PA-DPH). These data suggest that TMA-DPH preferentially partitions into a specific lipid environment associated with, or which exerts an influence on, the Cl- transport proteins and that increases in the fluidity of this environment are associated with higher transport rates. Data from other types of plasma membranes indicate that TMA-DPH partitions much more than PA-DPH into the cytoplasmic leaflet, suggesting that this part of the granule membrane is involved in the observed fluidity changes. Furthermore, increasing the bulk membrane fluidity with the local anesthetics benzyl alcohol and n-alkanols increased the Cl- transport rates up to 10-fold. This increase was apparently through specific transporters as anion selectivity was maintained in spite of the higher absolute rates.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Aberration in epigenetic gene regulation in hippocampal neurogenesis by developmental exposure to manganese chloride in mice.

    PubMed

    Wang, Liyun; Shiraki, Ayako; Itahashi, Megu; Akane, Hirotoshi; Abe, Hajime; Mitsumori, Kunitoshi; Shibutani, Makoto

    2013-11-01

    We have shown that maternal manganese (Mn) exposure caused sustained disruption of hippocampal neurogenesis of mouse offspring. To clarify the effects of maternal Mn exposure on epigenetic gene regulation contributing to the sustained disruption of hippocampal neurogenesis, we treated pregnant ICR mice with MnCl₂ in diet from gestational day 10 through day 21 after delivery on weaning and searched epigenetically downregulated genes by global promoter methylation analysis in the hippocampal dentate gyrus of male offspring on postnatal day (PND) 21 and PND 77. By CpG promoter microarray analysis on PND 21 following 800-ppm Mn exposure, sustained promoter hypermethylation and transcript downregulation through PND 77 were confirmed with Mid1, Atp1a3, and Nr2f1, whereas Pvalb showed a transient hypermethylation only on weaning. The numbers of Pvalb⁺ and ATP1a3⁺ neurons suggestive of γ-aminobutyric acid (GABA)ergic interneurons, Mid1⁺ cells suggestive of late-stage granule cell lineage and GABAergic interneurons, and COUP-TF1⁺ cells suggestive of early-stage granule cell lineage were all reduced on PND 21, and reductions were sustained on PND 77 except for no change in Pvalb⁺ cells. Mid1⁺ cells showed asymmetric distribution with right-side predominance, and Mn exposure abolished it by promoter hypermethylation of the right side. These findings indicate epigenetic mechanisms as mediators, through which Mn exposure modulates neurogenesis involving both granule cell lineage and GABAergic interneurons with long-lasting and stable repercussions. Disruption of asymmetric cellular distribution of Mid1 suggests that higher brain functions specialized in the left or right side of the brain were affected.

  10. Regulation of chloride self exchange by cAMP in cortical collecting tubule

    SciTech Connect

    Tago, K.; Schuster, V.L.; Stokes, J.B.

    1986-07-01

    The hormonal control of Cl transport was examined in rabbit cortical collecting tubules using the lumen-to-bath /sup 36/Cl tracer rate coefficient (K/sub Cl/, nm/s). Tracer movement via Sl-HCO/sub 3/ exchange was minimized by using HCO/sub 3/-CO/sub 2/-free solutions. The electrical driving force was minimized by treating with amiloride. Under these conditions, net Cl transport was zero, yet there was a large K/sub Cl/ that fell 88% on removing bath (trans) Cl. These results are consistent with the mechanism of tracer flux being predominantly Cl self exchange. K/sub Cl/ fell spontaneously with time in vitro; after this decline K/sub Cl/ could be stimulated with 8-bromo-cAMP. cAMP present from the onset of perfusion prevented the time-dependent fall in K/sub Cl/. When tracer movement was restricted to diffusion by eliminating Cl self exchange (0 Cl bath), cAMP had no effect on K/sub Cl/. Although both isoproterenol and vasopressin are known to stimulate adenylate cyclase in this epithelium, only isoproterenol mimicked the cAMP effect on K/sub Cl/. The isoproterenol effect was blocked by either propranolol or prostaglandin E/sub 2/. Lumen addition of the disulfonic stilbene DIDS had no effect on K/sub Cl/. Lumen addition of furosemide or trichloromethiazide had minimal or no effect. Taken together, these results indicate that Cl self exchange is regulated by ..beta..-adrenergic agents acting via cAMP. The lack of an effect of vasopressin suggests cellular heterogeneity in this response to cAMP.

  11. 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.

  12. Dietary flavonoid fisetin regulates aluminium chloride-induced neuronal apoptosis in cortex and hippocampus of mice brain.

    PubMed

    Prakash, Dharmalingam; Sudhandiran, Ganapasam

    2015-12-01

    Dietary flavonoids have been suggested to promote brain health by protecting brain parenchymal cells. Recently, understanding the possible mechanism underlying neuroprotective efficacy of flavonoids is of great interest. Given that fisetin exerts neuroprotection, we have examined the mechanisms underlying fisetin in regulating Aβ aggregation and neuronal apoptosis induced by aluminium chloride (AlCl3) administration in vivo. Male Swiss albino mice were induced orally with AlCl3 (200 mg/kg. b.wt./day/8 weeks). Fisetin (15 mg/Kg. b.wt. orally) was administered for 4 weeks before AlCl3-induction and administered simultaneously for 8 weeks during AlCl3-induction. We found aggregation of Amyloid beta (Aβ 40-42), elevated expressions of Apoptosis stimulating kinase (ASK-1), p-JNK (c-Jun N-terminal Kinase), p53, cytochrome c, caspases-9 and 3, with altered Bax/Bcl-2 ratio in favour of apoptosis in cortex and hippocampus of AlCl3-administered mice. Furthermore, TUNEL and fluoro-jade C staining demonstrate neurodegeneration in cortex and hippocampus. Notably, treatment with fisetin significantly (P<0.05) reduced Aβ aggregation, ASK-1, p-JNK, p53, cytochrome c, caspase-9 and 3 protein expressions and modulated Bax/Bcl-2 ratio. TUNEL-positive and fluoro-jade C stained cells were also significantly reduced upon fisetin treatment. We have identified the involvement of fisetin in regulating ASK-1 and p-JNK as possible mediator of Aβ aggregation and subsequent neuronal apoptosis during AlCl3-induced neurodegeneration. These findings define the possibility that fisetin may slow or prevent neurodegneration and can be utilised as neuroprotective agent against Alzheimer's and Parkinson's disease.

  13. Mouse cystic fibrosis transmembrane conductance regulator forms cAMP-PKA-regulated apical chloride channels in cortical collecting duct.

    PubMed

    Lu, Ming; Dong, Ke; Egan, Marie E; Giebisch, Gerhard H; Boulpaep, Emile L; Hebert, Steven C

    2010-03-30

    The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in many segments of the mammalian nephron, where it may interact with and modulate the activity of a variety of apical membrane proteins, including the renal outer medullary potassium (ROMK) K(+) channel. However, the expression of CFTR in apical cell membranes or its function as a Cl(-) channel in native renal epithelia has not been demonstrated. Here, we establish that CFTR forms protein kinase A (PKA)-activated Cl(-) channels in the apical membrane of principal cells from the cortical collecting duct obtained from mice. These Cl(-) channels were observed in cell-attached apical patches of principal cells after stimulation by forskolin/3-isobutyl-1-methylxanthine. Quiescent Cl(-) channels were present in patches excised from untreated tubules because they could be activated after exposure to Mg-ATP and the catalytic subunit of PKA. The single-channel conductance, kinetics, and anion selectivity of these Cl(-) channels were the same as those of recombinant mouse CFTR channels expressed in Xenopus laevis oocytes. The CFTR-specific closed-channel blocker CFTR(inh)-172 abolished apical Cl(-) channel activity in excised patches. Moreover, apical Cl(-) channel activity was completely absent in principal cells from transgenic mice expressing the DeltaF508 CFTR mutation but was present and unaltered in ROMK-null mice. We discuss the physiologic implications of open CFTR Cl(-) channels on salt handling by the collecting duct and on the functional CFTR-ROMK interactions in modulating the metabolic ATP-sensing of ROMK. PMID:20231442

  14. 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

  15. Inositol 1,4,5-trisphosphate receptor and dSTIM function in Drosophila insulin-producing neurons regulates systemic intracellular calcium homeostasis and flight.

    PubMed

    Agrawal, Neha; Venkiteswaran, Gayatri; Sadaf, Sufia; Padmanabhan, Nisha; Banerjee, Santanu; Hasan, Gaiti

    2010-01-27

    Calcium (Ca(2+)) signaling is known to regulate the development, maintenance and modulation of activity in neuronal circuits that underlie organismal behavior. In Drosophila, intracellular Ca(2+) signaling by the inositol 1,4,5-trisphosphate receptor and the store-operated channel (dOrai) regulates the formation and function of neuronal circuits that control flight. Here, we show that restoring InsP(3)R activity in insulin-producing neurons of flightless InsP(3)R mutants (itpr) during pupal development can rescue systemic flight ability. Expression of the store operated Ca(2+) entry (SOCE) regulator dSTIM in insulin-producing neurons also suppresses compromised flight ability of InsP(3)R mutants suggesting that SOCE can compensate for impaired InsP(3)R function. Despite restricted expression of wild-type InsP(3)R and dSTIM in insulin-producing neurons, a global restoration of SOCE and store Ca(2+) is observed in primary neuronal cultures from the itpr mutant. These results suggest that restoring InsP(3)R-mediated Ca(2+) release and SOCE in a limited subset of neuromodulatory cells can influence systemic behaviors such as flight by regulating intracellular Ca(2+) homeostasis in a large population of neurons through a non-cell-autonomous mechanism. PMID:20107057

  16. The C-terminal tail of protein kinase D2 and protein kinase D3 regulates their intracellular distribution

    SciTech Connect

    Papazyan, Romeo; Rozengurt, Enrique; Rey, Osvaldo . E-mail: orey@mednet.ucla.edu

    2006-04-14

    We generated a set of GFP-tagged chimeras between protein kinase D2 (PKD2) and protein kinase D3 (PKD3) to examine in live cells the contribution of their C-terminal region to their intracellular localization. We found that the catalytic domain of PKD2 and PKD3 can localize to the nucleus when expressed without other kinase domains. However, when the C-terminal tail of PKD2 was added to its catalytic domain, the nuclear localization of the resulting protein was inhibited. In contrast, the nuclear localization of the CD of PKD3 was not inhibited by its C-terminal tail. Furthermore, the exchange of the C-terminal tail of PKD2 and PKD3 in the full-length proteins was sufficient to exchange their intracellular localization. Collectively, these data demonstrate that the short C-terminal tail of these kinases plays a critical role in determining their cytoplasmic/nuclear localization.

  17. Antisense oligodeoxynucleotides to the cystic fibrosis transmembrane conductance regulator inhibit cAMP-activated but not calcium-activated chloride currents.

    PubMed Central

    Wagner, J A; McDonald, T V; Nghiem, P T; Lowe, A W; Schulman, H; Gruenert, D C; Stryer, L; Gardner, P

    1992-01-01

    Phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) by cAMP-dependent protein kinase leads to chloride flux in epithelial cells. Is CFTR also required for the calcium-dependent activation of chloride channels? We used antisense oligodeoxynucleotides to CFTR to reduce the expression of CFTR in colonic and tracheal epithelial cells. The antisense oligomers were a pair of adjacent 18-mers complementary to nucleotides 1-18 and 19-36 of CFTR mRNA. Sense and misantisense oligomers served as controls. A 48-h antisense treatment reduced the expression of CFTR protein as assayed by immunoprecipitation and autoradiography to 26% of the level in sense-treated T84 cells. Whole-cell patch clamp revealed that a 48-h antisense treatment of T84 and 56FHTE-8o- fetal tracheal epithelial cells reduced the cAMP-activated chloride current to approximately 10% of that in sense-treated cells. The half-life of functional CFTR is less than 24 h in these cells. In contrast, the calcium-activated chloride current was not affected by antisense treatment. Hence, the cAMP and calcium pathways are separate. CFTR is required for the cAMP pathway but not for the calcium pathway. Images PMID:1379720

  18. Exendin-4 antagonizes Aβ1-42-induced suppression of long-term potentiation by regulating intracellular calcium homeostasis in rat hippocampal neurons.

    PubMed

    Wang, Xiaohui; Wang, Li; Jiang, Ruirui; Yuan, Yuan; Yu, Qianqian; Li, Yameng

    2015-11-19

    An imbalance of intracellular calcium homeostasis induced by amyloid β-protein (Aβ) contributes to the pathogenesis of Alzheimer's disease (AD), such as deficits in learning and memory. Therefore, regulation of calcium homeostasis may represent a new strategy for treatment of AD. Growing evidence suggests that type 2 diabetes mellitus (T2DM) and AD are closely related in pathogenesis. Thus, drugs used in treatment of T2DM may modify the pathogenesis of AD. This study demonstrated that Exendin-4, which is a glucagon-like peptide-1 (GLP-1) analog used as a therapeutic drug for T2DM, significantly antagonized suppression of long-term potentiation (LTP) induced by Aβ1-42 in the rat hippocampal CA1 region in vivo. This neuroprotection may be mediated by regulation of calcium homeostasis. Pretreatment with Exendin-4 suppressed Aβ1-42-induced elevation in intracellular calcium concentration ([Ca(2+)]i) through L-type voltage-dependent calcium channels (L-VDCCs) and N-methyl-D-aspartate receptors (NMDARs). Furthermore, Exendin-4 antagonized the decrease in p-Ca(2+)/calmodulin-dependent protein kinase IIα (p-CaMKIIα) induced by Aβ1-42 in the rat hippocampal CA1 region. Thus, the neuroprotective effects of Exendin-4, which likely involve regulation of calcium homeostasis, provide theoretical support for using Exendin-4 to treat and prevent AD in the future.

  19. Exendin-4 antagonizes Aβ1-42-induced suppression of long-term potentiation by regulating intracellular calcium homeostasis in rat hippocampal neurons.

    PubMed

    Wang, Xiaohui; Wang, Li; Jiang, Ruirui; Yuan, Yuan; Yu, Qianqian; Li, Yameng

    2015-11-19

    An imbalance of intracellular calcium homeostasis induced by amyloid β-protein (Aβ) contributes to the pathogenesis of Alzheimer's disease (AD), such as deficits in learning and memory. Therefore, regulation of calcium homeostasis may represent a new strategy for treatment of AD. Growing evidence suggests that type 2 diabetes mellitus (T2DM) and AD are closely related in pathogenesis. Thus, drugs used in treatment of T2DM may modify the pathogenesis of AD. This study demonstrated that Exendin-4, which is a glucagon-like peptide-1 (GLP-1) analog used as a therapeutic drug for T2DM, significantly antagonized suppression of long-term potentiation (LTP) induced by Aβ1-42 in the rat hippocampal CA1 region in vivo. This neuroprotection may be mediated by regulation of calcium homeostasis. Pretreatment with Exendin-4 suppressed Aβ1-42-induced elevation in intracellular calcium concentration ([Ca(2+)]i) through L-type voltage-dependent calcium channels (L-VDCCs) and N-methyl-D-aspartate receptors (NMDARs). Furthermore, Exendin-4 antagonized the decrease in p-Ca(2+)/calmodulin-dependent protein kinase IIα (p-CaMKIIα) induced by Aβ1-42 in the rat hippocampal CA1 region. Thus, the neuroprotective effects of Exendin-4, which likely involve regulation of calcium homeostasis, provide theoretical support for using Exendin-4 to treat and prevent AD in the future. PMID:26390937

  20. TRIM30α Is a Negative-Feedback Regulator of the Intracellular DNA and DNA Virus-Triggered Response by Targeting STING

    PubMed Central

    Yang, Bo; Yan, Shanshan; Zhou, Haiyan; He, Lan; Lin, Guomei; Lian, Zhexiong; Jiang, Zhengfan; Sun, Bing

    2015-01-01

    Uncontrolled immune responses to intracellular DNA have been shown to induce autoimmune diseases. Homeostasis regulation of immune responses to cytosolic DNA is critical for limiting the risk of autoimmunity and survival of the host. Here, we report that the E3 ubiquitin ligase tripartite motif protein 30α (TRIM30α) was induced by herpes simplex virus type 1 (HSV-1) infection in dendritic cells (DCs). Knockdown or genetic ablation of TRIM30α augmented the type I IFNs and interleukin-6 response to intracellular DNA and DNA viruses. Trim30α-deficient mice were more resistant to infection by DNA viruses. Biochemical analyses showed that TRIM30α interacted with the stimulator of interferon genes (STING), which is a critical regulator of the DNA-sensing response. Overexpression of TRIM30α promoted the degradation of STING via K48-linked ubiquitination at Lys275 through a proteasome-dependent pathway. These findings indicate that E3 ligase TRIM30α is an important negative-feedback regulator of innate immune responses to DNA viruses by targeting STING. PMID:26114947

  1. 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

  2. Intracellular pH and its regulation in isolated type I carotid body cells of the neonatal rat.

    PubMed Central

    Buckler, K J; Vaughan-Jones, R D; Peers, C; Nye, P C

    1991-01-01

    1. The dual-emission pH-sensitive fluoroprobe carboxy-SNARF-1 (carboxy-seminaptharhodofluor) was used to measure pHi in type I cells enzymically dispersed from the neonatal rat carotid body. 2. Steady-state pHi in cells bathed in a HEPES-buffered Tyrode solution (pH 7.4) was found to be remarkably alkaline (pHi = 7.77) whereas cells bathed in a CO2-HCO3(-)-buffered Tyrode solution (pH 7.4) had a more 'normal' pHi (pHi = 7.28). These observations were further substantiated by using an independent nullpoint test method to determine pHi. 3. Intracellular intrinsic buffering (beta, determined by acidifying the cell using an NH4Cl pre-pulse) was in the range 7-20 mM per pH unit and appeared to be dependent upon pHi with beta increasing as pHi decreased. 4. In cells bathed in a HEPES-buffered Tyrode solution, pHi recovery from an induced intracellular acid load (10 mM-NH4Cl pre-pulse) was inhibited by the Na(+)-H+ exchange inhibitor ethyl isopropyl amiloride (EIPA; 150 microM) or substitution of Nao+ with N-methyl-D-glucamine (NMG). Both EIPA and Nao+ removal also caused a rapid intracellular acidification, which in the case of Nao+ removal, was readily reversible. The rate of this acidification was similar for both Nao+ removal and EIPA addition. 5. Transferring cells from a HEPES-buffered Tyrode solution to one buffered with 5% CO2-HCO3- resulted in an intracellular acidification which was partially, or wholly, sustained. The rate of acidification upon transfer to CO2-HCO3- was considerably slowed by the membrane permeant carbonic anhydrase inhibitor, acetazolamide, thus indicating the presence of the enzyme in these cells. 6. In CO2-HCO3(-)-buffered Tyrode solution, pHi recovery from an intracellular acidosis (NH4+ pre-pulse) was only partially inhibited by EIPA or amiloride whereas Nao+ removal completely inhibited the recovery. The stilbene DIDS (4,4-diisothiocyanatostilbenedisulphonic acid, 200 microM) also partially inhibited pHi recovery following an induced

  3. An intracellular anion channel critical for pigmentation

    PubMed Central

    Bellono, Nicholas W; Escobar, Iliana E; Lefkovith, Ariel J; Marks, Michael S; Oancea, Elena

    2014-01-01

    Intracellular ion channels are essential regulators of organellar and cellular function, yet the molecular identity and physiological role of many of these channels remains elusive. In particular, no ion channel has been characterized in melanosomes, organelles that produce and store the major mammalian pigment melanin. Defects in melanosome function cause albinism, characterized by vision and pigmentation deficits, impaired retinal development, and increased susceptibility to skin and eye cancers. The most common form of albinism is caused by mutations in oculocutaneous albinism II (OCA2), a melanosome-specific transmembrane protein with unknown function. Here we used direct patch-clamp of skin and eye melanosomes to identify a novel chloride-selective anion conductance mediated by OCA2 and required for melanin production. Expression of OCA2 increases organelle pH, suggesting that the chloride channel might regulate melanin synthesis by modulating melanosome pH. Thus, a melanosomal anion channel that requires OCA2 is essential for skin and eye pigmentation. DOI: http://dx.doi.org/10.7554/eLife.04543.001 PMID:25513726

  4. An intracellular anion channel critical for pigmentation.

    PubMed

    Bellono, Nicholas W; Escobar, Iliana E; Lefkovith, Ariel J; Marks, Michael S; Oancea, Elena

    2014-01-01

    Intracellular ion channels are essential regulators of organellar and cellular function, yet the molecular identity and physiological role of many of these channels remains elusive. In particular, no ion channel has been characterized in melanosomes, organelles that produce and store the major mammalian pigment melanin. Defects in melanosome function cause albinism, characterized by vision and pigmentation deficits, impaired retinal development, and increased susceptibility to skin and eye cancers. The most common form of albinism is caused by mutations in oculocutaneous albinism II (OCA2), a melanosome-specific transmembrane protein with unknown function. Here we used direct patch-clamp of skin and eye melanosomes to identify a novel chloride-selective anion conductance mediated by OCA2 and required for melanin production. Expression of OCA2 increases organelle pH, suggesting that the chloride channel might regulate melanin synthesis by modulating melanosome pH. Thus, a melanosomal anion channel that requires OCA2 is essential for skin and eye pigmentation. PMID:25513726

  5. Preferential intracellular pH regulation represents a general pattern of pH homeostasis during acid-base disturbances in the armoured catfish, Pterygoplichthys pardalis.

    PubMed

    Harter, T S; Shartau, R B; Baker, D W; Jackson, D C; Val, A L; Brauner, C J

    2014-08-01

    Preferential intracellular pH (pHi) regulation, where pHi is tightly regulated in the face of a blood acidosis, has been observed in a few species of fish, but only during elevated blood PCO2. To determine whether preferential pHi regulation may represent a general pattern for acid-base regulation during other pH disturbances we challenged the armoured catfish, Pterygoplichthys pardalis, with anoxia and exhaustive exercise, to induce a metabolic acidosis, and bicarbonate injections to induce a metabolic alkalosis. Fish were terminally sampled 2-3 h following the respective treatments and extracellular blood pH, pHi of red blood cells (RBC), brain, heart, liver and white muscle, and plasma lactate and total CO2 were measured. All treatments resulted in significant changes in extracellular pH and RBC pHi that likely cover a large portion of the pH tolerance limits of this species (pH 7.15-7.86). In all tissues other than RBC, pHi remained tightly regulated and did not differ significantly from control values, with the exception of a decrease in white muscle pHi after anoxia and an increase in liver pHi following a metabolic alkalosis. Thus preferential pHi regulation appears to be a general pattern for acid-base homeostasis in the armoured catfish and may be a common response in Amazonian fishes.

  6. Structure and function of NAD kinase and NADP phosphatase: key enzymes that regulate the intracellular balance of NAD(H) and NADP(H).

    PubMed

    Kawai, Shigeyuki; Murata, Kousaku

    2008-04-01

    The functions of NAD(H) (NAD(+) and NADH) and NADP(H) (NADP(+) and NADPH) are undoubtedly significant and distinct. Hence, regulation of the intracellular balance of NAD(H) and NADP(H) is important. The key enzymes involved in the regulation are NAD kinase and NADP phosphatase. In 2000, we first succeeded in identifying the gene for NAD kinase, thereby facilitating worldwide studies of this enzyme from various organisms, including eubacteria, archaea, yeast, plants, and humans. Molecular biological study has revealed the physiological function of this enzyme, that is to say, the significance of NADP(H), in some model organisms. Structural research has elucidated the tertiary structure of the enzyme, the details of substrate-binding sites, and the catalytic mechanism. Research on NAD kinase also led to the discovery of archaeal NADP phosphatase. In this review, we summarize the physiological functions, applications, and structure of NAD kinase, and the way we discovered archaeal NADP phosphatase.

  7. Chloride Test

    MedlinePlus

    ... Addison disease, or increased salt intake. If both chloride and sodium levels are high in a person on a ... anything else I should know? Drugs that affect sodium blood levels will also cause changes in chloride. In addition, swallowing large amounts of baking soda ...

  8. Altered chloride metabolism in cultured cystic fibrosis skin fibroblasts

    SciTech Connect

    Mattes, P.M.; Maloney, P.C.; Littlefield, J.W.

    1987-05-01

    An abnormal regulation of chloride permeability has been described for epithelial cells from patients with cystic fibrosis (CF). To learn more about the biochemical basis of this inherited disease, the authors have studied chloride metabolism in cultured CF fibroblasts by comparing the efflux of /sup 36/Cl/sup -/ from matched pairs of CF and normal fibroblasts. The rate constants describing /sup 36/Cl/sup -/ efflux did not differ between the two cell types, but in each of the four pairs tested the amount of /sup 36/Cl/sup -/ contained within CF cells was consistently reduced, by 25-30%, relative to normal cells. Comparisons of cell water content and /sup 22/Na/sup +/ efflux showed no differences between the two cell types, suggesting that overall intracellular chloride concentration is lower than normal in CF fibroblasts. Such data suggest that the CF gene defect is expressed in skin fibroblasts and that this defect may alter the regulation of intracellular Cl/sup -/ concentration, perhaps through changes in Cl/sup -/ permeability.

  9. Intracellular pH and its relationship to regulation of ion transport in normal and cystic fibrosis human nasal epithelia.

    PubMed

    Willumsen, N J; Boucher, R C

    1992-09-01

    1. Intracellular pH (pHi) of cultured human airway epithelial cells from normal and cystic fibrosis (CF) subjects were measured with double-barrelled pH-sensitive liquid exchanger microelectrodes. The cells, which were grown to confluence on a permeable collagen matrix support, were mounted in a modified miniature Ussing chamber. All studies were conducted under open circuit conditions. Values are given as means +/- S.E.M. and n refers to the number of preparations. 2. Normal preparations (n = 15) were characterized by a transepithelial potential difference (Vt) of -18 +/- 2 mV, an apical membrane potential (Va) of -19 +/- 2 mV, a basolateral membrane potential (Vb) of -37 +/- 2 mV, a transepithelial resistance (Rt) of 253 +/- 15 omega cm2, a fractional apical membrane resistance (fRa) of 0.40 +/- 0.04 and an equivalent short circuit current (Ieq) of -73 +/- 7 microA cm-2. 3. CF preparations (n = 13) were characterized by a Vt of -46 +/- 7 mV, a Va of 3 +/- 5 mV, a Vb of -43 +/- 3 mV, Rt of 373 +/- 47 omega cm2, fRa of 0.44 +/- 0.04 and an Ieq of -130 +/- 16 microA cm-2. All parameters except Vb and fRa were significantly different (P < 0.025) from those of normal preparations. 4. Despite large differences in electrochemical driving force for proton flow across the apical cell membranes between normal and CF preparations (-4 +/- 3 mV and 20 +/- 7 mV, respectively), pHi was similar (7.15 +/- 0.02 and 7.11 +/- 0.05, respectively). The driving force across the basolateral membrane was similar in normal and CF preparations (22 +/- 3 and 26 +/- 3 mV, respectively). 5. Intracellular alkalinization achieved by removal of CO2 from the luminal Ringer solution or by luminal ammonium prepulse led to stimulation of Ieq in both normal (from -58 to -70 microA cm-2, n = 4; P < 0.05) and CF (from -144 to -163 microA cm-2, n = 4; P < 0.005) preparations. The increase in Ieq was associated with a reduction of Rt, increase in fRa, and hyperpolarization of Vb. All changes in

  10. The gene for congenital chloride diarrhea maps close to but is distinct from the gene for cystic fibrosis transmembrane conductance regulator.

    PubMed Central

    Kere, J; Sistonen, P; Holmberg, C; de la Chapelle, A

    1993-01-01

    Congenital chloride diarrhea (CLD) is characterized by watery stools with high chloride content beginning prenatally and is inherited as an autosomal recessive trait. Perfusion studies have established a basic defect in ileal and colonic Cl-/HCO3- transport, resulting in defective chloride absorption. The protein and its gene defects have, however, remained uncharacterized. We attempted to exclude candidate genes by considering linkage disequilibrium as well as genetic linkage in a small number of Finnish families. Initial results were suggestive of linkage between CLD and the cystic fibrosis transmembrane regulator gene (CFTR). Extended analysis in eight families confirmed close linkage to chromosome 7 markers proximal of CFTR, with maximum logarithm of odds scores of 5.11 and 5.06 for D7S501 and D7S496, respectively, at zero recombination. Allelic associations were observed that were striking between CLD and D7S496 and weaker between CLD and D7S501. Multipoint analyses mapped CLD unequivocally at D7S496 with a maximum logarithm of odds score of 9.33. We conclude that the CLD gene maps close to, but is distinct from, CFTR. PMID:7504277

  11. Cyanoquinolines with Independent Corrector and Potentiator Activities Restore ΔPhe508-Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Function in Cystic Fibrosis

    PubMed Central

    Phuan, Puay-Wah; Yang, Baoxue; Knapp, John M.; Wood, Alex B.; Lukacs, Gergely L.; Kurth, Mark J.

    2011-01-01

    The ΔPhe508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) protein impairs its folding, stability, and chloride channel gating. Although small molecules that separately correct defective ΔPhe508-CFTR folding/cellular processing (“correctors”) or chloride channel gating (“potentiators”) have been discovered and are in clinical trials, single compounds with bona fide dual corrector and potentiator activities have not been identified. Here, screening of ∼110,000 small molecules not tested previously revealed a cyanoquinoline class of compounds with independent corrector and potentiator activities (termed CoPo). Analysis of 180 CoPo analogs revealed 6 compounds with dual corrector and potentiator activities and 13 compounds with only potentiator activity. N-(2-((3-Cyano-5,7-dimethylquinolin-2-yl)amino)ethyl)-3-methoxybenzamide (CoPo-22), which was synthesized in six steps in 52% overall yield, had low micromolar EC50 for ΔPhe508-CFTR corrector and potentiator activities by short-circuit current assay. Maximal corrector and potentiator activities were comparable with those conferred by the bithiazole Corr-4a and the flavone genistein, respectively. CoPo-22 also activated wild-type and G551D CFTR chloride conductance within minutes in a forskolin-dependent manner. Compounds with dual corrector and potentiator activities may be useful for single-drug treatment of cystic fibrosis caused by ΔPhe508 mutation. PMID:21730204

  12. 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

  13. 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

  14. Apoptosis induced by trimethyltin chloride in human neuroblastoma cells SY5Y is regulated by a balance and cross-talk between NF-κB and MAPKs signaling pathways.

    PubMed

    Qing, Yan; Liang, Yanfang; Du, Qingqing; Fan, Pan; Xu, Hangong; Xu, Yiping; Shi, Nian

    2013-07-01

    Trimethyltin chloride (TMT) has been known as a classic neurotoxicant which can cause serious neuronal degeneration diseases. Nuclear factor κB (NF-κB) and mitogen-activated protein kinases (MAPKs) signaling pathways play pivotal role in the central nerves system. In the present study, the intracellular pathways involved in TMT-induced apoptosis on human neuroblastoma cells SY5Y (SH-SY5Y) were investigated. We observed high level of nuclear NF-κB p65 submit, activated JNK, ERK, and p38 by TMT exposure. In contrast, low level of Bcl-2 and XIAP (two known NF-κB-regulated endogenous anti-apoptotic molecules) was present. To further investigate the role of these pathways and the relationship between them, specific inhibitors were used and the alteration of each pathway was evaluated. Pretreatment with MG132, an inhibitor of proteasome activity, and BAY11-7082, an inhibitor of IκBα phosphorylation, both inhibited NF-κB p65 translocation and significantly promoted apoptosis. NF-κB inhibition also induced down-expression of Bcl-2 and XIAP, exaggerated JNK phosphorylation, and ERK inhibition. SP600125 and U0126, by blocking the phosphorylation of c-Jun and MEK1/2, inhibited JNK and ERK phosphorylation, respectively, and attenuated apoptosis significantly. JNK and ERK inhibition also induced IκBα degradation and NF-κB p65 translocation, leading to expression of Bcl-2 and XIAP. The detrimental role of MG132 and BAY11-7082 appears related to the exaggerated JNK phosphorylation. The SP600125 and U0126 neuroprotection appears related to NF-κB-regulated transcriptional control of Bcl-2 and XIAP. These results suggest that the cross-talk and a balance between NF-κB and MAPKs may be involved in TMT-induced apoptosis on SH-SY5Y cells.

  15. Tomato QM-Like Protein Protects Saccharomyces cerevisiae Cells against Oxidative Stress by Regulating Intracellular Proline Levels

    PubMed Central

    Chen, Changbin; Wanduragala, Srimevan; Becker, Donald F.; Dickman, Martin B.

    2006-01-01

    Exogenous proline can protect cells of Saccharomyces cerevisiae from oxidative stress. We altered intracellular proline levels by overexpressing the proline dehydrogenase gene (PUT1) of S. cerevisiae. Put1p performs the first enzymatic step of proline degradation in S. cerevisiae. Overexpression of Put1p results in low proline levels and hypersensitivity to oxidants, such as hydrogen peroxide and paraquat. A put1-disrupted yeast mutant deficient in Put1p activity has increased protection from oxidative stress and increased proline levels. Following a conditional life/death screen in yeast, we identified a tomato (Lycopersicon esculentum) gene encoding a QM-like protein (tQM) and found that stable expression of tQM in the Put1p-overexpressing strain conferred protection against oxidative damage from H2O2, paraquat, and heat. This protection was correlated with reactive oxygen species (ROS) reduction and increased proline accumulation. A yeast two-hybrid system assay was used to show that tQM physically interacts with Put1p in yeast, suggesting that tQM is directly involved in modulating proline levels. tQM also can rescue yeast from the lethality mediated by the mammalian proapoptotic protein Bax, through the inhibition of ROS generation. Our results suggest that tQM is a component of various stress response pathways and may function in proline-mediated stress tolerance in plants. PMID:16751508

  16. Regulation of NF-κB oscillation by spatial parameters in true intracellular space (TiCS)

    NASA Astrophysics Data System (ADS)

    Ohshima, Daisuke; Sagara, Hiroshi; Ichikawa, Kazuhisa

    2013-10-01

    Transcription factor NF-κB is activated by cytokine stimulation, viral infection, or hypoxic environment leading to its translocation to the nucleus. The nuclear NF-κB is exported from the nucleus to the cytoplasm again, and by repetitive import and export, NF-κB shows damped oscillation with the period of 1.5-2.0 h. Oscillation pattern of NF-κB is thought to determine the gene expression profile. We published a report on a computational simulation for the oscillation of nuclear NF-κB in a 3D spherical cell, and showed the importance of spatial parameters such as diffusion coefficient and locus of translation for determining the oscillation pattern. Although the value of diffusion coefficient is inherent to protein species, its effective value can be modified by organelle crowding in intracellular space. Here we tested this possibility by computer simulation. The results indicate that the effective value of diffusion coefficient is significantly changed by the organelle crowding, and this alters the oscillation pattern of nuclear NF-κB.

  17. A single conserved leucine residue on the first intracellular loop regulates ER export of G protein-coupled receptors.

    PubMed

    Duvernay, Matthew T; Dong, Chunmin; Zhang, Xiaoping; Robitaille, Mélanie; Hébert, Terence E; Wu, Guangyu

    2009-05-01

    The intrinsic structural determinants for export trafficking of G protein-coupled receptors (GPCRs) have been mainly identified in the termini of the receptors. In this report, we determined the role of the first intracellular loop (ICL1) in the transport from the endoplasmic reticulum (ER) to the cell surface of GPCRs. The alpha(2B)-adrenergic receptor (AR) mutant lacking the ICL1 is unable to traffic to the cell surface and to initiate signaling measured as ERK1/2 activation. Mutagenesis studies identify a single Leu48 residue in the ICL1 modulates alpha(2B)-AR export from the ER. The ER export function of the Leu48 residue can be substituted by Phe, but not Ile, Val, Tyr and Trp, and is unlikely involved in correct folding or dimerization of alpha(2B)-AR in the ER. Importantly, the isolated Leu residue is remarkably conserved in the center of the ICL1s among the family A GPCRs and is also required for the export to the cell surface of beta(2)-AR, alpha(1B)-AR and angiotensin II type 1 receptor. These data indicate a crucial role for a single Leu residue within the ICL1 in ER export of GPCRs.

  18. Nitric oxide regulates cardiac intracellular Na+ and Ca2 + by modulating Na/K ATPase via PKCε and phospholemman-dependent mechanism☆

    PubMed Central

    Pavlovic, Davor; Hall, Andrew R.; Kennington, Erika J.; Aughton, Karen; Boguslavskyi, Andrii; Fuller, William; Despa, Sanda; Bers, Donald M.; Shattock, Michael J.

    2013-01-01

    In the heart, Na/K-ATPase regulates intracellular Na+ and Ca2 + (via NCX), thereby preventing Na+ and Ca2 + overload and arrhythmias. Here, we test the hypothesis that nitric oxide (NO) regulates cardiac intracellular Na+ and Ca2 + and investigate mechanisms and physiological consequences involved. Effects of both exogenous NO (via NO-donors) and endogenously synthesized NO (via field-stimulation of ventricular myocytes) were assessed in this study. Field stimulation of rat ventricular myocytes significantly increased endogenous NO (18 ± 2 μM), PKCε activation (82 ± 12%), phospholemman phosphorylation (at Ser-63 and Ser-68) and Na/K-ATPase activity (measured by DAF-FM dye, western-blotting and biochemical assay, respectively; p < 0.05, n = 6) and all were abolished by Ca2 +-chelation (EGTA 10 mM) or NOS inhibition l-NAME (1 mM). Exogenously added NO (spermine-NONO-ate) stimulated Na/K-ATPase (EC50 = 3.8 μM; n = 6/grp), via decrease in Km, in PLMWT but not PLMKO or PLM3SA myocytes (where phospholemman cannot be phosphorylated) as measured by whole-cell perforated-patch clamp. Field-stimulation with l-NAME or PKC-inhibitor (2 μM Bis) resulted in elevated intracellular Na+ (22 ± 1.5 and 24 ± 2 respectively, vs. 14 ± 0.6 mM in controls) in SBFI-AM-loaded rat myocytes. Arrhythmia incidence was significantly increased in rat hearts paced in the presence of l-NAME (and this was reversed by l-arginine), as well as in PLM3SA mouse hearts but not PLMWT and PLMKO. We provide physiological and biochemical evidence for a novel regulatory pathway whereby NO activates Na/K-ATPase via phospholemman phosphorylation and thereby limits Na+ and Ca2 + overload and arrhythmias. This article is part of a Special Issue entitled “Na+ Regulation in Cardiac Myocytes”. PMID:23612119

  19. Nitric oxide regulates cardiac intracellular Na⁺ and Ca²⁺ by modulating Na/K ATPase via PKCε and phospholemman-dependent mechanism.

    PubMed

    Pavlovic, Davor; Hall, Andrew R; Kennington, Erika J; Aughton, Karen; Boguslavskyi, Andrii; Fuller, William; Despa, Sanda; Bers, Donald M; Shattock, Michael J

    2013-08-01

    In the heart, Na/K-ATPase regulates intracellular Na(+) and Ca(2+) (via NCX), thereby preventing Na(+) and Ca(2+) overload and arrhythmias. Here, we test the hypothesis that nitric oxide (NO) regulates cardiac intracellular Na(+) and Ca(2+) and investigate mechanisms and physiological consequences involved. Effects of both exogenous NO (via NO-donors) and endogenously synthesized NO (via field-stimulation of ventricular myocytes) were assessed in this study. Field stimulation of rat ventricular myocytes significantly increased endogenous NO (18 ± 2 μM), PKCε activation (82 ± 12%), phospholemman phosphorylation (at Ser-63 and Ser-68) and Na/K-ATPase activity (measured by DAF-FM dye, western-blotting and biochemical assay, respectively; p<0.05, n=6) and all were abolished by Ca(2+)-chelation (EGTA 10mM) or NOS inhibition l-NAME (1mM). Exogenously added NO (spermine-NONO-ate) stimulated Na/K-ATPase (EC50=3.8 μM; n=6/grp), via decrease in Km, in PLM(WT) but not PLM(KO) or PLM(3SA) myocytes (where phospholemman cannot be phosphorylated) as measured by whole-cell perforated-patch clamp. Field-stimulation with l-NAME or PKC-inhibitor (2 μM Bis) resulted in elevated intracellular Na(+) (22 ± 1.5 and 24 ± 2 respectively, vs. 14 ± 0.6mM in controls) in SBFI-AM-loaded rat myocytes. Arrhythmia incidence was significantly increased in rat hearts paced in the presence of l-NAME (and this was reversed by l-arginine), as well as in PLM(3SA) mouse hearts but not PLM(WT) and PLM(KO). We provide physiological and biochemical evidence for a novel regulatory pathway whereby NO activates Na/K-ATPase via phospholemman phosphorylation and thereby limits Na(+) and Ca(2+) overload and arrhythmias. This article is part of a Special Issue entitled "Na(+) Regulation in Cardiac Myocytes".

  20. Determination of intracellular nitrate.

    PubMed Central

    Romero, J M; Lara, C; Guerrero, M G

    1989-01-01

    A sensitive procedure has been developed for the determination of intracellular nitrate. The method includes: (i) preparation of cell lysates in 2 M-H3PO4 after separation of cells from the outer medium by rapid centrifugation through a layer of silicone oil, and (ii) subsequent nitrate analysis by ion-exchange h.p.l.c. with, as mobile phase, a solution containing 50 mM-H3PO4 and 2% (v/v) tetrahydrofuran, adjusted to pH 1.9 with NaOH. The determination of nitrate is subjected to interference by chloride and sulphate when present in the samples at high concentrations. Nitrite also interferes, but it is easily eliminated by treatment of the samples with sulphamic acid. The method has been successfully applied to the study of nitrate transport in the unicellular cyanobacterium Anacystis nidulans. PMID:2497740

  1. 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

  2. Abnormalities in intracellular calcium regulation and contractile function in myocardium from dogs with pacing-induced heart failure

    NASA Technical Reports Server (NTRS)

    Perreault, C. L.; Shannon, R. P.; Komamura, K.; Vatner, S. F.; Morgan, J. P.

    1992-01-01

    24 d of rapid ventricular pacing induced dilated cardiomyopathy with both systolic and diastolic dysfunction in conscious, chronically instrumented dogs. We studied mechanical properties and intracellular calcium (Ca2+i) transients of trabeculae carneae isolated from 15 control dogs (n = 32) and 11 dogs with pacing-induced cardiac failure (n = 26). Muscles were stretched to maximum length at 30 degrees C and stimulated at 0.33 Hz; a subset (n = 17 control, n = 17 myopathic) was loaded with the [Ca2+]i indicator aequorin. Peak tension was depressed in the myopathic muscles, even in the presence of maximally effective (i.e., 16 mM) [Ca2+] in the perfusate. However, peak [Ca2+]i was similar (0.80 +/- 0.13 vs. 0.71 +/- 0.05 microM; [Ca2+]o = 2.5 mM), suggesting that a decrease in Cai2+ availability was not responsible for the decreased contractility. The time for decline from the peak of the Cai2+ transient was prolonged in the myopathic group, which correlated with prolongation of isometric contraction and relaxation. However, similar end-diastolic [Ca2+]i was achieved in both groups (0.29 +/- 0.05 vs. 0.31 +/- 0.02 microM), indicating that Cai2+ homeostasis can be maintained in myopathic hearts. The inotropic response of the myopathic muscles to milrinone was depressed compared with the controls. However, when cAMP production was stimulated by pretreatment with forskolin, the response of the myopathic muscles to milrinone was improved. Our findings provide direct evidence that abnormal [Ca2+]i handling is an important cause of contractile dysfunction in dogs with pacing-induced heart failure and suggest that deficient production of cAMP may be an important cause of these changes in excitation-contraction coupling.

  3. Role of Snf1p in regulation of intracellular sorting of the lactose and galactose transporter Lac12p in Kluyveromyces lactis.

    PubMed

    Wiedemuth, Christian; Breunig, Karin D

    2005-04-01

    The protein kinase Snf1/AMPK plays a central role in carbon and energy homeostasis in yeasts and higher eukaryotes. To work out which aspects of the Snf1-controlled regulatory network are conserved in evolution, the Snf1 requirement in galactose metabolism was analyzed in the yeast Kluyveromyces lactis. Whereas galactose induction was only delayed, K. lactis snf1 mutants failed to accumulate the lactose/galactose H+ symporter Lac12p in the plasma membran,e as indicated by Lac12-green fluorescent protein fusions. In contrast to wild-type cells, the fusion protein was mostly intracellular in the mutant. Growth on galactose and galactose uptake could be restored by the KHT3 gene, which encodes a new transporter of the HXT subfamily of major facilitators These findings indicate a new role of Snf1p in regulation of sugar transport in K. lactis. PMID:15821131

  4. Vinyl chloride and polyvinyl chloride.

    PubMed

    Lewis, R

    1999-01-01

    Polyvinyl chloride (PVC) is an important plastic resin for construction, pipe and tubing, siding, and other uses. Exposures to vinyl chloride monomer during the early years of production resulted in an important sentinel health event: the recognition of an excess of a rare liver cancer, hepatic angiosarcoma, at facilities throughout the world. Several other syndromes, including acro-osteolysis, also have been associated with PVC, but less clearly with vinyl chloride. Extensive research ranging from large-scale epidemiologic studies to biomarker research into molecular mechanisms continues to provide valuable insight into the pathogenesis of occupational cancer.

  5. Mechanism of riboflavin uptake by cultured human retinal pigment epithelial ARPE-19 cells: possible regulation by an intracellular Ca2+-calmodulin-mediated pathway.

    PubMed

    Said, Hamid M; Wang, Shuling; Ma, Thomas Y

    2005-07-15

    In mammalian cells (including those of the ocular system), the water-soluble vitamin B2 (riboflavin, RF) assumes an essential role in a variety of metabolic reactions and is critical for normal cellular functions, growth and development. Cells of the human retinal pigment epithelium (hRPE) play an important role in providing a sufficient supply of RF to the retina, but nothing is known about the mechanism of the vitamin uptake by these cells and its regulation. Our aim in the present study was to address this issue using the hRPE ARPE-19 cells as the retinal epithelial model. Our results show RF uptake in the hRPE to be: (1) energy and temperature dependent and occurring without metabolic alteration in the transported substrate, (2) pH but not Na+ dependent, (3) saturable as a function of concentration with an apparent Km of 80 +/- 14 nM, (4) trans-stimulated by unlabelled RF and its structural analogue lumiflavine, (5) cis-inhibited by the RF structural analogues lumiflavine and lumichrome but not by unrelated compounds, and (6) inhibited by the anion transport inhibitors 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) as well as by the Na+ -H+ exchange inhibitor amiloride and the sulfhydryl group inhibitor p-chloromercuriphenylsulphonate (p-CMPS). Maintaining the hRPE cells in a RF-deficient medium led to a specific and significant up-regulation in RF uptake which was mediated via changes in the number and affinity of the RF uptake carriers. While modulating the activities of intracellular protein kinase A (PKA)-, protein kinase C (PKC)-, protein tyrosine kinase (PTK)-, and nitric oxide (NO)-mediated pathways were found to have no role in regulating RF uptake, a role for the Ca2+ -calmodulin-mediated pathway was observed. These studies demonstrate for the first time the involvement of a specialized carrier-mediated mechanism for RF uptake by hRPE cells and show that the process is

  6. 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.

  7. The heme exporter Flvcr1 regulates expansion and differentiation of committed erythroid progenitors by controlling intracellular heme accumulation.

    PubMed

    Mercurio, Sonia; Petrillo, Sara; Chiabrando, Deborah; Bassi, Zuni Irma; Gays, Dafne; Camporeale, Annalisa; Vacaru, Andrei; Miniscalco, Barbara; Valperga, Giulio; Silengo, Lorenzo; Altruda, Fiorella; Baron, Margaret H; Santoro, Massimo Mattia; Tolosano, Emanuela

    2015-06-01

    Feline leukemia virus subgroup C receptor 1 (Flvcr1) encodes two heme exporters: FLVCR1a, which localizes to the plasma membrane, and FLVCR1b, which localizes to mitochondria. Here, we investigated the role of the two Flvcr1 isoforms during erythropoiesis. We showed that, in mice and zebrafish, Flvcr1a is required for the expansion of committed erythroid progenitors but cannot drive their terminal differentiation, while Flvcr1b contributes to the expansion phase and is required for differentiation. FLVCR1a-down-regulated K562 cells have defective proliferation, enhanced differentiation, and heme loading in the cytosol, while FLVCR1a/1b-deficient K562 cells show impairment in both proliferation and differentiation, and accumulate heme in mitochondria. These data support a model in which the coordinated expression of Flvcr1a and Flvcr1b contributes to control the size of the cytosolic heme pool required to sustain metabolic activity during the expansion of erythroid progenitors and to allow hemoglobinization during their terminal maturation. Consistently, reduction or increase of the cytosolic heme rescued the erythroid defects in zebrafish deficient in Flvcr1a or Flvcr1b, respectively. Thus, heme export represents a tightly regulated process that controls erythropoiesis.

  8. The neuroprotective action of the mood stabilizing drugs lithium chloride and sodium valproate is mediated through the up-regulation of the homeodomain protein Six1.

    PubMed

    Plant, Kathryn E; Anderson, Elizabeth; Simecek, Nicole; Brown, Richard; Forster, Sam; Spinks, Jenny; Toms, Nick; Gibson, G Gordon; Lyon, Jon; Plant, Nick

    2009-02-15

    The mood stabilizing agents lithium chloride (LiCl) and sodium valproate (VPA) have recently gained interest as potential neuroprotective therapeutics. However, exploitation of these therapeutic applications is hindered by both a lack of molecular understanding of the mode of action, and a number of sub-optimal properties, including a relatively small therapeutic window and variable patient response. Human neuroblastoma cells (SH-SY5Y) were exposed to 1 mM lithium chloride or 1 mM sodium valproate for 6 h or 72 h, and transcriptomes measured by Affymetrix U133A/B microarray. Statistically significant gene expression changes were identified using SAM software, with selected changes confirmed at transcript (TaqMan) and protein (Western blotting) levels. Finally, anti-apoptotic action was measured by an in vitro fluorescent assay. Exposure of SH-SY5Y cells to therapeutically relevant concentrations of either lithium chloride or sodium valproate elicited 936 statistically significant changes in gene expression. Amongst these changes we observed a large (maximal 31.3-fold) increase in the expression of the homeodomain protein Six1, and have characterized the time- and dose-dependent up-regulation of this gene in response to both drugs. In addition, we demonstrate that, like LiCl or VPA treatment, Six1 over-expression protects SH-SY5Y cells from staurosporine-induced apoptosis via the blockade of caspsase-3 activation, whereas removal of Six1 protein via siRNA antagonises the ability of LiCl and VPA to protect SH-SY5Y cells from STS-induced apoptosis. These results provide a novel mechanistic rationale underlying the neuroprotective mechanism of LiCl and VPA, suggesting exciting possibilities for the development of novel therapeutic agents against neurodegenerative diseases such as Alzheimer's or Parkinsonism.

  9. The neuroprotective action of the mood stabilizing drugs lithium chloride and sodium valproate is mediated through the up-regulation of the homeodomain protein Six1

    SciTech Connect

    Plant, Kathryn E.; Anderson, Elizabeth; Simecek, Nicole; Brown, Richard; Forster, Sam; Spinks, Jenny; Toms, Nick; Gibson, G. Gordon; Lyon, Jon; Plant, Nick

    2009-02-15

    The mood stabilizing agents lithium chloride (LiCl) and sodium valproate (VPA) have recently gained interest as potential neuroprotective therapeutics. However, exploitation of these therapeutic applications is hindered by both a lack of molecular understanding of the mode of action, and a number of sub-optimal properties, including a relatively small therapeutic window and variable patient response. Human neuroblastoma cells (SH-SY5Y) were exposed to 1 mM lithium chloride or 1 mM sodium valproate for 6 h or 72 h, and transcriptomes measured by Affymetrix U133A/B microarray. Statistically significant gene expression changes were identified using SAM software, with selected changes confirmed at transcript (TaqMan) and protein (Western blotting) levels. Finally, anti-apoptotic action was measured by an in vitro fluorescent assay. Exposure of SH-SY5Y cells to therapeutically relevant concentrations of either lithium chloride or sodium valproate elicited 936 statistically significant changes in gene expression. Amongst these changes we observed a large (maximal 31.3-fold) increase in the expression of the homeodomain protein Six1, and have characterized the time- and dose-dependent up-regulation of this gene in response to both drugs. In addition, we demonstrate that, like LiCl or VPA treatment, Six1 over-expression protects SH-SY5Y cells from staurosporine-induced apoptosis via the blockade of caspsase-3 activation, whereas removal of Six1 protein via siRNA antagonises the ability of LiCl and VPA to protect SH-SY5Y cells from STS-induced apoptosis. These results provide a novel mechanistic rationale underlying the neuroprotective mechanism of LiCl and VPA, suggesting exciting possibilities for the development of novel therapeutic agents against neurodegenerative diseases such as Alzheimer's or Parkinsonism.

  10. A new level of regulation in gluconeogenesis: metabolic state modulates the intracellular localization of aldolase B and its interaction with liver fructose-1,6-bisphosphatase.

    PubMed

    Droppelmann, Cristian A; Sáez, Doris E; Asenjo, Joel L; Yáñez, Alejandro J; García-Rocha, Mar; Concha, Ilona I; Grez, Manuel; Guinovart, Joan J; Slebe, Juan C

    2015-12-01

    Understanding how glucose metabolism is finely regulated at molecular and cellular levels in the liver is critical for knowing its relationship to related pathologies, such as diabetes. In order to gain insight into the regulation of glucose metabolism, we studied the liver-expressed isoforms aldolase B and fructose-1,6-bisphosphatase-1 (FBPase-1), key enzymes in gluconeogenesis, analysing their cellular localization in hepatocytes under different metabolic conditions and their protein-protein interaction in vitro and in vivo. We observed that glucose, insulin, glucagon and adrenaline differentially modulate the intracellular distribution of aldolase B and FBPase-1. Interestingly, the in vitro protein-protein interaction analysis between aldolase B and FBPase-1 showed a specific and regulable interaction between them, whereas aldolase A (muscle isozyme) and FBPase-1 showed no interaction. The affinity of the aldolase B and FBPase-1 complex was modulated by intermediate metabolites, but only in the presence of K(+). We observed a decreased association constant in the presence of adenosine monophosphate, fructose-2,6-bisphosphate, fructose-6-phosphate and inhibitory concentrations of fructose-1,6-bisphosphate. Conversely, the association constant of the complex increased in the presence of dihydroxyacetone phosphate (DHAP) and non-inhibitory concentrations of fructose-1,6-bisphosphate. Notably, in vivo FRET studies confirmed the interaction between aldolase B and FBPase-1. Also, the co-expression of aldolase B and FBPase-1 in cultured cells suggested that FBPase-1 guides the cellular localization of aldolase B. Our results provide further evidence that metabolic conditions modulate aldolase B and FBPase-1 activity at the cellular level through the regulation of their interaction, suggesting that their association confers a catalytic advantage for both enzymes. PMID:26417114

  11. Nuclear Localization of the Autism Candidate Gene Neurobeachin and Functional Interaction with the NOTCH1 Intracellular Domain Indicate a Role in Regulating Transcription

    PubMed Central

    Tuand, Krizia; Stijnen, Pieter; Volders, Karolien; Declercq, Jeroen; Nuytens, Kim; Meulemans, Sandra; Creemers, John

    2016-01-01

    Background Neurobeachin (NBEA) is an autism spectrum disorders (ASD) candidate gene. NBEA deficiency affects regulated secretion, receptor trafficking, synaptic architecture and protein kinase A (PKA)-mediated phosphorylation. NBEA is a large multidomain scaffolding protein. From N- to C-terminus, NBEA has a concanavalin A-like lectin domain flanked by armadillo repeats (ACA), an A-kinase anchoring protein domain that can bind to PKA, a domain of unknown function (DUF1088) and a BEACH domain, preceded by a pleckstrin homology-like domain and followed by WD40 repeats (PBW). Although most of these domains mediate protein-protein interactions, no interaction screen has yet been performed. Methods Yeast two-hybrid screens with the ACA and PBW domain modules of NBEA gave a list of interaction partners, which were analyzed for Gene Ontology (GO) enrichment. Neuro-2a cells were used for confocal microscopy and nuclear extraction analysis. NOTCH-mediated transcription was studied with luciferase reporter assays and qRT-PCR, combined with NBEA knockdown or overexpression. Results Both domain modules showed a GO enrichment for the nucleus. PBW almost exclusively interacted with transcription regulators, while ACA interacted with a number of PKA substrates. NBEA was partially localized in the nucleus of Neuro-2a cells, albeit much less than in the cytoplasm. A nuclear localization signal was found in the DUF1088 domain, which was shown to contribute to the nuclear localization of an EGFP-DPBW fusion protein. Yeast two-hybrid identified the Notch1 intracellular domain as a physical interactor of the PBW domain and a role for NBEA as a negative regulator in Notch-mediated transcription was demonstrated. Conclusion Defining novel interaction partners of conserved NBEA domain modules identified a role for NBEA as transcriptional regulator in the nucleus. The physical interaction of NBEA with NOTCH1 is most relevant for ASD pathogenesis because NOTCH signaling is essential for

  12. A new level of regulation in gluconeogenesis: metabolic state modulates the intracellular localization of aldolase B and its interaction with liver fructose-1,6-bisphosphatase.

    PubMed

    Droppelmann, Cristian A; Sáez, Doris E; Asenjo, Joel L; Yáñez, Alejandro J; García-Rocha, Mar; Concha, Ilona I; Grez, Manuel; Guinovart, Joan J; Slebe, Juan C

    2015-12-01

    Understanding how glucose metabolism is finely regulated at molecular and cellular levels in the liver is critical for knowing its relationship to related pathologies, such as diabetes. In order to gain insight into the regulation of glucose metabolism, we studied the liver-expressed isoforms aldolase B and fructose-1,6-bisphosphatase-1 (FBPase-1), key enzymes in gluconeogenesis, analysing their cellular localization in hepatocytes under different metabolic conditions and their protein-protein interaction in vitro and in vivo. We observed that glucose, insulin, glucagon and adrenaline differentially modulate the intracellular distribution of aldolase B and FBPase-1. Interestingly, the in vitro protein-protein interaction analysis between aldolase B and FBPase-1 showed a specific and regulable interaction between them, whereas aldolase A (muscle isozyme) and FBPase-1 showed no interaction. The affinity of the aldolase B and FBPase-1 complex was modulated by intermediate metabolites, but only in the presence of K(+). We observed a decreased association constant in the presence of adenosine monophosphate, fructose-2,6-bisphosphate, fructose-6-phosphate and inhibitory concentrations of fructose-1,6-bisphosphate. Conversely, the association constant of the complex increased in the presence of dihydroxyacetone phosphate (DHAP) and non-inhibitory concentrations of fructose-1,6-bisphosphate. Notably, in vivo FRET studies confirmed the interaction between aldolase B and FBPase-1. Also, the co-expression of aldolase B and FBPase-1 in cultured cells suggested that FBPase-1 guides the cellular localization of aldolase B. Our results provide further evidence that metabolic conditions modulate aldolase B and FBPase-1 activity at the cellular level through the regulation of their interaction, suggesting that their association confers a catalytic advantage for both enzymes.

  13. NOS1AP modulates intracellular Ca2+ in cardiac myocytes and is up-regulated in dystrophic cardiomyopathy

    PubMed Central

    Treuer, Adriana V; Gonzalez, Daniel R

    2014-01-01

    NOS1AP gene (nitric oxide synthase 1-adaptor protein) is strongly associated with abnormalities in the QT interval of the electrocardiogram and with sudden cardiac death. To determine the role of NOS1AP in the physiology of the cardiac myocyte, we assessed the impact of silencing NOS1AP, using siRNA, on [Ca2+]i transients in neonatal cardiomyocytes. In addition, we examined the co-localization of NOS1AP with cardiac ion channels, and finally, evaluated the expression of NOS1AP in a mouse model of dystrophic cardiomyopathy. Using siRNA, NOS1AP levels were reduced to ~30% of the control levels (p<0.05). NOS1AP silencing in cardiac myocytes reduced significantly the amplitude of electrically evoked calcium transients (p<0.05) and the degree of S-nitrosylation of the cells (p<0.05). Using confocal microscopy, we evaluated NOS1AP subcellular location and interactions with other proteins by co-localization analysis. NOS1AP showed a high degree of co-localization with the L-type calcium channel and the inwardly rectifying potassium channel Kir3.1, a low degree of co-localization with the ryanodine receptor (RyR2) and alfa-sarcomeric actin and no co-localization with connexin 43, suggesting functionally relevant interactions with the ion channels that regulate the action potential duration. Finally, using immunofluorescence and Western blotting, we observed that in mice with dystrophic cardiomyopathy, NOS1AP was significantly up-regulated (p<0.05). These results suggest for a role of NOS1AP on cardiac arrhythmias, acting on the L-type calcium channel, and potassium channels, probably through S-nitrosylation. PMID:24665357

  14. Intracellular Acidosis Promotes Mitochondrial Apoptosis Pathway: Role of EMMPRIN Down-regulation via Specific Single-chain Fv Intrabody.

    PubMed

    Thammasit, Patcharin; Sangboonruang, Sirikwan; Suwanpairoj, Supattara; Khamaikawin, Wannisa; Intasai, Nutjeera; Kasinrerk, Watchara; Tayapiwatana, Chatchai; Tragoolpua, Khajornsak

    2015-01-01

    Extracellular matrix metalloproteinase inducer (EMMPRIN) is a human leukocyte surface molecule that is enriched on the surface of many cancer cells, and it plays an important role in proliferation and metastasis. In this study, we utilized the chimeric adenoviral vector Ad5/F35 carrying gene encoding scFv against EMMPRIN (scFv-M6-1B9) to down-regulate EMMPRIN cell surface expression and investigated programmed cell death response in colorectal cancer (CRC) cell, Caco-2. The scFv-M6-1B9 intrabody exhibits robust activity in reducing EMMPRIN cell surface expression. This approach led to the inducing of apoptosis, which was relative to the increasing of apoptotic bodies in sub-G1 peak, phosphatidylserine externalization, as well as TUNEL-positive cells. In addition, real-time RT-PCR and western blotting analysis indicated that apoptosis was enhanced through the mitochondrial pathway, a marked reduction of Bcl-2, leading to the translocation of cytochrome c and also the dramatic activation of caspase-3. Moreover, carcinoembryonic antigen (CEA), a tumor marker for CRC, was found to have significantly diminished in both secreted protein and mRNA levels. In conclusion, these findings suggest that EMMPRIN down-regulation by scFv-M6-1B9 intrabody has great potential in enhancing the efficacy of apoptosis induction through the mitochondrial pathway and in effecting a decline in the CEA level. Thus, its benefits could be applied to project the future prospects for targeted gene therapy and therapeutic application in monitoring colorectal cancer.

  15. Intracellular Acidosis Promotes Mitochondrial Apoptosis Pathway: Role of EMMPRIN Down-regulation via Specific Single-chain Fv Intrabody

    PubMed Central

    Thammasit, Patcharin; Sangboonruang, Sirikwan; Suwanpairoj, Supattara; Khamaikawin, Wannisa; Intasai, Nutjeera; Kasinrerk, Watchara; Tayapiwatana, Chatchai; Tragoolpua, Khajornsak

    2015-01-01

    Extracellular matrix metalloproteinase inducer (EMMPRIN) is a human leukocyte surface molecule that is enriched on the surface of many cancer cells, and it plays an important role in proliferation and metastasis. In this study, we utilized the chimeric adenoviral vector Ad5/F35 carrying gene encoding scFv against EMMPRIN (scFv-M6-1B9) to down-regulate EMMPRIN cell surface expression and investigated programmed cell death response in colorectal cancer (CRC) cell, Caco-2. The scFv-M6-1B9 intrabody exhibits robust activity in reducing EMMPRIN cell surface expression. This approach led to the inducing of apoptosis, which was relative to the increasing of apoptotic bodies in sub-G1 peak, phosphatidylserine externalization, as well as TUNEL-positive cells. In addition, real-time RT-PCR and western blotting analysis indicated that apoptosis was enhanced through the mitochondrial pathway, a marked reduction of Bcl-2, leading to the translocation of cytochrome c and also the dramatic activation of caspase-3. Moreover, carcinoembryonic antigen (CEA), a tumor marker for CRC, was found to have significantly diminished in both secreted protein and mRNA levels. In conclusion, these findings suggest that EMMPRIN down-regulation by scFv-M6-1B9 intrabody has great potential in enhancing the efficacy of apoptosis induction through the mitochondrial pathway and in effecting a decline in the CEA level. Thus, its benefits could be applied to project the future prospects for targeted gene therapy and therapeutic application in monitoring colorectal cancer. PMID:25663946

  16. The regulation of intracellular pH studied by 31P- and 1H-NMR spectroscopy in superfused guinea-pig cerebral cortex slices.

    PubMed

    Brooks, K J; Bachelard, H S

    1992-10-01

    (1) The intracellular pH (pHi) of superfused slices of guinea-pig cerebral cortex was measured in 31P-NMR spectra using the chemical shifts of intracellular inorganic phosphate (Pi) and of 2-deoxyglucose 6-phosphate (DOG6P). The pHi was found to be 7.30 +/- 0.04 (SD, n = 15) in bicarbonate-buffered medium and 7.20 +/- 0.05 (n = 10, P < 0.001) in bicarbonate-free HEPES buffer of the same pH (7.4). (2) Decreases in pHe below 7.05 resulted in pHi falling to similar values, with a decrease in the energy state. There was no change in intracellular lactate as assessed by 1H-NMR. (3) The tissues showed an ability to buffer higher pH: increasing pHe to 8.0 had no effect on pHi, PCr or lactate. (4) In order to characterize possible mechanisms of pH regulation in the tissue, the recovery from acid insult was investigated under various conditions. Initially pHi was decreased to 6.44 +/- 0.15 (n = 15) by exposure to media containing 6 mM bicarbonate gassed with O2/CO2, 80:20 (pHe 6.4). When this medium was replaced by normal bicarbonate buffer (pH 7.4) there was full recovery of pHi to 7.31 +/- 0.05 (n = 15), whereas replacing the buffer with HEPES resulted in incomplete recovery of pHi to 6.88 +/- 0.15 (n = 15, P < 0.001). (5) In the presence of the carbonic anhydrase inhibitor, acetazolamide (1 mM), or the sodium/proton exchange inhibitor, amiloride (1 mM), there was an incomplete return of pHi to the control value (pHi 6.90 +/- 0.20, n = 5, P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1303163

  17. SPAK isoforms and OSR1 regulate sodium-chloride co-transporters in a nephron-specific manner.

    PubMed

    Grimm, P Richard; Taneja, Tarvinder K; Liu, Jie; Coleman, Richard; Chen, Yang-Yi; Delpire, Eric; Wade, James B; Welling, Paul A

    2012-11-01

    STE20/SPS-1-related proline-alanine-rich protein kinase (SPAK) and oxidative stress-related kinase (OSR1) activate the potassium-dependent sodium-chloride co-transporter, NKCC2, and thiazide-sensitive sodium-chloride cotransporter, NCC, in vitro, and both co-localize with a kinase regulatory molecule, Cab39/MO25α, at the apical membrane of the thick ascending limb (TAL) and distal convoluted tubule (DCT). Yet genetic ablation of SPAK in mice causes a selective loss of NCC function, whereas NKCC2 becomes hyperphosphorylated. Here, we explore the underlying mechanisms in wild-type and SPAK-null mice. Unlike in the DCT, OSR1 remains at the TAL apical membrane of KO mice where it is accompanied by an increase in the active, phosphorylated form of AMP-activated kinase. We found an alterative SPAK isoform (putative SPAK2 form), which modestly inhibits co-transporter activity in vitro, is more abundant in the medulla than the cortex. Thus, enhanced NKCC2 phosphorylation in the SPAK knock-out may be explained by removal of inhibitory SPAK2, sustained activity of OSR1, and activation of other kinases. By contrast, the OSR1/SPAK/M025α signaling apparatus is disrupted in the DCT. OSR1 becomes largely inactive and displaced from M025α and NCC at the apical membrane, and redistributes to dense punctate structures, containing WNK1, within the cytoplasm. These changes are paralleled by a decrease in NCC phosphorylation and a decrease in the mass of the distal convoluted tubule, exclusive to DCT1. As a result of the dependent nature of OSR1 on SPAK in the DCT, NCC is unable to be activated. Consequently, SPAK(-/-) mice are highly sensitive to dietary salt restriction, displaying prolonged negative sodium balance and hypotension.

  18. 7 CFR 58.434 - Calcium chloride.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 3 2010-01-01 2010-01-01 false Calcium chloride. 58.434 Section 58.434 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Standards... Material § 58.434 Calcium chloride. Calcium chloride, when used, shall meet the requirements of the...

  19. 7 CFR 58.434 - Calcium chloride.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 3 2014-01-01 2014-01-01 false Calcium chloride. 58.434 Section 58.434 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Standards... Material § 58.434 Calcium chloride. Calcium chloride, when used, shall meet the requirements of the...

  20. 7 CFR 58.434 - Calcium chloride.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 3 2013-01-01 2013-01-01 false Calcium chloride. 58.434 Section 58.434 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Standards... Material § 58.434 Calcium chloride. Calcium chloride, when used, shall meet the requirements of the...

  1. 7 CFR 58.434 - Calcium chloride.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 3 2012-01-01 2012-01-01 false Calcium chloride. 58.434 Section 58.434 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Standards... Material § 58.434 Calcium chloride. Calcium chloride, when used, shall meet the requirements of the...

  2. 7 CFR 58.434 - Calcium chloride.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 3 2011-01-01 2011-01-01 false Calcium chloride. 58.434 Section 58.434 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Standards... Material § 58.434 Calcium chloride. Calcium chloride, when used, shall meet the requirements of the...

  3. A Novel Metal Transporter Mediating Manganese Export (MntX) Regulates the Mn to Fe Intracellular Ratio and Neisseria meningitidis Virulence

    PubMed Central

    Veyrier, Frédéric J.; Boneca, Ivo G.; Cellier, Mathieu F.; Taha, Muhamed-Kheir

    2011-01-01

    Neisseria meningitidis (Nm) and N. gonorrhoeae (Ng) are adapted to different environments within their human host. If the basis of this difference has not yet been fully understood, previous studies (including our own data) have reported that, unlike Ng, Nm tolerates high manganese concentrations. As transition metals are essential regulators of cell growth and host pathogen interactions, we aimed to address mechanisms of Nm Mn2+ tolerance and its pathogenic consequences. Using bioinformatics, gene deletion and heterologous expression we identified a conserved bacterial manganese resistance factor MntX (formerly YebN). The predicted structure suggests that MntX represents a new family of transporters exporting Mn. In the Neisseria genus, this exporter is present and functional in all Nm isolates but it is mutated in a majority of Ng strains and commonly absent in nonpathogenic species. In Nm, Mn2+ export via MntX regulates the intracellular Mn/Fe ratio and protects against manganese toxicity that is exacerbated in low iron conditions. MntX is also important for N. meningitidis to resist killing by human serum and for survival in mice blood during septicemia. The present work thus points to new clues about Mn homeostasis, its interplay with Fe metabolism and the influence on N. meningitidis physiology and pathogenicity. PMID:21980287

  4. Mepiquat chloride

    Integrated Risk Information System (IRIS)

    Mepiquat chloride ; CASRN 24307 - 26 - 4 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogen

  5. Methyl chloride

    Integrated Risk Information System (IRIS)

    Methyl chloride ; CASRN 74 - 87 - 3 ( 07 / 17 / 2001 ) Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for

  6. Ethyl chloride

    Integrated Risk Information System (IRIS)

    Ethyl chloride ; CASRN 75 - 00 - 3 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Eff

  7. Benzyl chloride

    Integrated Risk Information System (IRIS)

    Benzyl chloride ; CASRN 100 - 44 - 7 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic E

  8. Vinyl chloride

    Integrated Risk Information System (IRIS)

    Vinyl chloride ; CASRN 75 - 01 - 4 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Eff

  9. Acetyl chloride

    Integrated Risk Information System (IRIS)

    Acetyl chloride ; CASRN 75 - 36 - 5 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Ef

  10. Hydrogen chloride

    Integrated Risk Information System (IRIS)

    Hydrogen chloride ; CASRN 7647 - 01 - 0 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogeni

  11. Allyl chloride

    Integrated Risk Information System (IRIS)

    Allyl chloride ; CASRN 107 - 05 - 1 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Ef

  12. The gene for congenital chloride diarrhea maps close to but is distinct from the gene for cystic fibrosis transmembrane conductance regulator

    SciTech Connect

    Kere, J.; de la Chapelle, A.; Holmberg, C. ); Sistonen, P. Finnish Red Cross Blood Transfusion Service, Helsinki )

    1993-11-15

    Congenital chloride diarrhea (CLD) is characterized by watery stools with high chloride content beginning prenatally and is inherited as an autosomal recessive trait. Perfusion studies have established a basic defect in ileal and colonic Cl[sup [minus

  13. Moderate activation of autophagy regulates the intracellular calcium ion concentration and mitochondrial membrane potential in beta-amyloid-treated PC12 cells.

    PubMed

    Xue, Zhongfeng; Guo, Yalei; Fang, Yongqi

    2016-04-01

    Alzheimer's disease (AD) is an age-related and progressive neurodegenerative disease. Aggregated beta-amyloid (Aβ) disturbs Ca(2+) homeostasis and causes mitochondrial dysfunction and finally underlies AD. Recent evidence suggests that autophagy initiation by Beclin-1 protein might be involved in the pathogenesis of AD. However, the effects of Beclin-1 dependent autophagy on intracellular calcium ion concentration ([Ca(2+)]i) and mitochondrial membrane potential (MMP) is unclear. The effects of Beclin-1 dependent autophagy that were activated by a gradient concentration of autophagy activator rapamycin or inhibited by autophagy inhibitor 3-methyladenine (3-MA) on cell viability and cell morphology were examined. Pretreatment with rapamycin significantly up-regulated the expression of Beclin-1 in response to Aβ1-42 application, but after pretreatment with 3-MA it was significantly down-regulated. Moderate activation of Beclin-1 dependent autophagy had an up regulation effect on cell viability and could maintain the original morphology of cells. Furthermore, rapamycin or 3-MA on [Ca(2+)]i and MMP in Aβ1-42 treatment of PC12 cells were evaluated. We also report that PC12 cells treated with Aβ1-42 showed an increase in [Ca(2+)]i but a decrease in MMP when compared to the normal control. However the application of rapamycin prior to this prevented the increase in [Ca(2+)]i and the decrease in MMP in response to Aβ1-42. When 3-MA was applied this exacerbated the effect of Aβ1-42 on the [Ca(2+)]i and the MMP. This shows that moderate activation of Beclin-1 dependent autophagy by rapamycin can modulate Ca(2+) homeostasis and maintain MMP in response to Aβ1-42 induced cytotoxicity and so may have a preventive function in AD. PMID:26923671

  14. Parallel down-regulation of chloride channel CLC-K1 and barttin mRNA in the thin ascending limb of the rat nephron by furosemide.

    PubMed

    Wolf, Konrad; Meier-Meitinger, Martina; Bergler, Tobias; Castrop, Hayo; Vitzthum, Helga; Riegger, Günter A J; Kurtz, Armin; Krämer, Bernhard K

    2003-09-01

    In the past few years the pivotal role of kidney Cl(-)channels (ClC-K) channels in maintaining salt and water homeostasis in the kidney has been established. The aim of the present study was to investigate the influence of the loop diuretic furosemide on the gene expression of the kidney chloride channel ClC-K1 and its recently described functional subunit barttin. Male Sprague Dawley rats received the loop diuretic furosemide (12 mg/kg/day) for 6 days. Rats had free access to 0.9% NaCl, 0.1%KCl solution to prevent volume depletion. Localisation and regulation of ClC-K1 and barttin mRNA was analysed by RNase protection and in situ hybridisation. Nephron-specific regulation was investigated by microdissection and real-time PCR quantification. In furosemide-treated rats ClC-K1 mRNA decreased to half in the inner medulla. In the renal cortex and outer medulla ClC-K1 mRNA levels were weak and did not change. Under furosemide treatment barttin mRNA was regulated in parallel with ClC-K1 mRNA. A significant mRNA decrease occurred after furosemide treatment in inner medulla (0.50 fold), whereas cortical and outer medulla levels remained unaffected. (35)S in situ hybridisation confirmed the regulation and distribution seen in the RNase protection assay experiments. Microdissection of the inner medullary collecting duct and thin limb of Henle's loop followed by real-time PCR revealed that CLC-K1 and barttin mRNA regulation in inner medulla was limited to the thin limb; mRNA levels in collecting ducts were not affected by furosemide treatment. Our findings imply that during furosemide treatment selective down-regulation of ClC-K1 and barttin mRNAs in thin limb plays a role in maintaining salt and water homeostasis. PMID:12759757

  15. Chloride-hydrogen antiporters ClC-3 and ClC-5 drive osteoblast mineralization and regulate fine-structure bone patterning in vitro.

    PubMed

    Larrouture, Quitterie C; Nelson, Deborah J; Robinson, Lisa J; Liu, Li; Tourkova, Irina; Schlesinger, Paul H; Blair, Harry C

    2015-11-01

    Osteoblasts form an epithelium-like layer with tight junctions separating bone matrix from extracellular fluid. During mineral deposition, calcium and phosphate precipitation in hydroxyapatite liberates 0.8 mole of H(+) per mole Ca(+2). Thus, acid export is needed for mineral formation. We examined ion transport supporting osteoblast vectorial mineral deposition. Previously we established that Na/H exchangers 1 and 6 are highly expressed at secretory osteoblast basolateral surfaces and neutralize massive acid loads. The Na/H exchanger regulatory factor-1 (NHERF1), a pdz-organizing protein, occurs at mineralizing osteoblast basolateral surfaces. We hypothesized that high-capacity proton transport from matrix into osteoblast cytosol must exist to support acid transcytosis for mineral deposition. Gene screening in mineralizing osteoblasts showed dramatic expression of chloride-proton antiporters ClC-3 and ClC-5. Antibody localization showed that ClC-3 and ClC-5 occur at the apical secretory surface facing the bone matrix and in membranes of buried osteocytes. Surprisingly, the Clcn3(-/-) mouse has only mildly disordered mineralization. However, Clcn3(-/-) osteoblasts have large compensatory increases in ClC-5 expression. Clcn3(-/-) osteoblasts mineralize in vitro in a striking and novel trabecular pattern; wild-type osteoblasts form bone nodules. In mesenchymal stem cells from Clcn3(-/-) mice, lentiviral ClC-5 shRNA created Clcn3(-/-), ClC-5 knockdown cells, validated by western blot and PCR. Osteoblasts from these cells produced no mineral under conditions where wild-type or Clcn3(-/-) cells mineralize well. We conclude that regulated acid export, mediated by chloride-proton exchange, is essential to drive normal bone mineralization, and that CLC transporters also regulate fine patterning of bone.

  16. Chloride-hydrogen antiporters ClC-3 and ClC-5 drive osteoblast mineralization and regulate fine-structure bone patterning in vitro.

    PubMed

    Larrouture, Quitterie C; Nelson, Deborah J; Robinson, Lisa J; Liu, Li; Tourkova, Irina; Schlesinger, Paul H; Blair, Harry C

    2015-11-01

    Osteoblasts form an epithelium-like layer with tight junctions separating bone matrix from extracellular fluid. During mineral deposition, calcium and phosphate precipitation in hydroxyapatite liberates 0.8 mole of H(+) per mole Ca(+2). Thus, acid export is needed for mineral formation. We examined ion transport supporting osteoblast vectorial mineral deposition. Previously we established that Na/H exchangers 1 and 6 are highly expressed at secretory osteoblast basolateral surfaces and neutralize massive acid loads. The Na/H exchanger regulatory factor-1 (NHERF1), a pdz-organizing protein, occurs at mineralizing osteoblast basolateral surfaces. We hypothesized that high-capacity proton transport from matrix into osteoblast cytosol must exist to support acid transcytosis for mineral deposition. Gene screening in mineralizing osteoblasts showed dramatic expression of chloride-proton antiporters ClC-3 and ClC-5. Antibody localization showed that ClC-3 and ClC-5 occur at the apical secretory surface facing the bone matrix and in membranes of buried osteocytes. Surprisingly, the Clcn3(-/-) mouse has only mildly disordered mineralization. However, Clcn3(-/-) osteoblasts have large compensatory increases in ClC-5 expression. Clcn3(-/-) osteoblasts mineralize in vitro in a striking and novel trabecular pattern; wild-type osteoblasts form bone nodules. In mesenchymal stem cells from Clcn3(-/-) mice, lentiviral ClC-5 shRNA created Clcn3(-/-), ClC-5 knockdown cells, validated by western blot and PCR. Osteoblasts from these cells produced no mineral under conditions where wild-type or Clcn3(-/-) cells mineralize well. We conclude that regulated acid export, mediated by chloride-proton exchange, is essential to drive normal bone mineralization, and that CLC transporters also regulate fine patterning of bone. PMID:26603451

  17. Exercise modulates chloride homeostasis after spinal cord injury.

    PubMed

    Côté, Marie-Pascale; Gandhi, Sapan; Zambrotta, Marina; Houlé, John D

    2014-07-01

    Activity-based therapies are routinely integrated in spinal cord injury (SCI) rehabilitation programs because they result in a reduction of hyperreflexia and spasticity. However, the mechanisms by which exercise regulates activity in spinal pathways to reduce spasticity and improve functional recovery are poorly understood. Persisting alterations in the action of GABA on postsynaptic targets is a signature of CNS injuries, including SCI. The action of GABA depends on the intracellular chloride concentration, which is determined largely by the expression of two cation-chloride cotransporters (CCCs), KCC2 and NKCC1, which serve as chloride exporters and importers, respectively. We hypothesized that the reduction in hyperreflexia with exercise after SCI relies on a return to chloride homeostasis. Sprague Dawley rats received a spinal cord transection at T12 and were assigned to SCI-7d, SCI-14d, SCI-14d+exercise, SCI-28d, SCI-28d+exercise, or SCI-56d groups. During a terminal experiment, H-reflexes were recorded from interosseus muscles after stimulation of the tibial nerve and the low-frequency-dependent depression (FDD) was assessed. We provide evidence that exercise returns spinal excitability and levels of KCC2 and NKCC1 toward normal levels in the lumbar spinal cord. Acutely altering chloride extrusion using the KCC2 blocker DIOA masked the effect of exercise on FDD, whereas blocking NKCC1 with bumetanide returned FDD toward intact levels after SCI. Our results indicate that exercise contributes to reflex recovery and restoration of endogenous inhibition through a return to chloride homeostasis after SCI. This lends support for CCCs as part of a pathway that could be manipulated to improve functional recovery when combined with rehabilitation programs.

  18. NKCC1 and NKCC2: The pathogenetic role of cation-chloride cotransporters in hypertension

    PubMed Central

    Orlov, Sergei N.; Koltsova, Svetlana V.; Kapilevich, Leonid V.; Gusakova, Svetlana V.; Dulin, Nickolai O.

    2015-01-01

    This review summarizes the data on the functional significance of ubiquitous (NKCC1) and renal-specific (NKCC2) isoforms of electroneutral sodium, potassium and chloride cotransporters. These carriers contribute to the pathogenesis of hypertension via regulation of intracellular chloride concentration in vascular smooth muscle and neuronal cells and via sensing chloride concentration in the renal tubular fluid, respectively. Both NKCC1 and NKCC2 are inhibited by furosemide and other high-ceiling diuretics widely used for attenuation of extracellular fluid volume. However, the chronic usage of these compounds for the treatment of hypertension and other volume-expanded disorders may have diverse side-effects due to suppression of myogenic response in microcirculatory beds. PMID:26114157

  19. Constitutive intracellular production of iNOS and NO in human melanoma: possible role in regulation of growth and resistance to apoptosis.

    PubMed

    Grimm, Elizabeth A; Ellerhorst, Julie; Tang, Chi-Hui; Ekmekcioglu, Suhendan

    2008-09-01

    Human melanoma tumors cells are known to express the enzyme, inducible nitric oxide synthase (iNOS), which is responsible for cytokine induced nitric oxide (NO) production during immune responses. This constitutive expression of iNOS in many patients' tumor cells, as well as its strong association with poor patient survival, have led to the consideration of iNOS as a molecular marker of poor prognosis, as well as a possible target for therapy. The expression of iNOS in patient tumors was found to associate with nitrotyrosine, COX2, pSTAT3, and arginase. Using human melanoma patients' samples as well as cell lines, we have further evidence supporting intracellular NO production by detection of nitrotyrosine and also by use of DAF-2DA staining. Experiments were performed to scavenge the endogenous NO (with c-PTIO) resulting in melanoma cell growth inhibition; this was restored with SIN-1 (NO and O2-donor) providing data to support a functional role of this gas. Our goal is to understand the aberrant biology leading to this curious phenomenon, and to regulate it in favor of patient treatments.

  20. The intracellular fate of zonula occludens 2 is regulated by the phosphorylation of SR repeats and the phosphorylation/O-GlcNAcylation of S257.

    PubMed

    Quiros, Miguel; Alarcón, Lourdes; Ponce, Arturo; Giannakouros, Thomas; González-Mariscal, Lorenza

    2013-08-01

    Zona occludens 2 (ZO-2) has a dual localization. In confluent epithelia, ZO-2 is present at tight junctions (TJs), whereas in sparse proliferating cells it is also found at the nucleus. Previously we demonstrated that in sparse cultures, newly synthesized ZO-2 travels to the nucleus before reaching the plasma membrane. Now we find that in confluent cultures newly synthesized ZO-2 goes directly to the plasma membrane. Epidermal growth factor induces through AKT activation the phosphorylation of the kinase for SR repeats, serine arginine protein kinase 1, which in turn phosphorylates ZO-2, which contains 16 SR repeats. This phosphorylation induces ZO-2 entry into the nucleus and accumulation in speckles. ZO-2 departure from the nucleus requires intact S257, and stabilizing the β-O-linked N-acetylglucosylation (O-GlcNAc) of S257 with O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate, an inhibitor of O-GlcNAcase, triggers nuclear exportation and proteosomal degradation of ZO-2. At the plasma membrane ZO-2 is not O-GlcNAc, and instead, as TJs mature, it becomes phosphorylated at S257 by protein kinase Cζ. This late phosphorylation of S257 is required for the correct cytoarchitecture to develop, as cells transfected with ZO-2 mutant S257A or S257E form aberrant cysts with multiple lumens. These results reveal novel posttranslational modifications of ZO-2 that regulate the intracellular fate of this protein. PMID:23804652

  1. Dual regulation of the native ClC-K2 chloride channel in the distal nephron by voltage and pH.

    PubMed

    Pinelli, Laurent; Nissant, Antoine; Edwards, Aurélie; Lourdel, Stéphane; Teulon, Jacques; Paulais, Marc

    2016-09-01

    ClC-K2, a member of the ClC family of Cl(-) channels and transporters, forms the major basolateral Cl(-) conductance in distal nephron epithelial cells and therefore plays a central role in renal Cl(-) absorption. However, its regulation remains largely unknown because of the fact that recombinant ClC-K2 has not yet been studied at the single-channel level. In the present study, we investigate the effects of voltage, pH, Cl(-), and Ca(2+) on native ClC-K2 in the basolateral membrane of intercalated cells from the mouse connecting tubule. The ∼10-pS channel shows a steep voltage dependence such that channel activity increases with membrane depolarization. Intracellular pH (pHi) and extracellular pH (pHo) differentially modulate the voltage dependence curve: alkaline pHi flattens the curve by causing an increase in activity at negative voltages, whereas alkaline pHo shifts the curve toward negative voltages. In addition, pHi, pHo, and extracellular Ca(2+) strongly increase activity, mainly because of an increase in the number of active channels with a comparatively minor effect on channel open probability. Furthermore, voltage alters both the number of active channels and their open probability, whereas intracellular Cl(-) has little influence. We propose that changes in the number of active channels correspond to them entering or leaving an inactivated state, whereas modulation of open probability corresponds to common gating by these channels. We suggest that pH, through the combined effects of pHi and pHo on ClC-K2, might be a key regulator of NaCl absorption and Cl(-)/HCO3 (-) exchange in type B intercalated cells. PMID:27574292

  2. A novel role for carbon monoxide as a potent regulator of intracellular Ca2+ and nitric oxide in rat pancreatic acinar cells.

    PubMed

    Moustafa, Amira; Habara, Yoshiaki

    2014-12-01

    Carbon monoxide (CO) is known as an essential gaseous messenger that regulates a wide array of physiological and pathological processes, similar to nitric oxide (NO) and hydrogen sulfide. The aim of the present study was to elucidate the potential role of CO in Ca(2+) homeostasis and to explore the underlying mechanisms in pancreatic acinar cells. The exogenous application of a CO-releasing molecule dose-dependently increased intracellular Ca(2+) concentration ([Ca(2+)]i). A heme oxygenase (HO) inducer increased [Ca(2+)]i in a concentration-dependent manner, and the increase was diminished by an HO inhibitor. The CO-induced [Ca(2+)]i increase persisted in the absence of extracellular Ca(2+), indicating that Ca(2+) release is the initial source for the increase. The inhibition of G protein, phospholipase C (PLC), and inositol 1,4,5-trisphosphate (IP3) receptor diminished the CO-induced [Ca(2+)]i increase. CO upregulated endothelial nitric oxide synthase (eNOS) expression and stimulated NO production, and NOS inhibitor, calmodulin inhibitor, or the absence of extracellular Ca(2+) eliminated the latter response. Blocking the phosphatidylinositol 3-kinase (PI3K)-Akt/protein kinase B (PKB) pathway abolished CO-induced NO production. Pretreatment with an NOS inhibitor, NO scavenger, or soluble guanylate cyclase inhibitor, did not affect the CO-induced [Ca(2+)]i increase, indicating that NO, soluble guanylate cyclase, and cyclic guanosine 5'-monophosphate are not involved in the CO-induced [Ca(2+)]i increase. CO inhibited the secretory responses to CCK-octapeptide or carbachol. We conclude that CO acts as a regulator not only for [Ca(2+)]i homeostasis via a PLC-IP3-IP3 receptor cascade but also for NO production via the calmodulin and PI3K-Akt/PKB pathway, and both CO and NO interact. Moreover, CO may provide potential therapy to ameliorate acute pancreatitis by inhibiting amylase secretion.

  3. Sarcolemmal localisation of Na+/H+ exchange and Na+-HCO3- co-transport influences the spatial regulation of intracellular pH in rat ventricular myocytes.

    PubMed

    Garciarena, Carolina D; Ma, Yu-ling; Swietach, Pawel; Huc, Laurence; Vaughan-Jones, Richard D

    2013-05-01

    Membrane acid extrusion by Na(+)/H(+) exchange (NHE1) and Na(+)-HCO3(-) co-transport (NBC) is essential for maintaining a low cytoplasmic [H(+)] (∼60 nm, equivalent to an intracellular pH (pHi) of 7.2). This protects myocardial function from the high chemical reactivity of H(+) ions, universal end-products of metabolism. We show here that, in rat ventricular myocytes, fluorescent antibodies map the NBC isoforms NBCe1 and NBCn1 to lateral sarcolemma, intercalated discs and transverse tubules (t-tubules), while NHE1 is absent from t-tubules. This unexpected difference matches functional measurements of pHi regulation (using AM-loaded SNARF-1, a pH fluorophore). Thus, myocyte detubulation (by transient exposure to 1.5 m formamide) reduces global acid extrusion on NBC by 40%, without affecting NHE1. Similarly, confocal pHi imaging reveals that NBC stimulation induces spatially uniform pHi recovery from acidosis, whereas NHE1 stimulation induces pHi non-uniformity during recovery (of ∼0.1 units, for 2-3 min), particularly at the ends of the cell where intercalated discs are commonly located, and where NHE1 immunostaining is prominent. Mathematical modelling shows that this induction of local pHi microdomains is favoured by low cytoplasmic H(+) mobility and long H(+) diffusion distances, particularly to surface NHE1 transporters mediating high membrane flux. Our results provide the first evidence for a spatial localisation of [H(+)]i regulation in ventricular myocytes, suggesting that, by guarding pHi, NHE1 preferentially protects gap junctional communication at intercalated discs, while NBC locally protects t-tubular excitation-contraction coupling.

  4. Inhibition of TREK-2 K(+) channels by PI(4,5)P2: an intrinsic mode of regulation by intracellular ATP via phosphatidylinositol kinase.

    PubMed

    Woo, Joohan; Shin, Dong Hoon; Kim, Hyun Jong; Yoo, Hae Young; Zhang, Yin-Hua; Nam, Joo Hyun; Kim, Woo Kyung; Kim, Sung Joon

    2016-08-01

    TWIK-related two-pore domain K(+) channels 1 and 2 (TREKs) are activated under various physicochemical conditions. However, the directions in which they are regulated by PI(4,5)P2 and intracellular ATP are not clearly presented yet. In this study, we investigated the effects of ATP and PI(4,5)P2 on overexpressed TREKs (HEK293T and COS-7) and endogenously expressed TREK-2 (mouse astrocytes and WEHI-231 B cells). In all of these cells, both TREK-1 and TREK-2 currents were spontaneously increased by dialysis with ATP-free pipette solution for whole-cell recording (ITREK-1,w-c and ITREK-2w-c) or by membrane excision for inside-out patch clamping without ATP (ITREK-1,i-o and ITREK-2,i-o). Steady state ITREK-2,i-o was reversibly decreased by 3 mM ATP applied to the cytoplasmic side, and this reduction was prevented by wortmannin, a PI-kinase inhibitor. An exogenous application of PI(4,5)P2 inhibited the spontaneously increased ITREKs,i-o, suggesting that intrinsic PI(4,5)P2 maintained by intracellular ATP and PI kinase may set the basal activity of TREKs in the intact cells. The inhibition of intrinsic TREK-2 by ATP was more prominent in WEHI-231 cells than astrocytes. Interestingly, unspecific screening of negative charges by poly-L-lysine also inhibited ITREK-2,i-o. Application of PI(4,5)P2 after the poly-L-lysine treatment showed dose-dependent dual effects, initial activation and subsequent inhibition of ITREK-2,i-o at low and high concentrations, respectively. In HEK293T cells coexpressing TREK-2 and a voltage-sensitive PI(4,5)P2 phosphatase, sustained depolarization increased ITREK-2,w-c initially (<5 s) but then decreased the current below the control level. In HEK293T cells coexpressing TREK-2 and type 3 muscarinic receptor, application of carbachol induced transient activation and sustained suppression of ITREK-2,w-c and cell-attached ITREK-2. The inhibition of TREK-2 by unspecific electrostatic quenching, extensive dephosphorylation, or sustained hydrolysis

  5. Inhibition of TREK-2 K(+) channels by PI(4,5)P2: an intrinsic mode of regulation by intracellular ATP via phosphatidylinositol kinase.

    PubMed

    Woo, Joohan; Shin, Dong Hoon; Kim, Hyun Jong; Yoo, Hae Young; Zhang, Yin-Hua; Nam, Joo Hyun; Kim, Woo Kyung; Kim, Sung Joon

    2016-08-01

    TWIK-related two-pore domain K(+) channels 1 and 2 (TREKs) are activated under various physicochemical conditions. However, the directions in which they are regulated by PI(4,5)P2 and intracellular ATP are not clearly presented yet. In this study, we investigated the effects of ATP and PI(4,5)P2 on overexpressed TREKs (HEK293T and COS-7) and endogenously expressed TREK-2 (mouse astrocytes and WEHI-231 B cells). In all of these cells, both TREK-1 and TREK-2 currents were spontaneously increased by dialysis with ATP-free pipette solution for whole-cell recording (ITREK-1,w-c and ITREK-2w-c) or by membrane excision for inside-out patch clamping without ATP (ITREK-1,i-o and ITREK-2,i-o). Steady state ITREK-2,i-o was reversibly decreased by 3 mM ATP applied to the cytoplasmic side, and this reduction was prevented by wortmannin, a PI-kinase inhibitor. An exogenous application of PI(4,5)P2 inhibited the spontaneously increased ITREKs,i-o, suggesting that intrinsic PI(4,5)P2 maintained by intracellular ATP and PI kinase may set the basal activity of TREKs in the intact cells. The inhibition of intrinsic TREK-2 by ATP was more prominent in WEHI-231 cells than astrocytes. Interestingly, unspecific screening of negative charges by poly-L-lysine also inhibited ITREK-2,i-o. Application of PI(4,5)P2 after the poly-L-lysine treatment showed dose-dependent dual effects, initial activation and subsequent inhibition of ITREK-2,i-o at low and high concentrations, respectively. In HEK293T cells coexpressing TREK-2 and a voltage-sensitive PI(4,5)P2 phosphatase, sustained depolarization increased ITREK-2,w-c initially (<5 s) but then decreased the current below the control level. In HEK293T cells coexpressing TREK-2 and type 3 muscarinic receptor, application of carbachol induced transient activation and sustained suppression of ITREK-2,w-c and cell-attached ITREK-2. The inhibition of TREK-2 by unspecific electrostatic quenching, extensive dephosphorylation, or sustained hydrolysis

  6. Applications of gene arrays in environmental toxicology: fingerprints of gene regulation associated with cadmium chloride, benzo(a)pyrene, and trichloroethylene.

    PubMed Central

    Bartosiewicz, M; Penn, S; Buckpitt, A

    2001-01-01

    Toxicity testing of unknown chemicals currently uses a number of short-term bioassays. These tests are costly and time consuming, require large numbers of animals, and generally focus on a single end point. The recent development of DNA arrays provides a potential mechanism for increasing the efficiency of standard toxicity testing through genome-wide assessments of gene regulation. In this study, we used DNA arrays containing 148 genes for xenobiotic metabolizing enzymes, DNA repair enzymes, heat shock proteins, cytokines, and housekeeping genes to examine gene expression patterns in the liver in response to cadmium chloride, benzo(a)pyrene (BaP), and trichloroethylene (TCE). Dose-response studies were carried out in mice for each chemical; each produced a unique pattern of gene induction. As expected, CdCl2 markedly up-regulated metallothionine I and II (5- to 10,000-fold at the highest doses) and several of the heat shock/stress response proteins and early response genes. In contrast, administration of BaP up-regulated only Cyp1a1 and Cyp1a2 genes and produced no significant increases in any of the stress response genes or any of the DNA repair genes present on the array. Likewise, TCE-induced gene induction was highly selective; only Hsp 25 and 86 and Cyp2a were up-regulated at the highest dose tested. Microarray analysis with a highly focused set of genes is capable of discriminating between different classes of toxicants and has potential for differentiating highly noxious versus more subtle toxic agents. These data suggest that use of microarrays to evaluate the potential hazards of unknown chemicals or chemical mixtures must include multiple doses and time points to provide effective assessments of potential toxicity of these substances. PMID:11171528

  7. Comparative Proteomics of Ovarian Cancer Aggregate Formation Reveals an Increased Expression of Calcium-activated Chloride Channel Regulator 1 (CLCA1)*

    PubMed Central

    Musrap, Natasha; Tuccitto, Alessandra; Karagiannis, George S.; Saraon, Punit; Batruch, Ihor; Diamandis, Eleftherios P.

    2015-01-01

    Ovarian cancer is a lethal gynecological disease that is characterized by peritoneal metastasis and increased resistance to conventional chemotherapies. This increased resistance and the ability to spread is often attributed to the formation of multicellular aggregates or spheroids in the peritoneal cavity, which seed abdominal surfaces and organs. Given that the presence of metastatic implants is a predictor of poor survival, a better understanding of how spheroids form is critical to improving patient outcome, and may result in the identification of novel therapeutic targets. Thus, we attempted to gain insight into the proteomic changes that occur during anchorage-independent cancer cell aggregation. As such, an ovarian cancer cell line, OV-90, was cultured in adherent and non-adherent conditions using stable isotope labeling with amino acids in cell culture (SILAC). Anchorage-dependent cells (OV-90AD) were grown in tissue culture flasks, whereas anchorage-independent cells (OV-90AI) were grown in suspension using the hanging-drop method. Cellular proteins from both conditions were then identified using LC-MS/MS, which resulted in the quantification of 1533 proteins. Of these, 13 and 6 proteins were up-regulated and down-regulated, respectively, in aggregate-forming cells compared with cells grown as monolayers. Relative gene expression and protein expression of candidates were examined in other cell line models of aggregate formation (TOV-112D and ES-2), which revealed an increased expression of calcium-activated chloride channel regulator 1 (CLCA1). Moreover, inhibitor and siRNA transfection studies demonstrated an apparent effect of CLCA1 on cancer cell aggregation. Further elucidation of the role of CLCA1 in the pathogenesis of ovarian cancer is warranted. PMID:26004777

  8. Comparative Proteomics of Ovarian Cancer Aggregate Formation Reveals an Increased Expression of Calcium-activated Chloride Channel Regulator 1 (CLCA1).

    PubMed

    Musrap, Natasha; Tuccitto, Alessandra; Karagiannis, George S; Saraon, Punit; Batruch, Ihor; Diamandis, Eleftherios P

    2015-07-10

    Ovarian cancer is a lethal gynecological disease that is characterized by peritoneal metastasis and increased resistance to conventional chemotherapies. This increased resistance and the ability to spread is often attributed to the formation of multicellular aggregates or spheroids in the peritoneal cavity, which seed abdominal surfaces and organs. Given that the presence of metastatic implants is a predictor of poor survival, a better understanding of how spheroids form is critical to improving patient outcome, and may result in the identification of novel therapeutic targets. Thus, we attempted to gain insight into the proteomic changes that occur during anchorage-independent cancer cell aggregation. As such, an ovarian cancer cell line, OV-90, was cultured in adherent and non-adherent conditions using stable isotope labeling with amino acids in cell culture (SILAC). Anchorage-dependent cells (OV-90AD) were grown in tissue culture flasks, whereas anchorage-independent cells (OV-90AI) were grown in suspension using the hanging-drop method. Cellular proteins from both conditions were then identified using LC-MS/MS, which resulted in the quantification of 1533 proteins. Of these, 13 and 6 proteins were up-regulated and down-regulated, respectively, in aggregate-forming cells compared with cells grown as monolayers. Relative gene expression and protein expression of candidates were examined in other cell line models of aggregate formation (TOV-112D and ES-2), which revealed an increased expression of calcium-activated chloride channel regulator 1 (CLCA1). Moreover, inhibitor and siRNA transfection studies demonstrated an apparent effect of CLCA1 on cancer cell aggregation. Further elucidation of the role of CLCA1 in the pathogenesis of ovarian cancer is warranted.

  9. Voltage-gated ion channelopathies: inherited disorders caused by abnormal sodium, chloride, and calcium regulation in skeletal muscle.

    PubMed

    Hoffman, E P

    1995-01-01

    The pathological genetic defects in the inherited myotonias and periodic paralyses were recently elucidated using molecular genetic studies. These disorders are usually transmitted as a dominant trait from an affected parent to a child. The many clinical symptoms include cold-induced uncontrollable contraction of muscle, potassium-induced contraction and paralysis, myotonia with dramatic muscular hypertrophy, muscle stiffness, and insulin-induced paralysis (in males). Horses afflicted with the disorder can suddenly collapse, despite an impressive physique. In the past three years, these clinically defined disorders have been shown to share a common etiology: subtle defects of ion channels in the muscle-fiber membrane. Although the specific ion channel involved varies depending on the disease, most patients have single amino acid changes in the channel proteins, with both normal and mutant channels present in each muscle fiber. For each patient, we can now establish a precise molecular diagnosis in the face of overlapping clinical symptoms and begin specific pharmacological treatment based on the primary problem. These studies have also provided insight into basic muscle biology and emphasize the careful regulation of ions in muscle excitation.

  10. Human potassium chloride cotransporter 1 (SLC12A4) promoter is regulated by AP-2 and contains a functional downstream promoter element.

    PubMed

    Zhou, Guo-Ping; Wong, Clara; Su, Robert; Crable, Scott C; Anderson, Kathleen P; Gallagher, Patrick G

    2004-06-01

    Most K-Cl cotransport in the erythrocyte is attributed to potassium chloride cotransporter 1 (KCC1). K-Cl cotransport is elevated in sickle erythrocytes, and the KCC1 gene has been proposed as a modifier gene in sickle cell disease. To provide insight into our understanding of the regulation of the human KCC1 gene, we mapped the 5' end of the KCC1 cDNA, cloned the corresponding genomic DNA, and identified the KCC1 gene promoter. The core promoter lacks a TATA box and is composed of an initiator element (InR) and a downstream promoter element (DPE), a combination found primarily in Drosophila gene promoters and rarely observed in mammalian gene promoters. Mutational analyses demonstrated that both the InR and DPE sites were critical for full promoter activity. In vitro DNase I footprinting, electrophoretic mobility shift assays, and reporter gene assays identified functional AP-2 and Sp1 sites in this region. The KCC1 promoter was transactivated by forced expression of AP-2 in heterologous cells. Sequences encoding the InR, DPE, AP-2, and Sp1 sites were 100% conserved between human and murine KCC1 genes. In vivo studies using chromatin immunoprecipitation assays with antihistone H3 and antihistone H4 antibodies demonstrated hyperacetylation of this core promoter region.

  11. Salt, chloride, bleach, and innate host defense.

    PubMed

    Wang, Guoshun; Nauseef, William M

    2015-08-01

    Salt provides 2 life-essential elements: sodium and chlorine. Chloride, the ionic form of chlorine, derived exclusively from dietary absorption and constituting the most abundant anion in the human body, plays critical roles in many vital physiologic functions, from fluid retention and secretion to osmotic maintenance and pH balance. However, an often overlooked role of chloride is its function in innate host defense against infection. Chloride serves as a substrate for the generation of the potent microbicide chlorine bleach by stimulated neutrophils and also contributes to regulation of ionic homeostasis for optimal antimicrobial activity within phagosomes. An inadequate supply of chloride to phagocytes and their phagosomes, such as in CF disease and other chloride channel disorders, severely compromises host defense against infection. We provide an overview of the roles that chloride plays in normal innate immunity, highlighting specific links between defective chloride channel function and failures in host defense.

  12. Salt, chloride, bleach, and innate host defense

    PubMed Central

    Wang, Guoshun; Nauseef, William M.

    2015-01-01

    Salt provides 2 life-essential elements: sodium and chlorine. Chloride, the ionic form of chlorine, derived exclusively from dietary absorption and constituting the most abundant anion in the human body, plays critical roles in many vital physiologic functions, from fluid retention and secretion to osmotic maintenance and pH balance. However, an often overlooked role of chloride is its function in innate host defense against infection. Chloride serves as a substrate for the generation of the potent microbicide chlorine bleach by stimulated neutrophils and also contributes to regulation of ionic homeostasis for optimal antimicrobial activity within phagosomes. An inadequate supply of chloride to phagocytes and their phagosomes, such as in CF disease and other chloride channel disorders, severely compromises host defense against infection. We provide an overview of the roles that chloride plays in normal innate immunity, highlighting specific links between defective chloride channel function and failures in host defense. PMID:26048979

  13. Salt, chloride, bleach, and innate host defense.

    PubMed

    Wang, Guoshun; Nauseef, William M

    2015-08-01

    Salt provides 2 life-essential elements: sodium and chlorine. Chloride, the ionic form of chlorine, derived exclusively from dietary absorption and constituting the most abundant anion in the human body, plays critical roles in many vital physiologic functions, from fluid retention and secretion to osmotic maintenance and pH balance. However, an often overlooked role of chloride is its function in innate host defense against infection. Chloride serves as a substrate for the generation of the potent microbicide chlorine bleach by stimulated neutrophils and also contributes to regulation of ionic homeostasis for optimal antimicrobial activity within phagosomes. An inadequate supply of chloride to phagocytes and their phagosomes, such as in CF disease and other chloride channel disorders, severely compromises host defense against infection. We provide an overview of the roles that chloride plays in normal innate immunity, highlighting specific links between defective chloride channel function and failures in host defense. PMID:26048979

  14. Regulation of the respiratory central pattern generator by chloride-dependent inhibition during development in the bullfrog (Rana catesbeiana).

    PubMed

    Broch, Lise; Morales, Rey D; Sandoval, Anthony V; Hedrick, Michael S

    2002-04-01

    Isolated brainstem preparations from larval (tadpole) and adult Rana catesbeiana were used to examine inhibitory mechanisms for developmental regulation of the respiratory central pattern generator (CPG). Preparations were superfused at 20-22 degrees C with Cl(-)-free artificial cerebrospinal fluid (aCSF) or with aCSF containing agonists/antagonists of gamma-aminobutyric acid (GABA) or glycine receptors. Respiratory motor output from the CPG, measured as neural activity from cranial nerve roots, was associated with fictive gill ventilation and lung ventilation in tadpoles and with fictive lung ventilation in adults. In tadpoles, fictive lung burst frequency was 0.8+/-0.2 min(-1) and did not change significantly with Cl(-)-free aCSF superfusion; however, lung burst amplitude increased by nearly 400 % (P<0.01). Fictive gill ventilation averaged 41.6+/-3.3 min(-1) and was reversibly abolished by Cl(-)-free aCSF. Superfusion with Cl(-)-free aCSF abolished lung bursts in two of seven adult preparations, and overall lung burst frequency decreased from 3.1+/-0.7 to 0.4+/-0.03 min(-1) (P<0.01), but burst amplitude was unchanged. Low concentrations of GABA (0.5 mmol l(-1)) produced a significant increase in lung burst frequency followed by almost complete inhibition at 5.0 mmol l(-1), accompanied by the abolition of gill ventilation at 2.5-5.0 mmol l(-1). By contrast, fictive lung ventilation in adults was inhibited in a dose-dependent manner by glycine and GABA, and inhibition occurred at approximately 10-fold lower concentrations compared with tadpoles. The glycine receptor antagonist strychnine (2.5-25.0 micromol l(-1)) and the GABA(A) receptor antagonist bicuculline (1-10 micromol l(-1)) inhibited fictive gill ventilation and increased fictive lung ventilation in tadpoles. However, bicuculline and strychnine inhibited fictive lung ventilation in adults. These results suggest that lung ventilation in the tadpole brainstem may be driven by a pacemaker-like mechanism since

  15. Effects of sodium chloride exposure on ion regulation in larvae (glochidia) of the freshwater mussel Lampsilis fasciola.

    PubMed

    Nogueira, Lygia S; Bianchini, Adalto; Wood, Chris M; Loro, Vania L; Higgins, Sarah; Gillis, Patricia L

    2015-12-01

    The salinization of freshwater can have negative effects on ecosystem health, with heightened effects in salt-sensitive biota such as glochidia, the larvae of freshwater mussels. However, the toxicological mechanism underlying this sensitivity is unknown. Therefore, Lampsilis fasciola glochidia were exposed to NaCl (nominally 0.25 and 1.0 g/L) prepared in reconstituted moderately-hard water (control), as well as to a dilution of that water (1:4) with ultrapure reference water (diluted control). Unidirectional Na(+) influx (measured with (22)Na) was evaluated after 1, 3 and 48 h of exposure. In addition, unidirectional Cl(-) influx (measured with (36)Cl), whole-body ion (Cl(-) and Na(+)) concentrations, and glochidia viability (measured as the ability to close valves) were assessed after 48 h of exposure. Significantly reduced glochidia viability (56%) was observed after exposure to 1.0 g/L NaCl. Na(+) influx was significantly higher in glochidia exposed to both 0.25 and 1.0 g/L NaCl for 1h than in those kept under control conditions. After 3 and 48 h of exposure, differences in Na(+) influx rate between salt-exposed and control glochidia were generally reduced, indicating that larvae may be able to, at least temporarily, recover their ability to regulate Na(+) influx when exposed to elevated NaCl concentration. Compared to the moderately-hard water control, whole-body Na(+) and Cl(-) concentrations were relatively unchanged in glochidia exposed to 0.25 g/L NaCl, but were significantly elevated in glochidia exposed to 1.0 g/L NaCl and the diluted control. While Na(+) influx rate had recovered to the control level after 48 h of exposure to 1.0 g/L NaCl, Cl(-) influx rate remained elevated, being ~7-fold higher than the Na(+) influx rate. These findings suggest that the loss of viability observed when glochidia were exposed to a high NaCl concentration (1.0 g/L) could be caused by ionoregulatory disturbances mainly associated with an elevated Cl(-) influx.

  16. The relationship between rumen acidosis resistance and expression of genes involved in regulation of intracellular pH and butyrate metabolism of ruminal epithelial cells in steers.

    PubMed

    Schlau, N; Guan, L L; Oba, M

    2012-10-01

    Past research has focused on the prevention and management of subacute rumen acidosis by manipulating the ration; however, the severity of acidosis varies even among animals fed a common high-grain diet. The objectives of this study were to compare the ruminal volatile fatty acid (VFA) profile and expression of genes involved in the metabolism of butyrate, the VFA most extensively metabolized by the ruminal epithelium, and intracellular pH regulation in ruminal epithelial cells between acidosis-resistant (AR) and acidosis-susceptible (AS) steers. Acidosis indexes (area per day under pH 5.8 divided by dry matter intake) were measured for 17 steers fed a common high-grain diet, and the 3 steers with the lowest (1.4 ± 1.2 pH∙min/kg) and the 3 with the highest values (23.9 ± 7.4 pH∙min/kg) were classified as AR and AS, respectively, and used in the subsequent study. The steers were force-fed a diet containing 85% grain at 60% of the expected daily intake (5.8 ± 0.8 and 5.6 ± 0.6 kg for AR and AS, respectively) within 30 min. Mean ruminal pH over the postprandial 6-h period was higher for AR compared with AS (6.02 vs. 5.55), and mean total VFA concentration was 74% for AR compared with AS (122 vs. 164 mM). Molar proportion of butyrate in the ruminal fluid was 139% higher for AR compared with AS (17.5 vs. 7.33 mol/100 mol of VFA). Expression of monocarboxylate cotransporter isoform 1, sodium hydrogen exchanger isoforms 1 and 2, and anion exchangers (downregulated in adenoma and putative anion exchanger, isoform 1) did not differ between AR and AS steers. However, expression of sodium hydrogen exchanger isoform 3, which imports Na(+) to the epithelial cell and exports H(+) to the rumen, was 176% higher in AR steers than in AS steers. Higher ruminal pH for AR might be partly due to a faster rate of VFA absorption, lower VFA production, or both.

  17. Mechanisms of pH regulation in lamprey (Lampetra fluviatilis) red blood cells.

    PubMed

    Nikinmaa, M; Kunnamo-Ojala, T; Railo, E

    1986-05-01

    Mechanisms regulating the red cell pH in lamprey (Lampetra fluviatilis) were studied using the ammonium chloride prepulse technique. The cells were initially incubated in a physiological saline containing 20 mmol l-1 ammonium chloride, and intracellular pH measured with the DMO technique. Ammonium chloride was then rapidly removed by centrifugation, and the changes in the intracellular pH followed. The intraerythrocytic pH is primarily regulated by an amiloride-sensitive sodium/proton exchange. When sodium is present in the incubation medium, the intracellular pH rapidly recovers from the acidification associated with the removal of ammonium chloride from the incubation. When sodium is removed from the incubation medium, intracellular pH does not recover, and when the cells are treated with 10(-3) mol l-1 amiloride in the presence of sodium, carbon dioxide and bicarbonate, the intracellular pH recovery is drastically reduced. The movements of carbon dioxide, its consecutive catalysed hydration and dissociation to protons and bicarbonate and, possibly, movements of bicarbonate out of the cell acidify the cell contents. This is shown by the observation that the steady-state intracellular pH is higher in a HEPES-buffered medium than in a CO2/HCO3(-)-buffered medium at the same extracellular pH. The acidification is dependent on cellular carbonic anhydrase activity, present in lamprey red cells, which speeds up the hydration reaction. When the action of carbonic anhydrase is inhibited by acetazolamide, removal of ammonium chloride from the incubation medium does not cause intracellular acidification. PMID:3088194

  18. Discovery of alpha-aminoazaheterocycle-methylglyoxal adducts as a new class of high-affinity inhibitors of cystic fibrosis transmembrane conductance regulator chloride channels.

    PubMed

    Routaboul, Christel; Norez, Caroline; Melin, Patricia; Molina, Marie-Carmen; Boucherle, Benjamin; Bossard, Florian; Noel, Sabrina; Robert, Renaud; Gauthier, Chantal; Becq, Frédéric; Décout, Jean-Luc

    2007-09-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) represents the main Cl(-) channel in the apical membrane of epithelial cells for cAMP-dependent Cl(-) secretion. Here we report on the synthesis and screening of a small library of nontoxic alpha-aminoazaheterocycle-methylglyoxal adducts, inhibitors of wild-type (WT) CFTR and G551D-, G1349D-, and F508del-CFTR Cl(-) channels. In whole-cell patch-clamp experiments of Chinese hamster ovary (CHO) cells expressing WT-CFTR, we recorded rapid and reversible inhibition of forskolin-activated CFTR currents in the presence of the adducts 5a and 8a,b at 10 pM concentrations. Using iodide efflux experiments, we compared concentration-dependent inhibition of CFTR with glibenclamide (IC(50) = 14.7 microM), 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl-)methylene]-2-thioxo-4-thiazolidinone (CFTR(inh)-172) (IC(50) = 1.2 microM), and alpha-aminoazaheterocycle-methylglyoxal adducts and identified compounds 5a (IC(50) = 71 pM), 8a,b (IC(50) = 2.5 nM), and 7a,b (IC(50) = 3.4 nM) as the most potent inhibitors of WT-CFTR channels. Similar ranges of inhibition were also found when these compounds were evaluated on CFTR channels with the cystic fibrosis mutations F508del (in temperature-corrected human airway epithelial F508del/F508del CF15 cells)-, G551D-, and G1349D-CFTR (expressed in CHO and COS-7 cells). No effect of compound 5a was detected on the volume-regulated or calcium-regulated iodide efflux. Picomolar inhibition of WT-CFTR with adduct 5a was also found using a 6-methoxy-N-(3-sulfopropyl)-quinolinium fluorescent probe applied to the human tracheobronchial epithelial cell line 16HBE14o-. Finally, we found comparable inhibition by 5a or by CFTR(inh)-172 of forskolin-dependent short-circuit currents in mouse colon. To the best of our knowledge, these new nontoxic alpha-aminoazaheterocycle-methylglyoxal adducts represent the most potent compounds reported to inhibit CFTR chloride channels.

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

    SciTech Connect

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

    1989-04-01

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

  20. 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

  1. Up-regulation of cyclooxygenase-2 by cobalt chloride-induced hypoxia is mediated by phospholipase D isozymes in human astroglioma cells.

    PubMed

    Ahn, Bong-Hyun; Park, Mi Hee; Lee, Young Han; Kwon, Taeg Kyu; Min, Do Sik

    2007-12-01

    Cyclooxygenase-2 (COX-2) is an isoform of prostaglandin H synthase induced by hypoxia and has been implicated in the growth and progression of a variety of human cancers. In the present study, we investigated the role of phospholipase D (PLD) isozymes in cobalt chloride (CoCl(2))-induced hypoxia-driven COX-2 expression in U87 MG human astroglioma cells. CoCl(2) stimulated PLD activity and synthesis of COX-2 protein in a dose and time-dependent manner. Moreover, elevated expression of PLD1 and PLD2 increased hypoxia-induced COX-2 expression and prostaglandin E2 (PGE(2)) production. Pretreatment of cells with 1-butanol, but not 3-butanol, suppressed CoCl(2)-induced COX-2 expression and PGE(2) formation. In addition, evidence that PLD activity was involved in the stimulation of COX-2 expression was provided by the observations that overexpression of wild type PLD isozymes, but not catalytically inactive PLD isozymes, stimulated CoCl(2)-induced COX-2 expression and PGE(2) production. PLD1 enhanced COX-2 expression by CoCl(2) via reactive oxygen species (ROS), p38 MAPK kinase, PKC-delta, and PKA, but not ERK, whereas PLD2 enhanced CoCl(2)-induced COX-2 expression via ROS and p38 MAPK, but not ERK, PKC-delta, and PKA. Differential regulation of COX-2 expression mediated through PLD isozymes was comparable with that of CoCl(2)-induced PLD activity in these two PLD isozymes. Taken together, our results demonstrate for the first time that PLD1 and PLD2 isozymes enhance CoCl(2)-induced COX-2 expression through differential signaling pathways in astroglioma cells.

  2. Intracellular pH in human arterial smooth muscle. Regulation by Na+/H+ exchange and a novel 5-(N-ethyl-N-isopropyl)amiloride-sensitive Na(+)- and HCO3(-)-dependent mechanism

    SciTech Connect

    Neylon, C.B.; Little, P.J.; Cragoe, E.J. Jr.; Bobik, A. )

    1990-10-01

    We investigated in a physiological salt solution (PSS) containing HCO3- the intracellular pH (pHi) regulating mechanisms in smooth muscle cells cultured from human internal mammary arteries, using the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and 22Na+ influx rates. The recovery of pHi from an equivalent intracellular acidosis was more rapid when the cells were incubated in CO2/HCO3(-)-buffered PSS than in HEPES-buffered PSS. Recovery of pHi was dependent on extracellular Na+ (Km, 13.1 mM); however, it was not attenuated by 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), indicating the absence of SITS-sensitive HCO3(-)-dependent mechanisms. Recovery instead appeared mostly dependent on processes sensitive to 5-(N-ethyl-N-isopropyl)amiloride (EIPA), indicating the involvement of Na+/H+ exchange and a previously undescribed EIPA-sensitive Na(+)- and HCO3(-)-dependent mechanism. Differentiation between this HCO3(-)-dependent mechanism and Na+/H+ exchange was achieved after depletion of cellular ATP. Under these conditions, the NH4Cl-induced 22Na+ influx rate stimulated by intracellular acidosis was markedly attenuated in HEPES-buffered PSS but not in CO2/HCO3(-)-buffered PSS. EIPA also appeared to inhibit the two mechanisms differentially. In HEPES-buffered PSS containing 20 mM Na+, the EIPA inhibition curve for the intracellular acidosis-induced 22Na+ influx was monophasic (IC50, 39 nM), whereas in an identical CO2/HCO3(-)-buffered PSS, the inhibition curve exhibited biphasic characteristics (IC50, 37.3 nM and 312 microM). Taken together, the results indicate that Na+/H+ exchange and a previously undescribed EIPA-sensitive Na(+)- and HCO3(-)-dependent mechanism play an important role in regulating the pHi of human vascular smooth muscle.

  3. Thioredoxin-1 promotes survival in cells exposed to S-nitrosoglutathione: Correlation with reduction of intracellular levels of nitrosothiols and up-regulation of the ERK1/2 MAP Kinases

    SciTech Connect

    Arai, Roberto J.; Debbas, Victor; Stern, Arnold; Monteiro, Hugo P.

    2008-12-01

    Accumulating evidence indicates that post-translational protein modifications by nitric oxide and its derived species are critical effectors of redox signaling in cells. These protein modifications are most likely controlled by intracellular reductants. Among them, the importance of the 12 kDa dithiol protein thioredoxin-1 (TRX-1) has been increasingly recognized. However, the effects of TRX-1 in cells exposed to exogenous nitrosothiols remain little understood. We investigated the levels of intracellular nitrosothiols and survival signaling in HeLa cells over-expressing TRX-1 and exposed to S-nitrosoglutahione (GSNO). A role for TRX-1 expression on GSNO catabolism and cell viability was demonstrated by the concentration-dependent effects of GSNO on decreasing TRX-1 expression, activation of caspase-3, and increasing cell death. The over-expression of TRX-1 in HeLa cells partially attenuated caspase-3 activation and enhanced cell viability upon GSNO treatment. This was correlated with reduction of intracellular levels of nitrosothiols and increasing levels of nitrite and nitrotyrosine. The involvement of ERK, p38 and JNK pathways were investigated in parental cells treated with GSNO. Activation of ERK1/2 MAP kinases was shown to be critical for survival signaling. In cells over-expressing TRX-1, basal phosphorylation levels of ERK1/2 MAP kinases were higher and further increased after GSNO treatment. These results indicate that the enhanced cell viability promoted by TRX-1 correlates with its capacity to regulate the levels of intracellular nitrosothiols and to up-regulate the survival signaling pathway mediated by the ERK1/2 MAP kinases.

  4. Rab27a negatively regulates CFTR chloride channel function in colonic epithelia: Involvement of the effector proteins in the regulatory mechanism

    SciTech Connect

    Saxena, Sunil K. . E-mail: ssaxena@stevens.edu; Kaur, Simarna

    2006-07-21

    Cystic fibrosis, an autosomal recessive disorder, is caused by the disruption of biosynthesis or function of CFTR. CFTR regulatory mechanisms include channel transport to plasma membrane and protein-protein interactions. Rab proteins are small GTPases involved in vesicle transport, docking, and fusion. The colorectal epithelial HT-29 cells natively express CFTR and respond to cAMP with an increase in CFTR-mediated currents. DPC-inhibited currents could be completely eliminated with CFTR-specific SiRNA. Over-expression of Rab27a inhibited, while isoform specific SiRNA and Rab27a antibody stimulated CFTR-mediated currents in HT-29 cells. CFTR activity is inhibited both by Rab27a (Q78L) (constitutive active GTP-bound form of Rab27a) and Rab27a (T23N) (constitutive negative form that mimics the GDP-bound form). Rab27a mediated effects could be reversed by Rab27a-binding proteins, the synaptotagmin-like protein (SLP-5) and Munc13-4 accessory protein (a putative priming factor for exocytosis). The SLP reversal of Rab27a effect was restricted to C2A/C2B domains while the SHD motif imparted little more inhibition. The CFTR-mediated currents remain unaffected by Rab3 though SLP-5 appears to weakly bind it. The immunoprecipitation experiments suggest protein-protein interactions between Rab27a and CFTR. Rab27a appears to impair CFTR appearance at the cell surface by trapping CFTR in the intracellular compartments. Munc13-4 and SLP-5, on the other hand, limit Rab27a availability to CFTR, thus minimizing its effect on channel function. These observations decisively prove that Rab27a is involved in CFTR channel regulation through protein-protein interactions involving Munc13-4 and SLP-5 effector proteins, and thus could be a potential target for cystic fibrosis therapy.

  5. EphB1 and EphB2 intracellular domains regulate the formation of the corpus callosum and anterior commissure.

    PubMed

    Robichaux, Michael A; Chenaux, George; Ho, Hsin-Yi Henry; Soskis, Michael J; Greenberg, Michael E; Henkemeyer, Mark; Cowan, Christopher W

    2016-04-01

    The two cortical hemispheres of the mammalian forebrain are interconnected by major white matter tracts, including the corpus callosum (CC) and the posterior branch of the anterior commissure (ACp), that bridge the telencephalic midline. We show here that the intracellular signaling domains of the EphB1 and EphB2 receptors are critical for formation of both the ACp and CC. We observe partial and complete agenesis of the corpus callosum, as well as highly penetrant ACp misprojection phenotypes in truncated EphB1/2 mice that lack intracellular signaling domains. Consistent with the roles for these receptors in formation of the CC and ACp, we detect expression of these receptors in multiple brain regions associated with the formation of these forebrain structures. Taken together, our findings suggest that a combination of forward and reverse EphB1/2 receptor-mediated signaling contribute to ACp and CC axon guidance.

  6. Superoxide radicals increase transforming growth factor-{beta}1 and collagen release from human lung fibroblasts via cellular influx through chloride channels

    SciTech Connect

    Qi Shufan Hartog, Gertjan J.M. den; Bast, Aalt

    2009-05-15

    Reactive oxygen species (ROS) have been implicated in the pathogenesis of fibrosis. However, it remains unclear which ROS is the major cause. We hypothesize that superoxide elicits specific toxicity to human lung fibroblasts and plays an important role in the development of pulmonary fibrosis. In this study, superoxide generated from xanthine and xanthine oxidase activated lung fibroblasts by increasing the release of TGF-{beta}1 and collagen. This was associated with increased levels of intracellular superoxide. SOD and tempol, by scavenging respectively extracellular and intracellular superoxide, prevented the activation of fibroblasts induced by exposure to exogenous superoxide, whereas catalase did not. Moreover, hydrogen peroxide did not activate fibroblasts. Apparently, superoxide rather than hydrogen peroxide is involved in the regulation of TGF-{beta}1 and collagen release in lung fibroblasts. The chloride channel blocker, DIDS, inhibited the increase of intracellular superoxide levels induced by exogenous superoxide and consequently prevented the activation of fibroblasts. This suggests that the cellular influx of superoxide through chloride channels is essential for superoxide-induced activation of fibroblasts. ERK1/2 and p38 MAPKs are involved in the intracellular pathway leading to superoxide-induced fibroblasts activation. Superoxide possesses until now undiscovered specific pro-fibrotic properties in human lung fibroblasts. This takes place via the cellular influx of superoxide through chloride channels rather than via the formation of hydrogen peroxide.

  7. Intracellular calcium levels are differentially regulated in T lymphocytes triggered by anti-CD2 and anti-CD3 monoclonal antibodies.

    PubMed

    Spinozzi, F; Agea, E; Bistoni, O; Belia, S; Travetti, A; Gerli, R; Muscat, C; Bertotto, A

    1995-03-01

    Antigen and/or mitogen-driven T-cell activation is mediated by a rise in intracellular free Ca2+, as second messenger. A regulatory key role for this process is represented by membrane-associated [Ca2+/Mg2+] ATP-ase that is mainly devoted to extrusion of intracellular ion excess. In the present study we have investigated the kinetics of CA2+ fluxes in both resting and already activated (Jurkat T-cell line) T lymphocytes after CD3 and CD2 (T11(2) and T11(3)) triggering and focused our attention on plasma membrane [Ca2+/Mg2+] ATP-ase activity. In both resting T cells and Jurkat cell line, the CD2 stimulation was able to determine a rise in intracellular free Ca2+ higher than that observed after CD3 triggering. In addition, this calcium signal was independent of negative feedback control exerted by [Ca2+/Mg2+] ATP-ase, as well as of IP3 generation. Thus the CD2 molecular system may, together with cell-adhesion properties, act as an amplifier of Ca2+ signals that, if delivered in the context of other molecular systems, such as CD3 or MHC class II antigens, are essentially devoted to the polyclonal co-stimulatory recruitment of a larger cellular repertoire. PMID:7662514

  8. WNK3 is a putative chloride-sensing kinase.

    PubMed

    Pacheco-Alvarez, Diana; Gamba, Gerardo

    2011-01-01

    The with-no-lysine kinase 3 (WNK3) is a serine/threonine kinase that modulates the activity of the electroneutral cation-coupled chloride cotransporters (CCC). Using the Xenopus laevis oocyte heterologous expression system, it has been shown that WNK3 activates the Na(+)-coupled chloride cotransporters NKCC1, NKCC2, and NCC and inhibits the K(+)-coupled chloride cotransporters KCC1 through KCC4. Interestingly, the effect of catalytically inactive WNK3 is opposite to that of wild type WNK3: inactive WNK3 inhibits NKCCs and activates KCCs. In doing so, wild type and catalytically inactive WNK3 bypass the tonicity requirement for activation/inhibition of the cotransporter. Thus, WNK3 modulation of the electroneutral cotransporters promotes Cl(-) influx and prevents Cl(-) efflux, thus fitting the profile for a putative "Cl(-)-sensing kinase". Other kinases that potentially have these properties are the Ste20-type kinases, SPAK/OSR1, which become phosphorylated in response to reductions in intracellular chloride concentration and regulate the activity of NKCC1. It has been demonstrated that WNKs lie upstream of SPAK/OSR1 and that the activity of these kinases is activated by phosphorylation of threonines in the T-loop by WNKs. It is possible that a protein phosphatase is also involved in the WNK3 effects on its associated cotransporters because activation of KCCs and inhibition of NKCCs by inactive WNK3 can be prevented by known inhibitors of protein phosphatases, such as calyculin A and cyclosporine, suggesting that a protein phosphatase is also involved in the protein complex. PMID:22179001

  9. Processes regulating groundwater chloride content in marshes under different environmental conditions: A comparative case study in Península Valdés and Samborombón Bay, Argentina

    NASA Astrophysics Data System (ADS)

    Carol, Eleonora; Alvarez, María del Pilar

    2016-03-01

    Salt marshes are some of the most important wetlands in many regions of the world. Soil and groundwater salinity plays an important role in coastal wetland ecosystems because of the differences in tolerances of plant species to salinity and tidal inundation. Given that the salinity of these environments is mostly dominated by the chloride anion, it is the aim of this study to identify the geochemical processes that regulate its content in groundwater. A comparison of two intertidal wetlands under different environmental conditions was carried out in Peninsula Valdés and in Samborombón Bay, both on the Atlantic coast of Argentina. The tidal influence over the groundwater marsh dynamics was analyzed from continuous records of groundwater levels and electrical conductivity. Besides, major ion and environmental isotope data were used to identify the geochemical processes that determine the chloride content, based on the study of ion ratios and analytical models. The results show that, despite the hydrological differences between the two studied marshes, the processes regulating the Cl- contents are similar: evaporation, transpiration and halite dissolution. Among them, evaporation/transpiration are the processes that could continuously increase the chloride concentration. However, it is expected that those are not processes that greatly increase the groundwater saline content if compared to the dissolution of halite.

  10. Brain-derived neurotrophic factor (BDNF) induces sustained intracellular Ca2+ elevation through the up-regulation of surface transient receptor potential 3 (TRPC3) channels in rodent microglia.

    PubMed

    Mizoguchi, Yoshito; Kato, Takahiro A; Seki, Yoshihiro; Ohgidani, Masahiro; Sagata, Noriaki; Horikawa, Hideki; Yamauchi, Yusuke; Sato-Kasai, Mina; Hayakawa, Kohei; Inoue, Ryuji; Kanba, Shigenobu; Monji, Akira

    2014-06-27

    Microglia are immune cells that release factors, including proinflammatory cytokines, nitric oxide (NO), and neurotrophins, following activation after disturbance in the brain. Elevation of intracellular Ca(2+) concentration ([Ca(2+)]i) is important for microglial functions such as the release of cytokines and NO from activated microglia. There is increasing evidence suggesting that pathophysiology of neuropsychiatric disorders is related to the inflammatory responses mediated by microglia. Brain-derived neurotrophic factor (BDNF) is a neurotrophin well known for its roles in the activation of microglia as well as in pathophysiology and/or treatment of neuropsychiatric disorders. In this study, we sought to examine the underlying mechanism of BDNF-induced sustained increase in [Ca(2+)]i in rodent microglial cells. We observed that canonical transient receptor potential 3 (TRPC3) channels contribute to the maintenance of BDNF-induced sustained intracellular Ca(2+) elevation. Immunocytochemical technique and flow cytometry also revealed that BDNF rapidly up-regulated the surface expression of TRPC3 channels in rodent microglial cells. In addition, pretreatment with BDNF suppressed the production of NO induced by tumor necrosis factor α (TNFα), which was prevented by co-adiministration of a selective TRPC3 inhibitor. These suggest that BDNF induces sustained intracellular Ca(2+) elevation through the up-regulation of surface TRPC3 channels and TRPC3 channels could be important for the BDNF-induced suppression of the NO production in activated microglia. We show that TRPC3 channels could also play important roles in microglial functions, which might be important for the regulation of inflammatory responses and may also be involved in the pathophysiology and/or the treatment of neuropsychiatric disorders.

  11. ClC-3 Chloride Channel Proteins Regulate the Cell Cycle by Up-regulating cyclin D1-CDK4/6 through Suppressing p21/p27 Expression in Nasopharyngeal Carcinoma Cells

    PubMed Central

    Ye, Dong; Luo, Hai; Lai, Zhouyi; Zou, Lili; Zhu, Linyan; Mao, Jianwen; Jacob, Tim; Ye, Wencai; Wang, Liwei; Chen, Lixin

    2016-01-01

    It was shown in this study that knockdown of ClC-3 expression by ClC-3 siRNA prevented the activation of hypotonicity-induced chloride currents, and arrested cells at the G0/G1 phase in nasopharyngeal carcinoma CNE-2Z cells. Reconstitution of ClC-3 expression with ClC-3 expression plasmids could rescue the cells from the cell cycle arrest caused by ClC-3 siRNA treatments. Transfection of cells with ClC-3 siRNA decreased the expression of cyclin D1, cyclin dependent kinase 4 and 6, and increased the expression of cyclin dependent kinase inhibitors (CDKIs), p21 and p27. Pretreatments of cells with p21 and p27 siRNAs depleted the inhibitory effects of ClC-3 siRNA on the expression of CDK4 and CDK6, but not on that of cyclin D1, indicating the requirement of p21 and p27 for the inhibitory effects of ClC-3 siRNA on CDK4 and CDK6 expression. ClC-3 siRNA inhibited cells to progress from the G1 phase to the S phase, but pretreatments of cells with p21 and p27 siRNAs abolished the inhibitory effects of ClC-3 siRNA on the cell cycle progress. Our data suggest that ClC-3 may regulate cell cycle transition between G0/G1 and S phases by up-regulation of the expression of CDK4 and CDK6 through suppression of p21 and p27 expression. PMID:27451945

  12. Intracellular Bacteria in Protozoa

    NASA Astrophysics Data System (ADS)

    Görtz, Hans-Dieter; Brigge, Theo

    Intracellular bacteria in humans are typically detrimental, and such infections are regarded by the patients as accidental and abnormal. In protozoa it seems obvious that many bacteria have coevolved with their hosts and are well adapted to the intracellular way of life. Manifold interactions between hosts and intracellular bacteria are found, and examples of antibacterial resistance of unknown mechanisms are observed. The wide diversity of intracellular bacteria in protozoa has become particularly obvious since they have begun to be classified by molecular techniques. Some of the bacteria are closely related to pathogens; others are responsible for the production of toxins.

  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. Regulation of the spvR gene of the Salmonella typhimurium virulence plasmid during exponential-phase growth in intracellular salts medium and at stationary phase in L broth.

    PubMed

    Wilson, J A; Gulig, P A

    1998-07-01

    The authors previously showed that the SpvR-regulated spvABCD operon of the Salmonella typhimurium virulence plasmid is highly induced during exponential-phase growth by salmonellae intracellularly in mammalian cells and in a medium designed to mimic the intracellular environment of mammalian cells, intracellular salts medium (ISM), as well as at stationary phase in L broth (LB). The most relevant signal(s) for spv gene expression in vivo is not known. To elucidate the means by which salmonellae regulate the spv genes in response to the environment during the disease process, expression of the spvR gene, encoding the positive regulatory protein SpvR, was examined under these same growth conditions by using RNAse-protection analysis. spvR was expressed at a low, basal level during exponential growth in LB but was induced during exponential growth in ISM and during stationary phase in LB, the same conditions that increased expression of the spvABCD operon. Basal expression of spvR during exponential growth in LB was independent of both SpvR and the alternative sigma factor RpoS, whereas maximal induction of spvR was dependent on both SpvR and RpoS. In an RpoS- background, spvR message was decreased in stationary phase, whereas spvR exhibited residual RpoS-independent induction during exponential growth in ISM. Deletion of spvA from the virulence plasmid of S. typhimurium increased expression of spvR during stationary phase in LB, but not during exponential growth in ISM. These results suggest that expression of spvR is controlled by different regulatory factors, depending on the growth conditions encountered by the salmonellae.

  15. Chloride in diet

    MedlinePlus

    ... found in table salt or sea salt as sodium chloride. It is also found in many vegetables. Foods ... Nutrition Board. Dietary Reference Intakes for Water, Potassium, Sodium, Chloride, and Sulfate. National Academy Press, Washington, DC: 2005. ...

  16. CALCIUM CHLORIDE PLANT LOOKING EAST. CALCIUM CHLORIDE BUILDING IN CENTER, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    CALCIUM CHLORIDE PLANT LOOKING EAST. CALCIUM CHLORIDE BUILDING IN CENTER, CALCIUM CHLORIDE STORAGE BUILDING ON RIGHT WITH SA (SODA ASH) BUILDING IN RIGHT BACKGROUND. - Solvay Process Company, Calcium Chloride Plant, Between Willis & Milton Avenues, Solvay, Onondaga County, NY

  17. Stochastic resonance in an intracellular genetic perceptron

    NASA Astrophysics Data System (ADS)

    Bates, Russell; Blyuss, Oleg; Zaikin, Alexey

    2014-03-01

    Intracellular genetic networks are more intelligent than was first assumed due to their ability to learn. One of the manifestations of this intelligence is the ability to learn associations of two stimuli within gene-regulating circuitry: Hebbian-type learning within the cellular life. However, gene expression is an intrinsically noisy process; hence, we investigate the effect of intrinsic and extrinsic noise on this kind of intracellular intelligence. We report a stochastic resonance in an intracellular associative genetic perceptron, a noise-induced phenomenon, which manifests itself in noise-induced increase of response in efficiency after the learning event under the conditions of optimal stochasticity.

  18. Annexin A4 induces platinum resistance in a chloride-and calcium-dependent manner.

    PubMed

    Morimoto, Akiko; Serada, Satoshi; Enomoto, Takayuki; Kim, Ayako; Matsuzaki, Shinya; Takahashi, Tsuyoshi; Ueda, Yutaka; Yoshino, Kiyoshi; Fujita, Masami; Fujimoto, Minoru; Kimura, Tadashi; Naka, Tetsuji

    2014-09-15

    Platinum resistance has long been a major issue in the treatment of various cancers. We previously reported that enhanced annexin A4 (ANXA4) expression, a Ca2+-regulated phospholipid-binding protein, induces chemoresistance to platinum-based drugs. In this study, we investigated the role of annexin repeats, a conserved structure of all the annexin family, responsible for platinum-resistance as well as the effect of knockdown of ANXA4. ANXA4 knockdown increased sensitivity to platinum-based drugs both in vitro and in vivo. To identify the domain responsible for chemoresistance, ANXA4 deletion mutants were constructed by deleting annexin repeats one by one from the C terminus. Platinum resistance was induced both in vitro and in vivo in cells expressing either full-length ANXA4 or the deletion mutants, containing at least one intact annexin repeat. However, cells expressing the mutant without any calcium-binding sites in the annexin repeated sequence, which is essential for ANXA4 translocation from the cytosol to plasma membrane, failed to acquire platinum resistance. After cisplatin treatment, the intracellular chloride ion concentration, whose channel is partly regulated by ANXA4, significantly increased in the platinum-resistant cells. These findings indicate that the calcium-binding site in the annexin repeat induces chemoresistance to the platinum-based drug by elevating the intracellular chloride concentration.

  19. CPB1 of Aedes aegypti interacts with DENV2 E protein and regulates intracellular viral accumulation and release from midgut cells.

    PubMed

    Tham, Hong-Wai; Balasubramaniam, Vinod R M T; Tejo, Bimo Ario; Ahmad, Hamdan; Hassan, Sharifah Syed

    2014-12-01

    Aedes aegypti is a principal vector responsible for the transmission of dengue viruses (DENV). To date, vector control remains the key option for dengue disease management. To develop new vector control strategies, a more comprehensive understanding of the biological interactions between DENV and Ae. aegypti is required. In this study, a cDNA library derived from the midgut of female adult Ae. aegypti was used in yeast two-hybrid (Y2H) screenings against DENV2 envelope (E) protein. Among the many interacting proteins identified, carboxypeptidase B1 (CPB1) was selected, and its biological interaction with E protein in Ae. aegypti primary midgut cells was further validated. Our double immunofluorescent assay showed that CPB1-E interaction occurred in the endoplasmic reticulum (ER) of the Ae. aegypti primary midgut cells. Overexpression of CPB1 in mosquito cells resulted in intracellular DENV2 genomic RNA or virus particle accumulation, with a lower amount of virus release. Therefore, we postulated that in Ae. aegypti midgut cells, CPB1 binds to the E protein deposited on the ER intraluminal membranes and inhibits DENV2 RNA encapsulation, thus inhibiting budding from the ER, and may interfere with immature virus transportation to the trans-Golgi network. PMID:25521592

  20. Salmonella SPI1 effector SipA persists after entry and cooperates with a SPI2 effector to regulate phagosome maturation and intracellular replication.

    PubMed

    Brawn, Lyndsey C; Hayward, Richard D; Koronakis, Vassilis

    2007-03-15

    Salmonellae employ two type III secretion systems (T3SSs), SPI1 and SPI2, to deliver virulence effectors into mammalian cells. SPI1 effectors, including actin-binding SipA, trigger initial bacterial uptake, whereas SPI2 effectors promote subsequent replication within customized Salmonella-containing vacuoles (SCVs). SCVs sequester actin filaments and subvert microtubule-dependent motors to migrate to the perinuclear region. We demonstrate that SipA delivery continues after Salmonella internalization, with dosage being restricted by host-mediated degradation. SipA is exposed on the cytoplasmic face of the SCV, from where it stimulates bacterial replication in both nonphagocytic cells and macrophages. Although SipA is sufficient to target and redistribute late endosomes, during infection it cooperates with the SPI2 effector SifA to modulate SCV morphology and ensure perinuclear positioning. Our findings define an unexpected additional function for SipA postentry and reveal precise intracellular communication between effectors deployed by distinct T3SSs underlying SCV biogenesis.

  1. Mammalian farnesyltransferase α subunit regulates vacuolar protein sorting-associated protein 4A (Vps4A)--dependent intracellular trafficking through recycling endosomes.

    PubMed

    Kubala, Marta H; Norwood, Suzanne J; Gomez, Guillermo A; Jones, Alun; Johnston, Wayne; Yap, Alpha S; Mureev, Sergey; Alexandrov, Kirill

    2015-12-25

    The protein farnesyltransferase (FTase) mediates posttranslational modification of proteins with isoprenoid lipids. FTase is a heterodimer and although the β subunit harbors the active site, it requires the α subunit for its activity. Here we explore the other functions of the FTase α subunit in addition to its established role in protein prenylation. We found that in the absence of the β subunit, the α subunit of FTase forms a stable autonomous dimeric structure in solution. We identify interactors of FTase α using mass spectrometry, followed by rapid in vitro analysis using the Leishmania tarentolae cell - free system. Vps4A was validated for direct binding to the FTase α subunit both in vitro and in vivo. Analysis of the interaction with Vps4A in Hek 293 cells demonstrated that FTase α controls trafficking of transferrin receptor upstream of this protein. These results point to the existence of previously undetected biological functions of the FTase α subunit that includes control of intracellular membrane trafficking. PMID:26551458

  2. Tyrosine Binding Protein Sites Regulate the Intracellular Trafficking and Processing of Amyloid Precursor Protein through a Novel Lysosome-Directed Pathway

    PubMed Central

    Tam, Joshua H. K.; Cobb, M. Rebecca; Seah, Claudia; Pasternak, Stephen H.

    2016-01-01

    The amyloid hypothesis posits that the production of β-amyloid (Aβ) aggregates leads to neurodegeneration and cognitive decline associated with AD. Aβ is produced by sequential cleavage of the amyloid precursor protein (APP) by β- and γ-secretase. While nascent APP is well known to transit to the endosomal/ lysosomal system via the cell surface, we have recently shown that APP can also traffic to lysosomes intracellularly via its interaction with AP-3. Because AP-3 interacts with cargo protein via interaction with tyrosine motifs, we mutated the three tyrosines motif in the cytoplasmic tail of APP. Here, we show that the YTSI motif interacts with AP-3, and phosphorylation of the serine in this motif disrupts the interaction and decreases APP trafficking to lysosomes. Furthermore, we show that phosphorylation at this motif can decrease the production of neurotoxic Aβ 42. This demonstrates that reducing APP trafficking to lysosomes may be a strategy to reduce Aβ 42 in Alzheimer’s disease. PMID:27776132

  3. CPB1 of Aedes aegypti interacts with DENV2 E protein and regulates intracellular viral accumulation and release from midgut cells.

    PubMed

    Tham, Hong-Wai; Balasubramaniam, Vinod R M T; Tejo, Bimo Ario; Ahmad, Hamdan; Hassan, Sharifah Syed

    2014-12-16

    Aedes aegypti is a principal vector responsible for the transmission of dengue viruses (DENV). To date, vector control remains the key option for dengue disease management. To develop new vector control strategies, a more comprehensive understanding of the biological interactions between DENV and Ae. aegypti is required. In this study, a cDNA library derived from the midgut of female adult Ae. aegypti was used in yeast two-hybrid (Y2H) screenings against DENV2 envelope (E) protein. Among the many interacting proteins identified, carboxypeptidase B1 (CPB1) was selected, and its biological interaction with E protein in Ae. aegypti primary midgut cells was further validated. Our double immunofluorescent assay showed that CPB1-E interaction occurred in the endoplasmic reticulum (ER) of the Ae. aegypti primary midgut cells. Overexpression of CPB1 in mosquito cells resulted in intracellular DENV2 genomic RNA or virus particle accumulation, with a lower amount of virus release. Therefore, we postulated that in Ae. aegypti midgut cells, CPB1 binds to the E protein deposited on the ER intraluminal membranes and inhibits DENV2 RNA encapsulation, thus inhibiting budding from the ER, and may interfere with immature virus transportation to the trans-Golgi network.

  4. 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

  5. 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

  6. 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.

  7. Intracellular calcium channels in protozoa.

    PubMed

    Docampo, Roberto; Moreno, Silvia N J; Plattner, Helmut

    2014-09-15

    Ca(2+)-signaling pathways and intracellular Ca(2+) channels are present in protozoa. Ancient origin of inositol 1,4,5-trisphosphate receptors (IP3Rs) and other intracellular channels predates the divergence of animals and fungi as evidenced by their presence in the choanoflagellate Monosiga brevicollis, the closest known relative to metazoans. The first protozoan IP3R cloned, from the ciliate Paramecium, displays strong sequence similarity to the rat type 3 IP3R. This ciliate has a large number of IP3- and ryanodine(Ry)-like receptors in six subfamilies suggesting the evolutionary adaptation to local requirements for an expanding diversification of vesicle trafficking. IP3Rs have also been functionally characterized in trypanosomatids, where they are essential for growth, differentiation, and establishment of infection. The presence of the mitochondrial calcium uniporter (MCU) in a number of protozoa indicates that mitochondrial regulation of Ca(2+) signaling is also an early appearance in evolution, and contributed to the discovery of the molecular nature of this channel in mammalian cells. There is only sequence evidence for the occurrence of two-pore channels (TPCs), transient receptor potential Ca(2+) channels (TRPCs) and intracellular mechanosensitive Ca(2+)-channels in Paramecium and in parasitic protozoa.

  8. Intracellular Calcium Channels in Protozoa

    PubMed Central

    Docampo, Roberto; Moreno, Silvia N.J.; Plattner, Helmut

    2014-01-01

    Ca2+-signaling pathways and intracellular Ca2+ channels are present in protozoa. Ancient origin of inositol 1,4,5-trisphosphate receptors (IP3Rs) and other intracellular channels predates the divergence of animals and fungi as evidenced by their presence in the choanoflagellate Monosiga brevicollis, the closest known relative to metazoans. The first protozoan IP3R cloned, from the ciliate Paramecium, displays strong sequence similarity to the rat type 3 IP3R. This ciliate has a large number of IP3- and ryanodine(Ry)-like receptors in 6 subfamilies suggesting the evolutionary adaptation to local requirements for an expanding diversification of vesicle trafficking. IP3Rs have also been functionally characterized in trypanosomatids, where they are essential for growth, differentiation, and establishment of infection. The presence of the mitochondrial calcium uniporter (MCU) in a number of protozoa indicates that mitochondrial regulation of Ca2+ signaling is also an early appearance in evolution, and contributed to the discovery of the molecular nature of this channel in mammalian cells. There is only sequence evidence for the occurrence of two-pore channels (TPCs), transient receptor potential Ca2+ channels (TRPCs) and intracellular mechanosensitive Ca2+-channels in Paramecium and in parasitic protozoa. PMID:24291099

  9. Intracellular pH (pHin) and cytosolic calcium ([Ca2+]cyt) regulation via ATPases: studies in cell populations, single cells, and subcellular compartments

    NASA Astrophysics Data System (ADS)

    Rojas, Jose D.; Sanka, Shankar C.; Gyorke, Sandor; Wesson, Donald E.; Minta, Akwasi; Martinez-Zaguilan, Raul

    1999-07-01

    Changes in pHin and (Ca2+)cyt are important in the signal transduction mechanisms leading to many physiological responses including cell growth, motility, secretion/exocytosis, etc. The concentrations of these ions are regulated via primary and secondary ion transporting mechanisms. In diabetes, specific pH and Ca2+ regulatory mechanism might be altered. To study these ions, we employ fluorescence spectroscopy, and cell imagin spectroscopy/confocal microscopy. pH and Ca2+ indicators are loaded in the cytosol with acetoxymethyl ester forms of dyes, and in endosomal/lysosomal (E/L) compartments by overnight incubation of cells with dextran- conjugated ion fluorescent probes. We focus on specific pH and Ca2+ regulatory systems: plasmalemmal vacuolar- type H+-ATPases (pm V-ATPases) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA). As experimental models, we employ vascular smooth muscle (VSM) and microvascular endothelial cells. We have chosen these cells because they are important in blood flow regulation and in angiogenesis. These processes are altered in diabetes. In many cell types, ion transport processes are dependent on metabolism of glucose for maximal activity. Our main findings are: (a) glycolysis coupling the activity of SERCA is required for cytosolic Ca2+ homeostasis in both VSM and microvascular endothelial cells; (b) E/L compartments are important for pH and Ca2+ regulation via H+-ATPases and SERCA, respectively; and (c) pm-V- ATPases are important for pHin regulation in microvascular endothelial cells.

  10. Pregnane X Receptor Regulates Pathogen-Induced Inflammation and Host Defense against an Intracellular Bacterial Infection through Toll-like Receptor 4

    PubMed Central

    Qiu, Zhijuan; Cervantes, Jorge L.; Cicek, Basak B.; Mukherjee, Subhajit; Venkatesh, Madhukumar; Maher, Leigh A.; Salazar, Juan C.; Mani, Sridhar; Khanna, Kamal M.

    2016-01-01

    The nuclear pregnane X receptor (PXR) plays a central role in regulating xenobiotic metabolism. We now report a novel role for PXR as a critical negative regulator of innate immunity after infection. Pxr−/− mice exhibited remarkably elevated pro-inflammatory cytokine and chemokine production following infection with Listeria monocytogenes (Lm). Despite the more robust innate immune response, Pxr−/− mice were highly susceptible to Lm infection. Surprisingly, disruption of the Toll-like receptor 4 (TLR4) but not TLR2 signaling restored the inflammation to normal levels and the ability to clear Lm in Pxr−/− mice. Mechanistically, the heightened inflammation in Pxr−/− mice resulted in the death of inflammatory monocytes that led to the enhanced susceptibility to Lm infection. These data demonstrated that PXR regulated pathogen-induced inflammation and host defense against Lm infection through modulating the TLR4 pathway. In summary, we discovered an apical role for PXR in regulating innate immunity. In addition, we uncovered a remarkable negative impact of the TLR4 pathway in controlling the quality of the inflammatory response and host defense against a gram-positive bacterial infection. PMID:27550658

  11. Pregnane X Receptor Regulates Pathogen-Induced Inflammation and Host Defense against an Intracellular Bacterial Infection through Toll-like Receptor 4.

    PubMed

    Qiu, Zhijuan; Cervantes, Jorge L; Cicek, Basak B; Mukherjee, Subhajit; Venkatesh, Madhukumar; Maher, Leigh A; Salazar, Juan C; Mani, Sridhar; Khanna, Kamal M

    2016-01-01

    The nuclear pregnane X receptor (PXR) plays a central role in regulating xenobiotic metabolism. We now report a novel role for PXR as a critical negative regulator of innate immunity after infection. Pxr(-/-) mice exhibited remarkably elevated pro-inflammatory cytokine and chemokine production following infection with Listeria monocytogenes (Lm). Despite the more robust innate immune response, Pxr(-/-) mice were highly susceptible to Lm infection. Surprisingly, disruption of the Toll-like receptor 4 (TLR4) but not TLR2 signaling restored the inflammation to normal levels and the ability to clear Lm in Pxr(-/-) mice. Mechanistically, the heightened inflammation in Pxr(-/-) mice resulted in the death of inflammatory monocytes that led to the enhanced susceptibility to Lm infection. These data demonstrated that PXR regulated pathogen-induced inflammation and host defense against Lm infection through modulating the TLR4 pathway. In summary, we discovered an apical role for PXR in regulating innate immunity. In addition, we uncovered a remarkable negative impact of the TLR4 pathway in controlling the quality of the inflammatory response and host defense against a gram-positive bacterial infection. PMID:27550658

  12. Regulation of L- and N-types of Ca2+ channels by intracellular ATP4- in frog dorsal root ganglion neurons.

    PubMed

    Yuki, T; Yamaoka, K; Seyama, I

    1999-07-01

    The roles of free Mg2+ ions, ATP4- ions and Mg-ATP complexes in the regulation of N- and L-types of Ca2+ channels were studied in frog dorsal root ganglion (DRG) neurons using the whole-cell patch-clamp technique. Because Mg2+ ions interact with ATP4- ions to form Mg-ATP complexes, addition of one species can influence the concentrations of the other two. In this study their concentrations were carefully controlled by varying the concentrations of two constituents at a time while keeping the third constant. The effects of each of the three species on barium currents through L-type (IBaL) and N-type (IBaN) Ca2+ channels were plotted against its concentrations. The dose-response curves for ATP4- show that IBaL and IBaN proportionally increased with ATP4- concentrations up to 1 mM at three different Mg2+ concentrations. At a fixed concentration of ATP4-, IBaL and IBaN remained unchanged even when pMg changed from 3 to 5. Dose-response curves for IBaL and IBaN plotted against Mg-ATP concentration did not show a consistent pattern. H-7 and Mg2+ ions did not exert any blocking effect on the activity of either Ca2+ channel type, and neither dibutyryl-cAMP nor NKH-477 had any stimulating effect, suggesting that phosphorylation is not likely to be involved in ATP-induced potentiation. From these observations, it is concluded that L-type and N-type Ca2+ channels in frog DRG neurons are regulated by ATP4- ions alone, and that the neuronal Ca2+ channels are regulated by mechanisms that are different from those regulating the cardiac Ca2+ channels. PMID:10370096

  13. Chloride transport in the cystic fibrosis enterocyte.

    PubMed

    Bijman, J; Veeze, H; Kansen, M; Tilly, B; Scholte, B; Hoogeveen, A; Halley, D; Sinaasappel, M; de Jonge, H

    1991-01-01

    Molecular mechanisms of intestinal chloride channel regulation and potential abnormalities in electrogenic chloride secretion in intestinal epithelium from cystic fibrosis (CF) patients were investigated by a combination of Ussing chamber, vesicle transport and off-cell patch-clamp analysis. Short circuit current (Isc) measurements in normal and CF rectal biopsies provided evidence for i) a defect in the cAMP-provoked activation of chloride secretion and a (hyper)expression of cAMP-dependent potassium secretion in all CF patients examined (n = 11); ii) a defect in the carbachol-provoked chloride secretion and a (hyper)expression of carbachol-induced potassium secretion in 6/11 patients; iii) a residual (but still impaired) carbachol-induced chloride secretion in 5/11 CF patients (including 2 sibs). The latter class of CF patients appeared to consist genetically of compound heterozygotes for the major delta-F508 deletion, suggesting a correlation between the nature of the mutation in the CF gene and the severity of the chloride secretory defect in CF intestine. In our search for a regulatory function of GTP-binding (G-) proteins detected previously in the luminal membrane of rat and human intestinal epithelial cells, evidence was found for the presence of a GTP[S]-activatable- and GDP[S]-inhibitable chloride conductance in the apical membrane of rat enterocytes and human colonocytes. In excised patches of human colonocyt membranes, this G-proteine-sensitive chloride conductance was identified further as a novel type of chloride channel (20pS; inwardly rectifying) that was different from the 33pS outwardly rectifying chloride channel activatable by cAMP-dependent proteinkinase (PK-A) and voltage depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)

  14. Curcumin inhibits apoptosis by regulating intracellular calcium release, reactive oxygen species and mitochondrial depolarization levels in SH-SY5Y neuronal cells.

    PubMed

    Uğuz, Abdülhadi Cihangir; Öz, Ahmi; Nazıroğlu, Mustafa

    2016-08-01

    Neurological diseases such as Alzheimer's and Parkinson's diseases are incurable progressive neurological disorders caused by the degeneration of neuronal cells and characterized by motor and non-motor symptoms. Curcumin, a turmeric product, is an anti-inflammatory agent and an effective reactive oxygen and nitrogen species scavenging molecule. Hydrogen peroxide (H2O2) is the main source of oxidative stress, which is claimed to be the major source of neurological disorders. Hence, in this study we aimed to investigate the effect of curcumin on Ca(2+) signaling, oxidative stress parameters, mitochondrial depolarization levels and caspase-3 and -9 activities that are induced by the H2O2 model of oxidative stress in SH-SY5Y neuronal cells. SH-SY5Y neuronal cells were divided into four groups namely, the control, curcumin, H2O2, and curcumin + H2O2 groups. The dose and duration of curcumin and H2O2 were determined from published data. The cells in the curcumin, H2O2, and curcumin + H2O2 groups were incubated for 24 h with 5 µM curcumin and 100 µM H2O2. Lipid peroxidation and cytosolic free Ca(2+) concentrations were higher in the H2O2 group than in the control group; however, their levels were lower in the curcumin and curcumin + H2O2 groups than in the H2O2 group alone. Reduced glutathione (GSH) and glutathione peroxidase (GSH-Px) values were lower in the H2O2 group although they were higher in the curcumin and curcumin + H2O2 groups than in the H2O2 group. Caspase-3 activity was lower in the curcumin group than in the H2O2 group. In conclusion, curcumin strongly induced modulator effects on oxidative stress, intracellular Ca(2+) levels, and the caspase-3 and -9 values in an experimental oxidative stress model in SH-SY5Y cells.

  15. Effects of the inflammatory cytokines TNF-α and IL-13 on stromal interaction molecule-1 aggregation in human airway smooth muscle intracellular Ca(2+) regulation.

    PubMed

    Jia, Li; Delmotte, Philippe; Aravamudan, Bharathi; Pabelick, Christina M; Prakash, Y S; Sieck, Gary C

    2013-10-01

    Inflammation elevates intracellular Ca(2+) ([Ca(2+)]i) concentrations in airway smooth muscle (ASM). Store-operated Ca(2+) entry (SOCE) is an important source of [Ca(2+)]i mediated by stromal interaction molecule-1 (STIM1), a sarcoplasmic reticulum (SR) protein. In transducing SR Ca(2+) depletion, STIM1 aggregates to form puncta, thereby activating SOCE via interactions with a Ca(2+) release-activated Ca(2+) channel protein (Orai1) in the plasma membrane. We hypothesized that STIM1 aggregation is enhanced by inflammatory cytokines, thereby augmenting SOCE in human ASM cells. We used real-time fluorescence microscopic imaging to assess the dynamics of STIM1 aggregation and SOCE after exposure to TNF-α or IL-13 in ASM cells overexpressing yellow fluorescent protein-tagged wild-type STIM1 (WT-STIM1) and STIM1 mutants lacking the Ca(2+)-sensing EF-hand (STIM1-D76A), or lacking the cytoplasmic membrane binding site (STIM1ΔK). STIM1 aggregation was analyzed by monitoring puncta size during the SR Ca(2+) depletion induced by cyclopiazonic acid (CPA). We found that puncta size was increased in cells expressing WT-STIM1 after CPA. However, STIM1-D76A constitutively formed puncta, whereas STIM1ΔK failed to form puncta. Furthermore, cytokines increased basal WT-STIM1 puncta size, and the SOCE triggered by SR Ca(2+) depletion was increased in cells expressing WT-STIM1 or STIM1-D76A. Meanwhile, SOCE in cells expressing STIM1ΔK and STIM1 short, interfering RNA (siRNA) was decreased. Similarly, in cells overexpressing STIM1, the siRNA knockdown of Orai1 blunted SOCE. However, exposure to cytokines increased SOCE in all cells, increased basal [Ca(2+)]i, and decreased SR Ca(2+) content. These data suggest that cytokines induce a constitutive increase in STIM1 aggregation that contributes to enhanced SOCE in human ASM after inflammation. Such effects of inflammation on STIM1 aggregations may contribute to airway hyperresponsiveness. PMID:23713409

  16. Skb5, an SH3 adaptor protein, regulates Pmk1 MAPK signaling by controlling the intracellular localization of the MAPKKK Mkh1.

    PubMed

    Kanda, Yuki; Satoh, Ryosuke; Matsumoto, Saki; Ikeda, Chisato; Inutsuka, Natsumi; Hagihara, Kanako; Matzno, Sumio; Tsujimoto, Sho; Kita, Ayako; Sugiura, Reiko

    2016-08-15

    The mitogen-activated protein kinase (MAPK) cascade is a highly conserved signaling module composed of MAPK kinase kinases (MAPKKKs), MAPK kinases (MAPKK) and MAPKs. The MAPKKK Mkh1 is an initiating kinase in Pmk1 MAPK signaling, which regulates cell integrity in fission yeast (Schizosaccharomyces pombe). Our genetic screen for regulators of Pmk1 signaling identified Shk1 kinase binding protein 5 (Skb5), an SH3-domain-containing adaptor protein. Here, we show that Skb5 serves as an inhibitor of Pmk1 MAPK signaling activation by downregulating Mkh1 localization to cell tips through its interaction with the SH3 domain. Consistent with this, the Mkh1(3PA) mutant protein, with impaired Skb5 binding, remained in the cell tips, even when Skb5 was overproduced. Intriguingly, Skb5 needs Mkh1 to localize to the growing ends as Mkh1 deletion and disruption of Mkh1 binding impairs Skb5 localization. Deletion of Pck2, an upstream activator of Mkh1, impaired the cell tip localization of Mkh1 and Skb5 as well as the Mkh1-Skb5 interaction. Interestingly, both Pck2 and Mkh1 localized to the cell tips at the G1/S phase, which coincided with Pmk1 MAPK activation. Taken together, Mkh1 localization to cell tips is important for transmitting upstream signaling to Pmk1, and Skb5 spatially regulates this process. PMID:27451356

  17. Two distinct intracellular Ca2+-release components act in opposite ways in the regulation of the auxin-dependent MIA biosynthesis in Catharanthus roseus cells.

    PubMed

    Poutrain, Pierre; Mazars, Christian; Thiersault, Martine; Rideau, Marc; Pichon, Olivier

    2009-01-01

    Calcium-mediated signalling is ubiquitous in both animals and plants. Changes in cytoplasmic free Ca(2+) concentration couple diverse arrays of stimuli to their specific responses, the specificity of the stimulus being determined by integrated actions between multiple Ca(2+) mobilization pathways. In this work, a pharmacological approach is reported, aimed at deciphering the role of calcium as a second messenger in the transduction pathway leading to the inhibitory effect of 2,4-dichlorophenoxyacetic acid (2,4-D), in regulating monoterpene indole alkaloid (MIA) biosynthesis in Catharanthus roseus cells. It is demonstrated here that auxin-dependent MIA biosynthesis is differentially regulated by two distinct calcium release components from internal stores in C. roseus showing pharmacological profiles similar to those displayed by animal RyR and IP3 channels. MIA biosynthesis is stimulated by caffeine (Ca(2+)-release activator through RyR channels) and by heparin and TMB8 (Ca(2+)-release inhibitors of IP3 channels) whereas MIA biosynthesis is inhibited by mastoparan (Ca(2+)-release activator of IP3 channels) and by ruthenium red and DHBP (Ca(2+)-release inhibitors of RyR channels). Furthermore, calcium, as 2,4-D, acts on MIA biosynthesis by regulating the monoterpene moiety of the MIA biosynthesis pathway since calcium channel modulators preferentially modulate g10h expression, the gene encoding the enzyme of the secoiridoid monoterpene pathway, that is the major target of 2,4-D action. In addition, the simultaneous use of caffeine (an activator of RyR channel in animals) and TMB8 (an inhibitor of the IP3 channel) in 2,4-D treated cells triggers a synergistic effect on MIA accumulation. This finding suggests an opposite and co-ordinated action of multiple Ca(2+)-release pathways in 2,4-D signal transduction, adding a new level of complexity to calcium signalling in plants and questioning the existence of RyR and IP3 channels in plants.

  18. Structural homologues P(II) and P(Z) of Azospirillum brasilense provide intracellular signalling for selective regulation of various nitrogen-dependent functions.

    PubMed

    de Zamaroczy, M

    1998-07-01

    P(II) (glnB) is a signal transduction protein that in Azospirillum brasilense is specifically required for nitrogen fixation. Little is known about whether and how its homologue P(Z) (glnZ) participates in the regulation of cellular functions. In this study, we have shown the regulatory action of the two proteins by analysing the relevant single and double null-mutant strains. The transcription of glnZ is monocistronic, and it starts mainly from a sigma54-dependent promoter, activated by NtrC. glnZ expression is dependent on the ntr system, even under conditions of nitrogen excess, and is greatly enhanced in the presence of aspartate. P(Z) is uridylylated in response to nitrogen limitation, like P(II), although different amounts of the two proteins are synthesized. P(II) is required for the dephosphorylation of NtrC. Thus, in the absence of P(II), the repression of nitrate assimilation is not promoted, which, in turn, leads to a high rate of ammonium excretion. Unexpectedly, P(II) and P(Z) proteins are not essential for the reversible modification of glutamine synthetase. (Methyl)ammonium transport into the cell is negatively regulated by P(Z). The growth of a double-mutant strain (glnB::kan; glnZ::omega) is drastically disabled, although wild-type growth is restored by complementation with either glnB or glnZ. We conclude that P(II) and P(Z), despite their structural similarity, are involved in different regulatory processes, except for that required for cell growth.

  19. 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.

  20. Ca2+ Binding/Permeation via Calcium Channel, CaV1.1, Regulates the Intracellular Distribution of the Fatty Acid Transport Protein, CD36, and Fatty Acid Metabolism.

    PubMed

    Georgiou, Dimitra K; Dagnino-Acosta, Adan; Lee, Chang Seok; Griffin, Deric M; Wang, Hui; Lagor, William R; Pautler, Robia G; Dirksen, Robert T; Hamilton, Susan L

    2015-09-25

    Ca(2+) permeation and/or binding to the skeletal muscle L-type Ca(2+) channel (CaV1.1) facilitates activation of Ca(2+)/calmodulin kinase type II (CaMKII) and Ca(2+) store refilling to reduce muscle fatigue and atrophy (Lee, C. S., Dagnino-Acosta, A., Yarotskyy, V., Hanna, A., Lyfenko, A., Knoblauch, M., Georgiou, D. K., Poché, R. A., Swank, M. W., Long, C., Ismailov, I. I., Lanner, J., Tran, T., Dong, K., Rodney, G. G., Dickinson, M. E., Beeton, C., Zhang, P., Dirksen, R. T., and Hamilton, S. L. (2015) Skelet. Muscle 5, 4). Mice with a mutation (E1014K) in the Cacna1s (α1 subunit of CaV1.1) gene that abolishes Ca(2+) binding within the CaV1.1 pore gain more body weight and fat on a chow diet than control mice, without changes in food intake or activity, suggesting that CaV1.1-mediated CaMKII activation impacts muscle energy expenditure. We delineate a pathway (Cav1.1→ CaMKII→ NOS) in normal skeletal muscle that regulates the intracellular distribution of the fatty acid transport protein, CD36, altering fatty acid metabolism. The consequences of blocking this pathway are decreased mitochondrial β-oxidation and decreased energy expenditure. This study delineates a previously uncharacterized CaV1.1-mediated pathway that regulates energy utilization in skeletal muscle.

  1. Ca2+ Binding/Permeation via Calcium Channel, CaV1.1, Regulates the Intracellular Distribution of the Fatty Acid Transport Protein, CD36, and Fatty Acid Metabolism*

    PubMed Central

    Georgiou, Dimitra K.; Dagnino-Acosta, Adan; Lee, Chang Seok; Griffin, Deric M.; Wang, Hui; Lagor, William R.; Pautler, Robia G.; Dirksen, Robert T.; Hamilton, Susan L.

    2015-01-01

    Ca2+ permeation and/or binding to the skeletal muscle L-type Ca2+ channel (CaV1.1) facilitates activation of Ca2+/calmodulin kinase type II (CaMKII) and Ca2+ store refilling to reduce muscle fatigue and atrophy (Lee, C. S., Dagnino-Acosta, A., Yarotskyy, V., Hanna, A., Lyfenko, A., Knoblauch, M., Georgiou, D. K., Poché, R. A., Swank, M. W., Long, C., Ismailov, I. I., Lanner, J., Tran, T., Dong, K., Rodney, G. G., Dickinson, M. E., Beeton, C., Zhang, P., Dirksen, R. T., and Hamilton, S. L. (2015) Skelet. Muscle 5, 4). Mice with a mutation (E1014K) in the Cacna1s (α1 subunit of CaV1.1) gene that abolishes Ca2+ binding within the CaV1.1 pore gain more body weight and fat on a chow diet than control mice, without changes in food intake or activity, suggesting that CaV1.1-mediated CaMKII activation impacts muscle energy expenditure. We delineate a pathway (Cav1.1→ CaMKII→ NOS) in normal skeletal muscle that regulates the intracellular distribution of the fatty acid transport protein, CD36, altering fatty acid metabolism. The consequences of blocking this pathway are decreased mitochondrial β-oxidation and decreased energy expenditure. This study delineates a previously uncharacterized CaV1.1-mediated pathway that regulates energy utilization in skeletal muscle. PMID:26245899

  2. 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

  3. [Low molecular weight regulators of the intracellular insulin signal transduction as a correction method of the insulin resistance in the treatment of type 2 diabetes].

    PubMed

    Galenova, T I; Kyznetsova, M Y; Savchuk, O N; Ostapchenco, L I

    2016-01-01

    Insulin resistance is the characteristic feature of type 2 diabetes. This condition is manifested in the reduction of peripheral tissues sensitivity to the biological action of insulin and is expressed in the inhibition of cellular glucose absorption and metabolism in response to hormonal stimulation. At the cellular level, disorders which are realized both at the receptor and the postreceptor levels can serve a prerequisite to the formation of insulin resistance and are associated with a change in the amount or dysfunction of major molecular signaling cascade. Thus, the insulin receptor, as well as the other related signaling molecules can be considered as ideal therapeutic targets for the correction of insulin resistance and thus low molecular weight effectors which act on the individual links of insulin signaling cascade may be positioned as a new generation of anti-diabetic agents. This report provides information on the regulators of insulin receptor cascade, main advantages and disadvantages of their impact on biological targets and prospects for their therapeutic use as anti-diabetic drugs. PMID:26973184

  4. Phosphonium chloride for thermal storage

    NASA Technical Reports Server (NTRS)

    Sutton, J. G.; Heimlich, P. F.; Tepper, E. H.

    1972-01-01

    Development of systems for storage of thermal energy is discussed. Application of phosphonium chloride for heat storage through reversible dissociation is described. Chemical, physical, and thermodynamic properties of phosphonium chloride are analyzed and dangers in using phosphonium chloride are explained.

  5. Intracellular parcel service: current issues in intracellular membrane trafficking.

    PubMed

    Herrmann, Johannes M; Spang, Anne

    2015-01-01

    Eukaryotic cells contain a multitude of membrane structures that are connected through a highly dynamic and complex exchange of their constituents. The vibrant instability of these structures challenges the classical view of defined, static compartments that are connected by different types of vesicles. Despite this astonishing complexity, proteins and lipids are accurately transported into the different intracellular membrane systems. Over the past few decades many factors have been identified that either mediate or regulate intracellular membrane trafficking. Like in a modern parcel sorting system of a logistics center, the cargo typically passes through several sequential sorting stations until it finally reaches the location that is specified by its individual address label. While each membrane system employs specific sets of factors, the transport processes typically operate on common principles. With the advent of genome- and proteome-wide screens, the availability of mutant collections, exciting new developments in microscope technology and sophisticated methods to study their dynamics, the future promises a broad and comprehensive picture of the processes by which eukaryotic cells sort their proteins.

  6. Regulation of DM-20 mRNA expression and intracellular translocation of glutathione-S-transferase pi isoform during oligodendrocyte differentiation in the adult rat spinal cord.

    PubMed

    Kitada, Masaaki; Takeda, Kazuya; Dezawa, Mari

    2016-07-01

    We previously demonstrated that NG2-positive oligodendrocyte precursor cells (OPCs) do not express DM-20 mRNA and identified a distinct DM-20 mRNA-positive cell population expressing glutathione-S-transferase pi isoform (GST-pi) in the nucleus (GST-pi(Nuc)) of the adult rat spinal cord. As GST-pi intranuclear localization correlates with progenitor cell properties, we examined the differentiation status of this cell population under the intensive 5-bromo-2'-deoxyuridine (BrdU) administration method, consisting of intraperitoneal BrdU injections every 2 h for 48 h. We observed that a certain population of proliferating/proliferated cells expressed DM-20 mRNA, and sometimes two proliferating/proliferated cells were observed still attached to each other. We performed triple staining for BrdU, DM-20 mRNA, and NG2 and found pairs of neighboring BrdU-positive cells, which were considered to originate from the same progenitor cells and where both cells expressed DM-20 mRNA. Triple staining for BrdU, DM-20 mRNA, and GST-pi detected proliferating/proliferated cells exhibiting the GST-pi(Nuc)/DM-20 mRNA-positive expression pattern. These findings suggested the presence of a GST-pi(Nuc)/DM-20 mRNA-positive oligodendrocyte-lineage progenitor cell population in the adult rat spinal cord. However, we did not find any pair of neighboring BrdU-positive cells with this expression pattern. These observations collectively support the idea that GST-pi(Nuc)/DM-20 mRNA-expressing cells are the progeny of NG2-positive OPCs rather than a novel type of oligodendrocyte-lineage progenitor cells and that DM-20 mRNA expression is dynamically regulated during differentiation of OPCs into oligodendrocytes.

  7. Photoperiodic regulation of insulin receptor mRNA and intracellular insulin signaling in the arcuate nucleus of the Siberian hamster, Phodopus sungorus.

    PubMed

    Tups, Alexander; Helwig, Michael; Stöhr, Sigrid; Barrett, Perry; Mercer, Julian G; Klingenspor, Martin

    2006-09-01

    During the last 5 years it has been well established that photoperiod-induced changes in body weight in the seasonal hamster, Phodopus sungorus, are accompanied by a marked seasonal cycle in leptin sensitivity. In the present study, we investigated the possible involvement of insulin signaling in seasonal body weight regulation. We analyzed the expression pattern and relative intensity of insulin receptor (IR), phosphatidylinositol 3-kinase (PI3-kinase), and protein tyrosine phosphatase 1B (PTP1B) mRNAs by in situ hybridization in the brains of juvenile female hamsters acclimated to either long- (LD) or short-day length (SD) for 8 wk, with or without superimposed food deprivation for 48 h. Furthermore, the hypothalamic concentration and distribution of phospho-AKT, a marker of PI3-kinase activity was determined by immunoblotting and immunohistochemistry. Eight weeks of acclimation to SD led to a substantial downregulation of IR, PTP1B gene expression, and phospho-AKT concentration in this brain region, whereas PI3-kinase mRNA was unchanged. Food deprivation induced a decrease in PTP1B and a trend toward lowered IR gene expression in LD but not in SD. Additionally, a striking increase in PTP1B gene expression in the thalamus was observed after food deprivation in both photoperiods. The direction of change in neuronal insulin signaling contrasts to the central catabolic nature of this pathway described in other species. SD-induced reduction in insulin signaling may be due to decline in body fat stores mediated by enhanced central leptin sensitivity. Increased anorexigenic tone of leptin may overwrite central insulin signaling to prevent catabolic overdrive.

  8. New evidence for coupled clock regulation of the normal automaticity of sinoatrial nodal pacemaker cells: bradycardic effects of ivabradine are linked to suppression of intracellular Ca²⁺ cycling.

    PubMed

    Yaniv, Yael; Sirenko, Syevda; Ziman, Bruce D; Spurgeon, Harold A; Maltsev, Victor A; Lakatta, Edward G

    2013-09-01

    Beneficial clinical bradycardic effects of ivabradine (IVA) have been interpreted solely on the basis of If inhibition, because IVA specifically inhibits If in sinoatrial nodal pacemaker cells (SANC). However, it has been recently hypothesized that SANC normal automaticity is regulated by crosstalk between an "M clock," the ensemble of surface membrane ion channels, and a "Ca(2+) clock," the sarcoplasmic reticulum (SR). We tested the hypothesis that crosstalk between the two clocks regulates SANC automaticity, and that indirect suppression of the Ca(2+) clock further contributes to IVA-induced bradycardia. IVA (3 μM) not only reduced If amplitude by 45 ± 6% in isolated rabbit SANC, but the IVA-induced slowing of the action potential (AP) firing rate was accompanied by reduced SR Ca(2+) load, slowed intracellular Ca(2+) cycling kinetics, and prolonged the period of spontaneous local Ca(2+) releases (LCRs) occurring during diastolic depolarization. Direct and specific inhibition of SERCA2 by cyclopiazonic acid (CPA) had effects similar to IVA on LCR period and AP cycle length. Specifically, the LCR period and AP cycle length shift toward longer times almost equally by either direct perturbations of the M clock (IVA) or the Ca(2+) clock (CPA), indicating that the LCR period reports the crosstalk between the clocks. Our numerical model simulations predict that entrainment between the two clocks that involves a reduction in INCX during diastolic depolarization is required to explain the experimentally AP firing rate reduction by IVA. In summary, our study provides new evidence that a coupled-clock system regulates normal cardiac pacemaker cell automaticity. Thus, IVA-induced bradycardia includes a suppression of both clocks within this system. PMID:23651631

  9. Intracellular pH in sperm physiology.

    PubMed

    Nishigaki, Takuya; José, Omar; González-Cota, Ana Laura; Romero, Francisco; Treviño, Claudia L; Darszon, Alberto

    2014-08-01

    Intracellular pH (pHi) regulation is essential for cell function. Notably, several unique sperm ion transporters and enzymes whose elimination causes infertility are either pHi dependent or somehow related to pHi regulation. Amongst them are: CatSper, a Ca(2+) channel; Slo3, a K(+) channel; the sperm-specific Na(+)/H(+) exchanger and the soluble adenylyl cyclase. It is thus clear that pHi regulation is of the utmost importance for sperm physiology. This review briefly summarizes the key components involved in pHi regulation, their characteristics and participation in fundamental sperm functions such as motility, maturation and the acrosome reaction.

  10. Gating the glutamate gate of CLC-2 chloride channel by pore occupancy.

    PubMed

    De Jesús-Pérez, José J; Castro-Chong, Alejandra; Shieh, Ru-Chi; Hernández-Carballo, Carmen Y; De Santiago-Castillo, José A; Arreola, Jorge

    2016-01-01

    CLC-2 channels are dimeric double-barreled chloride channels that open in response to hyperpolarization. Hyperpolarization activates protopore gates that independently regulate the permeability of the pore in each subunit and the common gate that affects the permeability through both pores. CLC-2 channels lack classic transmembrane voltage-sensing domains; instead, their protopore gates (residing within the pore and each formed by the side chain of a glutamate residue) open under repulsion by permeant intracellular anions or protonation by extracellular H(+). Here, we show that voltage-dependent gating of CLC-2: (a) is facilitated when permeant anions (Cl(-), Br(-), SCN(-), and I(-)) are present in the cytosolic side; (b) happens with poorly permeant anions fluoride, glutamate, gluconate, and methanesulfonate present in the cytosolic side; (c) depends on pore occupancy by permeant and poorly permeant anions; (d) is strongly facilitated by multi-ion occupancy; (e) is absent under likely protonation conditions (pHe = 5.5 or 6.5) in cells dialyzed with acetate (an impermeant anion); and (f) was the same at intracellular pH 7.3 and 4.2; and (g) is observed in both whole-cell and inside-out patches exposed to increasing [Cl(-)]i under unlikely protonation conditions (pHe = 10). Thus, based on our results we propose that hyperpolarization activates CLC-2 mainly by driving intracellular anions into the channel pores, and that protonation by extracellular H(+) plays a minor role in dislodging the glutamate gate. PMID:26666914

  11. Gating the glutamate gate of CLC-2 chloride channel by pore occupancy.

    PubMed

    De Jesús-Pérez, José J; Castro-Chong, Alejandra; Shieh, Ru-Chi; Hernández-Carballo, Carmen Y; De Santiago-Castillo, José A; Arreola, Jorge

    2016-01-01

    CLC-2 channels are dimeric double-barreled chloride channels that open in response to hyperpolarization. Hyperpolarization activates protopore gates that independently regulate the permeability of the pore in each subunit and the common gate that affects the permeability through both pores. CLC-2 channels lack classic transmembrane voltage-sensing domains; instead, their protopore gates (residing within the pore and each formed by the side chain of a glutamate residue) open under repulsion by permeant intracellular anions or protonation by extracellular H(+). Here, we show that voltage-dependent gating of CLC-2: (a) is facilitated when permeant anions (Cl(-), Br(-), SCN(-), and I(-)) are present in the cytosolic side; (b) happens with poorly permeant anions fluoride, glutamate, gluconate, and methanesulfonate present in the cytosolic side; (c) depends on pore occupancy by permeant and poorly permeant anions; (d) is strongly facilitated by multi-ion occupancy; (e) is absent under likely protonation conditions (pHe = 5.5 or 6.5) in cells dialyzed with acetate (an impermeant anion); and (f) was the same at intracellular pH 7.3 and 4.2; and (g) is observed in both whole-cell and inside-out patches exposed to increasing [Cl(-)]i under unlikely protonation conditions (pHe = 10). Thus, based on our results we propose that hyperpolarization activates CLC-2 mainly by driving intracellular anions into the channel pores, and that protonation by extracellular H(+) plays a minor role in dislodging the glutamate gate.

  12. Gating the glutamate gate of CLC-2 chloride channel by pore occupancy

    PubMed Central

    De Jesús-Pérez, José J.; Castro-Chong, Alejandra; Shieh, Ru-Chi; Hernández-Carballo, Carmen Y.; De Santiago-Castillo, José A.

    2016-01-01

    CLC-2 channels are dimeric double-barreled chloride channels that open in response to hyperpolarization. Hyperpolarization activates protopore gates that independently regulate the permeability of the pore in each subunit and the common gate that affects the permeability through both pores. CLC-2 channels lack classic transmembrane voltage–sensing domains; instead, their protopore gates (residing within the pore and each formed by the side chain of a glutamate residue) open under repulsion by permeant intracellular anions or protonation by extracellular H+. Here, we show that voltage-dependent gating of CLC-2: (a) is facilitated when permeant anions (Cl−, Br−, SCN−, and I−) are present in the cytosolic side; (b) happens with poorly permeant anions fluoride, glutamate, gluconate, and methanesulfonate present in the cytosolic side; (c) depends on pore occupancy by permeant and poorly permeant anions; (d) is strongly facilitated by multi-ion occupancy; (e) is absent under likely protonation conditions (pHe = 5.5 or 6.5) in cells dialyzed with acetate (an impermeant anion); and (f) was the same at intracellular pH 7.3 and 4.2; and (g) is observed in both whole-cell and inside-out patches exposed to increasing [Cl−]i under unlikely protonation conditions (pHe = 10). Thus, based on our results we propose that hyperpolarization activates CLC-2 mainly by driving intracellular anions into the channel pores, and that protonation by extracellular H+ plays a minor role in dislodging the glutamate gate. PMID:26666914

  13. Influence of sodium chloride on the regulation of Krebs cycle intermediates and enzymes of respiratory chain in mungbean (Vigna radiata L. Wilczek) seedlings.

    PubMed

    Saha, Papiya; Kunda, Pranamita; Biswas, Asok K

    2012-11-01

    The effect of common salt (NaCl) on ion contents, Krebs cycle intermediates and its regulatory enzymes was investigated in growing mungbean (Vigna radiata L. Wilczek, B 105) seedlings. Sodium and chloride ion contents increased in both root and shoot whereas potassium ion content decreased in shoot of test seedlings with increasing concentrations of NaCl. Organic acids like pyruvate and citrate levels increased whereas malate level decreased under stress in both roots and shoots. Salt stress also variedly affected the activities of different enzymes of respiratory chain. The activity of pyruvate dehydrogenase (E.C. 1.2.4.1) decreased in 50 mM NaCl but increased in 100 mM and 150 mM concentrations, in both root and shoot samples. Succinate dehydrogenase (E.C. 1.3.5.1) activity was reduced in root whereas stimulated in shoot under increasing concentrations of salt. The activity of isocitrate dehydrogenase (E.C. 1.1.1.41) and malate dehydrogenase (E.C. 1.1.1.37) decreased in both root and shoot samples under salt stress. On the contrary, pretreatment of mungbean seeds with sublethal dose of NaCl was able to overcome the adverse effects of stress imposed by NaCl to variable extents with significant alterations of all the tested parameters, resulting in better growth and efficient respiration in mungbean seedlings. Thus, plants can acclimate to lethal level of salinity by pretreatment of seeds with sublethal level of NaCl, which serves to improve their health and production under saline condition, but the sublethal concentration of NaCl should be carefully chosen.

  14. CALCIUM CHLORIDE PLANT LOOKING EAST. CALCIUM CHLORIDE BUILDING ON LEFT, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    CALCIUM CHLORIDE PLANT LOOKING EAST. CALCIUM CHLORIDE BUILDING ON LEFT, CALCIUM CHLORIDE STORAGE BUILDING ON RIGHT OF CENTER WITH TOP OF SA (SODA ASH) BUILDING IN RIGHT BACKGROUND. - Solvay Process Company, Calcium Chloride Plant, Between Willis & Milton Avenues, Solvay, Onondaga County, NY

  15. Strontium-89 Chloride

    MedlinePlus

    ... ask your doctor or pharmacist for more information.Strontium-89 chloride is in a class of drugs known as radioisotopes. It delivers radiation to cancer sites and ultimately decreases bone pain. The length of treatment depends on the ...

  16. Mercuric chloride poisoning

    MedlinePlus

    ... Mercuric chloride is a very poisonous form of mercury. It is a type of mercury salt. There are different types of mercury poisonings . This article discusses poisoning from swallowing mercuric ...

  17. Hydrogen chloride test set

    NASA Technical Reports Server (NTRS)

    Workman, G. L.

    1976-01-01

    Detector uses tertiary amine, which makes reaction fairly specific for relatively small highly polarized hydrogen chloride molecule. Reaction is monitored by any microbalance capable of measuring extremely small mass differences in real time.

  18. Cell density and growth-dependent down-regulation of both intracellular calcium responses to agonist stimuli and expression of smooth-surfaced endoplasmic reticulum in MC3T3-E1 osteoblast-like cells.

    PubMed

    Koizumi, Toshiyuki; Hikiji, Hisako; Shin, Wee Soo; Takato, Tsuyoshi; Fukuda, Satoru; Abe, Takahiro; Koshikiya, Noboru; Iwasawa, Kuniaki; Toyo-oka, Teruhiko

    2003-02-21

    A two-dimensional intracellular Ca(2+) ([Ca(2+)](i)) imaging system was used to examine the relationship between [Ca(2+)](i) handling and the proliferation of MC3T3-E1 osteoblast-like cells. The resting [Ca(2+)](i) level in densely cultured cells was 1.5 times higher than the [Ca(2+)](i) level in sparsely cultured cells or in other cell types (mouse fibroblasts, rat vascular smooth muscle cells, and bovine endothelial cells). A high resting [Ca(2+)](i) level may be specific for MC3T3-E1 cells. MC3T3-E1 cells were stimulated with ATP (10 microM), caffeine (10 mM), thapsigargin (1 microM), or ionomycin (10 microM), and the effect on the [Ca(2+)](i) level of MC3T3-E1 cells was studied. The percentage of responding cells and the degree of [Ca(2+)](i) elevation were high in the sparsely cultured cells and low in densely cultured cells. The rank order for the percentage of responding cells and magnitude of the Ca(2+) response to the stimuli was ionomycin > thapsigargin = ATP > caffeine and suggests the existence of differences among the various [Ca(2+)](i) channels. All Ca(2+) responses in the sparsely cultured MC3T3-E1 cells, unlike in other cell types, disappeared after the cells reached confluence. Heptanol treatment of densely cultured cells restored the Ca(2+) response, suggesting that cell-cell contact is involved with the confluence-dependent disappearance of the Ca(2+) response. Immunohistological analysis of type 1 inositol trisphosphate receptors and electron microscopy showed distinct expression of inositol trisphosphate receptor proteins and smooth-surfaced endoplasmic reticulum in sparsely cultured cells but reduced levels in densely cultured cells. These results indicate that the underlying basis of confluence-dependent [Ca(2+)](i) regulation is down-regulation of smooth-surfaced endoplasmic reticulum by cell-cell contacts.

  19. 29 CFR 1915.1017 - Vinyl chloride.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 7 2010-07-01 2010-07-01 false Vinyl chloride. 1915.1017 Section 1915.1017 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR (CONTINUED) OCCUPATIONAL SAFETY AND HEALTH STANDARDS FOR SHIPYARD EMPLOYMENT Toxic and Hazardous...

  20. 29 CFR 1915.1017 - Vinyl chloride.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 29 Labor 7 2011-07-01 2011-07-01 false Vinyl chloride. 1915.1017 Section 1915.1017 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR (CONTINUED) OCCUPATIONAL SAFETY AND HEALTH STANDARDS FOR SHIPYARD EMPLOYMENT Toxic and Hazardous...

  1. Spinal cord injury-induced attenuation of GABAergic inhibition in spinal dorsal horn circuits is associated with down-regulation of the chloride transporter KCC2 in rat.

    PubMed

    Lu, Yan; Zheng, Jihong; Xiong, Lize; Zimmermann, Manfred; Yang, Jing

    2008-12-01

    Most spinal cord injury (SCI) patients suffer from chronic pain. Effective therapy for this pain is lacking, and the underlying mechanisms are poorly understood. The spinal superficial dorsal horn (SDH) contains neuronal circuits capable of modulating primary afferent information involved in pain processing. KCC2 is an isoform of the K(+)-Cl(-) cotransporter that contributes to the regulation of transmembrane anion gradient which plays a key role in shaping GABA(A) receptor-mediated signalling in the CNS. We tested the hypothesis that SCI causes down-regulation of KCC2 distal to the injury and contributes to the neuronal hyperresponsiveness and pain-related behaviours. SCI was a hemisection at T(13) level of adult Sprague-Dawley rats. Spinal sagittal slices with attached dorsal roots (DR) were prepared from L(4) to L(6) level. The reversal potentials of GABA responses (E(GABA)) and DR-evoked IPSPs and EPSPs of L(4-6) SDH neurones in sham-operated and SCI rats were compared using gramicidin-perforated patch-clamp recordings. Here we report that thoracic SCI-induced down-regulation of KCC2 in the lumbar SDH parallels the development of allodynia. The subsequent changes of E(GABA) in SDH neurones attenuate the GABA(A) receptor-mediated inhibitory synaptic transmission. These changes cause certain normally subthreshold primary A and C fibre inputs to evoke action potential output in SDH neurones. We conclude that SCI induces KCC2 down-regulation and subsequent changes of E(GABA) in the SDH below the injury site. The resulting disinhibition unmasks normally ineffective SDH neuronal circuits and may contribute to the below-level central pain-related behaviours after incomplete SCI. PMID:18845615

  2. Depolarizing chloride gradient in developing cochlear nucleus neurons: underlying mechanism and implication for calcium signaling.

    PubMed

    Witte, M; Reinert, T; Dietz, B; Nerlich, J; Rübsamen, R; Milenkovic, I

    2014-03-01

    Precise regulation of the chloride homeostasis crucially determines the action of inhibitory transmitters GABA and glycine and thereby endows neurons or even discrete neuronal compartments with distinct physiological responses to the same transmitters. In mammals, the signaling mediated by GABAA/glycine receptors shifts during early postnatal life from depolarization to hyperpolarization, due to delayed maturation of the chloride homeostasis system. While the activity of the secondary active, K(+)-Cl(-)-extruding cotransporter KCC2, renders GABA/glycine hyperpolarizing in auditory brainstem nuclei of altricial rodents, the mechanisms contributing to the initially depolarizing transmembrane gradient for Cl(-) in respective neurons remained unknown. Here we used gramicidin-perforated patch recordings, non-invasive Cl(-) and Ca(2+) imaging, and immunohistochemistry to identify the Cl(-)-loading transporter that renders depolarizing effects of GABA/glycine in early postnatal life of spherical bushy cells in the cochlear nucleus of gerbil. Our data identify the 1Na(+):1K(+):2Cl(-) cotransporter 1 (NKCC1) as the major Cl(-)-loader responsible for depolarizing action of GABA/glycine at postnatal days 3-5 (P3-5). Extracellular GABA/muscimol elicited calcium signaling through R-, L-, and T-type channels, which was dependent on bumetanide- and [Na(+)]e-sensitive Cl(-) accumulation. The "adult like", low intracellular Cl(-) concentration is established during the second postnatal week, through a mechanism engaging the NKCC1-down regulation between P5 and P15 and ongoing KCC2-mediated Cl(-)-extrusion.

  3. Depolarizing chloride gradient in developing cochlear nucleus neurons: underlying mechanism and implication for calcium signaling.

    PubMed

    Witte, M; Reinert, T; Dietz, B; Nerlich, J; Rübsamen, R; Milenkovic, I

    2014-03-01

    Precise regulation of the chloride homeostasis crucially determines the action of inhibitory transmitters GABA and glycine and thereby endows neurons or even discrete neuronal compartments with distinct physiological responses to the same transmitters. In mammals, the signaling mediated by GABAA/glycine receptors shifts during early postnatal life from depolarization to hyperpolarization, due to delayed maturation of the chloride homeostasis system. While the activity of the secondary active, K(+)-Cl(-)-extruding cotransporter KCC2, renders GABA/glycine hyperpolarizing in auditory brainstem nuclei of altricial rodents, the mechanisms contributing to the initially depolarizing transmembrane gradient for Cl(-) in respective neurons remained unknown. Here we used gramicidin-perforated patch recordings, non-invasive Cl(-) and Ca(2+) imaging, and immunohistochemistry to identify the Cl(-)-loading transporter that renders depolarizing effects of GABA/glycine in early postnatal life of spherical bushy cells in the cochlear nucleus of gerbil. Our data identify the 1Na(+):1K(+):2Cl(-) cotransporter 1 (NKCC1) as the major Cl(-)-loader responsible for depolarizing action of GABA/glycine at postnatal days 3-5 (P3-5). Extracellular GABA/muscimol elicited calcium signaling through R-, L-, and T-type channels, which was dependent on bumetanide- and [Na(+)]e-sensitive Cl(-) accumulation. The "adult like", low intracellular Cl(-) concentration is established during the second postnatal week, through a mechanism engaging the NKCC1-down regulation between P5 and P15 and ongoing KCC2-mediated Cl(-)-extrusion. PMID:24388924

  4. Revealing the activation pathway for TMEM16A chloride channels from macroscopic currents and kinetic models.

    PubMed

    Contreras-Vite, Juan A; Cruz-Rangel, Silvia; De Jesús-Pérez, José J; Figueroa, Iván A Aréchiga; Rodríguez-Menchaca, Aldo A; Pérez-Cornejo, Patricia; Hartzell, H Criss; Arreola, Jorge

    2016-07-01

    TMEM16A (ANO1), the pore-forming subunit of calcium-activated chloride channels, regulates several physiological and pathophysiological processes such as smooth muscle contraction, cardiac and neuronal excitability, salivary secretion, tumour growth and cancer progression. Gating of TMEM16A is complex because it involves the interplay between increases in intracellular calcium concentration ([Ca(2+)]i), membrane depolarization, extracellular Cl(-) or permeant anions and intracellular protons. Our goal here was to understand how these variables regulate TMEM16A gating and to explain four observations. (a) TMEM16A is activated by voltage in the absence of intracellular Ca(2+). (b) The Cl(-) conductance is decreased after reducing extracellular Cl(-) concentration ([Cl(-)]o). (c) ICl is regulated by physiological concentrations of [Cl(-)]o. (d) In cells dialyzed with 0.2 μM [Ca(2+)]i, Cl(-) has a bimodal effect: at [Cl(-)]o <30 mM TMEM16A current activates with a monoexponential time course, but above 30 mM, [Cl(-)]o ICl activation displays fast and slow kinetics. To explain the contribution of Vm, Ca(2+) and Cl(-) to gating, we developed a 12-state Markov chain model. This model explains TMEM16A activation as a sequential, direct, and Vm-dependent binding of two Ca(2+) ions coupled to a Vm-dependent binding of an external Cl(-) ion, with Vm-dependent transitions between states. Our model predicts that extracellular Cl(-) does not alter the apparent Ca(2+) affinity of TMEM16A, which we corroborated experimentally. Rather, extracellular Cl(-) acts by stabilizing the open configuration induced by Ca(2+) and by contributing to the Vm dependence of activation. PMID:27138167

  5. Revealing the activation pathway for TMEM16A chloride channels from macroscopic currents and kinetic models.

    PubMed

    Contreras-Vite, Juan A; Cruz-Rangel, Silvia; De Jesús-Pérez, José J; Figueroa, Iván A Aréchiga; Rodríguez-Menchaca, Aldo A; Pérez-Cornejo, Patricia; Hartzell, H Criss; Arreola, Jorge

    2016-07-01

    TMEM16A (ANO1), the pore-forming subunit of calcium-activated chloride channels, regulates several physiological and pathophysiological processes such as smooth muscle contraction, cardiac and neuronal excitability, salivary secretion, tumour growth and cancer progression. Gating of TMEM16A is complex because it involves the interplay between increases in intracellular calcium concentration ([Ca(2+)]i), membrane depolarization, extracellular Cl(-) or permeant anions and intracellular protons. Our goal here was to understand how these variables regulate TMEM16A gating and to explain four observations. (a) TMEM16A is activated by voltage in the absence of intracellular Ca(2+). (b) The Cl(-) conductance is decreased after reducing extracellular Cl(-) concentration ([Cl(-)]o). (c) ICl is regulated by physiological concentrations of [Cl(-)]o. (d) In cells dialyzed with 0.2 μM [Ca(2+)]i, Cl(-) has a bimodal effect: at [Cl(-)]o <30 mM TMEM16A current activates with a monoexponential time course, but above 30 mM, [Cl(-)]o ICl activation displays fast and slow kinetics. To explain the contribution of Vm, Ca(2+) and Cl(-) to gating, we developed a 12-state Markov chain model. This model explains TMEM16A activation as a sequential, direct, and Vm-dependent binding of two Ca(2+) ions coupled to a Vm-dependent binding of an external Cl(-) ion, with Vm-dependent transitions between states. Our model predicts that extracellular Cl(-) does not alter the apparent Ca(2+) affinity of TMEM16A, which we corroborated experimentally. Rather, extracellular Cl(-) acts by stabilizing the open configuration induced by Ca(2+) and by contributing to the Vm dependence of activation.

  6. Nanovehicular Intracellular Delivery Systems

    PubMed Central

    PROKOP, ALES; DAVIDSON, JEFFREY M.

    2013-01-01

    This article provides an overview of principles and barriers relevant to intracellular drug and gene transport, accumulation and retention (collectively called as drug delivery) by means of nanovehicles (NV). The aim is to deliver a cargo to a particular intracellular site, if possible, to exert a local action. Some of the principles discussed in this article apply to noncolloidal drugs that are not permeable to the plasma membrane or to the blood–brain barrier. NV are defined as a wide range of nanosized particles leading to colloidal objects which are capable of entering cells and tissues and delivering a cargo intracelullarly. Different localization and targeting means are discussed. Limited discussion on pharmacokinetics and pharmacodynamics is also presented. NVs are contrasted to micro-delivery and current nanotechnologies which are already in commercial use. Newer developments in NV technologies are outlined and future applications are stressed. We also briefly review the existing modeling tools and approaches to quantitatively describe the behavior of targeted NV within the vascular and tumor compartments, an area of particular importance. While we list “elementary” phenomena related to different level of complexity of delivery to cancer, we also stress importance of multi-scale modeling and bottom-up systems biology approach. PMID:18200527

  7. Analysis of the Proteome of Intracellular Shigella flexneri Reveals Pathways Important for Intracellular Growth

    PubMed Central

    Pieper, Rembert; Fisher, C. R.; Suh, Moo-Jin; Huang, S.-T.; Parmar, P.

    2013-01-01

    Global proteomic analysis was performed with Shigella flexneri strain 2457T in association with three distinct growth environments: S. flexneri growing in broth (in vitro), S. flexneri growing within epithelial cell cytoplasm (intracellular), and S. flexneri that were cultured with, but did not invade, Henle cells (extracellular). Compared to in vitro and extracellular bacteria, intracellular bacteria had increased levels of proteins required for invasion and cell-to-cell spread, including Ipa, Mxi, and Ics proteins. Changes in metabolic pathways in response to the intracellular environment also were evident. There was an increase in glycogen biosynthesis enzymes, altered expression of sugar transporters, and a reduced amount of the carbon storage regulator CsrA. Mixed acid fermentation enzymes were highly expressed intracellularly, while tricarboxylic acid (TCA) cycle oxidoreductive enzymes and most electron transport chain proteins, except CydAB, were markedly decreased. This suggested that fermentation and the CydAB system primarily sustain energy generation intracellularly. Elevated levels of PntAB, which is responsible for NADPH regeneration, suggested a shortage of reducing factors for ATP synthesis. These metabolic changes likely reflect changes in available carbon sources, oxygen levels, and iron availability. Intracellular bacteria showed strong evidence of iron starvation. Iron acquisition systems (Iut, Sit, FhuA, and Feo) and the iron starvation, stress-associated Fe-S cluster assembly (Suf) protein were markedly increased in abundance. Mutational analysis confirmed that the mixed-acid fermentation pathway was required for wild-type intracellular growth and spread of S. flexneri. Thus, iron stress and changes in carbon metabolism may be key factors in the S. flexneri transition from the extra- to the intracellular milieu. PMID:24101689

  8. Capsaicin induces NKCC1 internalization and inhibits chloride secretion in colonic epithelial cells independently of TRPV1.

    PubMed

    Bouyer, Patrice G; Tang, Xu; Weber, Christopher R; Shen, Le; Turner, Jerrold R; Matthews, Jeffrey B

    2013-01-15

    Colonic chloride secretion is regulated via the neurohormonal and immune systems. Exogenous chemicals (e.g., butyrate, propionate) can affect chloride secretion. Capsaicin, the pungent ingredient of the chili peppers, exerts various effects on gastrointestinal function. Capsaicin is known to activate the transient receptor potential vanilloid type 1 (TRPV1), expressed in the mesenteric nervous system. Recent studies have also demonstrated its presence in epithelial cells but its role remains uncertain. Because capsaicin has been reported to inhibit colonic chloride secretion, we tested whether this effect of capsaicin could occur by direct action on epithelial cells. In mouse colon and model T84 human colonic epithelial cells, we found that capsaicin inhibited forskolin-dependent short-circuit current (FSK-I(sc)). Using PCR and Western blot, we demonstrated the presence of TRPV1 in colonic epithelial cells. In T84 cells, TRPV1 localized at the basolateral membrane and in vesicular compartments. In permeabilized monolayers, capsaicin activated apical chloride conductance, had no effect on basolateral potassium conductance, but induced NKCC1 internalization demonstrated by immunocytochemistry and basolateral surface biotinylation. AMG-9810, a potent inhibitor of TRPV1, did not prevent the inhibition of the FSK-I(sc) by capsaicin. Neither resiniferatoxin nor N-oleoyldopamine, two selective agonists of TRPV1, blocked the FSK-I(sc). Conversely capsaicin, resiniferatoxin, and N-oleoyldopamine raised intracellular calcium ([Ca(2+)](i)) in T84 cells and AMG-9810 blocked the rise in [Ca(2+)](i) induced by capsaicin and resiniferatoxin suggesting the presence of a functional TRPV1 channel. We conclude that capsaicin inhibits chloride secretion in part by causing NKCC1 internalization, but by a mechanism that appears to be independent of TRPV1. PMID:23139219

  9. Oscillatory Chloride Efflux at the Pollen Tube Apex Has a Role in Growth and Cell Volume Regulation and Is Targeted by Inositol 3,4,5,6-Tetrakisphosphate

    PubMed Central

    Zonia, Laura; Cordeiro, Sofia; Tupý, Jaroslav; Feijó, José A.

    2002-01-01

    overlaps the phase of cell elongation during the growth cycle. In summary, these investigations indicate that Cl− ion dynamics are an important component in the network of events that regulate pollen tube homeostasis and growth. PMID:6457666

  10. Comment on "Local impermeant anions establish the neuronal chloride concentration".

    PubMed

    Luhmann, Heiko J; Kirischuk, Sergei; Kilb, Werner

    2014-09-01

    Glykys et al. (Reports, 7 February 2014, p. 670) proposed that cytoplasmic impermeant anions and polyanionic extracellular matrix glycoproteins establish the local neuronal intracellular chloride concentration, [Cl(-)]i, and thereby the polarity of γ-aminobutyric acid type A (GABAA) receptor signaling. The experimental procedures and results in this study are insufficient to support these conclusions. Contradictory results previously published by these authors and other laboratories are not referred to.

  11. 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

  12. Decreased chloride channel expression in the dorsolateral prefrontal cortex in schizophrenia.

    PubMed

    Sullivan, Courtney R; Funk, Adam J; Shan, Dan; Haroutunian, Vahram; McCullumsmith, Robert E

    2015-01-01

    Alterations in GABAergic neurotransmission are implicated in several psychiatric illnesses, including schizophrenia. The Na-K-Cl and K-Cl cotransporters regulate intracellular chloride levels. Abnormalities in cotransporter expression levels could shift the chloride electrochemical gradient and impair GABAergic transmission. In this study, we performed Western blot analysis to investigate whether the Na-K-Cl and K-Cl cotransporter protein is abnormally expressed in the dorsal lateral prefrontal cortex and the anterior cingulate cortex in patients with schizophrenia versus a control group. We found decreased K-Cl cotransporter protein expression in the dorsal lateral prefrontal cortex, but not the anterior cingulate cortex, in subjects with schizophrenia, supporting the hypothesis of region level abnormal GABAergic function in the pathophysiology of schizophrenia. Subjects with schizophrenia off antipsychotic medication at the time of death had decreased K-Cl cotransporter protein expression compared to both normal controls and subjects with schizophrenia on antipsychotics. Our results provide evidence for KCC2 protein abnormalities in schizophrenia and suggest that antipsychotic medications might reverse deficits of this protein in the illness. PMID:25826365

  13. Glucose stimulates calcium-activated chloride secretion in small intestinal cells.

    PubMed

    Yin, Liangjie; Vijaygopal, Pooja; MacGregor, Gordon G; Menon, Rejeesh; Ranganathan, Perungavur; Prabhakaran, Sreekala; Zhang, Lurong; Zhang, Mei; Binder, Henry J; Okunieff, Paul; Vidyasagar, Sadasivan

    2014-04-01

    The sodium-coupled glucose transporter-1 (SGLT1)-based oral rehydration solution (ORS) used in the management of acute diarrhea does not substantially reduce stool output, despite the fact that glucose stimulates the absorption of sodium and water. To explain this phenomenon, we investigated the possibility that glucose might also stimulate anion secretion. Transepithelial electrical measurements and isotope flux measurements in Ussing chambers were used to study the effect of glucose on active chloride and fluid secretion in mouse small intestinal cells and human Caco-2 cells. Confocal fluorescence laser microscopy and immunohistochemistry measured intracellular changes in calcium, sodium-glucose linked transporter, and calcium-activated chloride channel (anoctamin 1) expression. In addition to enhancing active sodium absorption, glucose increased intracellular calcium and stimulated electrogenic chloride secretion. Calcium imaging studies showed increased intracellular calcium when intestinal cells were exposed to glucose. Niflumic acid, but not glibenclamide, inhibited glucose-stimulated chloride secretion in mouse small intestines and in Caco-2 cells. Glucose-stimulated chloride secretion was not seen in ileal tissues incubated with the intracellular calcium chelater BAPTA-AM and the sodium-potassium-2 chloride cotransporter 1 (NKCC1) blocker bumetanide. These observations establish that glucose not only stimulates active Na absorption, a well-established phenomenon, but also induces a Ca-activated chloride secretion. This may explain the failure of glucose-based ORS to markedly reduce stool output in acute diarrhea. These results have immediate potential to improve the treatment outcomes for acute and/or chronic diarrheal diseases by replacing glucose with compounds that do not stimulate chloride secretion.

  14. Intracellular pH in sperm physiology.

    PubMed

    Nishigaki, Takuya; José, Omar; González-Cota, Ana Laura; Romero, Francisco; Treviño, Claudia L; Darszon, Alberto

    2014-08-01

    Intracellular pH (pHi) regulation is essential for cell function. Notably, several unique sperm ion transporters and enzymes whose elimination causes infertility are either pHi dependent or somehow related to pHi regulation. Amongst them are: CatSper, a Ca(2+) channel; Slo3, a K(+) channel; the sperm-specific Na(+)/H(+) exchanger and the soluble adenylyl cyclase. It is thus clear that pHi regulation is of the utmost importance for sperm physiology. This review briefly summarizes the key components involved in pHi regulation, their characteristics and participation in fundamental sperm functions such as motility, maturation and the acrosome reaction. PMID:24887564

  15. Intracellular pH in Sperm Physiology

    PubMed Central

    Nishigaki, Takuya; José, Omar; González-Cota, Ana Laura; Romero, Francisco; Treviño, Claudia L.; Darszon, Alberto

    2014-01-01

    Intracellular pH (pHi) regulation is essential for cell function. Notably, several unique sperm ion transporters and enzymes whose elimination causes infertility are either pHi dependent or somehow related to pHi regulation. Amongst them are: CatSper, a Ca2+ channel; Slo3, a K+ channel; the sperm-specific Na+/H+ exchanger and the soluble adenylyl cyclase. It is thus clear that pHi regulation is of the utmost importance for sperm physiology. This review briefly summarizes the key components involved in pHi regulation, their characteristics and participation in fundamental sperm functions such as motility, maturation and the acrosome reaction. PMID:24887564

  16. Mechanisms of intracellular ice formation.

    PubMed Central

    Muldrew, K; McGann, L E

    1990-01-01

    The phenomenon of intracellular freezing in cells was investigated by designing experiments with cultured mouse fibroblasts on a cryomicroscope to critically assess the current hypotheses describing the genesis of intracellular ice: (a) intracellular freezing is a result of critical undercooling; (b) the cytoplasm is nucleated through aqueous pores in the plasma membrane; and (c) intracellular freezing is a result of membrane damage caused by electrical transients at the ice interface. The experimental data did not support any of these theories, but was consistent with the hypothesis that the plasma membrane is damaged at a critical gradient in osmotic pressure across the membrane, and intracellular freezing occurs as a result of this damage. An implication of this hypothesis is that mathematical models can be used to design protocols to avoid damaging gradients in osmotic pressure, allowing new approaches to the preservation of cells, tissues, and organs by rapid cooling. PMID:2306499

  17. Fungal colonization with Pneumocystis correlates to increasing chloride channel accessory 1 (hCLCA1) suggesting a pathway for up-regulation of airway mucus responses, in infant lungs

    PubMed Central

    Pérez, Francisco J.; Ponce, Carolina A.; Rojas, Diego A.; Iturra, Pablo A.; Bustamante, Rebeca I.; Gallo, Myriam; Hananias, Karime; Vargas, Sergio L.

    2014-01-01

    Fungal colonization with Pneumocystis is associated with increased airway mucus in infants during their primary Pneumocystis infection, and to severity of COPD in adults. The pathogenic mechanisms are under investigation. Interestingly, increased levels of hCLCA1 – a member of the calcium-sensitive chloride conductance family of proteins that drives mucus hypersecretion – have been associated with increased mucus production in patients diagnosed with COPD and in immunocompetent rodents with Pneumocystis infection. Pneumocystis is highly prevalent in infants; therefore, the contribution of Pneumocystis to hCLCA1 expression was examined in autopsied infant lungs. Respiratory viruses that may potentially increase mucus, were also examined. hCLCA1 expression was measured using actin-normalized Western-blot, and the burden of Pneumocystis organisms was quantified by qPCR in 55 autopsied lungs from apparently healthy infants who died in the community. Respiratory viruses were diagnosed using RT-PCR for RSV, metapneumovirus, influenza, and parainfluenza viruses; and by PCR for adenovirus. hCLCA1 levels in virus positive samples were comparable to those in virus-negative samples. An association between Pneumocystis and increased hCLCA1 expression was documented (P=0.028). Additionally, increasing Pneumocystis burden correlated with increasing hCLCA1 protein expression levels (P=0.017). Results strengthen the evidence of Pneumocystis-associated up-regulation of mucus-related airway responses in infant lungs. Further characterization of this immunocompetent host-Pneumocystis-interaction, including assessment of potential clinical significance, is warranted. PMID:25379375

  18. Imaging and controlling intracellular reactions: Lysosome transport as a function of diameter and the intracellular synthesis of conducting polymers

    NASA Astrophysics Data System (ADS)

    Payne, Christine

    2014-03-01

    Eukaryotic cells are the ultimate complex environment with intracellular chemical reactions regulated by the local cellular environment. For example, reactants are sequestered into specific organelles to control local concentration and pH, motor proteins transport reactants within the cell, and intracellular vesicles undergo fusion to bring reactants together. Current research in the Payne Lab in the School of Chemistry and Biochemistry at Georgia Tech is aimed at understanding and utilizing this complex environment to control intracellular chemical reactions. This will be illustrated using two examples, intracellular transport as a function of organelle diameter and the intracellular synthesis of conducting polymers. Using single particle tracking fluorescence microscopy, we measured the intracellular transport of lysosomes, membrane-bound organelles, as a function of diameter as they underwent transport in living cells. Both ATP-dependent active transport and diffusion were examined. As expected, diffusion scales with the diameter of the lysosome. However, active transport is unaffected suggesting that motor proteins are insensitive to cytosolic drag. In a second example, we utilize intracellular complexity, specifically the distinct micro-environments of different organelles, to carry out chemical reactions. We show that catalase, found in the peroxisomes of cells, can be used to catalyze the polymerization of the conducting polymer PEDOT:PSS. More importantly, we have found that a range of iron-containing biomolecules are suitable catalysts with different iron-containing biomolecules leading to different polymer properties. These experiments illustrate the advantage of intracellular complexity for the synthesis of novel materials.

  19. Dynamic [Cl-]i measurement with chloride sensing quantum dots nanosensor in epithelial cells

    NASA Astrophysics Data System (ADS)

    Wang, Yuchi; Mao, Hua; Wong, Lid B.

    2010-02-01

    We have synthesized a chloride sensing quantum dots (QD) nanosensor, Cl-QD, for the dynamic measurements of chloride ion concentration in the millimolar range, a sensitivity that is applicable to most physiological intracellular chloride ion concentration ([Cl-]i) measurements in epithelial cells. The Cl-QD is synthesized by conjugating an anion receptor, 1-(2-mercapto-ethyl)-3-phenyl-thiourea (MEPTU) to a water soluble CdSe/ZnS QD at an emission wavelength of 620 nm. Upon binding of chloride ions to the Cl-QD, a photo-induced electron transfer mechanism caused the fluorescence of the QD to quench. This resulted in an inversely proportional relationship between the chloride ion concentration and the fluorescence intensity of the Cl-QD. We have utilized this Cl-QD to measure [Cl-]i in T84 and CF-PAC cultured cells, with either the C1C-2 or CFTR chloride channels being manipulated by pharmacological chloride channel activators and inhibitors. Activations of C1C-2 and CFTR chloride channels in T84 by the respective lubiprostone and genistein caused predictive increases in the fluorescence of the Cl-QD, i.e., a decrease of [Cl-]i. Conversely, glibenclamide, a chloride channel inhibitor, applied to the CF-PAC cells caused a predictable decrease in the fluorescence of Cl-QD due to the increase of [Cl-]i. These are the first data in using QD-based chloride ion sensors for dynamic measurements of intracellular chloride ion concentrations in epithelial cells.

  20. Transport of butyrate across the isolated bovine rumen epithelium--interaction with sodium, chloride and bicarbonate.

    PubMed

    Sehested, J; Diernaes, L; Møller, P D; Skadhauge, E

    1999-08-01

    The Ussing chamber technique was used for studying unidirectional fluxes of 14C-butyrate across the bovine rumen epithelium in vitro. Significant amounts of butyrate were absorbed across the bovine rumen epithelium in vitro, without any external driving force. The paracellular pathway was quantitatively insignificant. The transcellular pathway was predominately voltage-insensitive. The serosal to mucosal (SM) pathway was regulated by mass action, whereas the mucosal to serosal (MS) pathway further includes a saturable process, which accounted for 30 to 55% of the MS flux. The studied transport process for 14C-butyrate across the epithelium could include metabolic processes and transport of 14C-labelled butyrate metabolites. The transport of butyrate interacted with Na+, Cl- and HCO3-, and there was a linear relationship between butyrate and sodium net transport. Lowering the sodium concentration from 140 to 10 mmol l-1 decreased the butyrate MS flux significantly. Amiloride (1 mmol l-1) did, however, not reduce the butyrate flux significantly. Chloride concentration in itself did not seem to influence the transport of butyrate, but chloride-free conditions tended to increase the MS and SM flux of butyrate by a DIDS-sensitive pathway. DIDS (bilateral 0.5 mmol l-1) did further decrease the butyrate SM flux significantly at all chloride concentrations. Removing bicarbonate from the experimental solutions decreased the MS and increased the SM flux of butyrate significantly, and abolished net butyrate flux. There were no significant effects of the carbonic anhydrase inhibitor Acetazolamide (bilateral 1.0 mmol l-1). The results can be explained by a model where butyrate and butyrate metabolites are transported both by passive diffusion and by an electroneutral anion-exchange with bicarbonate. The model couples sodium and butyrate via CO2 from metabolism of butyrate, and intracellular pH.

  1. An Apical-Membrane Chloride Channel in Human Tracheal Epithelium

    NASA Astrophysics Data System (ADS)

    Welsh, Michael J.

    1986-06-01

    The mechanism of chloride transport by airway epithelia has been of substantial interest because airway and sweat gland-duct epithelia are chloride-impermeable in cystic fibrosis. The decreased chloride permeability prevents normal secretion by the airway epithelium, thereby interfering with mucociliary clearance and contributing to the morbidity and mortality of the disease. Because chloride secretion depends on and is regulated by chloride conductance in the apical cell membrane, the patch-clamp technique was used to directly examine single-channel currents in primary cultures of human tracheal epithelium. The cells contained an anion-selective channel that was not strongly voltage-gated or regulated by calcium in cell-free patches. The channel was also blocked by analogs of carboxylic acid that decrease apical chloride conductance in intact epithelia. When attached to the cell, the channel was activated by isoproterenol, although the channel was also observed to open spontaneously. However, in some cases, the channel was only observed after the patch was excised from the cell. These results suggest that this channel is responsible for the apical chloride conductance in airway epithelia.

  2. Electrochemical Visualization of Intracellular Hydrogen Peroxide at Single Cells.

    PubMed

    He, Ruiqin; Tang, Huifen; Jiang, Dechen; Chen, Hong-yuan

    2016-02-16

    In this Letter, the electrochemical visualization of hydrogen peroxide inside one cell was achieved first using a comprehensive Au-luminol-microelectrode and electrochemiluminescence. The capillary with a tip opening of 1-2 μm was filled with the mixture of chitosan and luminol, which was coated with the thin layers of polyvinyl chloride/nitrophenyloctyl ether (PVC/NPOE) and gold as the microelectrode. Upon contact with the aqueous hydrogen peroxide, hydrogen peroxide and luminol in contact with the gold layer were oxidized under the positive potential resulting in luminescence for the imaging. Due to the small diameter of the electrode, the microelectrode tip was inserted into one cell and the bright luminescence observed at the tip confirmed the visualization of intracellular hydrogen peroxide. The further coupling of oxidase on the electrode surface could open the field in the electrochemical imaging of intracellular biomolecules at single cells, which benefited the single cell electrochemical detection. PMID:26879364

  3. Electrochemical Visualization of Intracellular Hydrogen Peroxide at Single Cells.

    PubMed

    He, Ruiqin; Tang, Huifen; Jiang, Dechen; Chen, Hong-yuan

    2016-02-16

    In this Letter, the electrochemical visualization of hydrogen peroxide inside one cell was achieved first using a comprehensive Au-luminol-microelectrode and electrochemiluminescence. The capillary with a tip opening of 1-2 μm was filled with the mixture of chitosan and luminol, which was coated with the thin layers of polyvinyl chloride/nitrophenyloctyl ether (PVC/NPOE) and gold as the microelectrode. Upon contact with the aqueous hydrogen peroxide, hydrogen peroxide and luminol in contact with the gold layer were oxidized under the positive potential resulting in luminescence for the imaging. Due to the small diameter of the electrode, the microelectrode tip was inserted into one cell and the bright luminescence observed at the tip confirmed the visualization of intracellular hydrogen peroxide. The further coupling of oxidase on the electrode surface could open the field in the electrochemical imaging of intracellular biomolecules at single cells, which benefited the single cell electrochemical detection.

  4. 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...

  5. Unique chloride-sensing properties of WNK4 permit the distal nephron to modulate potassium homeostasis

    PubMed Central

    Terker, Andrew S.; Zhang, Chong; Erspamer, Kayla J.; Gamba, Gerardo; Yang, Chao-Ling; Ellison, David H.

    2015-01-01

    Dietary potassium deficiency activates thiazide-sensitive sodium chloride cotransport along the distal nephron. This may explain, in part, the hypertension and cardiovascular mortality observed in individuals who consume a low potassium diet. Recent data suggest plasma potassium affects the distal nephron directly by influencing intracellular chloride, an inhibitor of the With no lysine kinase (WNK)-Ste20p-related proline-and alanine-rich kinase (SPAK) pathway. Since previous studies used extreme dietary manipulations, we sought to determine if the relationship between potassium and NCC is physiologically relevant and clarify the mechanisms involved. We report that modest changes in both dietary and plasma potassium affect the thiazide-sensitive sodium-chloride cotransporter, NCC, in vivo. Kinase assay studies showed that chloride inhibits WNK4 kinase activity at lower concentrations than it inhibits activity of WNK1 or WNK3. Also, chloride inhibited WNK4 within the range of distal cell chloride. Mutation of a previously identified WNK chloride-binding motif converted WNK4 effects on SPAK from inhibitory to stimulatory in mammalian cells. Disruption of this motif in WNKs 1, 3 and 4 had different effects on NCC, consistent with the three WNKs having different chloride sensitivities. Thus, potassium effects on NCC are graded within the physiological range, which explains how unique chloride-sensing properties of WNK4 enable kinase mediating effects of potassium on NCC in vivo. PMID:26422504

  6. Are extracellular osmolality and sodium concentration determined by Donnan effects of intracellular protein charges and of pumped sodium?

    PubMed

    Kurbel, Sven

    2008-06-21

    Although we are used to attribute almost identical extracellular fluid (ECF) sodium concentrations in birds, amphibians, reptiles, and mammals to the composition of the primordial oceans in which, presumably, all life originated, this interpretation is not supported by geological data suggesting that the ocean salinity was never much lower than the present-day values, still four times higher than our plasma sodium. Here presented interpretation is that the similar ECF salt concentrations are dictated by the opposed Donnan effects on the cell membrane. The only way for the cell to reach the osmotic equilibrium is to alter cell volume, until concentration of nondiffusible intracellular ions (mainly charges on intracellular proteins) is equal to the ECF restricted ions (mainly Na+ ions, restricted by pumping out of cells). The achievement of electroneutrality requires that the sum of all anions equals concentration of positive ions in the cell (mainly K+). Negative charges on cytoplasmic proteins are the most stable component among ionized particles and other ions have to adapt to their concentration. Positive and negative soluble intracellular ions are all osmotically active and to achieve balance of osmotic forces on the cell membrane, the sum of their intracellular concentrations must equal the concentration of osmotically active extracellular particles. Since almost half the osmotically active ECF particles are sodium ions, the ECF sodium concentration seems related to concentration of charges on cytoplasmic proteins and concentration of intracellular phosphates. Our ancestors could not leave the salty ocean and move to brackish, or even fresh waters, without adequate regulation of their ECF sodium concentration and osmolality. Concentration of charges on cytoplasmic proteins or of intracellular phosphate buffers could not be altered, since this would compromise cell functioning. The remaining solution was to maintain the lowest ECF Na+ concentration effective in

  7. Chlormequat chloride retards rat embryo growth in vitro.

    PubMed

    Xiagedeer, Bayindala; Wu, Shuang; Liu, Yingjuan; Hao, Weidong

    2016-08-01

    Chlormequat chloride is the most widely used plant growth regulator in agriculture to promote sturdier growth of grain crops by avoidance of lodging. Therefore, human exposure to chlormequat chloride is very common, but its developmental toxicity has not been studied. Thus, we investigated the developmental toxicity of chlormequat chloride by applying rat whole embryo culture (WEC) model, limb bud micromass culture and 3T3 fibroblast cytotoxicity test. Chlormequat chloride at 150μg/ml (0.93mM) retarded the rat embryo growth without causing significant morphological malformations and at 500μg/ml (3.1mM) caused both retardation and morphological malformation of the embryos. However, the proliferation and differentiation of limb bud cells were not affected by chlormequat chloride at as high as up to 1000μg/ml (6.2mM) applied. This concentration of chlormequat chloride did not affect the cell viability as examined by 3T3 fibroblast cytotoxicity test either, suggesting that cellular toxicity may not play a role in chlormequat induced inhibition of rat embryo growth. Collectively, our results demonstrated that chlormequat chloride may affect embryo growth and development without inhibiting cell viability.

  8. 21 CFR 184.1297 - Ferric chloride.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... hydrogen chloride. The pure material occurs as hydroscopic, hexagonal, dark crystals. Ferric chloride... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Ferric chloride. 184.1297 Section 184.1297 Food... Specific Substances Affirmed as GRAS § 184.1297 Ferric chloride. (a) Ferric chloride (iron (III)...

  9. 21 CFR 184.1297 - Ferric chloride.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... GRAS § 184.1297 Ferric chloride. (a) Ferric chloride (iron (III) chloride, FeC13, CAS Reg. No. 7705-08-0) may be prepared from iron and chlorine or from ferric oxide and hydrogen chloride. The pure... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Ferric chloride. 184.1297 Section 184.1297...

  10. 21 CFR 184.1297 - Ferric chloride.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... hydrogen chloride. The pure material occurs as hydroscopic, hexagonal, dark crystals. Ferric chloride... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Ferric chloride. 184.1297 Section 184.1297 Food... Specific Substances Affirmed as GRAS § 184.1297 Ferric chloride. (a) Ferric chloride (iron (III)...

  11. 21 CFR 184.1297 - Ferric chloride.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... hydrogen chloride. The pure material occurs as hydroscopic, hexagonal, dark crystals. Ferric chloride... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Ferric chloride. 184.1297 Section 184.1297 Food... Specific Substances Affirmed as GRAS § 184.1297 Ferric chloride. (a) Ferric chloride (iron (III)...

  12. Benzalkonium Chloride and Glaucoma

    PubMed Central

    Kaufman, Paul L.; Kiland, Julie A.

    2014-01-01

    Abstract Glaucoma patients routinely take multiple medications, with multiple daily doses, for years or even decades. Benzalkonium chloride (BAK) is the most common preservative in glaucoma medications. BAK has been detected in the trabecular meshwork (TM), corneal endothelium, lens, and retina after topical drop installation and may accumulate in those tissues. There is evidence that BAK causes corneal and conjunctival toxicity, including cell loss, disruption of tight junctions, apoptosis and preapoptosis, cytoskeleton changes, and immunoinflammatory reactions. These same effects have been reported in cultured human TM cells exposed to concentrations of BAK found in common glaucoma drugs and in the TM of primary open-angle glaucoma donor eyes. It is possible that a relationship exists between chronic exposure to BAK and glaucoma. The hypothesis that BAK causes/worsens glaucoma is being tested experimentally in an animal model that closely reflects human physiology. PMID:24205938

  13. Temperature dependence of chloride, bromide, iodide, thiocyanate and salicylate transport in human red cells

    PubMed Central

    Dalmark, Mads; Wieth, Jens Otto

    1972-01-01

    1. The temperature dependence of the steady-state self-exchange of chloride between human red cells and a plasma-like electrolyte medium has been studied by measuring the rate of 36Cl- efflux from radioactively labelled cells. Between 0 and 10° C the rate increased by a factor of eight corresponding to an Arrhenius activation energy of 33 kcal/mole. 2. The rate of chloride exchange decreased significantly in experiments where 95% of the chloride ions in cells and medium were replaced by other monovalent anions of a lyotropic series. The rate of chloride self-exchange was increasingly reduced by bromide, bicarbonate, nitrate, iodide, thiocyanate, and salicylate. The latter aromatic anion was by far the most potent inhibitor, reducing the rate of chloride self-exchange to 0·2% of the value found in a chloride medium. 3. The temperature sensitivity of the chloride self-exchange was not affected significantly by the anionic inhibitors. The Arrhenius activation energies of chloride exchange were between 30 and 40 kcal/mole in the presence of the six inhibitory anions mentioned above. 4. The rate of self-exchange of bromide, thiocyanate, and iodide between human red cells and media was determined after washing and labelling cells in media containing 120 mM bromide, thiocyanate, or iodide respectively. The rate of self-exchange of the three anions were 12, 3, and 0·4% of the rate of chloride self-exchange found in the chloride medium. 5. The Arrhenius activation energies of the self-exchange of bromide, iodide, and thiocyanate were all between 29 and 37 kcal/mole, the same magnitude as found for the self-exchange of chloride. 6. Although approximately 40% of the intracellular iodide and salicylate ions appeared to be adsorbed to intracellular proteins, the rate of tracer anion efflux followed first order kinetics until at least 98% of the intracellular anions had been exchanged. 7. The self-exchange of salicylate across the human red cell membrane occurred by a

  14. Chloride Channel 3 Channels in the Activation and Migration of Human Blood Eosinophils in Allergic Asthma

    PubMed Central

    Gaurav, Rohit; Bewtra, Againdra K.

    2015-01-01

    Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is responsible for respiratory burst in immune cells. Chloride channel 3 (CLC3) has been linked to the respiratory burst in eosinophils and neutrophils. The effect of cytokines and the involvement of CLC3 in the regulation of NADPH-dependent oxidative stress and on cytokine-mediated migration of eosinophils are not known. Human peripheral blood eosinophils were isolated from healthy individuals and from individuals with asthma by negative selection. Real-time PCR was used to detect the expression of NADPH oxidases in eosinophils. Intracellular reactive oxygen species (ROS) measurement was done with flow cytometry. Superoxide generation was measured with transforming growth factor (TGF)-β, eotaxin, and CLC3 blockers. CLC3 dependence of eosinophils in TGF-β– and eotaxin-induced migration was also examined. The messenger RNA (mRNA) transcripts of NADPH oxidase (NOX) 2, dual oxidase (DUOX) 1, and DUOX2 were detected in blood eosinophils, with very low expression of NOX1, NOX3, and NOX5 and no NOX4 mRNA. The level of NOX2 mRNA transcripts increased with disease severity in the eosinophils of subjects with asthma compared with healthy nonatopic volunteers. Change in granularity and size in eosinophils, but no change in intracellular ROS, was observed with phorbol myristate acetate (PMA). PMA, TGF-β, and eotaxin used the CLC3-dependent pathway to increase superoxide radicals. TGF-β and eotaxin induced CLC3-dependent chemotaxis of eosinophils. These findings support the requirement of CLC3 in the activation and migration of human blood eosinophils and may provide a potential novel therapeutic target to regulate eosinophil hyperactivity in allergic airway inflammation in asthma. PMID:25514499

  15. Guanine nucleotide exchange factor 2 for Rab5 proteins coordinated with GLUP6/GEF regulates the intracellular transport of the proglutelin from the Golgi apparatus to the protein storage vacuole in rice endosperm

    PubMed Central

    Wen, Liuying; Fukuda, Masako; Sunada, Mariko; Ishino, Sonoko; Ishino, Yoshizumi; Okita, Thomas W.; Ogawa, Masahiro; Ueda, Takashi; Kumamaru, Toshihiro

    2015-01-01

    Rice glutelin polypeptides are initially synthesized on the endoplasmic reticulum (ER) membrane as a proglutelin, which are then transported to the protein storage vacuole (PSV) via the Golgi apparatus. Rab5 and its cognate activator guanine nucleotide exchange factor (GEF) are essential for the intracellular transport of proglutelin from the Golgi apparatus to the PSV. Results from previous studies showed that the double recessive type of glup4/rab5a and glup6/gef mutant accumulated much higher amounts of proglutelin than either parent line. The present study demonstrates that the double recessive type of glup4/rab5a and glup6/gef mutant showed not only elevated proglutelin levels and much larger paramural bodies but also reduced the number and size of PSVs, indicating a synergistic mutation effect. These observations led us to the hypothesis that other isoforms of Rab5 and GEF also participate in the intracellular transport of rice glutelin. A database search identified a novel guanine nucleotide exchange factor, Rab5-GEF2. Like GLUP6/GEF, Rab5-GEF2 was capable of activating Rab5a and two other Rab5 isoforms in in vitro GTP/GDP exchange assays. GEF proteins consist of the helical bundle (HB) domain at the N-terminus, Vps9 domain, and a C-terminal region. By the deletion analysis of GEFs, the HB domain was found essential for the activation of Rab5 proteins. PMID:26136263

  16. A tyrosine-based motif and a casein kinase II phosphorylation site regulate the intracellular trafficking of the varicella-zoster virus glycoprotein I, a protein localized in the trans-Golgi network.

    PubMed Central

    Alconada, A; Bauer, U; Hoflack, B

    1996-01-01

    We have studied the intracellular trafficking of the envelope glycoprotein I (gpI) of the varicella-zoster virus, a human herpes virus whose assembly is believed to occur in the trans-Golgi network (TGN) and/or in endocytic compartments. When expressed in HeLa cells in the absence of additional virally encoded factors, this type-I membrane protein localizes to the TGN and cycles between this compartment and the cell surface. The expression of gpI promotes the recruitment of the AP-1 Golgi-specific assembly proteins onto TGN membranes, strongly suggesting that gpI, like the mannose 6-phosphate receptors, can leave the TGN in clathrin-coated vesicles for subsequent transport to endosomes. Its return from the cell surface to the TGN also occurs through endosomes. The transfer of the gpI cytoplasmic domain onto a reporter molecule shows that this domain is sufficient to confer TGN localization. Mutational analysis of this domain indicates that proper subcellular localization and cycling of gpI depend on two different determinants, a tyrosine-containing tetrapeptide related to endocytosis sorting signals and a cluster of acidic amino acids containing casein kinase II phosphorylatable residues. Thus, the VZV gpI and the mannose 6-phosphate receptors, albeit localized in different intracellular compartments at steady-state, follow similar trafficking pathways and share similar sorting mechanisms. Images PMID:8947032

  17. Guanine nucleotide exchange factor 2 for Rab5 proteins coordinated with GLUP6/GEF regulates the intracellular transport of the proglutelin from the Golgi apparatus to the protein storage vacuole in rice endosperm.

    PubMed

    Wen, Liuying; Fukuda, Masako; Sunada, Mariko; Ishino, Sonoko; Ishino, Yoshizumi; Okita, Thomas W; Ogawa, Masahiro; Ueda, Takashi; Kumamaru, Toshihiro

    2015-10-01

    Rice glutelin polypeptides are initially synthesized on the endoplasmic reticulum (ER) membrane as a proglutelin, which are then transported to the protein storage vacuole (PSV) via the Golgi apparatus. Rab5 and its cognate activator guanine nucleotide exchange factor (GEF) are essential for the intracellular transport of proglutelin from the Golgi apparatus to the PSV. Results from previous studies showed that the double recessive type of glup4/rab5a and glup6/gef mutant accumulated much higher amounts of proglutelin than either parent line. The present study demonstrates that the double recessive type of glup4/rab5a and glup6/gef mutant showed not only elevated proglutelin levels and much larger paramural bodies but also reduced the number and size of PSVs, indicating a synergistic mutation effect. These observations led us to the hypothesis that other isoforms of Rab5 and GEF also participate in the intracellular transport of rice glutelin. A database search identified a novel guanine nucleotide exchange factor, Rab5-GEF2. Like GLUP6/GEF, Rab5-GEF2 was capable of activating Rab5a and two other Rab5 isoforms in in vitro GTP/GDP exchange assays. GEF proteins consist of the helical bundle (HB) domain at the N-terminus, Vps9 domain, and a C-terminal region. By the deletion analysis of GEFs, the HB domain was found essential for the activation of Rab5 proteins. PMID:26136263

  18. Reactor-chromatographic determination of vinyl chloride in polyvinyl chloride

    SciTech Connect

    Berezkin, V.G.

    1986-08-01

    The authors carry out a chromatographic study of the volatile products that evolve when various grades of domestic polyvinyl chloride are heated, to determine the concentration of residual monomer. To find vinyl chloride in complex mixtures of air pollutants the authors used sorptive reaction concentration of impurities. This new combination of methods is based on preliminary separation at the sampling stage of impurities that interfere in the analysis, followed by concentration of the desired components in a trap with an adsorbent, and chromatographic determination of the concentrated trace materials. The method obtains low vinyl chloride concentrations (down to 10/sup -4/-10/sup -5/ wt. %) with +/-5 relative error.

  19. The Contribution of Raised Intraneuronal Chloride to Epileptic Network Activity

    PubMed Central

    Alfonsa, Hannah; Merricks, Edward M.; Codadu, Neela K.; Cunningham, Mark O.; Deisseroth, Karl; Racca, Claudia

    2015-01-01

    Altered inhibitory function is an important facet of epileptic pathology. A key concept is that GABAergic activity can become excitatory if intraneuronal chloride rises. However, it has proved difficult to separate the role of raised chloride from other contributory factors in complex network phenomena, such as epileptic pathology. Therefore, we asked what patterns of activity are associated with chloride dysregulation by making novel use of Halorhodopsin to load clusters of mouse pyramidal cells artificially with Cl−. Brief (1–10 s) activation of Halorhodopsin caused substantial positive shifts in the GABAergic reversal potential that were proportional to the charge transfer during the illumination and in adult neocortical pyramidal neurons decayed with a time constant of τ = 8.0 ± 2.8s. At the network level, these positive shifts in EGABA produced a transient rise in network excitability, with many distinctive features of epileptic foci, including high-frequency oscillations with evidence of out-of-phase firing (Ibarz et al., 2010). We show how such firing patterns can arise from quite small shifts in the mean intracellular Cl− level, within heterogeneous neuronal populations. Notably, however, chloride loading by itself did not trigger full ictal events, even with additional electrical stimulation to the underlying white matter. In contrast, when performed in combination with low, subepileptic levels of 4-aminopyridine, Halorhodopsin activation rapidly induced full ictal activity. These results suggest that chloride loading has at most an adjunctive role in ictogenesis. Our simulations also show how chloride loading can affect the jitter of action potential timing associated with imminent recruitment to an ictal event (Netoff and Schiff, 2002). PMID:25995461

  20. Unique chloride-sensing properties of WNK4 permit the distal nephron to modulate potassium homeostasis.

    PubMed

    Terker, Andrew S; Zhang, Chong; Erspamer, Kayla J; Gamba, Gerardo; Yang, Chao-Ling; Ellison, David H

    2016-01-01

    Dietary potassium deficiency activates thiazide-sensitive sodium chloride cotransport along the distal nephron. This may explain, in part, the hypertension and cardiovascular mortality observed in individuals who consume a low-potassium diet. Recent data suggest that plasma potassium affects the distal nephron directly by influencing intracellular chloride, an inhibitor of the with-no-lysine kinase (WNK)-Ste20p-related proline- and alanine-rich kinase (SPAK) pathway. As previous studies used extreme dietary manipulations, we sought to determine whether the relationship between potassium and NaCl cotransporter (NCC) is physiologically relevant and clarify the mechanisms involved. We report that modest changes in both dietary and plasma potassium affect NCC in vivo. Kinase assay studies showed that chloride inhibits WNK4 kinase activity at lower concentrations than it inhibits activity of WNK1 or WNK3. Also, chloride inhibited WNK4 within the range of distal cell chloride concentration. Mutation of a previously identified WNK chloride-binding motif converted WNK4 effects on SPAK from inhibitory to stimulatory in mammalian cells. Disruption of this motif in WNKs 1, 3, and 4 had different effects on NCC, consistent with the three WNKs having different chloride sensitivities. Thus, potassium effects on NCC are graded within the physiological range, which explains how unique chloride-sensing properties of WNK4 enable it to mediate effects of potassium on NCC in vivo.

  1. Epithelial Cell Gene Expression Induced by Intracellular Staphylococcus aureus

    PubMed Central

    Li, Xianglu; Fusco, William G.; Seo, Keun S.; Bayles, Kenneth W.; Mosley, Erin E.; McGuire, Mark A.; Bohach, Gregory A.

    2009-01-01

    HEp-2 cell monolayers were cocultured with intracellular Staphylococcus aureus, and changes in gene expression were profiled using DNA microarrays. Intracellular S. aureus affected genes involved in cellular stress responses, signal transduction, inflammation, apoptosis, fibrosis, and cholesterol biosynthesis. Transcription of stress response and signal transduction-related genes including atf3, sgk, map2k1, map2k3, arhb, and arhe was increased. In addition, elevated transcription of proinflammatory genes was observed for tnfa, il1b, il6, il8, cxcl1, ccl20, cox2, and pai1. Genes involved in proapoptosis and fibrosis were also affected at transcriptional level by intracellular S. aureus. Notably, intracellular S. aureus induced strong transcriptional down-regulation of several cholesterol biosynthesis genes. These results suggest that epithelial cells respond to intracellular S. aureus by inducing genes affecting immunity and in repairing damage caused by the organism, and are consistent with the possibility that the organism exploits an intracellular environment to subvert host immunity and promote colonization. PMID:20016671

  2. Sirtuin inhibitor sirtinol is an intracellular iron chelator

    PubMed Central

    Gautam, R.; Akam, E. A.; Astashkin, A. V.; Loughrey, J. J.

    2015-01-01

    Sirtinol is a known inhibitor of sirtuin proteins, a family of deacetylases involved in the pathophysiology of aging. Spectroscopic and structural data reveal that this compound is also an iron chelator forming high-spin ferric species in vitro and in cultured leukemia cells. Interactions with the highly regulated iron pool therefore contribute to its overall intracellular agenda. PMID:25715179

  3. Inhibition of ANO1/TMEM16A Chloride Channel by Idebenone and Its Cytotoxicity to Cancer Cell Lines.

    PubMed

    Seo, Yohan; Park, Jinhong; Kim, Minseo; Lee, Ho K; Kim, Jin-Hee; Jeong, Jin-Hyun; Namkung, Wan

    2015-01-01

    The expression levels of anoctamin 1 (ANO1, TMEM16A), a calcium-activated chloride channel (CaCC), are significantly increased in several tumors, and inhibition of ANO1 is known to reduce cell proliferation and migration. Here, we performed cell-based screening of a collection of natural products and drug-like compounds to identify inhibitors of ANO1. As a result of the screening, idebenone, miconazole and plumbagin were identified as novel ANO1 inhibitors. Electrophysiological studies showed that idebenone, a synthetic analog of coenzyme Q10, completely blocked ANO1 activity in FRT cells expressing ANO1 without any effect on intracellular calcium signaling and CFTR, a cAMP-regulated chloride channel. The CaCC activities in PC-3 and CFPAC-1 cells expressing abundant endogenous ANO1 were strongly blocked by idebenone. Idebenone inhibited cell proliferation and induced apoptosis in PC-3 and CFPAC-1 cells, but not in A549 cells, which do not express ANO1. These data suggest that idebenone, a novel ANO1 inhibitor, has potential for use in cancer therapy. PMID:26196390

  4. Four basic residues critical for the ion selectivity and pore blocker sensitivity of TMEM16A calcium-activated chloride channels.

    PubMed

    Peters, Christian J; Yu, Haibo; Tien, Jason; Jan, Yuh Nung; Li, Min; Jan, Lily Yeh

    2015-03-17

    TMEM16A (transmembrane protein 16) (Anoctamin-1) forms a calcium-activated chloride channel (CaCC) that regulates a broad array of physiological properties in response to changes in intracellular calcium concentration. Although known to conduct anions according to the Eisenman type I selectivity sequence, the structural determinants of TMEM16A anion selectivity are not well-understood. Reasoning that the positive charges on basic residues are likely contributors to anion selectivity, we performed whole-cell recordings of mutants with alanine substitution for basic residues within the putative pore region and identified four residues on four different putative transmembrane segments that significantly increased the permeability of the larger halides and thiocyanate relative to that of chloride. Because TMEM16A permeation properties are known to shift with changes in intracellular calcium concentration, we further examined the calcium dependence of anion selectivity. We found that WT TMEM16A but not mutants with alanine substitution at those four basic residues exhibited a clear decline in the preference for larger anions as intracellular calcium was increased. Having implicated these residues as contributing to the TMEM16A pore, we scrutinized candidate small molecules from a high-throughput CaCC inhibitor screen to identify two compounds that act as pore blockers. Mutations of those four putative pore-lining basic residues significantly altered the IC50 of these compounds at positive voltages. These findings contribute to our understanding regarding anion permeation of TMEM16A CaCC and provide valuable pharmacological tools to probe the channel pore.

  5. The effect of intracellular pH on the regulation of the Rab 16A and the alpha-amylase 1/6-4 promoter by abscisic acid and gibberellia.

    PubMed

    Heimovaara-Dijkstra, S; Mundy, J; Wang, M

    1995-02-01

    Intracellular pH (pHi) of barley aleurone cells is known to be affected by hormones and plant growth conditions. The possible mechanisms by which these pHi shifts influence the actions of abscisic acid (ABA) or gibberellin (GA) is being investigated. Here we report an attempt to study the effect of pHi on hormone-induced gene expression. We used weak acids and weak bases to artificially mimic the pHi changes brought about by ABA and GA and found that chloramphenicol acetyltransferase (CAT) expression controlled by the Rab promoter was affected while the alpha-amylase promoter seemed insensitive. CAT fused to the 35S promoter was used as a control which is not inducible by ABA or GA3. The expression of this construct was not significantly affected by artificial pHi changes.

  6. Rate-dependent force, intracellular calcium, and action potential voltage alternans are modulated by sarcomere length and heart failure induced-remodeling of thin filament regulation in human heart failure: A myocyte modeling study.

    PubMed

    Zile, Melanie A; Trayanova, Natalia A

    2016-01-01

    Microvolt T-wave alternans (MTWA) testing identifies heart failure patients at risk for lethal ventricular arrhythmias at near-resting heart rates (<110 beats per minute). Since pressure alternans occurs simultaneously with MTWA and has a higher signal to noise ratio, it may be a better predictor of arrhythmia, although the mechanism remains unknown. Therefore, we investigated the relationship between force alternans (FORCE-ALT), the cellular manifestation of pressure alternans, and action potential voltage alternans (APV-ALT), the cellular driver of MTWA. Our goal was to uncover the mechanisms linking APV-ALT and FORCE-ALT in failing human myocytes and to investigate how the link between those alternans was affected by pacing rate and by physiological conditions such as sarcomere length and heart failure induced-remodeling of mechanical parameters. To achieve this, a mechanically-based, strongly coupled human electromechanical myocyte model was constructed. Reducing the sarcoplasmic reticulum calcium uptake current (Iup) to 27% was incorporated to simulate abnormal calcium handling in human heart failure. Mechanical remodeling was incorporated to simulate altered thin filament activation and crossbridge (XB) cycling rates. A dynamical pacing protocol was used to investigate the development of intracellular calcium concentration ([Ca]i), voltage, and active force alternans at different pacing rates. FORCE-ALT only occurred in simulations incorporating reduced Iup, demonstrating that alternans in the intracellular calcium concentration (CA-ALT) induced FORCE-ALT. The magnitude of FORCE-ALT was found to be largest at clinically relevant pacing rates (<110 bpm), where APV-ALT was smallest. We found that the magnitudes of FORCE-ALT, CA-ALT and APV-ALT were altered by heart failure induced-remodeling of mechanical parameters and sarcomere length due to the presence of myofilament feedback. These findings provide important insight into the relationship between heart

  7. Studies Update Vinyl Chloride Hazards.

    ERIC Educational Resources Information Center

    Rawls, Rebecca

    1980-01-01

    Extensive study affirms that vinyl chloride is a potent animal carcinogen. Epidemiological studies show elevated rates of human cancers in association with extended contact with the compound. (Author/RE)

  8. Intracellular localization of the Menkes and Wilson's disease proteins and their role in intracellular copper transport.

    PubMed

    Suzuki, M; Gitlin, J D

    1999-08-01

    Copper is a heavy metal ion essential for the activity of a variety of enzymes in the body. In excess, copper is a very toxic ion and therefore efficient regulation of its metabolism is required. This is dramatically illustrated by the genetic disorders X-linked Menkes disease and autosomal recessive Wilson's disease. In 1993, both the Menkes and Wilson's genes were isolated and it was found that these genes encode homologous cation copper transporting P-type ATPase proteins. The Menkes protein (ATP7A) is expressed in most tissues, except liver. In contrast, the Wilson's protein (ATP7B) is abundantly expressed in liver. Intracellular localization of those proteins was investigated. Both ATP7A and ATP7B are localized in the trans-Golgi network and post-Golgi vesicular compartment (PGVC) in the cell. This intracellular localization was altered by the copper content present in the cell. This result may support the hypothesis that ATP7A and ATP7B are involved in cellular copper transport and those proteins could be suitable models for elucidating intracellular copper metabolism.

  9. 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.

  10. With no lysine L-WNK1 isoforms are negative regulators of the K+-Cl- cotransporters.

    PubMed

    Mercado, Adriana; de Los Heros, Paola; Melo, Zesergio; Chávez-Canales, María; Murillo-de-Ozores, Adrián R; Moreno, Erika; Bazúa-Valenti, Silvana; Vázquez, Norma; Hadchouel, Juliette; Gamba, Gerardo

    2016-07-01

    The K(+)-Cl(-) cotransporters (KCC1-KCC4) encompass a branch of the SLC12 family of electroneutral cation-coupled chloride cotransporters that translocate ions out of the cell to regulate various factors, including cell volume and intracellular chloride concentration, among others. L-WNK1 is an ubiquitously expressed kinase that is activated in response to osmotic stress and intracellular chloride depletion, and it is implicated in two distinct hereditary syndromes: the renal disease pseudohypoaldosteronism type II (PHAII) and the neurological disease hereditary sensory neuropathy 2 (HSN2). The effect of L-WNK1 on KCC activity is unknown. Using Xenopus laevis oocytes and HEK-293 cells, we show that the activation of KCCs by cell swelling was prevented by L-WNK1 coexpression. In contrast, the activity of the Na(+)-K(+)-2Cl(-) cotransporter NKCC1 was remarkably increased with L-WNK1 coexpression. The negative effect of L-WNK1 on the KCCs is kinase dependent. Elimination of the STE20 proline-alanine rich kinase (SPAK)/oxidative stress-responsive kinase (OSR1) binding site or the HQ motif required for the WNK-WNK interaction prevented the effect of L-WNK1 on KCCs, suggesting a required interaction between L-WNK1 molecules and SPAK. Together, our data support that NKCC1 and KCCs are coordinately regulated by L-WNK1 isoforms.

  11. An XAFS study of nickel chloride in the ionic liquid 1-ethyl-3-methyl imidazolium chloride/ aluminum chloride

    SciTech Connect

    D Roeper; G Cheek; K Pandya; W OGrady

    2011-12-31

    Nickel chloride was studied with cyclic voltammetry and X-ray absorption spectroscopy in acidic and basic aluminum chloride/1-ethyl-3-methyl imidazolium chloride (EMIC) ionic liquids. Acidic melts display metal stripping peaks which are not observed in the basic melt. EXAFS analysis shows that the nickel is tetrahedrally coordinated with chloride ions in the basic solution. In the acidic solution the nickel is coordinated by six chloride ions that are also associated with aluminum ions.

  12. Magnetic tweezers for intracellular applications

    NASA Astrophysics Data System (ADS)

    Hosu, Basarab G.; Jakab, Károly; Bánki, Péter; Tóth, Ferenc I.; Forgacs, Gabor

    2003-09-01

    We have designed and constructed a versatile magnetic tweezer primarily for intracellular investigations. The micromanipulator uses only two coils to simultaneously magnetize to saturation micron-size superparamagnetic particles and generate high magnitude constant field gradients over cellular dimensions. The apparatus resembles a miniaturized Faraday balance, an industrial device used to measure magnetic susceptibility. The device operates in both continuous and pulse modes. Due to its compact size, the tweezers can conveniently be mounted on the stage of an inverted microscope and used for intracellular manipulations. A built-in temperature control unit maintains the sample at physiological temperatures. The operation of the tweezers was tested by moving 1.28 μm diameter magnetic beads inside macrophages with forces near 500 pN.

  13. 21 CFR 184.1622 - Potassium chloride.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Potassium chloride. 184.1622 Section 184.1622 Food... Specific Substances Affirmed as GRAS § 184.1622 Potassium chloride. (a) Potassium chloride (KCl, CAS Reg... levels not to exceed current good manufacturing practice. Potassium chloride may be used in...

  14. 21 CFR 184.1622 - Potassium chloride.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Potassium chloride. 184.1622 Section 184.1622 Food... GRAS § 184.1622 Potassium chloride. (a) Potassium chloride (KCl, CAS Reg. No. 7447-40-7) is a white... manufacturing practice. Potassium chloride may be used in infant formula in accordance with section 412(g)...

  15. 21 CFR 184.1622 - Potassium chloride.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Potassium chloride. 184.1622 Section 184.1622 Food... Specific Substances Affirmed as GRAS § 184.1622 Potassium chloride. (a) Potassium chloride (KCl, CAS Reg... levels not to exceed current good manufacturing practice. Potassium chloride may be used in...

  16. 21 CFR 184.1622 - Potassium chloride.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Potassium chloride. 184.1622 Section 184.1622 Food... Specific Substances Affirmed as GRAS § 184.1622 Potassium chloride. (a) Potassium chloride (KCl, CAS Reg... levels not to exceed current good manufacturing practice. Potassium chloride may be used in...

  17. 21 CFR 184.1138 - Ammonium chloride.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... from the filtrate on cooling. Alternatively, hydrogen chloride formed by the burning of hydrogen in... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Ammonium chloride. 184.1138 Section 184.1138 Food... Specific Substances Affirmed as GRAS § 184.1138 Ammonium chloride. (a) Ammonium chloride (NH4Cl, CAS...

  18. 21 CFR 184.1138 - Ammonium chloride.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... from the filtrate on cooling. Alternatively, hydrogen chloride formed by the burning of hydrogen in... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Ammonium chloride. 184.1138 Section 184.1138 Food... Specific Substances Affirmed as GRAS § 184.1138 Ammonium chloride. (a) Ammonium chloride (NH4Cl, CAS...

  19. 21 CFR 184.1138 - Ammonium chloride.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... from the filtrate on cooling. Alternatively, hydrogen chloride formed by the burning of hydrogen in... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Ammonium chloride. 184.1138 Section 184.1138 Food... Specific Substances Affirmed as GRAS § 184.1138 Ammonium chloride. (a) Ammonium chloride (NH4Cl, CAS...

  20. 21 CFR 184.1138 - Ammonium chloride.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... from the filtrate on cooling. Alternatively, hydrogen chloride formed by the burning of hydrogen in... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Ammonium chloride. 184.1138 Section 184.1138 Food... Specific Substances Affirmed as GRAS § 184.1138 Ammonium chloride. (a) Ammonium chloride (NH4Cl, CAS...

  1. 21 CFR 184.1138 - Ammonium chloride.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    .... Alternatively, hydrogen chloride formed by the burning of hydrogen in chlorine is dissolved in water and then... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Ammonium chloride. 184.1138 Section 184.1138 Food... GRAS § 184.1138 Ammonium chloride. (a) Ammonium chloride (NH4Cl, CAS Reg. No. 12125-02-9) is...

  2. Regeneration of zinc chloride hydrocracking catalyst

    DOEpatents

    Zielke, Clyde W.

    1979-01-01

    Improved rate of recovery of zinc values from the solids which are carried over by the effluent vapors from the oxidative vapor phase regeneration of spent zinc chloride catalyst is achieved by treatment of the solids with both hydrogen chloride and calcium chloride to selectively and rapidly recover the zinc values as zinc chloride.

  3. 21 CFR 184.1622 - Potassium chloride.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Potassium chloride. 184.1622 Section 184.1622 Food... Specific Substances Affirmed as GRAS § 184.1622 Potassium chloride. (a) Potassium chloride (KCl, CAS Reg... levels not to exceed current good manufacturing practice. Potassium chloride may be used in...

  4. Direct Measurement of Intracellular Pressure

    PubMed Central

    Petrie, Ryan J.; Koo, Hyun

    2014-01-01

    A method to directly measure the intracellular pressure of adherent, migrating cells is described in the Basic Protocol. This approach is based on the servo-null method where a microelectrode is introduced into the cell to directly measure the physical pressure of the cytoplasm. We also describe the initial calibration of the microelectrode as well as the application of the method to cells migrating inside three-dimensional (3D) extracellular matrix (ECM). PMID:24894836

  5. 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.

  6. 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

  7. Intracellular sphingosine releases calcium from lysosomes.

    PubMed

    Höglinger, Doris; Haberkant, Per; Aguilera-Romero, Auxiliadora; Riezman, Howard; Porter, Forbes D; Platt, Frances M; Galione, Antony; Schultz, Carsten

    2015-11-27

    To elucidate new functions of sphingosine (Sph), we demonstrate that the spontaneous elevation of intracellular Sph levels via caged Sph leads to a significant and transient calcium release from acidic stores that is independent of sphingosine 1-phosphate, extracellular and ER calcium levels. This photo-induced Sph-driven calcium release requires the two-pore channel 1 (TPC1) residing on endosomes and lysosomes. Further, uncaging of Sph leads to the translocation of the autophagy-relevant transcription factor EB (TFEB) to the nucleus specifically after lysosomal calcium release. We confirm that Sph accumulates in late endosomes and lysosomes of cells derived from Niemann-Pick disease type C (NPC) patients and demonstrate a greatly reduced calcium release upon Sph uncaging. We conclude that sphingosine is a positive regulator of calcium release from acidic stores and that understanding the interplay between Sph homeostasis, calcium signaling and autophagy will be crucial in developing new therapies for lipid storage disorders such as NPC.

  8. Stochastic models of intracellular transport

    NASA Astrophysics Data System (ADS)

    Bressloff, Paul C.; Newby, Jay M.

    2013-01-01

    The interior of a living cell is a crowded, heterogenuous, fluctuating environment. Hence, a major challenge in modeling intracellular transport is to analyze stochastic processes within complex environments. Broadly speaking, there are two basic mechanisms for intracellular transport: passive diffusion and motor-driven active transport. Diffusive transport can be formulated in terms of the motion of an overdamped Brownian particle. On the other hand, active transport requires chemical energy, usually in the form of adenosine triphosphate hydrolysis, and can be direction specific, allowing biomolecules to be transported long distances; this is particularly important in neurons due to their complex geometry. In this review a wide range of analytical methods and models of intracellular transport is presented. In the case of diffusive transport, narrow escape problems, diffusion to a small target, confined and single-file diffusion, homogenization theory, and fractional diffusion are considered. In the case of active transport, Brownian ratchets, random walk models, exclusion processes, random intermittent search processes, quasi-steady-state reduction methods, and mean-field approximations are considered. Applications include receptor trafficking, axonal transport, membrane diffusion, nuclear transport, protein-DNA interactions, virus trafficking, and the self-organization of subcellular structures.

  9. CHLORIDE WASHER PERFORMACE TESTING

    SciTech Connect

    Coughlin, J; David Best, D; Robert Pierce, R

    2007-11-30

    Testing was performed to determine the chloride (Cl-) removal capabilities of the Savannah River National Laboratory (SRNL) designed and built Cl- washing equipment intended for HB-Line installation. The equipment to be deployed was tested using a cerium oxide (CeO2) based simulant in place of the 3013 plutonium oxide (PuO2) material. Two different simulant mixtures were included in this testing -- one having higher Cl- content than the other. The higher Cl- simulant was based on K-Area Interim Surveillance Inspection Program (KIS) material with Cl- content approximately equal to 70,000 ppm. The lower Cl- level simulant was comparable to KIS material containing approximately 8,000-ppm Cl- content. The performance testing results indicate that the washer is capable of reducing the Cl- content of both surrogates to below 200 ppm with three 1/2-liter washes of 0.1M sodium hydroxide (NaOH) solution. Larger wash volumes were used with similar results - all of the prescribed test parameters consistently reduced the Cl- content of the surrogate to a value below 200 ppm Cl- in the final washed surrogate material. The washer uses a 20-micron filter to retain the surrogate solids. Tests showed that 0.16-0.41% of the insoluble fraction of the starting mass passed through the 20-micron filter. The solids retention performance indicates that the fissile masses passing through the 20-micron filter should not exceed the waste acceptance criteria for discard in grout to TRU waste. It is recommended that additional testing be pursued for further verification and optimization purposes. It is likely that wash volumes smaller than those tested could still reduce the Cl- values to acceptable levels. Along with reduced wash volumes, reuse of the third wash volume (in the next run processed) should be tested as a wash solution minimization plan. A 67% reduction in the number of grouted paint pails could be realized if wash solution minimization testing returned acceptable results.

  10. Cell–cell and intracellular lactate shuttles

    PubMed Central

    Brooks, George A

    2009-01-01

    Once thought to be the consequence of oxygen lack in contracting skeletal muscle, the glycolytic product lactate is formed and utilized continuously in diverse cells under fully aerobic conditions. ‘Cell–cell’ and ‘intracellular lactate shuttle’ concepts describe the roles of lactate in delivery of oxidative and gluconeogenic substrates as well as in cell signalling. Examples of the cell–cell shuttles include lactate exchanges between between white-glycolytic and red-oxidative fibres within a working muscle bed, and between working skeletal muscle and heart, brain, liver and kidneys. Examples of intracellular lactate shuttles include lactate uptake by mitochondria and pyruvate for lactate exchange in peroxisomes. Lactate for pyruvate exchanges affect cell redox state, and by itself lactate is a ROS generator. In vivo, lactate is a preferred substrate and high blood lactate levels down-regulate the use of glucose and free fatty acids (FFA). As well, lactate binding may affect metabolic regulation, for instance binding to G-protein receptors in adipocytes inhibiting lipolysis, and thus decreasing plasma FFA availability. In vitro lactate accumulation upregulates expression of MCT1 and genes coding for other components of the mitochondrial reticulum in skeletal muscle. The mitochondrial reticulum in muscle and mitochondrial networks in other aerobic tissues function to establish concentration and proton gradients necessary for cells with high mitochondrial densities to oxidize lactate. The presence of lactate shuttles gives rise to the realization that glycolytic and oxidative pathways should be viewed as linked, as opposed to alternative, processes, because lactate, the product of one pathway, is the substrate for the other. PMID:19805739

  11. Intracellular proton access in a Cl(-)/H(+) antiporter.

    PubMed

    Lim, Hyun-Ho; Shane, Tania; Miller, Christopher

    2012-01-01

    Chloride-transporting membrane proteins of the CLC family appear in two distinct mechanistic flavors: H(+)-gated Cl(-) channels and Cl(-)/H(+) antiporters. Transmembrane H(+) movement is an essential feature of both types of CLC. X-ray crystal structures of CLC antiporters show the Cl(-) ion pathway through these proteins, but the H(+) pathway is known only inferentially by two conserved glutamate residues that act as way-stations for H(+) in its path through the protein. The extracellular-facing H(+) transfer glutamate becomes directly exposed to aqueous solution during the transport cycle, but the intracellular glutamate E203, Glu(in), is buried within the protein. Two regions, denoted "polar" and "interfacial," at the intracellular surface of the bacterial antiporter CLC-ec1 are examined here as possible pathways by which intracellular aqueous protons gain access to Glu(in). Mutations at multiple residues of the polar region have little effect on antiport rates. In contrast, mutation of E202, a conserved glutamate at the protein-water boundary of the interfacial region, leads to severe slowing of the Cl(-)/H(+) antiport rate. An X-ray crystal structure of E202Y, the most strongly inhibited of these substitutions, shows an aqueous portal leading to Glu(in) physically blocked by cross-subunit interactions; moreover, this mutation has only minimal effect on a monomeric CLC variant, which necessarily lacks such interactions. The several lines of experiments presented argue that E202 acts as a water-organizer that creates a proton conduit connecting intracellular solvent with Glu(in). PMID:23239938

  12. 21 CFR 184.1297 - Ferric chloride.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Substances Affirmed as GRAS § 184.1297 Ferric chloride. (a) Ferric chloride (iron (III) chloride, FeC13, CAS Reg. No. 7705-08-0) may be prepared from iron and chlorine or from ferric oxide and hydrogen chloride... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Ferric chloride. 184.1297 Section 184.1297 Food...

  13. Intracellular transport of fat-soluble vitamins A and E.

    PubMed

    Kono, Nozomu; Arai, Hiroyuki

    2015-01-01

    Vitamins are compounds that are essential for the normal growth, reproduction and functioning of the human body. Of the 13 known vitamins, vitamins A, D, E and K are lipophilic compounds and are therefore called fat-soluble vitamins. Because of their lipophilicity, fat-soluble vitamins are solubilized and transported by intracellular carrier proteins to exert their actions and to be metabolized properly. Vitamin A and its derivatives, collectively called retinoids, are solubilized by intracellular retinoid-binding proteins such as cellular retinol-binding protein (CRBP), cellular retinoic acid-binding protein (CRABP) and cellular retinal-binding protein (CRALBP). These proteins act as chaperones that regulate the metabolism, signaling and transport of retinoids. CRALBP-mediated intracellular retinoid transport is essential for vision in human. α-Tocopherol, the main form of vitamin E found in the body, is transported by α-tocopherol transfer protein (α-TTP) in hepatic cells. Defects of α-TTP cause vitamin E deficiency and neurological disorders in humans. Recently, it has been shown that the interaction of α-TTP with phosphoinositides plays a critical role in the intracellular transport of α-tocopherol and is associated with familial vitamin E deficiency. In this review, we summarize the mechanisms and biological significance of the intracellular transport of vitamins A and E.

  14. Development of cadmium-free quantum dot for intracellular labelling through electroporation or lipid-calcium-phosphate

    NASA Astrophysics Data System (ADS)

    Liu, Ying-Feng; Hung, Wei-Ling; Hou, Tzh-Yin; Huang, Hsiu-Ying; Lin, Cheng-An J.

    2016-04-01

    Traditional fluorescent labelling techniques has severe photo-bleaching problem such as organic dyes and fluorescent protein. Quantum dots made up of traditional semiconductor (CdSe/ZnS) material has sort of biological toxicity. This research has developed novel Cd-free quantum dots divided into semiconductor (Indium phosphide, InP) and noble metal (Gold). Former has lower toxicity compared to traditional quantum dots. Latter consisting of gold (III) chloride (AuCl3) and toluene utilizes sonochemical preparation and different stimulus to regulate fluorescent wavelength. Amphoteric macromolecule surface technology and ligand Exchange in self-Assembled are involved to develop hydrophilic nanomaterials which can regulate the number of grafts per molecule of surface functional groups. Calcium phosphate (CaP) nanoparticle (NP) with an asymmetric lipid bilayer coating technology developed for intracellular delivery and labelling has synthesized Cd-free quantum dots possessing high brightness and multi-fluorescence successfully. Then, polymer coating and ligand exchange transfer to water-soluble materials to produce liposome nanomaterials as fluorescent probes and enhancing medical applications of nanotechnology.

  15. Chemical Analysis of Drug Biocrystals: A Role for Counterion Transport Pathways in Intracellular Drug Disposition.

    PubMed

    Keswani, Rahul K; Baik, Jason; Yeomans, Larisa; Hitzman, Chuck; Johnson, Allison M; Pawate, Ashtamurthy S; Kenis, Paul J A; Rodriguez-Hornedo, Nair; Stringer, Kathleen A; Rosania, Gus R

    2015-07-01

    In mammals, highly lipophilic small molecule chemical agents can accumulate as inclusions within resident tissue macrophages. In this context, we characterized the biodistribution, chemical composition, and structure of crystal-like drug inclusions (CLDIs) formed by clofazimine (CFZ), a weakly basic lipophilic drug. With prolonged oral dosing, CFZ exhibited a significant partitioning with respect to serum and fat due to massive bioaccumulation and crystallization in the liver and spleen. The NMR, Raman, and powder X-ray diffraction (p-XRD) spectra of CLDIs isolated from the spleens of CFZ-treated mice matched the spectra of pure, CFZ hydrochloride crystals (CFZ-HCl). Elemental analysis revealed a 237-fold increase in chlorine content in CLDIs compared to untreated tissue samples and a 5-fold increase in chlorine content compared to CFZ-HCl, suggesting that the formation of CLDIs occurs through a chloride mediated crystallization mechanism. Single crystal analysis revealed that CFZ-HCl crystals had a densely packed orthorhombic lattice configuration. In vitro, CFZ-HCl formed at a pH of 4-5 only if chloride ions were present at sufficiently high concentrations (>50:1 Cl(-)/CFZ), indicating that intracellular chloride transport mechanisms play a key role in the formation of CLDIs. While microscopy and pharmacokinetic analyses clearly revealed crystallization and intracellular accumulation of the drug in vivo, the chemical and structural characterization of CLDIs implicates a concentrative, chloride transport mechanism, paralleling and thermodynamically stabilizing the massive bioaccumulation of a weakly basic drug.

  16. Worker exposure to vinyl chloride and poly(vinyl chloride).

    PubMed

    Jones, J H

    1981-10-01

    The National Institute for Occupational Safety and Health (NIOSH) in early 1974 began industrial hygiene studies of vinyl chloride exposed workers. Three VC monomer plants, three VC polymerization plants, and seven PVC fabrication plants were surveyed. V polymerization plant workers and workers in one job category in VC monomer plants were exposed to average levels above 1 ppm. The highest average exposure was 22 ppm. NIOSH health hazard evaluation studies since these initial surveys have primarily shown nondetectable levels of vinyl chloride. A NIOSH control technology study in 1977 showed that exposure levels in VC polymerization plants had been drastically reduced but exposure levels above 1 ppm were still found in several cases.

  17. 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

  18. Cystic Fibrosis Gene Encodes a cAMP-Dependent Chloride Channel in Heart

    NASA Astrophysics Data System (ADS)

    Hart, Padraig; Warth, John D.; Levesque, Paul C.; Collier, Mei Lin; Geary, Yvonne; Horowitz, Burton; Hume, Joseph R.

    1996-06-01

    cAMP-dependent chloride channels in heart contribute to autonomic regulation of action potential duration and membrane potential and have been inferred to be due to cardiac expression of the epithelial cystic fibrosis transmembrane conductance regulator (C