WorldWideScience

Sample records for intracellular nucleotide-sensing receptors

  1. Intracellular recording from a spider vibration receptor.

    Science.gov (United States)

    Gingl, Ewald; Burger, Anna-M; Barth, Friedrich G

    2006-05-01

    The present study introduces a new preparation of a spider vibration receptor that allows intracellular recording of responses to natural mechanical or electrical stimulation of the associated mechanoreceptor cells. The spider vibration receptor is a lyriform slit sense organ made up of 21 cuticular slits located on the distal end of the metatarsus of each walking leg. The organ is stimulated when the tarsus receives substrate vibrations, which it transmits to the organ's cuticular structures, reducing the displacement to about one tenth due to geometrical reasons. Current clamp recording was used to record action potentials generated by electrical or mechanical stimuli. Square pulse stimulation identified two groups of sensory cells, the first being single-spike cells which generated only one or two action potentials and the second being multi-spike cells which produced bursts of action potentials. When the more natural mechanical sinusoidal stimulation was applied, differences in adaptation rate between the two cell types remained. In agreement with prior extracellular recordings, both cell types showed a decrease in the threshold tarsus deflection with increasing stimulus frequency. Off-responses to mechanical stimuli have also been seen in the metatarsal organ for the first time.

  2. Intracellular angiotensin II inhibits heterologous receptor stimulated Ca2+ entry

    NARCIS (Netherlands)

    Filipeanu, CM; Brailoiu, E; Henning, RH; Deelman, LE; de Zeeuw, D; Nelemans, SA

    2001-01-01

    Recent studies show that angiotensin II (AngII) can act from within the cell, possibly via intracellular receptors pharmacologically different from typical plasma membrane AngII receptors. The role of this intracellular AngII (AngII(i)) is unclear. Besides direct effects of AngII(i) on cellular

  3. Surveillance for Intracellular Antibody by Cytosolic Fc Receptor TRIM21

    Directory of Open Access Journals (Sweden)

    William A. McEwan

    2016-11-01

    Full Text Available TRIM21 has emerged as an atypical Fc receptor that is broadly conserved and widely expressed in the cytoplasm of mammalian cells. Viruses that traffic surface-bound antibodies into the cell during infection recruit TRIM21 via a high affinity interaction between Fc and TRIM21 PRYSPRY domain. Following binding of intracellular antibody, TRIM21 acts as both antiviral effector and sensor for innate immune signalling. These activities serve to reduce viral replication by orders of magnitude in vitro and contribute to host survival during in vivo infection. Neutralization occurs rapidly after detection and requires the activity of the ubiquitin-proteasome system. The microbial targets of this arm of intracellular immunity are still being identified: TRIM21 activity has been reported following infection by several non-enveloped viruses and intracellular bacteria. These findings extend the sphere of influence of antibodies to the intracellular domain and have broad implications for immunity. TRIM21 has been implicated in the chronic auto-immune condition systemic lupus erythematosus and is itself an auto-antigen in Sjögren’s syndrome. This review summarises our current understanding of TRIM21’s role as a cytosolic Fc receptor and briefly discusses pathological circumstances where intracellular antibodies have been described, or are hypothesized to occur, and may benefit from further investigations of the role of TRIM21.

  4. DMPD: NOD-like receptors (NLRs): bona fide intracellular microbial sensors. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18585455 NOD-like receptors (NLRs): bona fide intracellular microbial sensors. Shaw...tml) (.csml) Show NOD-like receptors (NLRs): bona fide intracellular microbial sensors. PubmedID 18585455 Ti...tle NOD-like receptors (NLRs): bona fide intracellular microbial sensors. Authors

  5. Sigma-1 receptor: The novel intracellular target of neuropsychotherapeutic drugs

    Directory of Open Access Journals (Sweden)

    Teruo Hayashi

    2015-01-01

    Full Text Available Sigma-1 receptor ligands have been long expected to serve as drugs for treatment of human diseases such as neurodegenerative disorders, depression, idiopathic pain, drug abuse, and cancer. Recent research exploring the molecular function of the sigma-1 receptor started unveiling underlying mechanisms of the therapeutic activity of those ligands. Via the molecular chaperone activity, the sigma-1 receptor regulates protein folding/degradation, ER/oxidative stress, and cell survival. The chaperone activity is activated or inhibited by synthetic sigma-1 receptor ligands in an agonist-antagonist manner. Sigma-1 receptors are localized at the endoplasmic reticulum (ER membranes that are physically associated with the mitochondria (MAM: mitochondria-associated ER membrane. In specific types of neurons (e.g., those at the spinal cord, sigma-1 receptors are also clustered at ER membranes that juxtapose postsynaptic plasma membranes. Recent studies indicate that sigma-1 receptors, partly in sake of its unique subcellular localization, regulate the mitochondria function that involves bioenergetics and free radical generation. The sigma-1 receptor may thus provide an intracellular drug target that enables controlling ER stress and free radical generation under pathological conditions.

  6. Xenoestrogens alter estrogen receptor (ER α intracellular levels.

    Directory of Open Access Journals (Sweden)

    Piergiorgio La Rosa

    Full Text Available 17β-estradiol (E2-dependent estrogen receptor (ER α intracellular concentration is a well recognized critical step in the pleiotropic effects elicited by E2 in several target tissues. Beside E2, a class of synthetic and plant-derived chemicals collectively named endocrine disruptors (EDs or xenoestrogens bind to and modify both nuclear and extra-nuclear ERα activities. However, at the present no information is available on the ability of EDs to hamper ERα intracellular concentration. Here, the effects of bisphenol A (BPA and naringenin (Nar, prototypes of synthetic and plant-derived ERα ligands, have been evaluated on ERα levels in MCF-7 cells. Both EDs mimic E2 in triggering ERα Ser118 phosphorylation and gene transcription. However, only E2 or BPA induce an increase of cell proliferation; whereas 24 hrs after Nar stimulation a dose-dependent decrease in cell number is reported. E2 or BPA treatment reduces ERα protein and mRNA levels after 24 hrs. Contrarily, Nar stimulation does not alter ERα content but reduces ERα mRNA levels like other ligands. Co-stimulation experiments indicate that 48 hrs of Nar treatment prevents the E2-induced ERα degradation and hijacks the physiological ability of E2:ERα complex to regulate gene transcription. Mechanistically, Nar induces ERα protein accumulation by preventing proteasomal receptor degradation via persistent activation of p38/MAPK pathway. As a whole these data demonstrate that ERα intracellular concentration is an important target through which EDs hamper the hormonal milieu of E2 target cells driving cells to different outcomes or mimicking E2 even in the absence of the hormone.

  7. Xenoestrogens alter estrogen receptor (ER) α intracellular levels.

    Science.gov (United States)

    La Rosa, Piergiorgio; Pellegrini, Marco; Totta, Pierangela; Acconcia, Filippo; Marino, Maria

    2014-01-01

    17β-estradiol (E2)-dependent estrogen receptor (ER) α intracellular concentration is a well recognized critical step in the pleiotropic effects elicited by E2 in several target tissues. Beside E2, a class of synthetic and plant-derived chemicals collectively named endocrine disruptors (EDs) or xenoestrogens bind to and modify both nuclear and extra-nuclear ERα activities. However, at the present no information is available on the ability of EDs to hamper ERα intracellular concentration. Here, the effects of bisphenol A (BPA) and naringenin (Nar), prototypes of synthetic and plant-derived ERα ligands, have been evaluated on ERα levels in MCF-7 cells. Both EDs mimic E2 in triggering ERα Ser118 phosphorylation and gene transcription. However, only E2 or BPA induce an increase of cell proliferation; whereas 24 hrs after Nar stimulation a dose-dependent decrease in cell number is reported. E2 or BPA treatment reduces ERα protein and mRNA levels after 24 hrs. Contrarily, Nar stimulation does not alter ERα content but reduces ERα mRNA levels like other ligands. Co-stimulation experiments indicate that 48 hrs of Nar treatment prevents the E2-induced ERα degradation and hijacks the physiological ability of E2:ERα complex to regulate gene transcription. Mechanistically, Nar induces ERα protein accumulation by preventing proteasomal receptor degradation via persistent activation of p38/MAPK pathway. As a whole these data demonstrate that ERα intracellular concentration is an important target through which EDs hamper the hormonal milieu of E2 target cells driving cells to different outcomes or mimicking E2 even in the absence of the hormone.

  8. LDL Receptors as Gateways for Intracellular Porphyrin Uptake

    International Nuclear Information System (INIS)

    Novick, S.; Laster, B.; Quastel, M.

    2004-01-01

    Boronated compounds are currently being studied for possible use in Boron Neutron Capture Therapy (BNCT). We found that one of these agents, BOPP (tetrakis-carborane-carboxylate, esters of 2,4-bis (a,b- dihydroxyethyl) deuteroporphyrin IX), could also be labeled with indium (In-BOPP) and, therefore, could also be used potentially to transport high Z atoms into tumor cell DNA for AET (Auger Electron Therapy). In order to assess the uptake of these agents into cells, the role of the LDL receptor in the intracellular accumulation of BOPP and In-BOPP was investigated. Pre-incubation of V-79 Chinese hamster cells in medium containing delipidized fetal bovine serum (FBS) markedly increased the subsequent uptake of intracellular boron transported by both BOPP and In-BOPP when compared with cells that had been pre-incubated with medium containing 10% normal FBS (lipidized). The increased uptake was characterized by elevated levels of receptor, and greater affinity was shown for both BOPP and In-BOPP, although less marked with the latter. Positive cooperativity was demonstrated by sigmoid saturation curves, Scatchard analysis and Hill plots. Increasing the amount of LDL in the incubation medium had a relatively small effect on the total accumulation of either indium or boron atoms inside the cell. Furthermore, chemical acetylation of LDL did not decrease the intracellular uptake of either boron or indium transported by BOPP or In-BOPP. It is thus concluded that BOPP and In-BOPP preferentially enter the cells directly by way of the LDL receptor and that only a small fraction of these molecules are transported into the cells indirectly using serum LDLs as their carriers. These data suggest a novel way of bringing greater amounts of boron and indium (and perhaps other agents) into tissues. Porphyrins can be used to transport different agents into tumor cells because they are tumor affinic molecules. Tumors express a higher number of LDL receptors than do most normal tissues

  9. Intracellular insulin-receptor dissociation and segregation in a rat fibroblast cell line transfected with a human insulin receptor gene

    International Nuclear Information System (INIS)

    Levy, J.R.; Olefsky, J.M.

    1988-01-01

    The cellular processing of insulin and insulin receptors was studied using a rat fibroblast cell line that had been transfected with a normal human insulin receptor gene, expressing approximately 500 times the normal number of native fibroblasts insulin receptors. These cells bind and internalize insulin normally. Biochemically assays based on the selective precipitation by polyethylene glycol of intact insulin-receptor complexes but not of free intracellular insulin were developed to study the time course of intracellular insulin-receptor dissociation. Fibroblasts were incubated with radiolabeled insulin at 4 0 C, and internalization of insulin-receptor complexes was initiated by warming the cells to 37 0 C. Within 2 min, 90% of the internalized radioactivity was composed of intact insulin-receptor complexes. The dissociation of insulin from internalized insulin-receptor complexes was markedly inhibited by monensin and chloroquine. Furthermore, chloroquine markedly increased the number of cross-linkable intracellular insulin-receptor complexes, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. These findings suggest that acidification of intracellular vesicles is responsible for insulin-receptor dissociation. Physical segregation of dissociated intracellular insulin from its receptor was monitored. The results are consistent with the view that segregation of insulin and receptor occurs 5-10 min after initiation of dissociation. These studies demonstrate the intracellular itinerary of insulin-receptor complexes, including internalization, dissociation of insulin from the internalized receptor within an acidified compartment, segregation of insulin from the receptor, and subsequent ligand degradation

  10. Downregulation of transferrin receptor surface expression by intracellular antibody

    International Nuclear Information System (INIS)

    Peng Jilin; Wu Sha; Zhao Xiaoping; Wang Min; Li Wenhan; Shen Xin; Liu Jing; Lei Ping; Zhu Huifen; Shen Guanxin

    2007-01-01

    To deplete cellular iron uptake, and consequently inhibit the proliferation of tumor cells, we attempt to block surface expression of transferrin receptor (TfR) by intracellular antibody technology. We constructed two expression plasmids (scFv-HAK and scFv-HA) coding for intracellular single-chain antibody against TfR with or without endoplasmic reticulum (ER) retention signal, respectively. Then they were transfected tumor cells MCF-7 by liposome. Applying RT-PCR, Western blotting, immunofluorescence microscopy and immunoelectron microscope experiments, we insure that scFv-HAK intrabody was successfully expressed and retained in ER contrasted to the secreted expression of scFv-HA. Flow cytometric analysis confirmed that the TfR surface expression was markedly decreased approximately 83.4 ± 2.5% in scFv-HAK transfected cells, while there was not significantly decrease in scFv-HA transfected cells. Further cell growth and apoptosis characteristics were evaluated by cell cycle analysis, nuclei staining and MTT assay. Results indicated that expression of scFv-HAK can dramatically induce cell cycle G1 phase arrest and apoptosis of tumor cells, and consequently significantly suppress proliferation of tumor cells compared with other control groups. For First time this study demonstrates the potential usage of anti-TfR scFv-intrabody as a growth inhibitor of TfR overexpressing tumors

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

    DEFF Research Database (Denmark)

    Billestrup, N; Allevato, G; Norstedt, G

    1994-01-01

    The growth hormone (GH) receptor belongs to the GH/prolactin/cytokine super-family of receptors. The signal transduction mechanism utilized by this class of receptors remains largely unknown. In order to identify functional domains in the intracellular region of the GH receptor we generated...... a number of GH receptor mutants and analyzed their function after transfection into various cell lines. A truncated GH receptor missing 184 amino acids at the C-terminus was unable to mediate GH effects on transcription of the Spi 2.1 and insulin genes. However, this mutant was fully active in mediating GH...... as well as metabolic effects. These results indicate that the intracellular part of the GH receptor can be divided into at least three functional domains: (i) for transcriptional activity, two domains are involved, one located in the C-terminal 184 amino acids and the other in the proline-rich domain; (ii...

  12. Interactions between Intracellular Domains as Key Determinants of the Quaternary Structure and Function of Receptor Heteromers*

    Science.gov (United States)

    Navarro, Gemma; Ferré, Sergi; Cordomi, Arnau; Moreno, Estefania; Mallol, Josefa; Casadó, Vicent; Cortés, Antoni; Hoffmann, Hanne; Ortiz, Jordi; Canela, Enric I.; Lluís, Carme; Pardo, Leonardo; Franco, Rafael; Woods, Amina S.

    2010-01-01

    G protein-coupled receptor (GPCR) heteromers are macromolecular complexes with unique functional properties different from those of its individual protomers. Little is known about what determines the quaternary structure of GPCR heteromers resulting in their unique functional properties. In this study, using resonance energy transfer techniques in experiments with mutated receptors, we provide for the first time clear evidence for a key role of intracellular domains in the determination of the quaternary structure of GPCR heteromers between adenosine A2A, cannabinoid CB1, and dopamine D2 receptors. In these interactions, arginine-rich epitopes form salt bridges with phosphorylated serine or threonine residues from CK1/2 consensus sites. Each receptor (A2A, CB1, and D2) was found to include two evolutionarily conserved intracellular domains to establish selective electrostatic interactions with intracellular domains of the other two receptors, indicating that these particular electrostatic interactions constitute a general mechanism for receptor heteromerization. Mutation experiments indicated that the interactions of the intracellular domains of the CB1 receptor with A2A and D2 receptors are fundamental for the correct formation of the quaternary structure needed for the function (MAPK signaling) of the A2A-CB1-D2 receptor heteromers. Analysis of MAPK signaling in striatal slices of CB1 receptor KO mice and wild-type littermates supported the existence of A1-CB1-D2 receptor heteromer in the brain. These findings allowed us to propose the first molecular model of the quaternary structure of a receptor heteromultimer. PMID:20562103

  13. Evaluation of Intracellular Signaling Downstream Chimeric Antigen Receptors.

    Directory of Open Access Journals (Sweden)

    Hannah Karlsson

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

  14. Cannabinoid Receptor Activation Modifies NMDA Receptor Mediated Release of Intracellular Calcium: Implications for Endocannabinoid Control of Hippocampal Neural Plasticity

    Science.gov (United States)

    Hampson, Robert E.; Miller, Frances; Palchik, Guillermo; Deadwyler, Sam A.

    2011-01-01

    Chronic activation or inhibition of cannabinoid receptors (CB1) leads to continuous suppression of neuronal plasticity in hippocampus and other brain regions, suggesting that endocannabinoids may have a functional role in synaptic processes that produce state-dependent transient modulation of hippocampal cell activity. In support of this, it has previously been shown in vitro that cannabinoid CB1 receptors modulate second messenger systems in hippocampal neurons that can modulate intracellular ion channels, including channels which release calcium from intracellular stores. Here we demonstrate in hippocampal slices a similar endocannabinoid action on excitatory glutamatergic synapses via modulation of NMDA-receptor mediated intracellular calcium levels in confocal imaged neurons. Calcium entry through glutamatergic NMDA-mediated ion channels increases intracellular calcium concentrations via modulation of release from ryanodine-sensitive channels in endoplasmic reticulum. The studies reported here show that NMDA-elicited increases in Calcium Green fluorescence are enhanced by CB1 receptor antagonists (i.e. rimonabant), and inhibited by CB1 agonists (i.e. WIN 55,212-2). Suppression of endocannabinoid breakdown by either reuptake inhibition (AM404) or fatty-acid amide hydrolase inhibition (URB597) produced suppression of NMDA elicited calcium increases comparable to WIN 55,212-2, while enhancement of calcium release provoked by endocannabinoid receptor antagonists (Rimonabant) was shown to depend on the blockade of CB1 receptor mediated de-phosphorylation of Ryanodine receptors. Such CB1 receptor modulation of NMDA elicited increases in intracellular calcium may account for the respective disruption and enhancement by CB1 agents of trial-specific hippocampal neuron ensemble firing patterns during performance of a short-term memory task, reported previously from this laboratory. PMID:21288475

  15. AMPA receptor pHluorin-GluA2 reports NMDA receptor-induced intracellular acidification in hippocampal neurons

    DEFF Research Database (Denmark)

    Rathje, Mette; Fang, Huaqiang; Bachman, Julia L

    2013-01-01

    NMDA receptor activation promotes endocytosis of AMPA receptors, which is an important mechanism underlying long-term synaptic depression. The pH-sensitive GFP variant pHluorin fused to the N terminus of GluA2 (pH-GluA2) has been used to assay NMDA-mediated AMPA receptor endocytosis and recycling...... recovery was eliminated in the presence of the NHE1 inhibitor zoniporide. Our results indicate that the pH-GluA2 recycling assay is an unreliable assay for studying AMPA receptor trafficking and also suggest a role for PICK1 in regulating intracellular pH via modulation of NHE activity....

  16. Structural rearrangement of the intracellular domains during AMPA receptor activation

    DEFF Research Database (Denmark)

    Zachariassen, Linda Grønborg; Katchan, Ljudmila; Jensen, Anna Guldvang

    2016-01-01

    changes that underlie receptor function is lacking. Here, we used single and dual insertion of GFP variants at various positions in AMPAR subunits to enable measurements of conformational changes using fluorescence resonance energy transfer (FRET) in live cells. We produced dual CFP/YFP-tagged GluA2...... subunit constructs that had normal activity and displayed intrareceptor FRET. We used fluorescence lifetime imaging microscopy (FLIM) in live HEK293 cells to determine distinct steady-state FRET efficiencies in the presence of different ligands, suggesting a dynamic picture of the resting state. Patch...

  17. An intracellular traffic jam: Fc receptor-mediated transport of immunoglobulin G.

    Science.gov (United States)

    Tesar, Devin B; Björkman, Pamela J

    2010-04-01

    Recent advances in imaging techniques along with more powerful in vitro and in vivo models of receptor-mediated ligand transport are facilitating advances in our understanding of how cells efficiently direct receptors and their cargo to target destinations within the cytoplasm and at the plasma membrane. Specifically, light and 3D electron microscopy studies examining the trafficking behavior of the neonatal Fc receptor (FcRn), a transport receptor for immunoglobulin G (IgG), have given us new insights into the dynamic interplay between the structural components of the cytosolic trafficking machinery, its protein regulators, and the receptors it directs to various locations within the cell. These studies build upon previous biochemical characterizations of FcRn transport and are allowing us to begin formulation of a more complete model for the intracellular trafficking of receptor-ligand complexes.

  18. AMPA receptor pHluorin-GluA2 reports NMDA receptor-induced intracellular acidification in hippocampal neurons

    OpenAIRE

    Rathje, Mette; Fang, Huaqiang; Bachman, Julia L.; Anggono, Victor; Gether, Ulrik; Huganir, Richard L.; Madsen, Kenneth L.

    2013-01-01

    NMDA receptor activation promotes endocytosis of AMPA receptors, which is an important mechanism underlying long-term synaptic depression. The pH-sensitive GFP variant pHluorin fused to the N terminus of GluA2 (pH-GluA2) has been used to assay NMDA-mediated AMPA receptor endocytosis and recycling. Here, we demonstrate that in somatic and dendritic regions of hippocampal neurons a large fraction of the fluorescent signal originates from intracellular pH-GluA2, and that the decline in fluoresce...

  19. Intracellular Ca2+ and TRPM5-mediated membrane depolarization produce ATP secretion from taste receptor cells

    Science.gov (United States)

    Huang, Yijen A; Roper, Stephen D

    2010-01-01

    ATP is a transmitter secreted from taste bud receptor (Type II) cells through ATP-permeable gap junction hemichannels most probably composed of pannexin 1. The elevation of intracellular Ca2+ and membrane depolarization are both believed to be involved in transmitter secretion from receptor cells, but their specific roles have not been fully elucidated. In the present study, we show that taste-evoked ATP secretion from mouse vallate receptor cells is evoked by the combination of intracellular Ca2+ release and membrane depolarization. Unexpectedly, ATP secretion is not blocked by tetrodotoxin, indicating that transmitter release from these cells still takes place in the absence of action potentials. Taste-evoked ATP secretion is absent in receptor cells isolated from TRPM5 knockout mice or in taste cells from wild type mice where current through TRPM5 channels has been eliminated. These findings suggest that membrane voltage initiated by TRPM5 channels is required for ATP secretion during taste reception. Nonetheless, even in the absence of TRPM5 channel activity, ATP release could be triggered by depolarizing cells with KCl. Collectively, the findings indicate that taste-evoked elevation of intracellular Ca2+ has a dual role: (1) Ca2+ opens TRPM5 channels to depolarize receptor cells and (2) Ca2+ plus membrane depolarization opens ATP-permeable gap junction hemichannels. PMID:20498227

  20. Intracellular Ca(2+) and TRPM5-mediated membrane depolarization produce ATP secretion from taste receptor cells.

    Science.gov (United States)

    Huang, Yijen A; Roper, Stephen D

    2010-07-01

    ATP is a transmitter secreted from taste bud receptor (Type II) cells through ATP-permeable gap junction hemichannels most probably composed of pannexin 1. The elevation of intracellular Ca(2+) and membrane depolarization are both believed to be involved in transmitter secretion from receptor cells, but their specific roles have not been fully elucidated. In the present study, we show that taste-evoked ATP secretion from mouse vallate receptor cells is evoked by the combination of intracellular Ca(2+) release and membrane depolarization. Unexpectedly, ATP secretion is not blocked by tetrodotoxin, indicating that transmitter release from these cells still takes place in the absence of action potentials. Taste-evoked ATP secretion is absent in receptor cells isolated from TRPM5 knockout mice or in taste cells from wild type mice where current through TRPM5 channels has been eliminated. These findings suggest that membrane voltage initiated by TRPM5 channels is required for ATP secretion during taste reception. Nonetheless, even in the absence of TRPM5 channel activity, ATP release could be triggered by depolarizing cells with KCl. Collectively, the findings indicate that taste-evoked elevation of intracellular Ca(2+) has a dual role: (1) Ca(2+) opens TRPM5 channels to depolarize receptor cells and (2) Ca(2+) plus membrane depolarization opens ATP-permeable gap junction hemichannels.

  1. Intracellular processing of growth hormone receptors by adipocytes in primary culture.

    Science.gov (United States)

    Roupas, P; Herington, A C

    1988-05-01

    Rat adipocytes in primary culture have been used to study the intracellular processing of growth hormone (GH) receptors. Pretreatment of adipocytes with 20 micrograms/ml cycloheximide resulted in a rapid decline (t1/2 approximately 45 min) of the 125I-human growth hormone (hGH) binding capacity of the cells. This decline occurred at a faster rate in the presence of extracellular unlabeled hGH (400 ng/ml) and was not due to receptor occupancy. These data suggest that GH receptors turn over rapidly and constitutively on the plasma membrane and in the absence of protein synthesis are not replaced. Dissociation of GH-receptor complexes was shown not to occur at pH 5.5, the pH encountered in the acidic pre-lysosomal compartments (endosomes) where intracellular dissociation of many hormone-receptor complexes takes place. These data, together, suggest that the majority of GH receptors are not recycled but instead suffer the same fate as the majority of GH, i.e. degradation. To determine the rate of appearance of GH receptors at the cell surface, adipocytes were first treated with trypsin and then incubated at 37 degrees C to permit incorporation of any available GH receptors into the plasma membrane. Binding of 125I-hGH recovered to pre-trypsin levels by 2 h. This recovery was completely blocked by concomitant treatment with monensin, cytochalasin B, colchicine and 2,4-dinitrophenol. NH4Cl had no effect on receptor recovery. These data suggest that once GH receptors are synthesized in the rough endoplasmic reticulum, they travel via the Golgi apparatus to the plasma membrane (by processes involving both microfilaments and microtubules) and are then inserted into the plasma membrane in an energy-dependent step.

  2. Enhanced NMDA receptor-mediated intracellular calcium signaling in magnocellular neurosecretory neurons in heart failure rats.

    Science.gov (United States)

    Stern, Javier E; Potapenko, Evgeniy S

    2013-08-15

    An enhanced glutamate excitatory function within the hypothalamic supraoptic and paraventricluar nuclei is known to contribute to increased neurosecretory and presympathetic neuronal activity, and hence, neurohumoral activation, during heart failure (HF). Still, the precise mechanisms underlying enhanced glutamate-driven neuronal activity in HF remain to be elucidated. Here, we performed simultaneous electrophysiology and fast confocal Ca²⁺ imaging to determine whether altered N-methyl-d-aspartate (NMDA) receptor-mediated changes in intracellular Ca²⁺ levels (NMDA-ΔCa²⁺) occurred in hypothalamic magnocellular neurosecretory cells (MNCs) in HF rats. We found that activation of NMDA receptors resulted in a larger ΔCa²⁺ in MNCs from HF when compared with sham rats. The enhanced NMDA-ΔCa²⁺ was neither dependent on the magnitude of the NMDA-mediated current (voltage clamp) nor on the degree of membrane depolarization or firing activity evoked by NMDA (current clamp). Differently from NMDA receptor activation, firing activity evoked by direct membrane depolarization resulted in similar changes in intracellular Ca²⁺ in sham and HF rats. Taken together, our results support a relatively selective alteration of intracellular Ca²⁺ homeostasis and signaling following activation of NMDA receptors in MNCs during HF. The downstream functional consequences of such altered ΔCa²⁺ signaling during HF are discussed.

  3. Killing of intracellular Mycobacterium tuberculosis by receptor-mediated drug delivery

    International Nuclear Information System (INIS)

    Majumdar, S.; Basu, S.K.

    1991-01-01

    p-Aminosalicylic acid (PAS) conjugated to maleylated bovine serum albumin (MBSA) was taken up efficiently through high-affinity MBSA-binding sites on macrophages. Binding of the radiolabeled conjugate to cultured mouse peritoneal macrophages at 4 degrees C was competed for by MBSA but not by PAS. At 37 degrees C, the radiolabeled conjugate was rapidly degraded by the macrophages, leading to release of acid-soluble degradation products in the medium. The drug conjugate was nearly 100 times as effective as free PAS in killing the intracellular mycobacteria in mouse peritoneal macrophages infected in culture with Mycobacterium tuberculosis. The killing of intracellular mycobacteria mediated by the drug conjugate was effectively prevented by simultaneous addition of excess MBSA (100 micrograms/ml) or chloroquine (3 microM) to the medium, whereas these agents did not affect the microbicidal action of free PAS. These results suggest that (i) uptake of the PAS-MBSA conjugate was mediated by cell surface receptors on macrophages which recognize MBSA and (ii) lysosomal hydrolysis of the internalized conjugate resulted in intracellular release of a pharmacologically active form of the drug, which led to selective killing of the M. tuberculosis harbored by mouse macrophages infected in culture. This receptor-mediated modality of delivering drugs to macrophages could contribute to greater therapeutic efficacy and minimization of toxic side effects in the management of tuberculosis and other intracellular mycobacterial infections

  4. Y1 receptors for neuropeptide Y are coupled to mobilization of intracellular calcium and inhibition of adenylate cyclase

    DEFF Research Database (Denmark)

    Aakerlund, L; Gether, U; Fuhlendorff, J

    1990-01-01

    Two types of binding sites have previously been described for neuropeptide Y (NPY), called Y1 and Y2 receptors. The intracellular events following Y1 receptor activation was studied in the human neuroblastoma cell line SK-N-MC. Both NPY and the specific Y1 receptor ligand, [Leu31,Pro34]-NPY, caused...

  5. AMPA receptor pHluorin-GluA2 reports NMDA receptor-induced intracellular acidification in hippocampal neurons.

    Science.gov (United States)

    Rathje, Mette; Fang, Huaqiang; Bachman, Julia L; Anggono, Victor; Gether, Ulrik; Huganir, Richard L; Madsen, Kenneth L

    2013-08-27

    NMDA receptor activation promotes endocytosis of AMPA receptors, which is an important mechanism underlying long-term synaptic depression. The pH-sensitive GFP variant pHluorin fused to the N terminus of GluA2 (pH-GluA2) has been used to assay NMDA-mediated AMPA receptor endocytosis and recycling. Here, we demonstrate that in somatic and dendritic regions of hippocampal neurons a large fraction of the fluorescent signal originates from intracellular pH-GluA2, and that the decline in fluorescence in response to NMDA and AMPA primarily describes an intracellular acidification, which quenches the pHluorin signal from intracellular receptor pools. Neurons expressing an endoplasmic reticulum-retained mutant of GluA2 (pH-GluA2 ΔC49) displayed a larger response to NMDA than neurons expressing wild-type pH-GluA2. A similar NMDA-elicited decline in pHluorin signal was observed by expressing cytosolic pHluorin alone without fusion to GluA2 (cyto-pHluorin). Intracellular acidification in response to NMDA was further confirmed by using the ratiometric pH indicator carboxy-SNARF-1. The NMDA-induced decline was followed by rapid recovery of the fluorescent signal from both cyto-pHluorin and pH-GluA2. The recovery was sodium-dependent and sensitive to Na(+)/H(+)-exchanger (NHE) inhibitors. Moreover, recovery was more rapid after shRNA-mediated knockdown of the GluA2 binding PDZ domain-containing protein interacting with C kinase 1 (PICK1). Interestingly, the accelerating effect of PICK1 knockdown on the fluorescence recovery was eliminated in the presence of the NHE1 inhibitor zoniporide. Our results indicate that the pH-GluA2 recycling assay is an unreliable assay for studying AMPA receptor trafficking and also suggest a role for PICK1 in regulating intracellular pH via modulation of NHE activity.

  6. Acetylcholine Attenuates Hydrogen Peroxide-Induced Intracellular Calcium Dyshomeostasis Through Both Muscarinic and Nicotinic Receptors in Cardiomyocytes.

    Science.gov (United States)

    Palee, Siripong; Apaijai, Nattayaporn; Shinlapawittayatorn, Krekwit; Chattipakorn, Siriporn C; Chattipakorn, Nipon

    2016-01-01

    Oxidative stress induced intracellular Ca2+ overload plays an important role in the pathophysiology of several heart diseases. Acetylcholine (ACh) has been shown to suppress reactive oxygen species generation during oxidative stress. However, there is little information regarding the effects of ACh on the intracellular Ca2+ regulation in the presence of oxidative stress. Therefore, we investigated the effects of ACh applied before or after hydrogen peroxide (H2O2) treatment on the intracellular Ca2+ regulation in isolated cardiomyocytes. Single ventricular myocytes were isolated from the male Wistar rats for the intracellular Ca2+ transient study by a fluorimetric ratio technique. H2O2 significantly decreased both of intracellular Ca2+ transient amplitude and decay rate. ACh applied before, but not after, H2O2 treatment attenuated the reduction of intracellular Ca2+ transient amplitude and decay rate. Both atropine (a muscarinic acetylcholine receptor blocker) and mecamylamine (a nicotinic acetylcholine receptor blocker) significantly decreased the protective effects of acetylcholine on the intracellular Ca2+ regulation. Moreover, the combination of atropine and mecamylamine completely abolished the protective effects of acetylcholine on intracellular Ca2+ transient amplitude and decay rate. ACh pretreatment attenuates H2O2-induced intracellular Ca2+ dyshomeostasis through both muscarinic and nicotinic receptors. © 2016 The Author(s) Published by S. Karger AG, Basel.

  7. Activation of intracellular angiotensin AT2 receptors induces rapid cell death in human uterine leiomyosarcoma cells

    DEFF Research Database (Denmark)

    Zhao, Yi; Lützen, Ulf; Fritsch, Jürgen

    2015-01-01

    of apoptosis and cell death in cultured human uterine leiomyosarcoma (SK-UT-1) cells and control human uterine smooth muscle cells (HutSMC). The intracellular levels of the AT2 receptor are low in proliferating SK-UT-1 cells but the receptor is substantially up-regulated in quiescent SK-UT-1 cells with high....... e. down-regulation of the Bcl-2 protein, induction of the Bax protein and activation of caspase-3. All quiescent SK-UT-1 cells died within 5 days after treatment with a single dose of C21. C21 was devoid of cytotoxic effects in proliferating SK-UT-1 cells and in quiescent HutSMC. Our results point...... to a new, unique approach enabling to eliminate non-cycling uterine leiomyosarcoma cells providing that they over-express the AT2 receptor....

  8. Tripeptidyl peptidase II regulates sperm function by modulating intracellular Ca(2+ stores via the ryanodine receptor.

    Directory of Open Access Journals (Sweden)

    Yuchuan Zhou

    Full Text Available Recent studies have identified Ca(2+ stores in sperm cells; however, it is not clear whether these Ca(2+ stores are functional and how they are mobilized. Here, in vitro and in vivo, we determined that tripeptidyl peptidase II antagonists strongly activated the cAMP/PKA signaling pathway that drives sperm capacitation-associated protein tyrosine phosphorylation. We demonstrated that in the absence of Ca(2+, TPIII antagonists elevated the intracellular Ca(2+ levels in sperm, resulting in a marked improvement in sperm movement, capacitation, acrosome reaction, and the in vitro fertilizing ability. This antagonist-induced release of intracellular Ca(2+ could be blocked by the inhibitors of ryanodine receptors (RyRs which are the main intracellular Ca(2+ channels responsible for releasing stored Ca(2+. Consistent with these results, indirect immunofluorescence assay using anti-RyR antibodies further validated the presence of RyR3 in the acrosomal region of mature sperm. Thus, TPPII can regulate sperm maturation by modulating intracellular Ca(2+ stores via the type 3 RyR.

  9. Intracellular calcium levels determine differential modulation of allosteric interactions within G protein-coupled receptor heteromers.

    Science.gov (United States)

    Navarro, Gemma; Aguinaga, David; Moreno, Estefania; Hradsky, Johannes; Reddy, Pasham P; Cortés, Antoni; Mallol, Josefa; Casadó, Vicent; Mikhaylova, Marina; Kreutz, Michael R; Lluís, Carme; Canela, Enric I; McCormick, Peter J; Ferré, Sergi

    2014-11-20

    The pharmacological significance of the adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromer is well established and it is being considered as an important target for the treatment of Parkinson’s disease and other neuropsychiatric disorders. However, the physiological factors that control its distinctive biochemical properties are still unknown. We demonstrate that different intracellular Ca2+ levels exert a differential modulation of A2AR-D2R heteromer-mediated adenylyl-cyclase and MAPK signaling in striatal cells. This depends on the ability of low and high Ca2+ levels to promote a selective interaction of the heteromer with the neuronal Ca2+-binding proteins NCS-1 and calneuron-1, respectively. These Ca2+-binding proteins differentially modulate allosteric interactions within the A2AR-D2R heteromer, which constitutes a unique cellular device that integrates extracellular (adenosine and dopamine) and intracellular (Ca+2) signals to produce a specific functional response.

  10. Intracellular metabotropic glutamate receptor 5 (mGluR5) activates signaling cascades distinct from cell surface counterparts.

    Science.gov (United States)

    Jong, Yuh-Jiin I; Kumar, Vikas; O'Malley, Karen L

    2009-12-18

    G-protein-coupled receptors are thought to transmit extracellular signals to the cytoplasm from their position on the cell surface. Some receptors, including the metabotropic glutamate receptor 5 (mGluR5), are also highly expressed on intracellular membranes where they serve unknown functions. Here, we show that activation of cell surface versus intracellular mGluR5 results in unique Ca(2+) signatures leading to unique cellular responses. Specifically, activation of either cell surface or intracellular mGluR5 leads to JNK, Ca(2+)/calmodulin-dependent protein kinase (CaMK), and cyclic adenosine 3',5'-monophosphate-responsive element-binding protein phosphorylation, whereas activation of only intracellular mGluR5 leads to ERK1/2 and Elk-1 phosphorylation. Using pharmacological and genetic approaches, the present findings support a role for CaMK kinase in mediating mGluR5-dependent cyclic adenosine 3',5'-monophosphate-responsive element-binding protein phosphorylation, whereas CaMKII is upstream of intracellular mGluR5-mediated Elk-1 phosphorylation. Consistent with models showing Elk-1 regulating cascades of gene expression, the known Elk-1 targets c-fos and egr1 were up-regulated following intracellular mGluR5 activation, whereas a representative non-Elk-1 target, c-jun, was not. These findings emphasize that glutamate not only serves as a neurotransmitter for cell surface receptors but, when transported into the cell, can also activate intracellular receptors such as mGluR5. Glutamate activation of intracellular mGluR5 serves an important role in the regulation of nuclear Ca(2+), transcriptional activation, and gene expression necessary for physiological processes such as synaptic plasticity.

  11. 14-3-3 Proteins Buffer Intracellular Calcium Sensing Receptors to Constrain Signaling.

    Directory of Open Access Journals (Sweden)

    Michael P Grant

    Full Text Available Calcium sensing receptors (CaSR interact with 14-3-3 binding proteins at a carboxyl terminal arginine-rich motif. Mutations identified in patients with familial hypocalciuric hypercalcemia, autosomal dominant hypocalcemia, pancreatitis or idiopathic epilepsy support the functional importance of this motif. We combined total internal reflection fluorescence microscopy and biochemical approaches to determine the mechanism of 14-3-3 protein regulation of CaSR signaling. Loss of 14-3-3 binding caused increased basal CaSR signaling and plasma membrane levels, and a significantly larger signaling-evoked increase in plasma membrane receptors. Block of core glycosylation with tunicamycin demonstrated that changes in plasma membrane CaSR levels were due to differences in exocytic rate. Western blotting to quantify time-dependent changes in maturation of expressed wt CaSR and a 14-3-3 protein binding-defective mutant demonstrated that signaling increases synthesis to maintain constant levels of the immaturely and maturely glycosylated forms. CaSR thus operates by a feed-forward mechanism, whereby signaling not only induces anterograde trafficking of nascent receptors but also increases biosynthesis to maintain steady state levels of net cellular CaSR. Overall, these studies suggest that 14-3-3 binding at the carboxyl terminus provides an important buffering mechanism to increase the intracellular pool of CaSR available for signaling-evoked trafficking, but attenuates trafficking to control the dynamic range of responses to extracellular calcium.

  12. Opposing Roles of Calcium and Intracellular ATP on Gating of the Purinergic P2X2 Receptor Channel

    Directory of Open Access Journals (Sweden)

    Milos B. Rokic

    2018-04-01

    Full Text Available P2X2 receptors (P2X2R exhibit a slow desensitization during the initial ATP application and a progressive, calcium-dependent increase in rates of desensitization during repetitive stimulation. This pattern is observed in whole-cell recordings from cells expressing recombinant and native P2X2R. However, desensitization is not observed in perforated-patched cells and in two-electrode voltage clamped oocytes. Addition of ATP, but not ATPγS or GTP, in the pipette solution also abolishes progressive desensitization, whereas intracellular injection of apyrase facilitates receptor desensitization. Experiments with injection of alkaline phosphatase or addition of staurosporine and ATP in the intracellular solution suggest a role for a phosphorylation-dephosphorylation in receptor desensitization. Mutation of residues that are potential phosphorylation sites identified a critical role of the S363 residue in the intracellular ATP action. These findings indicate that intracellular calcium and ATP have opposing effects on P2X2R gating: calcium allosterically facilitates receptor desensitization and ATP covalently prevents the action of calcium. Single cell measurements further revealed that intracellular calcium stays elevated after washout in P2X2R-expressing cells and the blockade of mitochondrial sodium/calcium exchanger lowers calcium concentrations during washout periods to basal levels, suggesting a role of mitochondria in this process. Therefore, the metabolic state of the cell can influence P2X2R gating.

  13. Antigen-B Cell Receptor Complexes Associate with Intracellular major histocompatibility complex (MHC) Class II Molecules*

    Science.gov (United States)

    Barroso, Margarida; Tucker, Heidi; Drake, Lisa; Nichol, Kathleen; Drake, James R.

    2015-01-01

    Antigen processing and MHC class II-restricted antigen presentation by antigen-presenting cells such as dendritic cells and B cells allows the activation of naïve CD4+ T cells and cognate interactions between B cells and effector CD4+ T cells, respectively. B cells are unique among class II-restricted antigen-presenting cells in that they have a clonally restricted antigen-specific receptor, the B cell receptor (BCR), which allows the cell to recognize and respond to trace amounts of foreign antigen present in a sea of self-antigens. Moreover, engagement of peptide-class II complexes formed via BCR-mediated processing of cognate antigen has been shown to result in a unique pattern of B cell activation. Using a combined biochemical and imaging/FRET approach, we establish that internalized antigen-BCR complexes associate with intracellular class II molecules. We demonstrate that the M1-paired MHC class II conformer, shown previously to be critical for CD4 T cell activation, is incorporated selectively into these complexes and loaded selectively with peptide derived from BCR-internalized cognate antigen. These results demonstrate that, in B cells, internalized antigen-BCR complexes associate with intracellular MHC class II molecules, potentially defining a site of class II peptide acquisition, and reveal a selective role for the M1-paired class II conformer in the presentation of cognate antigen. These findings provide key insights into the molecular mechanisms used by B cells to control the source of peptides charged onto class II molecules, allowing the immune system to mount an antibody response focused on BCR-reactive cognate antigen. PMID:26400081

  14. Estradiol coupling to human monocyte nitric oxide release is dependent on intracellular calcium transients: evidence for an estrogen surface receptor.

    Science.gov (United States)

    Stefano, G B; Prevot, V; Beauvillain, J C; Fimiani, C; Welters, I; Cadet, P; Breton, C; Pestel, J; Salzet, M; Bilfinger, T V

    1999-10-01

    We tested the hypothesis that estrogen acutely stimulates constitutive NO synthase (cNOS) activity in human peripheral monocytes by acting on an estrogen surface receptor. NO release was measured in real time with an amperometric probe. 17beta-estradiol exposure to monocytes stimulated NO release within seconds in a concentration-dependent manner, whereas 17alpha-estradiol had no effect. 17beta-estradiol conjugated to BSA (E2-BSA) also stimulated NO release, suggesting mediation by a membrane surface receptor. Tamoxifen, an estrogen receptor inhibitor, antagonized the action of both 17beta-estradiol and E2-BSA, whereas ICI 182,780, a selective inhibitor of the nuclear estrogen receptor, had no effect. We further showed, using a dual emission microfluorometry in a calcium-free medium, that the 17beta-estradiol-stimulated release of monocyte NO was dependent on the initial stimulation of intracellular calcium transients in a tamoxifen-sensitive process. Leeching out the intracellular calcium stores abolished the effect of 17beta-estradiol on NO release. RT-PCR analysis of RNA obtained from the cells revealed a strong estrogen receptor-alpha amplification signal and a weak beta signal. Taken together, a physiological dose of estrogen acutely stimulates NO release from human monocytes via the activation of an estrogen surface receptor that is coupled to increases in intracellular calcium.

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

    Science.gov (United States)

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

    2015-10-01

    During early development of the peripheral nervous system, Schwann cell precursors proliferate, migrate, and differentiate into premyelinating Schwann cells. After birth, Schwann cells envelop neuronal axons with myelin sheaths. Although some molecular mechanisms underlying myelination by Schwann cells have been identified, the whole picture remains unclear. Here we show that signaling through Tyro3 receptor tyrosine kinase and its binding partner, Fyn nonreceptor cytoplasmic tyrosine kinase, is involved in myelination by Schwann cells. Impaired formation of myelin segments is observed in Schwann cell neuronal cultures established from Tyro3-knockout mouse dorsal root ganglia (DRG). Indeed, Tyro3-knockout mice exhibit reduced myelin thickness. By affinity chromatography, Fyn was identified as the binding partner of the Tyro3 intracellular domain, and activity of Fyn is down-regulated in Tyro3-knockout mice, suggesting that Tyro3, acting through Fyn, regulates myelination. Ablating Fyn in mice results in reduced myelin thickness. Decreased myelin formation is observed in cultures established from Fyn-knockout mouse DRG. Furthermore, decreased kinase activity levels and altered expression of myelination-associated transcription factors are observed in these knockout mice. These results suggest the involvement of Tyro3 receptor and its binding partner Fyn in Schwann cell myelination. This constitutes a newly recognized receptor-linked signaling mechanism that can control Schwann cell myelination. © 2015 Miyamoto et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  16. Growth hormone induction of lactogenic receptors at intracellular sites in male rat liver

    International Nuclear Information System (INIS)

    Norstedt, G.; Andersson, G.; Gustafsson, J.A.

    1984-01-01

    Male and female rat livers were fractionated by density gradient centrifugation into Golgi I (mainly secretory vesicles), Golgi II (mainly cisternal elements), and lysosomes. Estimations of fraction purity and representativity were made by marker enzyme and electron microscopic analyses. The binding of [ 125 I]iodo-human GH ( [ 125 I]iodo-hGH) to different subcellular liver fractions were studied. In Golgi I and II the binding specificity was similar in both sexes and indicated that [ 125 I] iodo-hGH binds to a lactogenic receptor. Scatchard analysis showed a larger number of binding sites in female Golgi I (5600 fmol/mg protein), Golgi II (3400 fmol/mg), and lysosomes (1300 fmol/mg) than in male Golgi I (240 fmol/mg), Golgi II (200 fmol/mg), and lysosomes (230 fmol/mg). The apparent dissociation constant was within a similar range (0.6-0.7 X 10(-9) M) in all fractions. Administration of hGH to male rats by continuous infusion (infusion rate, 5 micrograms/h) resulted, after 5 days of treatment, in an increase in the number of lactogenic binding sites in Golgi I and II to levels similar to the binding in the corresponding female Golgi fractions. When rat GH was given to hypophysectomized male rats (infusion rate, 10 micrograms/h) for 1 week, the binding of [ 125 I]iodo-hGH in lysosomal and Golgi fractions was increased to a female level. The present results suggest that lactogenic receptors are located in the Golgi complex as well as the lysosomal compartment and that these receptors can be induced at these intracellular sites with both a somatotropic-lactogenic hormone (human GH) and a pure somatotropic hormone (rat GH)

  17. The human gonadotropin releasing hormone type I receptor is a functional intracellular GPCR expressed on the nuclear membrane.

    Directory of Open Access Journals (Sweden)

    Michelle Re

    Full Text Available The mammalian type I gonadotropin releasing hormone receptor (GnRH-R is a structurally unique G protein-coupled receptor (GPCR that lacks cytoplasmic tail sequences and displays inefficient plasma membrane expression (PME. Compared to its murine counterparts, the primate type I receptor is inefficiently folded and retained in the endoplasmic reticulum (ER leading to a further reduction in PME. The decrease in PME and concomitant increase in intracellular localization of the mammalian GnRH-RI led us to characterize the spatial distribution of the human and mouse GnRH receptors in two human cell lines, HEK 293 and HTR-8/SVneo. In both human cell lines we found the receptors were expressed in the cytoplasm and were associated with the ER and nuclear membrane. A molecular analysis of the receptor protein sequence led us to identify a putative monopartite nuclear localization sequence (NLS in the first intracellular loop of GnRH-RI. Surprisingly, however, neither the deletion of the NLS nor the addition of the Xenopus GnRH-R cytoplasmic tail sequences to the human receptor altered its spatial distribution. Finally, we demonstrate that GnRH treatment of nuclei isolated from HEK 293 cells expressing exogenous GnRH-RI triggers a significant increase in the acetylation and phosphorylation of histone H3, thereby revealing that the nuclear-localized receptor is functional. Based on our findings, we conclude that the mammalian GnRH-RI is an intracellular GPCR that is expressed on the nuclear membrane. This major and novel discovery causes us to reassess the signaling potential of this physiologically and clinically important receptor.

  18. Intracellular expression of toll-like receptor 4 in neuroblastoma cells and their unresponsiveness to lipopolysaccharide

    Directory of Open Access Journals (Sweden)

    Mori Isamu

    2006-12-01

    Full Text Available Abstract Background Recently it has been reported that, toll-like receptors (TLRs are expressed on a series of tumor cells, such as colon cancer, breast cancer, prostate cancer, melanoma and lung cancer. Although some cancer cells like melanoma cells are known to respond to lipopolysaccharide (LPS via TLR4, not all cancer cells are positive for TLR4. There is little information on the expression and function of TLR4 in neuroblastoma cells. In this study, we investigated the expression of TLR4 in human neuroblastoma NB-1 cell line. Methods Expression and localization of TLR4 were detected by reverse transcription-polymerase chain reaction (RT-PCR and flow cytometric analysis, respectively. Activation of nuclear factor (NF-κB by LPS was detected by degradation of IκB-α and NF-κB luciferase assay. Activation and expression of mitogen-activated protein (MAP kinase and interferon regulatory factor (IRF-3 was detected by immunoblot analysis. Results Human NB-1 neuroblastoma cells expressed intracellular form of TLR4, but not the cell surface form. Further, NB-1 cells express CD14, MD2 and MyD88, which are required for LPS response. However, LPS did not significantly induce NF-κB activation in NB-1 cells although it slightly degraded IκB-α. NB-1 cells expressed no IRF-3, which plays a pivotal role on the MyD88-independent pathway of LPS signaling. Collectively, NB-1 cells are capable to avoid their response to LPS. Conclusion Although human NB-1 neuroblastoma cells possessed all the molecules required for LPS response, they did not respond to LPS. It might be responsible for intracellular expression of TLR4 or lack of IRF-3.

  19. Honey bee dopamine and octopamine receptors linked to intracellular calcium signaling have a close phylogenetic and pharmacological relationship.

    Directory of Open Access Journals (Sweden)

    Kyle T Beggs

    Full Text Available BACKGROUND: Three dopamine receptor genes have been identified that are highly conserved among arthropod species. One of these genes, referred to in honey bees as Amdop2, shows a close phylogenetic relationship to the a-adrenergic-like octopamine receptor family. In this study we examined in parallel the functional and pharmacological properties of AmDOP2 and the honey bee octopamine receptor, AmOA1. For comparison, pharmacological properties of the honey bee dopamine receptors AmDOP1 and AmDOP3, and the tyramine receptor AmTYR1, were also examined. METHODOLOGY/PRINCIPAL FINDINGS: Using HEK293 cells heterologously expressing honey bee biogenic amine receptors, we found that activation of AmDOP2 receptors, like AmOA1 receptors, initiates a rapid increase in intracellular calcium levels. We found no evidence of calcium signaling via AmDOP1, AmDOP3 or AmTYR1 receptors. AmDOP2- and AmOA1-mediated increases in intracellular calcium were inhibited by 10 µM edelfosine indicating a requirement for phospholipase C-β activity in this signaling pathway. Edelfosine treatment had no effect on AmDOP2- or AmOA1-mediated increases in intracellular cAMP. The synthetic compounds mianserin and epinastine, like cis-(Z-flupentixol and spiperone, were found to have significant antagonist activity on AmDOP2 receptors. All 4 compounds were effective antagonists also on AmOA1 receptors. Analysis of putative ligand binding sites offers a possible explanation for why epinastine acts as an antagonist at AmDOP2 receptors, but fails to block responses mediated via AmDOP1. CONCLUSIONS/SIGNIFICANCE: Our results indicate that AmDOP2, like AmOA1, is coupled not only to cAMP, but also to calcium-signalling and moreover, that the two signalling pathways are independent upstream of phospholipase C-β activity. The striking similarity between the pharmacological properties of these 2 receptors suggests an underlying conservation of structural properties related to receptor

  20. Activation of muscarinic acetylcholine receptors elevates intracellular Ca(2+) concentrations in accessory lobe neurons of the chick.

    Science.gov (United States)

    Takahashi, Keita; Kitamura, Naoki; Suzuki, Yuki; Yamanaka, Yuko; Shinohara, Hikaru; Shibuya, Izumi

    2015-04-01

    Accessory lobes are protrusions located at the lateral sides of the spinal cord of chicks and it has been proposed that they play a role as a sensory organ for equilibrium during walking. We have reported that functional neurons exist in the accessory lobe. As there is histological evidence that synaptic terminals of cholinergic nerves exist near the somata of accessory lobe neurons, we examined the effects of acetylcholine on changes in intracellular Ca2+ concentrations ([Ca2+]i), as an index of cellular activities. Acetylcholine (0.1-100 µM) caused a transient rise in the [Ca2+]i. Acetylcholine-evoked [Ca2+]i rises were observed in the absence of extracellular Ca2+, and they were abolished in the presence of cyclopiazonic acid, an inhibitor of Ca2+-ATPase of intracellular Ca2+ stores or atropine, a muscarinic receptor antagonist. mRNAs coding M3 and M5 isoforms of the muscarinic receptors were detected in accessory lobes by the RT-PCR. These results indicate that chick accessory lobe neurons express functional muscarinic acetylcholine receptors, and that acetylcholine stimulates Ca2+ mobilization from intracellular Ca2+ stores, which elevates the [Ca2+]i in the somata of accessory lobe neurons, through activation of these receptors. Cholinergic synaptic transmission to the accessory lobe neurons may regulate some cellular functions through muscarinic receptors.

  1. Proximal tubule-dominant transfer of AT1a receptors induces blood pressure responses to intracellular angiotensin II in AT1a receptor-deficient mice

    Science.gov (United States)

    Li, Xiao C.

    2013-01-01

    The role of intracellular ANG II in proximal tubules of the kidney remains poorly understood. We tested the hypothesis that proximal tubule-dominant transfer of AT1a receptors in the cortex mediates intracellular ANG II-induced blood pressure responses in AT1a receptor-deficient (Agtr1a-/-) mice. A GFP-tagged AT1a receptor, AT1aR/GFP, and an enhanced cyan fluorescent intracellular ANG II fusion protein, ECFP/ANG II, were expressed in proximal tubules of Agtr1a-/- mouse kidneys via the adenoviral transfer using a sodium and glucose cotransporter 2 promoter. Transfer of AT1aR/GFP alone or with ECFP/ANG II induced proximal tubule-dominant expression of AT1aR/GFP and/or ECFP/ANG II with a peak response at 2 wk. No significant AT1aR/GFP and/or ECFP/ANG II expression was observed in the glomeruli, medulla, or extrarenal tissues. Transfer of AT1aR/GFP alone, but not ECFP/ANG II, increased systolic blood pressure by 12 ± 2 mmHg by day 14 (n = 9, P tubule-dominant transfer of AT1aR/GFP alone (P tubules (P tubules had no effect on all indices. These results suggest that AT1a receptors and intracellular ANG II in proximal tubules of the kidney play an important physiological role in blood pressure regulation. PMID:23427083

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

    Science.gov (United States)

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

    2015-01-01

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

  3. Minireview: Role of Intracellular Scaffolding Proteins in the Regulation of Endocrine G Protein-Coupled Receptor Signaling

    Science.gov (United States)

    Walther, Cornelia

    2015-01-01

    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. PMID:25942107

  4. Intracellular colocalization of HAP1/STBs with steroid hormone receptors and its enhancement by a proteasome inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Fujinaga, Ryutaro; Takeshita, Yukio; Yoshioka, Kazuhiro; Nakamura, Hiroyuki; Shinoda, Shuhei; Islam, Md. Nabiul; Jahan, Mir Rubayet; Yanai, Akie; Kokubu, Keiji; Shinoda, Koh, E-mail: shinoda@yamaguchi-u.ac.jp

    2011-07-15

    The stigmoid body (STB) is a cytoplasmic inclusion containing huntingtin-associated protein 1 (HAP1), and HAP1/STB formation is induced by transfection of the HAP1 gene into cultured cells. In the present study, we examined the intracellular colocalization of HAP1/STBs with steroid hormone receptors (SHRs), including the androgen receptor (AR), estrogen receptor, glucocorticoid receptor (GR), and mineralocorticoid receptor, in COS-7 cells cotransfected with HAP1 and each receptor. We found that C-terminal ligand-binding domains of all SHRs had potential for colocalization with HAP1/STBs, whereas only AR and GR were clearly colocalized with HAP1/STBs when each full-length SHR was coexpressed with HAP1. In addition, it appeared that HAP1/STBs did not disrupt GR and AR functions because the receptors on HAP1/STBs maintained nuclear translocation activity in response to their specific ligands. When the cells were treated with a proteasome inhibitor, GR and AR localized outside HAP1/STBs translocated into the nucleus, whereas the receptors colocalized with HAP1/STBs persisted in their colocalization even after treatment with their ligands. Therefore, HAP1/STBs may be involved in cytoplasmic modifications of the nuclear translocation of GR and AR in a ubiquitin-proteasome system.

  5. Ontogenic and sexual differences in pituitary GnRH receptors and intracellular Ca2+ mobilization induced by GnRH.

    Science.gov (United States)

    Lacau-Mengido, I M; González Iglesias, A; Lux-Lantos, V; Libertun, C; Becú-Villalobos, D

    1998-04-01

    The present experiments were designed in order to elucidate the participation of the developing hypophysis in determining the changing sensitivity of gonadotrophins to gonadotropin-releasing hormone (GnRH) during ontogeny in the rat. To that end, we chose two well defined developmental ages that differ markedly in sexual and ontogenic characteristics of hypophyseal sensitivity to GnRH, 15 and 30 d. In order to study sex differences and the role of early sexual organization of the hypothalamus, experiments were carried out in males, females, and neonatally androgenized females (TP females). We evaluated (1) the characteristics of pituitary GnRH receptors, and (2) associated changes in GnRH-induced mobilization of intracellular Ca2+ (a second messenger involved in gonadotropins exocytosis). We measured binding characteristics of the GnRH analog D-Ser(TBu)6-des-Gly10-GnRH ethylamide in pituitary homogenates. We found that Kds did not vary among the different sex groups. Total number and concentration of receptors decreased in the female rat from 15-30 d of age, whereas in the male and TP female, receptors/pituitary increased, and the concentration/mg tissue did not change. Also, at 30 days of age, males presented higher content and concentration of receptors than females, and higher content than TP females. In order to evaluate if developmental and sexual differences in pituitary sensitivity to GnRH might be expressed through variations in the intracellular Ca2+ signal, we studied the mobilization of intracellular Ca2+ induced by GnRH (1 x 10(-8) to 1 x 10(-11) M) in a suspension of dispersed pituitary cells in the six groups. In cells from 15-d-old females, Ca2+ response was greater than in 30-d-old females at the doses of 10(-8) to 10(-10) M, indicating that in the infantile female rat activation of highly concentrated GnRH receptors is reflected in an increase in signal transduction mediated by Ca2+. In males and in female rats androgenized at birth, there was also

  6. Internalisation of the protease-activated receptor 1: role of the third intracellular loop and of the cytoplasmic tail.

    Science.gov (United States)

    Chen, X; Berrou, J; Vigneau, C; Delarue, F; Rondeau, E

    2001-06-01

    To analyse the mechanisms of PAR-1 internalisation, we constructed several PAR-1 mutants and stably expressed them in CHO cells. Our study shows that the Ser(306)-->Ala mutation (S306A), which eliminates a potential site of phosphorylation by PKC in the third intracellular loop of PAR-1, did not change the rate of phosphorylation but reduced the rate of thrombin-induced internalisation of the PAR-1 mutant (58 versus 78% of membrane PAR-1 in 15 min, pinternalisation upon activation. This deletion also inhibited the PMA-induced and the agonist-independent internalisation of the receptor. The Tyr(371)--> Ala mutation (Y371A), in a NPXXY motif of the seventh transmembrane domain of the receptor had no effect on the receptor behaviour. Our results indicate that both the C-tail and the third intracellular loop are involved in PAR-1 internalisation induced by thrombin while only the C-tail plays a role in the PMA-induced and in the agonist-independent PAR-1 internalisation.

  7. Weekly Treatment of 2-Hydroxypropyl-β-cyclodextrin Improves Intracellular Cholesterol Levels in LDL Receptor Knockout Mice

    Directory of Open Access Journals (Sweden)

    Sofie M. A. Walenbergh

    2015-09-01

    Full Text Available Recently, the importance of lysosomes in the context of the metabolic syndrome has received increased attention. Increased lysosomal cholesterol storage and cholesterol crystallization inside macrophages have been linked to several metabolic diseases, such as atherosclerosis and non-alcoholic fatty liver disease (NAFLD. Two-hydroxypropyl-β-cyclodextrin (HP-B-CD is able to redirect lysosomal cholesterol to the cytoplasm in Niemann-Pick type C1 disease, a lysosomal storage disorder. We hypothesize that HP-B-CD ameliorates liver cholesterol and intracellular cholesterol levels inside Kupffer cells (KCs. Hyperlipidemic low-density lipoprotein receptor knockout (Ldlr−/− mice were given weekly, subcutaneous injections with HP-B-CD or control PBS. In contrast to control injections, hyperlipidemic mice treated with HP-B-CD demonstrated a shift in intracellular cholesterol distribution towards cytoplasmic cholesteryl ester (CE storage and a decrease in cholesterol crystallization inside KCs. Compared to untreated hyperlipidemic mice, the foamy KC appearance and liver cholesterol remained similar upon HP-B-CD administration, while hepatic campesterol and 7α-hydroxycholesterol levels were back increased. Thus, HP-B-CD could be a useful tool to improve intracellular cholesterol levels in the context of the metabolic syndrome, possibly through modulation of phyto- and oxysterols, and should be tested in the future. Additionally, these data underline the existence of a shared etiology between lysosomal storage diseases and NAFLD.

  8. Intracellular calcium levels determine differential modulation of allosteric interactions within G protein-coupled receptor heteromers

    NARCIS (Netherlands)

    Navarro, G.; Aguinaga, D.; Hradsky, J.; Moreno, E.; Reddy, P.P.; Cortés, A.; Mallol, J.; Casadó, V.; Mikhaylova, Marina; Kreutz, M.R.; Lluís, C.; Canela, E.I.; McCormick, P.J.; Ferreira, S.; Ferré, S.

    2014-01-01

    The pharmacological significance of the adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromer is well established and it is being considered as an important target for the treatment of Parkinson’s disease and other neuropsychiatric disorders. However, the physiological factors that

  9. Nod factor receptors form heteromeric complexes and are essential for intracellular infection in Medicago nodules

    NARCIS (Netherlands)

    Moling, S.; Pietraszewska-Bogiel, A.; Postma, M.; Fedorova, E.E.; Hink, M.A.; Limpens, E.H.M.; Gadella, T.W.J.; Bisseling, T.

    2014-01-01

    Rhizobial Nod factors are the key signaling molecules in the legume-rhizobium nodule symbiosis. In this study, the role of the Nod factor receptors NOD FACTOR PERCEPTION (NFP) and LYSIN MOTIF RECEPTOR-LIKE KINASE3 (LYK3) in establishing the symbiotic interface in root nodules was investigated. It

  10. Nod factor receptors form heteromeric complexes and are essential for intracellular infection in medicago nodules

    NARCIS (Netherlands)

    Moling, S.; Pietraszewska-Bogiel, A.; Postma, M.; Fedorova, E.; Hink, M.A.; Limpens, E.; Gadella, T.W.J.; Bisseling, T.

    2014-01-01

    Rhizobial Nod factors are the key signaling molecules in the legume-rhizobium nodule symbiosis. In this study, the role of the Nod factor receptors NOD FACTOR PERCEPTION (NFP) and LYSIN MOTIF RECEPTOR-LIKE KINASE3 (LYK3) in establishing the symbiotic interface in root nodules was investigated. It

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

    Directory of Open Access Journals (Sweden)

    Belén Mezquita

    2014-02-01

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

  12. The cannabinoid agonist WIN55,212-2 increases intracellular calcium via CB1 receptor coupling to Gq/11 G proteins

    OpenAIRE

    Lauckner, Jane E.; Hille, Bertil; Mackie, Ken

    2005-01-01

    Central nervous system responses to cannabis are primarily mediated by CB1 receptors, which couple preferentially to Gi/o G proteins. Here, we used calcium photometry to monitor the effect of CB1 activation on intracellular calcium concentration. Perfusion with 5 μM CB1 aminoalkylindole agonist, WIN55,212-2 (WIN), increased intracellular calcium by several hundred nanomolar in human embryonic kidney 293 cells stably expressing CB1 and in cultured hippocampal neurons. The increase was blocked ...

  13. Discovery and mapping of an intracellular antagonist binding site at the chemokine receptor CCR2

    DEFF Research Database (Denmark)

    Zweemer, Annelien J M; Bunnik, Julia; Veenhuizen, Margo

    2014-01-01

    The chemokine receptor CCR2 is a G protein-coupled receptor that is involved in many diseases characterized by chronic inflammation, and therefore a large variety of CCR2 small molecule antagonists has been developed. On the basis of their chemical structures these antagonists can roughly...... be divided into two groups with most likely two topographically distinct binding sites. The aim of the current study was to identify the binding site of one such group of ligands, exemplified by three allosteric antagonists, CCR2-RA-[R], JNJ-27141491, and SD-24. We first used a chimeric CCR2/CCR5 receptor...... approach to obtain insight into the binding site of the allosteric antagonists and additionally introduced eight single point mutations in CCR2 to further characterize the putative binding pocket. All constructs were studied in radioligand binding and/or functional IP turnover assays, providing evidence...

  14. Importance of constitutive activity and arrestin-independent mechanisms for intracellular trafficking of the ghrelin receptor

    DEFF Research Database (Denmark)

    Holliday, Nicholas D; Holst, Birgitte; Rodionova, Elena A

    2007-01-01

    and substantially decreased agonist-induced internalization in transiently transfected HEK293 cells. Internalized GhrelinR and GhR-39 were predominantly localized to recycling compartments, identified with transferrin and the monomeric G proteins Rab5 and Rab11. Both the inverse agonist [d-Arg(1), d-Phe(5), d-Trp(7....... Furthermore the interaction between phosphorylated receptors and beta-arrestin adaptor proteins has been examined. Replacement of the FLAG-tagged GhrelinR C tail with the equivalent GPR39 domain (GhR-39 chimera) preserved G(q) signaling. However in contrast to the GhrelinR, GhR-39 receptors exhibited no basal....... In contrast, agonist-stimulated GhrelinRs recruited the clathrin adaptor green fluorescent protein-tagged beta-arrestin2 to endosomes, coincident with increased receptor phosphorylation. Thus, GhrelinR internalization to recycling compartments depends on C-terminal motifs and constitutive activity...

  15. Evidence that leishmania donovani utilizes a mannose receptor on human mononuclear phagocytes to establish intracellular parasitism

    International Nuclear Information System (INIS)

    Wilson, M.E.; Pearson, R.D.

    1986-01-01

    The pathogenic protozoan Leishmania donovani must gain entrance into mononuclear phagocytes to successfully parasitize man. The parasite's extracellular promastigote stage is ingested by human peripheral blood monocytes or monocyte-derived macrophages in the absence of serum, in a manner characteristic of receptor-mediated endocytosis. Remarkable similarities have been found between the macrophage receptor(s) for promastigotes and a previously characterized eucaryotic receptor system, the mannose/fucose receptor (MFR), that mediates the binding of zymosan particles and mannose- or fucose-terminal glycoconjugates to macrophages. Ingestion of promastigotes by monocyte-derived macrophages was inhibited by several MFR ligands; that is mannan, mannose-BSA and fucose-BSA. In contrast, promastigote ingestion by monocytes was unaffected by MFR ligands. Furthermore, attachment of promastigotes to macrophages, assessed by using cytochalasin D to prevent phagocytosis, was reduced 49.8% by mannan. Reorientation of the MFR to the ventral surface of the cell was achieved by plating macrophages onto mannan-coated coverslips, reducing MFR activity on the exposed cell surface by 94% as assessed by binding of 125 I-mannose-BSA. Under these conditions, ingestion of promastigotes was inhibited by 71.4%. Internalization of the MFR by exposure of macrophages to zymosan before infection with promastigotes resulted in a 62.3% decrease in parasite ingestion. Additionally, NH 4 Cl decreased macrophage ingestion of promastigotes by 38.2%. Subinhibitory concentration of NH 4 Cl (10 mM) and of mannan (0.25 mg/ml) together inhibited parsite ingestion by 76.4%

  16. Hiding from intracellular pattern recognition receptors, a passive strategy of flavivirus immune evasion.

    Science.gov (United States)

    Overby, Anna K; Weber, Friedemann

    2011-01-01

    Tick-borne encephalitis virus (TBEV) is a medically important flavivirus in Europe and Asia, causing meningitis and encephalitis in thousands of people annually. Despite its relevance for public health, the interaction of TBEV with the type I interferon (IFN) system is poorly characterized. Induction of these antiviral cytokines is normally triggered by cytoplasmic recognition of viral signature molecules such as double-stranded (ds) RNA. In a recent paper, we showed that TBEV infection leads to formation of intracellular membrane vesicles which protect the viral dsRNA from cellular recognition. This delays the onset of antiviral IFN production sufficiently enough for an unhindered release of progeny viruses over 24 h. Thus, TBEV has evolved a stealth strategy to outrun the antiviral IFN response.

  17. Membrane Estrogen Receptor-α Interacts with Metabotropic Glutamate Receptor Type 1a to Mobilize Intracellular Calcium in Hypothalamic Astrocytes

    Science.gov (United States)

    Kuo, John; Hariri, Omid R.; Bondar, Galyna; Ogi, Julie; Micevych, Paul

    2009-01-01

    Estradiol, acting on a membrane-associated estrogen receptor-α (mERα), induces an increase in free cytoplasmic calcium concentration ([Ca2+]i) needed for progesterone synthesis in hypothalamic astrocytes. To determine whether rapid estradiol signaling involves an interaction of mERα with metabotropic glutamate receptor type 1a (mGluR1a), changes in [Ca2+]i were monitored with the calcium indicator, Fluo-4 AM, in primary cultures of female postpubertal hypothalamic astrocytes. 17β-Estradiol over a range of 1 nm to 100 nm induced a maximal increase in [Ca2+]i flux measured as a change in relative fluorescence [ΔF Ca2+ = 615 ± 36 to 641 ± 47 relative fluorescent units (RFU)], whereas 0.1 nm of estradiol stimulated a moderate [Ca2+]i increase (275 ± 16 RFU). The rapid estradiol-induced [Ca2+]i flux was blocked with 1 μm of the estrogen receptor antagonist ICI 182,780 (635 ± 24 vs. 102 ± 11 RFU, P estradiol-induced membrane signaling in astrocytes. PMID:18948402

  18. The Role of Sigma-1 Receptor, an Intracellular Chaperone in Neurodegenerative Diseases.

    Science.gov (United States)

    Penke, Botond; Fulop, Livia; Szucs, Maria; Frecska, Ede

    2018-01-01

    Widespread protein aggregation occurs in the living system under stress or during aging, owing to disturbance of endoplasmic reticulum (ER) proteostasis. Many neurodegenerative diseases may have a common mechanism: the failure of protein homeostasis. Perturbation of ER results in unfolded protein response (UPR). Prolonged chronical UPR may activate apoptotic pathways and cause cell death. Research articles on Sigma-1 receptor were reviewed. ER is associated to mitochondria by the mitochondria-associated ER-membrane, MAM. The sigma-1 receptor (Sig-1R), a well-known ER-chaperone localizes in the MAM. It serves for Ca2+-signaling between the ER and mitochondria, involved in ion channel activities and especially important during neuronal differentiation. Sig-1R acts as central modulator in inter-organelle signaling. Sig-1R helps cell survival by attenuating ER-stress. According to sequence based predictions Sig-1R is a 223 amino acid protein with two transmembrane (2TM) domains. The X-ray structure of the Sig-1R [1] showed a membrane-bound trimeric assembly with one transmembrane (1TM) region. Despite the in vitro determined assembly, the results of in vivo studies are rather consistent with the 2TM structure. The receptor has unique and versatile pharmacological profile. Dimethyl tryptamine (DMT) and neuroactive steroids are endogenous ligands that activate Sig-1R. The receptor has a plethora of interacting client proteins. Sig-1R exists in oligomeric structures (dimer-trimer-octamer-multimer) and this fact may explain interaction with diverse proteins. Sig-1R agonists have been used in the treatment of different neurodegenerative diseases, e.g. Alzheimer's and Parkinson's diseases (AD and PD) and amyotrophic lateral sclerosis. Utilization of Sig-1R agents early in AD and similar other diseases has remained an overlooked therapeutic opportunity. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  19. Structural analysis of the intracellular domain of (pro)renin receptor fused to maltose-binding protein

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yanfeng; Gao, Xiaoli [Department of Biochemistry and Molecular Biology, Michigan State University (United States); Michael Garavito, R., E-mail: garavito@msu.edu [Department of Biochemistry and Molecular Biology, Michigan State University (United States)

    2011-04-22

    Highlights: {yields} Crystal structure of the intracellular domain of (pro)renin receptor (PRR-IC) as MBP fusion protein at 2.0 A (maltose-free) and 2.15 A (maltose-bound). {yields} MBP fusion protein is a dimer in crystals in the presence and absence of maltose. {yields} PRR-IC domain is responsible for the dimerization of the fusion protein. {yields} Residues in the PRR-IC domain, particularly two tyrosines, dominate the intermolecular interactions, suggesting a role for the PRR-IC domain in PRR dimerization. -- Abstract: The (pro)renin receptor (PRR) is an important component of the renin-angiotensin system (RAS), which regulates blood pressure and cardiovascular function. The integral membrane protein PRR contains a large extracellular domain ({approx}310 amino acids), a single transmembrane domain ({approx}20 amino acids) and an intracellular domain ({approx}19 amino acids). Although short, the intracellular (IC) domain of the PRR has functionally important roles in a number of signal transduction pathways activated by (pro)renin binding. Meanwhile, together with the transmembrane domain and a small portion of the extracellular domain ({approx}30 amino acids), the IC domain is also involved in assembly of V{sub 0} portion of the vacuolar proton-translocating ATPase (V-ATPase). To better understand structural and multifunctional roles of the PRR-IC, we report the crystal structure of the PRR-IC domain as maltose-binding protein (MBP) fusion proteins at 2.0 A (maltose-free) and 2.15 A (maltose-bound). In the two separate crystal forms having significantly different unit-cell dimensions and molecular packing, MBP-PRR-IC fusion protein was found to be a dimer, which is different with the natural monomer of native MBP. The PRR-IC domain appears as a relatively flexible loop and is responsible for the dimerization of MBP fusion protein. Residues in the PRR-IC domain, particularly two tyrosines, dominate the intermonomer interactions, suggesting a role for the PRR

  20. Hypertonic saline enhances host response to bacterial challenge by augmenting receptor-independent neutrophil intracellular superoxide formation.

    LENUS (Irish Health Repository)

    Shields, Conor J

    2012-02-03

    OBJECTIVE: This study sought to determine whether hypertonic saline (HTS) infusion modulates the host response to bacterial challenge. METHODS: Sepsis was induced in 30 Balb-C mice by intraperitoneal injection of Escherichia coli (5 x 107 organisms per animal). In 10 mice, resuscitation was performed at 0 and 24 hours with a 4 mL\\/kg bolus of HTS (7.5% NaCl), 10 animals received 4 mL\\/kg of normal saline (0.9% NaCl), and the remaining animals received 30 mL\\/kg of normal saline. Samples of blood, spleen, and lung were cultured at 8 and 36 hours. Polymorphonucleocytes were incubated in isotonic or hypertonic medium before culture with E. coli. Phagocytosis was assessed by flow cytometry, whereas intracellular bacterial killing was measured after inhibition of phagocytosis with cytochalasin B. Intracellular formation of free radicals was assessed by the molecular probe CM-H(2)DCFDA. Mitogen-activated protein (MAP) kinase p38 and ERK-1 phosphorylation, and nuclear factor kappa B (NFkappaB) activation were determined. Data are represented as means (SEM), and an analysis of variance test was performed to gauge statistical significance. RESULTS: Significantly reduced bacterial culture was observed in the animals resuscitated with HTS when compared with their NS counterparts, in blood (51.8 +\\/- 4.3 vs. 82.0 +\\/- 3.3 and 78.4 +\\/- 4.8, P = 0.005), lung (40.0 +\\/- 4.1 vs. 93.2 +\\/- 2.1 and 80.9 +\\/- 4.7, P = 0.002), and spleen (56.4 +\\/- 3.8 vs. 85.4 +\\/- 4.2 and 90.1 +\\/- 5.9, P = 0.05). Intracellular killing of bacteria increased markedly (P = 0.026) and superoxide generation was enhanced upon exposure to HTS (775.78 +\\/- 23.6 vs. 696.57 +\\/- 42.2, P = 0.017) despite inhibition of MAP kinase and NFkappaB activation. CONCLUSIONS: HTS significantly enhances intracellular killing of bacteria while attenuating receptor-mediated activation of proinflammatory cascades.

  1. Three Basic Residues of Intracellular Loop 3 of the Beta-1 Adrenergic Receptor Are Required for Golgin-160-Dependent Trafficking

    Directory of Open Access Journals (Sweden)

    Catherine E. Gilbert

    2014-02-01

    Full Text Available Golgin-160 is a member of the golgin family of proteins, which have been implicated in the maintenance of Golgi structure and in vesicle tethering. Golgin-160 is atypical; it promotes post-Golgi trafficking of specific cargo proteins, including the β-1 adrenergic receptor (β1AR, a G protein-coupled receptor. Here we show that golgin-160 binds directly to the third intracellular loop of β1AR and that this binding depends on three basic residues in this loop. Mutation of the basic residues does not affect trafficking of β1AR from the endoplasmic reticulum through the Golgi complex, but results in reduced steady-state levels at the plasma membrane. We hypothesize that golgin-160 promotes incorporation of β1AR into specific transport carriers at the trans-Golgi network to ensure efficient delivery to the cell surface. These results add to our understanding of the biogenesis of β1AR, and suggest a novel point of regulation for its delivery to the plasma membrane.

  2. Identification of tyrosine residues in the intracellular domain of the growth hormone receptor required for transcriptional signaling and Stat5 activation

    DEFF Research Database (Denmark)

    Hansen, L. H.; Wang, X.; Kopchick, J J

    1996-01-01

    phosphorylation in intracellular signaling, we constructed GH receptors in which combinations of tyrosines were mutated to phenylalanines. We identified three tyrosine residues at positions 534, 566, and 627 that were required for activation of GH-stimulated transcription of the serine protease inhibitor (Spi) 2...

  3. Growth hormone receptor C-terminal domains required for growth hormone-induced intracellular free Ca2+ oscillations and gene transcription

    DEFF Research Database (Denmark)

    Billestrup, N; Bouchelouche, P; Allevato, G

    1995-01-01

    The biological effects of growth hormone (GH) are initiated by its binding to the GH receptor (GHR) followed by association and activation of the tyrosine kinase JAK2. Here we report that GH can stimulate an increase in intracellular free Ca2+ concentration ([Ca2+]i) in cells expressing wild-type...

  4. IL-6/IL-12 Cytokine Receptor Shuffling of Extra- and Intracellular Domains Reveals Canonical STAT Activation via Synthetic IL-35 and IL-39 Signaling.

    Science.gov (United States)

    Floss, D M; Schönberg, M; Franke, M; Horstmeier, F C; Engelowski, E; Schneider, A; Rosenfeldt, E M; Scheller, J

    2017-11-09

    IL-35 and IL-39 are recently discovered shared members of the IL-6- and IL-12-type cytokine family with immune-suppressive capacity. IL-35 has been reported to induce the formation of four different receptor complexes: gp130:IL-12β2, gp130:gp130, IL-12β2:IL-12β2, and IL-12β2:WSX-1. IL-39 was proposed to form a gp130:IL-23R receptor complex. IL-35, but not IL-39, has been reported to activate non-conventional STAT signaling, depending on the receptor complex and target cell. Analyses of IL-35 and IL-39 are, however, hampered by the lack of biologically active recombinant IL-35 and IL-39 proteins. Therefore, we engineered chimeric cytokine receptors to accomplish synthetic IL-35 and IL- 39 signaling by shuffling the extra- and intracellular domains of IL-6/IL-12-type cytokine receptors, resulting in biological activity for all previously described IL-35 receptor complexes. Moreover, we found that the proposed IL-39 receptor complex is biologically active and discovered two additional biologically active synthetic receptor combinations, gp130/IL-12Rβ1 and IL-23R/IL-12Rβ2. Surprisingly, synthetic IL-35 activation led to more canonical STAT signaling of all receptor complexes. In summary, our receptor shuffling approach highlights an interchangeable, modular domain structure among IL-6- and IL-12-type cytokine receptors and enabled synthetic IL-35 and IL-39 signaling.

  5. Targeting of pegylated liposomal mitomycin-C prodrug to the folate receptor of cancer cells: Intracellular activation and enhanced cytotoxicity.

    Science.gov (United States)

    Patil, Yogita; Amitay, Yasmine; Ohana, Patricia; Shmeeda, Hilary; Gabizon, Alberto

    2016-03-10

    Mitomycin C (MMC) is a powerful anti-bacterial, anti-fungal and anti-tumor antibiotic, often active against multidrug resistant cells. Despite a broad spectrum of antitumor activity, MMC clinical use is relatively limited due to its fast clearance and dose-limiting toxicity. To exploit the potential antitumor activity of MMC and reduce its toxicity we have previously developed a formulation of pegylated liposomes with a lipophilic prodrug of MMC (PL-MLP), activated by endogenous reducing agents which are abundant in the tumor cell environment in the form of different thiols. PL-MLP has minimal in vitro cytotoxicity unless reducing agents are added to the cell culture to activate the prodrug. In the present study, we hypothesized that targeting PL-MLP via folate receptors will facilitate intracellular activation of prodrug and enhance cytotoxic activity without added reducing agents. We grafted a lipophilic folate conjugate (folate-PEG(5000)-DSPE) to formulate folate targeted liposomes (FT-PL-MLP) and examined in vitro cell uptake and cytotoxic activity in cancer cell lines with high folate receptors (HiFR). 3H-cholesterol-hexadecyl ether (3H-Chol)-radiolabeled liposomes were prepared to study liposome-cell binding in parallel to cellular uptake of prodrug MLP. 3H-Chol and MLP cell uptake levels were 4-fold and 9-fold greater in KB HiFR cells when FT-PL-MLP is compared to non-targeted PL-MLP liposomes. The cytotoxic activity of FT-PL-MLP liposomes was significantly increased up to ~5-fold compared with PL-MLP liposomes in all tested HiFR expressing cell lines. The enhanced uptake and intracytoplasmic liposome delivery was confirmed by confocal fluorescence studies with Rhodamine-labeled liposomes. In vivo, no significant differences in pharmacokinetics and biodistribution were observed when PL-MLP was compared to FT-PL-MLP by the intravenous route. However, when liposomes were directly injected into the peritoneal cavity of mice with malignant ascites of J6456 Hi

  6. Mitofusin 2 decreases intracellular lipids in macrophages by regulating peroxisome proliferator-activated receptor

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Chun; Ge, Beihai [Department of Cardiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan 430030 (China); He, Chao [Department of Cardiology, China Three Gorges University, Yichang 433000 (China); Zhang, Yi; Liu, Xiaowen [Department of Cardiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan 430030 (China); Liu, Kejian [Department of Cardiology, The First Affiliated Hospital of Medical College, Shihezi University (China); Qian, Cuiping; Zhang, Yu; Peng, Wenzhong [Department of Cardiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan 430030 (China); Guo, Xiaomei, E-mail: xmguo@tjh.tjmu.edu.cn [Department of Cardiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan 430030 (China)

    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.

  7. Mitofusin 2 decreases intracellular lipids in macrophages by regulating peroxisome proliferator-activated receptor

    International Nuclear Information System (INIS)

    Liu, Chun; Ge, Beihai; He, Chao; Zhang, Yi; Liu, Xiaowen; Liu, Kejian; Qian, Cuiping; Zhang, Yu; Peng, Wenzhong; Guo, Xiaomei

    2014-01-01

    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

  8. Rapid expression of RASD1 is regulated by estrogen receptor-dependent intracellular signaling pathway in the mouse uterus.

    Science.gov (United States)

    Kim, Hye-Ryun; Cho, Kil-Sang; Kim, Eunhye; Lee, Ok-Hee; Yoon, Hyemin; Lee, Sangho; Moon, Sohyeon; Park, Miseon; Hong, Kwonho; Na, Younghwa; Shin, Ji-Eun; Kwon, Hwang; Song, Haengseok; Choi, Dong Hee; Choi, Youngsok

    2017-05-05

    Dexamethasone-induced RAS-related protein 1 (RASD1) is a signaling protein that is involved in various cellular processes. In a previous study, we found that RASD1 expression was down-regulated in the uterine endometrium of repeated implantation failure patients. The study aim was to determine whether RASD1 is expressed in the endometrium of mouse uterus and how it is regulated by steroid hormones during the estrous cycle. In this study, we investigated RASD1 expression and regulation in an ovariectomized female mouse model. Rasd1 mRNA was highly expressed in mouse reproductive tissues, including the uterus. Rasd1 expression was detected exclusively in the endometrial epithelium at the proestrus stage of the estrous cycle. Rasd1 expression in uteri increased with administration of estradiol, but not progesterone. Its expression was rapidly induced within 2 h after E 2 treatment. Pretreatment with ICI 182,780, an estrogen receptor antagonist, reduced RASD1 protein expression. In addition, we identified that rapid expression of Rasd1 was mediated by the estrogen intracellular signaling including both p38-mitogen-activated protein kinase and the extracellular signal-regulated kinase. These findings suggest that RASD1 acts as a novel signaling molecule and plays an important role in regulating dynamic uterine remodeling during the estrous cycle in the uterus. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Functional fusions of T4 lysozyme in the third intracellular loop of a G protein-coupled receptor identified by a random screening approach in yeast.

    Science.gov (United States)

    Mathew, Elizabeth; Ding, Fa-Xiang; Naider, Fred; Dumont, Mark E

    2013-01-01

    The insertion of a stable soluble protein into loops of transmembrane proteins has proved to be a successful approach for enhancing their stabilities and crystallization, and may also be useful in contexts where the inserted proteins can modulate or report on the activities of membrane proteins. While the use of T4 lysozyme to replace portions of the third intracellular loops of G protein-coupled receptors (GPCRs) has allowed determination of the structures of members of this important class of receptors, the creation of such fusion proteins generally leads to loss of signaling function of the resulting fusion protein, since the third intracellular loops of GPCRs play critical roles in their interactions with G proteins. We describe here a random screening approach allowing insertion of T4 lysozyme into diverse positions in the third loop of the yeast α-pheromone receptor, a GPCR encoded by the yeast STE2 gene. Insertions were accompanied by varying extents of deletion or duplication of the loop. A set of phenotypic screens allow detection of potentially rare variant receptors that are expressed, bind to agonist and are capable of signal transduction via activation of the cognate G protein. A large fraction of screened full-length receptor variants containing at least partial duplications of the loop on either side of the inserted T4 lysozyme retain the ability to activate the downstream signaling pathway in response to binding of ligand. However, we were unable to identify any receptors with truncated C-termini that retain significant signaling function in the presence of inserted T4 lysozyme. Our results establish the feasibility of creating functional receptors containing insertions of T4 lysozyme in their third intracellular loops.

  10. The cannabinoid agonist WIN55,212-2 increases intracellular calcium via CB1 receptor coupling to Gq/11 G proteins.

    Science.gov (United States)

    Lauckner, Jane E; Hille, Bertil; Mackie, Ken

    2005-12-27

    Central nervous system responses to cannabis are primarily mediated by CB(1) receptors, which couple preferentially to G(i/o) G proteins. Here, we used calcium photometry to monitor the effect of CB(1) activation on intracellular calcium concentration. Perfusion with 5 microM CB(1) aminoalkylindole agonist, WIN55,212-2 (WIN), increased intracellular calcium by several hundred nanomolar in human embryonic kidney 293 cells stably expressing CB(1) and in cultured hippocampal neurons. The increase was blocked by coincubation with the CB(1) antagonist, SR141716A, and was absent in nontransfected human embryonic kidney 293 cells. The calcium rise was WIN-specific, being essentially absent in cells treated with other classes of cannabinoid agonists, including Delta(9)-tetrahydrocannabinol, HU-210, CP55,940, 2-arachidonoylglycerol, methanandamide, and cannabidiol. The increase in calcium elicited by WIN was independent of G(i/o), because it was present in pertussis toxin-treated cells. Indeed, pertussis toxin pretreatment enhanced the potency and efficacy of WIN to increase intracellular calcium. The calcium increases appeared to be mediated by G(q) G proteins and phospholipase C, because they were markedly attenuated in cells expressing dominant-negative G(q) or treated with the phospholipase C inhibitors U73122 and ET-18-OCH(3) and were accompanied by an increase in inositol phosphates. The calcium increase was blocked by the sarco/endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin, the inositol trisphosphate receptor inhibitor xestospongin D, and the ryanodine receptor inhibitors dantrolene and 1,1'-diheptyl-4,4'-bipyridinium dibromide, but not by removal of extracellular calcium, showing that WIN releases calcium from intracellular stores. In summary, these results suggest that WIN stabilizes CB(1) receptors in a conformation that enables G(q) signaling, thus shifting the G protein specificity of the receptor.

  11. Intracellular signaling of the Ufo/Axl receptor tyrosine kinase is mediated mainly by a multi-substrate docking-site.

    Science.gov (United States)

    Braunger, J; Schleithoff, L; Schulz, A S; Kessler, H; Lammers, R; Ullrich, A; Bartram, C R; Janssen, J W

    1997-06-05

    Ufo/Axl belongs to a new family of receptor tyrosine kinases with an extracellular structure similar to that of neural cell adhesion molecules. In order to elucidate intracellular signaling, the cytoplasmic moiety of Ufo/Axl was used to screen an expression library according to the CORT (cloning of receptor targets) method. Three putative Ufo substrates were identified: phospholipase Cgamma1 (PLCgamma), as well as p85alpha and p85beta subunits of phosphatidylinositol 3'-kinase (PI3-kinase). Subsequently, chimeric EGFR/Ufo receptors consisting of the extracellular domains of the epidermal growth factor receptor (EGFR) and the transmembrane and intracellular moiety of Ufo were engineered. Using different far-Western blot analyses and coimmunoprecipitation assays, receptor binding of PLCgamma and p85 proteins as well as GRB2, c-src and lck was examined in vitro and in vivo. Competitive inhibition of substrate binding and mutagenesis experiments with EGFR/Ufo constructs revealed C-terminal tyrosine 821 (EILpYVNMDEG) as a docking site for multiple effectors, namely PLCgamma, p85 proteins, GRB2, c-src and lck. Tyrosine 779 (DGLpYALMSRC) demonstrated an additional, but lower binding affinity for the p85 proteins in vitro. In addition, binding of PLCgamma occurred through tyrosine 866 (AGRpYVLCPST). Moreover, our in vivo data indicate that further direct or indirect binding sites for PLCgamma, GRB2, c-src and lck on the human Ufo receptor may exist.

  12. Antibody-mediated immunity to the obligate intracellular bacterial pathogen Coxiella burnetii is Fc receptor- and complement-independent

    Directory of Open Access Journals (Sweden)

    Heinzen Robert A

    2009-05-01

    Full Text Available Abstract Background The obligate intracellular bacterial pathogen Coxiella burnetii causes the zoonosis Q fever. The intracellular niche of C. burnetii has led to the assumption that cell-mediated immunity is the most important immune component for protection against this pathogen. However, passive immunization with immune serum can protect naïve animals from challenge with virulent C. burnetii, indicating a role for antibody (Ab in protection. The mechanism of this Ab-mediated protection is unknown. Therefore, we conducted a study to determine whether Fc receptors (FcR or complement contribute to Ab-mediated immunity (AMI to C. burnetii. Results Virulent C. burnetii infects and replicates within human dendritic cells (DC without inducing their maturation or activation. We investigated the effects of Ab opsonized C. burnetii on human monocyte-derived and murine bone marrow-derived DC. Infection of DC with Ab-opsonized C. burnetii resulted in increased expression of maturation markers and inflammatory cytokine production. Bacteria that had been incubated with naïve serum had minimal effect on DC, similar to virulent C. burnetii alone. The effect of Ab opsonized C. burnetii on DC was FcR dependent as evidenced by a reduced response of DC from FcR knockout (FcR k/o compared to C57Bl/6 (B6 mice. To address the potential role of FcR in Ab-mediated protection in vivo, we compared the response of passively immunized FcR k/o mice to the B6 controls. Interestingly, we found that FcR are not essential for AMI to C. burnetii in vivo. We subsequently examined the role of complement in AMI by passively immunizing and challenging several different strains of complement-deficient mice and found that AMI to C. burnetii is also complement-independent. Conclusion Despite our data showing FcR-dependent stimulation of DC in vitro, Ab-mediated immunity to C. burnetii in vivo is FcR-independent. We also found that passive immunity to this pathogen is independent of

  13. Impact of human D398N single nucleotide polymorphism on intracellular calcium response mediated by α3β4α5 nicotinic acetylcholine receptors

    Science.gov (United States)

    Tammimäki, Anne; Herder, Penelope; Li, Ping; Esch, Caroline; Laughlin, James R.; Akk, Gustav; Stitzel, Jerry A.

    2013-01-01

    The human CHRNA5 D398N polymorphism (rs16969968) causes an aspartic acid to asparagine change in the nicotinic acetylcholine receptor (nAChR) α5 subunit gene. The N398 variant of CHRNA5 is linked to increased risk for nicotine dependence. In this study, we explored the effect of the CHRNA5 D398N polymorphism on the properties of human α3β4* nicotinic acetylcholine receptors in human embryonic kidney (HEK) cells. Addition of either D398 or N398 variant of α5 subunit in the α3β4* receptor did not affect total [125I]-epibatidine binding or surface expression of the receptor. However, addition of α5D398 into α3β4* receptor decreased the maximal response to agonist without significantly affecting EC50 in aequorin intracellular calcium assay. α3β4α5N398 nAChRs showed further decreased maximal response. The differences in agonist efficacy between the receptor subtypes were found to be dependent upon the concentration of external calcium but independent of external sodium. Moreover, activation of α3β4α5 nAChRs led to significantly greater intracellular calcium release from IP3 stores relative to α3β4 nAChRs although no effect of the α5 polymorphism was observed. Finally, inclusion of the α5 variant caused a small shift to the left in IC50 for some of the antagonists tested, depending upon α5 variant but did not affect sensitivity of α3β4* receptors to desensitization in response to incubation with nicotine. In conclusion, addition of either variant of a5 into an α3β4α5 receptor similarly effects receptor pharmacology and function. However, the N398 variant exhibits a reduced response to agonists when extracellular calcium is high and it may lead to distinct downstream cellular signaling. PMID:22820273

  14. Elements in nucleotide sensing and hydrolysis of the AAA+ disaggregation machine ClpB: a structure-based mechanistic dissection of a molecular motor

    Energy Technology Data Exchange (ETDEWEB)

    Zeymer, Cathleen, E-mail: cathleen.zeymer@mpimf-heidelberg.mpg.de; Barends, Thomas R. M.; Werbeck, Nicolas D.; Schlichting, Ilme; Reinstein, Jochen, E-mail: cathleen.zeymer@mpimf-heidelberg.mpg.de [Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg (Germany)

    2014-02-01

    High-resolution crystal structures together with mutational analysis and transient kinetics experiments were utilized to understand nucleotide sensing and the regulation of the ATPase cycle in an AAA+ molecular motor. ATPases of the AAA+ superfamily are large oligomeric molecular machines that remodel their substrates by converting the energy from ATP hydrolysis into mechanical force. This study focuses on the molecular chaperone ClpB, the bacterial homologue of Hsp104, which reactivates aggregated proteins under cellular stress conditions. Based on high-resolution crystal structures in different nucleotide states, mutational analysis and nucleotide-binding kinetics experiments, the ATPase cycle of the C-terminal nucleotide-binding domain (NBD2), one of the motor subunits of this AAA+ disaggregation machine, is dissected mechanistically. The results provide insights into nucleotide sensing, explaining how the conserved sensor 2 motif contributes to the discrimination between ADP and ATP binding. Furthermore, the role of a conserved active-site arginine (Arg621), which controls binding of the essential Mg{sup 2+} ion, is described. Finally, a hypothesis is presented as to how the ATPase activity is regulated by a conformational switch that involves the essential Walker A lysine. In the proposed model, an unusual side-chain conformation of this highly conserved residue stabilizes a catalytically inactive state, thereby avoiding unnecessary ATP hydrolysis.

  15. Macrophage ABCA2 deletion modulates intracellular cholesterol deposition, affects macrophage apoptosis, and decreases early atherosclerosis in LDL receptor knockout mice.

    Science.gov (United States)

    Calpe-Berdiel, Laura; Zhao, Ying; de Graauw, Marjo; Ye, Dan; van Santbrink, Peter J; Mommaas, A Mieke; Foks, Amanda; Bot, Martine; Meurs, Illiana; Kuiper, Johan; Mack, Jody T; Van Eck, Miranda; Tew, Kenneth D; van Berkel, Theo J C

    2012-08-01

    The ABCA2 transporter shares high structural homology to ABCA1, which is crucial for the removal of excess cholesterol from macrophages and, by extension, in atherosclerosis. It has been suggested that ABCA2 sequesters cholesterol inside the lysosomes, however, little is known of the macrophage-specific role of ABCA2 in regulating lipid homeostasis in vivo and in modulating susceptibility to atherosclerosis. Chimeras with dysfunctional macrophage ABCA2 were generated by transplantation of bone marrow from ABCA2 knockout (KO) mice into irradiated LDL receptor (LDLr) KO mice. Interestingly, lack of ABCA2 in macrophages resulted in a diminished lesion size in the aortic root (-24.5%) and descending thoracic aorta (-36.6%) associated with a 3-fold increase in apoptotic cells, as measured by both caspase 3 and TUNEL. Upon oxidized LDL exposure, macrophages from wildtype (WT) transplanted animals developed filipin-positive droplets in lysosomal-like compartments, corresponding to free cholesterol (FC) accumulation. In contrast, ABCA2-deficient macrophages displayed an abnormal diffuse distribution of FC over peripheral regions. The accumulation of neutral sterols in lipid droplets was increased in ABCA2-deficient macrophages, but primarily in cytoplasmic clusters and not in lysosomes. Importantly, apoptosis of oxLDL loaded macrophages lacking ABCA2 was increased 2.7-fold, probably as a consequence of the broad cellular distribution of FC. Lack of functional ABCA2 generates abnormalities in intracellular lipid distribution/trafficking in macrophages consistent with its lysosomal sequestering role, leading to an increased susceptibility to apoptosis in response to oxidized lipids and reduced atherosclerotic lesion development. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  16. Butyrate increases intracellular calcium levels and enhances growth hormone release from rat anterior pituitary cells via the G-protein-coupled receptors GPR41 and 43.

    Directory of Open Access Journals (Sweden)

    Maria Consolata Miletta

    Full Text Available Butyrate is a short-chain fatty acid (SCFA closely related to the ketone body ß-hydroxybutyrate (BHB, which is considered to be the major energy substrate during prolonged exercise or starvation. During fasting, serum growth hormone (GH rises concomitantly with the accumulation of BHB and butyrate. Interactions between GH, ketone bodies and SCFA during the metabolic adaptation to fasting have been poorly investigated to date. In this study, we examined the effect of butyrate, an endogenous agonist for the two G-protein-coupled receptors (GPCR, GPR41 and 43, on non-stimulated and GH-releasing hormone (GHRH-stimulated hGH secretion. Furthermore, we investigated the potential role of GPR41 and 43 on the generation of butyrate-induced intracellular Ca2+ signal and its ultimate impact on hGH secretion. To study this, wt-hGH was transfected into a rat pituitary tumour cell line stably expressing the human GHRH receptor. Treatment with butyrate promoted hGH synthesis and improved basal and GHRH-induced hGH-secretion. By acting through GPR41 and 43, butyrate enhanced intracellular free cytosolic Ca2+. Gene-specific silencing of these receptors led to a partial inhibition of the butyrate-induced intracellular Ca2+ rise resulting in a decrease of hGH secretion. This study suggests that butyrate is a metabolic intermediary, which contributes to the secretion and, therefore, to the metabolic actions of GH during fasting.

  17. Butyrate increases intracellular calcium levels and enhances growth hormone release from rat anterior pituitary cells via the G-protein-coupled receptors GPR41 and 43.

    Science.gov (United States)

    Miletta, Maria Consolata; Petkovic, Vibor; Eblé, Andrée; Ammann, Roland A; Flück, Christa E; Mullis, Primus-E

    2014-01-01

    Butyrate is a short-chain fatty acid (SCFA) closely related to the ketone body ß-hydroxybutyrate (BHB), which is considered to be the major energy substrate during prolonged exercise or starvation. During fasting, serum growth hormone (GH) rises concomitantly with the accumulation of BHB and butyrate. Interactions between GH, ketone bodies and SCFA during the metabolic adaptation to fasting have been poorly investigated to date. In this study, we examined the effect of butyrate, an endogenous agonist for the two G-protein-coupled receptors (GPCR), GPR41 and 43, on non-stimulated and GH-releasing hormone (GHRH)-stimulated hGH secretion. Furthermore, we investigated the potential role of GPR41 and 43 on the generation of butyrate-induced intracellular Ca2+ signal and its ultimate impact on hGH secretion. To study this, wt-hGH was transfected into a rat pituitary tumour cell line stably expressing the human GHRH receptor. Treatment with butyrate promoted hGH synthesis and improved basal and GHRH-induced hGH-secretion. By acting through GPR41 and 43, butyrate enhanced intracellular free cytosolic Ca2+. Gene-specific silencing of these receptors led to a partial inhibition of the butyrate-induced intracellular Ca2+ rise resulting in a decrease of hGH secretion. This study suggests that butyrate is a metabolic intermediary, which contributes to the secretion and, therefore, to the metabolic actions of GH during fasting.

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

    Energy Technology Data Exchange (ETDEWEB)

    Sugita, Kazuya; Yamamura, Chiaki; Tabata, Ken-ichi [Laboratory of Pharmacoinformatics, Graduate School of Ritsumeikan University, Kusatsu, Shiga 525-8577 (Japan); Fujita, Norihisa, E-mail: nori@ph.ritsumei.ac.jp [Laboratory of Pharmacoinformatics, Graduate School of Ritsumeikan University, Kusatsu, Shiga 525-8577 (Japan); School of Pharmacy, Ristumeikan University, Kusatsu, Shiga 525-8577 (Japan)

    2013-01-04

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

  19. The carboxy-terminal tail or the intracellular loop 3 is required for β-arrestin-dependent internalization of a mammalian type II GnRH receptor.

    Science.gov (United States)

    Madziva, Michael T; Mkhize, Nonhlanhla N; Flanagan, Colleen A; Katz, Arieh A

    2015-08-15

    The type II GnRH receptor (GnRH-R2) in contrast to mammalian type I GnRH receptor (GnRH-R1) has a cytosolic carboxy-terminal tail. We investigated the role of β-arrestin 1 in GnRH-R2-mediated signalling and mapped the regions in GnRH-R2 required for recruitment of β-arrestin, employing internalization assays. We show that GnRH-R2 activation of ERK is dependent on β-arrestin and protein kinase C. Appending the tail of GnRH-R2 to GnRH-R1 enabled GRK- and β-arrestin-dependent internalization of the chimaeric receptor. Surprisingly, carboxy-terminally truncated GnRH-R2 retained β-arrestin and GRK-dependent internalization, suggesting that β-arrestin interacts with additional elements of GnRH-R2. Mutating serine and threonine or basic residues of intracellular loop 3 did not abolish β-arrestin 1-dependent internalization but a receptor lacking these basic residues and the carboxy-terminus showed no β-arrestin 1-dependent internalization. Our results suggest that basic residues at the amino-terminal end of intracellular loop 3 or the carboxy-terminal tail are required for β-arrestin dependent internalization. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  20. Spinal intracellular metabotropic glutamate receptor 5 (mGluR5) contributes to pain and c-fos expression in a rat model of inflammatory pain.

    Science.gov (United States)

    Vincent, Kathleen; Wang, Shu Fan; Laferrière, André; Kumar, Naresh; Coderre, Terence J

    2017-04-01

    Metabotropic glutamate receptor 5 (mGluR5) is an excitatory G-protein-coupled receptor (GPCR) present in the spinal cord dorsal horn (SCDH) where it has a well-established role in pain. In addition to its traditional location on the cytoplasmic membrane, recent evidence shows that these receptors are present intracellularly on the nuclear membrane in the spinal cord dorsal horn and are implicated in neuropathic pain. Nuclear mGluR5 is a functional receptor that binds glutamate entering the cell through the neuronal glutamate transporter (GT) EAAT3 and activates transcription factor c-fos, whereas plasma membrane mGluR5 is responsible for c-jun activation. Here, we extend these findings to a model of inflammatory pain using complete Freund's adjuvant (CFA) and show that nuclear mGluR5 is also upregulated in the spinal cord dorsal horn following inflammation. We also show that pretreatment with an excitatory amino acid transporter (EAAT) inhibitor attenuates pain and decreases Fos, but not Jun, expression in complete Freund's adjuvant rats. In contrast, selective glial glutamate transporter inhibitors are pronociceptive and increase spinal glutamate concentrations. Additionally, we found that permeable mGluR5 antagonists are more effective at attenuating pain and Fos expression than nonpermeable group I mGluR antagonists. Taken together, these results suggest that under inflammatory conditions, intracellular mGluR5 is actively involved in the relay of nociceptive information in the spinal cord.

  1. LH and hCG Action on the Same Receptor Results in Quantitatively and Qualitatively Different Intracellular Signalling

    Science.gov (United States)

    Casarini, Livio; Lispi, Monica; Longobardi, Salvatore; Milosa, Fabiola; La Marca, Antonio; Tagliasacchi, Daniela; Pignatti, Elisa; Simoni, Manuela

    2012-01-01

    Human luteinizing hormone (hLH) and chorionic gonadotropin (hCG) act on the same receptor (LHCGR) but it is not known whether they elicit the same cellular and molecular response. This study compares for the first time the activation of cell-signalling pathways and gene expression in response to hLH and hCG. Using recombinant hLH and recombinant hCG we evaluated the kinetics of cAMP production in COS-7 and hGL5 cells permanently expressing LHCGR (COS-7/LHCGR, hGL5/LHCGR), as well as cAMP, ERK1/2, AKT activation and progesterone production in primary human granulosa cells (hGLC). The expression of selected target genes was measured in the presence or absence of ERK- or AKT-pathways inhibitors. In COS-7/LHCGR cells, hCG is 5-fold more potent than hLH (cAMP ED50: 107.1±14.3 pM and 530.0±51.2 pM, respectively). hLH maximal effect was significantly faster (10 minutes by hLH; 1 hour by hCG). In hGLC continuous exposure to equipotent doses of gonadotropins up to 36 hours revealed that intracellular cAMP production is oscillating and significantly higher by hCG versus hLH. Conversely, phospho-ERK1/2 and -AKT activation was more potent and sustained by hLH versus hCG. ERK1/2 and AKT inhibition removed the inhibitory effect on NRG1 (neuregulin) expression by hLH but not by hCG; ERK1/2 inhibition significantly increased hLH- but not hCG-stimulated CYP19A1 (aromatase) expression. We conclude that: i) hCG is more potent on cAMP production, while hLH is more potent on ERK and AKT activation; ii) hGLC respond to equipotent, constant hLH or hCG stimulation with a fluctuating cAMP production and progressive progesterone secretion; and iii) the expression of hLH and hCG target genes partly involves the activation of different pathways depending on the ligand. Therefore, the LHCGR is able to differentiate the activity of hLH and hCG. PMID:23071612

  2. P2Y13 receptor-mediated rapid increase in intracellular calcium induced by ADP in cultured dorsal spinal cord microglia.

    Science.gov (United States)

    Zeng, Junwei; Wang, Gaoxia; Liu, Xiaohong; Wang, Chunmei; Tian, Hong; Liu, Aidong; Jin, Huan; Luo, Xiaomei; Chen, Yuanshou

    2014-11-01

    P2Y receptors have been implicated in the calcium mobilization by the response to neuroexcitatory substances in neurons and astrocytes, but little is known about P2Y receptors in microglia cells. In the present study, the effects of ADP on the intracellular calcium concentration ([Ca(2+)]i) in cultured dorsal spinal cord microglia were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescence indicator that could monitor real-time alterations of [Ca(2+)]i. Here we show that ADP (0.01-100 μM) causes a rapid increase in [Ca(2+)]i with a dose-dependent manner in cultured microglia. The action of ADP on [Ca(2+)]i was significantly blocked by MRS2211 (a selective P2Y13 receptor antagonist), but was unaffected by MRS2179 (a selective P2Y1 receptor antagonist) or MRS2395 (a selective P2Y12 receptor antagonist), which suggest that P2Y13 receptor may be responsible for ADP-evoked Ca(2+) mobilization in cultured microglia. P2Y13-evoked Ca(2+) response can be obviously inhibited by BAPTA-AM and U-73122, respectively. Moreover, removal of extracellular Ca(2+) (by EGTA) also can obvious suppress the Ca(2+) mobilization. These results means both intracellular calcium and extracellular calcium are potentially important mechanisms in P2Y13 receptor-evoked Ca(2+) mobilization. However, P2Y13 receptor-evoked Ca(2+) response was not impaired after CdCl2 and verapamil administration, which suggest that voltage-operated Ca(2+) channels may be not related with P2Y13-evoked Ca(2+) response. In addition, Ca(2+) mobilization induced by ADP was abolished by different store-operated Ca(2+) channels (SOCs) blocker, 2-APB (50 μM) and SKF-96365 (1 mM), respectively. These observations suggest that the activation of P2Y13 receptor might be involved in the effect of ADP on [Ca(2+)]i in cultured dorsal spinal cord microglia. Furthermore, our results raise a possibility that P2Y13 receptor activation causes Ca(2+) release from Ca(2+) store, which leads to the

  3. β adrenergic receptor/cAMP/PKA signaling contributes to the intracellular Ca2+release by tentacle extract from the jellyfish Cyanea capillata.

    Science.gov (United States)

    Wang, Qianqian; Zhang, Hui; Wang, Bo; Wang, Chao; Xiao, Liang; Zhang, Liming

    2017-07-25

    Intracellular Ca 2+ overload induced by extracellular Ca 2+ entry has previously been confirmed to be an important mechanism for the cardiotoxicity as well as the acute heart dysfunction induced by jellyfish venom, while the underlying mechanism remains to be elucidated. Under extracellular Ca 2+ -free or Ca 2+ -containing conditions, the Ca 2+ fluorescence in isolated adult mouse cardiomyocytes pre-incubated with tentacle extract (TE) from the jellyfish Cyanea capillata and β blockers was scanned by laser scanning confocal microscope. Then, the cyclic adenosine monophosphate (cAMP) concentration and protein kinase A (PKA) activity in primary neonatal rat ventricular cardiomyocytes were determined by ELISA assay. Furthermore, the effect of propranolol against the cardiotoxicity of TE was evaluated in Langendorff-perfused rat hearts and intact rats. The increase of intracellular Ca 2+ fluorescence signal by TE was significantly attenuated and delayed when the extracellular Ca 2+ was removed. The β adrenergic blockers, including propranolol, atenolol and esmolol, partially inhibited the increase of intracellular Ca 2+ in the presence of 1.8 mM extracellular Ca 2+ and completely abolished the Ca 2+ increase under an extracellular Ca 2+ -free condition. Both cAMP concentration and PKA activity were stimulated by TE, and were inhibited by the β adrenergic blockers. Cardiomyocyte toxicity of TE was antagonized by β adrenergic blockers and the PKA inhibitor H89. Finally, the acute heart dysfuction by TE was antagonized by propranolol in Langendorff-perfused rat hearts and intact rats. Our findings indicate that β adrenergic receptor/cAMP/PKA signaling contributes to the intracellular Ca 2+ overload through intracellular Ca 2+ release by TE from the jellyfish C. capillata.

  4. Highly specific detection of muscarinic M3 receptor, G protein interaction and intracellular trafficking in human detrusor using Proximity Ligation Assay (PLA).

    Science.gov (United States)

    Berndt-Paetz, Mandy; Herbst, Luise; Weimann, Annett; Gonsior, Andreas; Stolzenburg, Jens-Uwe; Neuhaus, Jochen

    2018-03-15

    Muscarinic acetylcholine receptors (mAChRs) regulate a number of important physiological functions. Alteration of mAChR expression or function has been associated in the etiology of several pathologies including functional bladder disorders (e.g bladder pain syndrome/interstitial cystitis - BPS/IC). In a previous study we found specific mAChR expression patterns associated with BPS/IC, while correlation between protein and gene expression was lacking. Posttranslational regulatory mechanisms, e.g. altered intracellular receptor trafficking, could explain those differences. In addition, alternative G protein (GP) coupling could add to the pathophysiology via modulation of muscarinic signaling. In our proof-of-principle study, we addressed these questions in situ. We established PLA in combination with confocal laserscanning microscopy (CLSM) and 3D object reconstruction for highly specific detection and analysis of muscarinic 3 receptors (M3), G protein (GP) coupling and intracellular trafficking in human detrusor samples. Paraffin sections of formalin-fixed bladder tissue (FFPE) of BPS/IC patients receiving transurethral biopsy were examined by Cy3-PLA for M3 expression, coupling of M3 to GPs (G αq/11 , G αs , G αi ) and interaction of M3 with endocytic regulator proteins. Membranes were labeled with wheat germ agglutinin-Alexa Fluor ® 488, nuclei were stained with DAPI. Object density and co-localization were analyzed in 3D-reconstruction of high resolution confocal z-stacks. Confocal image stack processing resulted in well demarcated objects. Calculated receptor densities correlated significantly with existing confocal expression data, while significantly improved specificity of M3 detection by PLA was verified using bladder tissue samples from transgenic mice. 50-60% of the M3 receptor complexes were plasma membrane associated in human bladder detrusor. Application of PLA for M3 and GPs allowed visualization of M3-GP interactions and revealed individual GP

  5. Individual differences in ethanol locomotor sensitization are associated with dopamine D1 receptor intra-cellular signaling of DARPP-32 in the nucleus accumbens.

    Directory of Open Access Journals (Sweden)

    Karina Possa Abrahao

    Full Text Available In mice there are clear individual differences in the development of behavioral sensitization to ethanol, a progressive potentiation of its psychomotor stimulant effect. Variability in the behavioral responses to ethanol has been associated with alcohol preference. Here we investigated if the functional hyperresponsiveness of D1 receptors observed in ethanol sensitized mice leads to an increased activation of DARPP-32, a central regulatory protein in medium spiny neurons, in the nucleus accumbens - a brain region known to play a role in drug reinforcement. Swiss Webster mice received ethanol (2.2 g/kg/day or saline i.p. administrations for 21 days and were weekly evaluated regarding their locomotor activity. From those treated with ethanol, the 33% with the highest levels of locomotor activity were classified as "sensitized" and the 33% with the lowest levels as "non-sensitized". The latter presented similar locomotor levels to those of saline-treated mice. Different subgroups of mice received intra-accumbens administrations of saline and, 48 h later, SKF-38393, D1 receptor agonist 0.1 or 1 µg/side. Indeed, sensitized mice presented functional hyperresponsiveness of D1 receptors in the accumbens. Two weeks following the ethanol treatment, other subgroups received systemic saline or SKF 10 mg/kg, 20 min before the euthanasia. The nucleus accumbens were dissected for the Western Blot analyses of total DARPP-32 and phospho-Thr34-DARPP-32 expression. D1 receptor activation induced higher phospho-Thr34-DARPP-32 expression in sensitized mice than in non-sensitized or saline. The functionally hyperresponsiveness of D1 receptors in the nucleus accumbens is associated with an increased phospho-Thr34-DARPP-32 expression after D1 receptor activation. These data suggest that an enduring increase in the sensitivity of the dopamine D1 receptor intracellular pathway sensitivity represents a neurobiological correlate associated with the development of

  6. Complement receptor 3 and Toll-like receptor 4 act sequentially in uptake and intracellular killing of unopsonized Salmonella enterica serovar Typhimurium by human neutrophils

    NARCIS (Netherlands)

    van Bruggen, Robin; Zweers, Debby; van Diepen, Angela; van Dissel, Jaap T.; Roos, Dirk; Verhoeven, Arthur J.; Kuijpers, Taco W.

    2007-01-01

    The uptake and subsequent killing of Salmonella enterica serovar Typhimurium by human neutrophils was studied. In particular, two pattern recognition receptors, complement receptor 3 (CR3) and Toll-like receptor 4 (TLR4), were found to be essential for the efficient uptake and activation,

  7. Chronic ethanol exposure induces SK-N-SH cell apoptosis by increasing N-methyl-D-aspartic acid receptor expression and intracellular calcium.

    Science.gov (United States)

    Wang, Hongbo; Wang, Xiaolong; Li, Yan; Yu, Hao; Wang, Changliang; Feng, Chunmei; Xu, Guohui; Chen, Jiajun; You, Jiabin; Wang, Pengfei; Wu, Xu; Zhao, Rui; Zhang, Guohua

    2018-04-01

    It has been identified that chronic ethanol exposure damages the nervous system, particularly neurons. There is scientific evidence suggesting that neuronal loss caused by chronic ethanol exposure has an association with neuron apoptosis and intracellular calcium oscillation is one of the primary inducers of apoptosis. Therefore, the present study aimed to investigate the inductive effects of intracellular calcium oscillation on apoptosis in SK-N-SH human neuroblastoma cells and the protective effects of the N-methyl-D-aspartic acid receptor (NMDAR) antagonist, memantine, on SK-N-SH cell apoptosis caused by chronic ethanol exposure. SK-N-SH cells were treated with 100 mM ethanol and memantine (4 µM) for 2 days. Protein expression of NR1 was downregulated by RNA interference (RNAi). Apoptosis was detected by Annexin V/propidium iodide (PI) double-staining and flow cytometry and cell viability was detected using an MTS kit. Fluorescence dual wavelength spectrophotometry was used to determine the intracellular calcium concentration and the levels of NR1 and caspase-3 were detected using western blotting. NR1 mRNA levels were also detected using qPCR. It was found that chronic ethanol exposure reduced neuronal cell viability and caused apoptosis of SK-N-SH cells, and the extent of damage in SK-N-SH cells was associated with ethanol exposure concentration and time. In addition, chronic ethanol exposure increased the concentration of intracellular calcium in SK-N-SH cells by inducing the expression of NMDAR, resulting in apoptosis, and memantine treatment reduced ethanol-induced cell apoptosis. The results of the present study indicate that the application of memantine may provide a novel strategy for the treatment of alcoholic dementia.

  8. Invasive Streptococcus mutans induces inflammatory cytokine production in human aortic endothelial cells via regulation of intracellular toll-like receptor 2 and nucleotide-binding oligomerization domain 2.

    Science.gov (United States)

    Nagata, E; Oho, T

    2017-04-01

    Streptococcus mutans, the primary etiologic agent of dental caries, can gain access to the bloodstream and has been associated with cardiovascular disease. However, the roles of S. mutans in inflammation in cardiovascular disease remain unclear. The aim of this study was to examine cytokine production induced by S. mutans in human aortic endothelial cells (HAECs) and to evaluate the participation of toll-like receptors (TLRs) and cytoplasmic nucleotide-binding oligomerization domain (NOD) -like receptors in HAECs. Cytokine production by HAECs was determined using enzyme-linked immunosorbent assays, and the expression of TLRs and NOD-like receptors was evaluated by real-time polymerase chain reaction, flow cytometry and immunocytochemistry. The involvement of TLR2 and NOD2 in cytokine production by invaded HAECs was examined using RNA interference. The invasion efficiencies of S. mutans strains were evaluated by means of antibiotic protection assays. Five of six strains of S. mutans of various serotypes induced interleukin-6, interleukin-8 and monocyte chemoattractant protein-1 production by HAECs. All S. mutans strains upregulated TLR2 and NOD2 mRNA levels in HAECs. Streptococcus mutans Xc upregulated the intracellular TLR2 and NOD2 protein levels in HAECs. Silencing of the TLR2 and NOD2 genes in HAECs invaded by S. mutans Xc led to a reduction in interleukin-6, interleukin-8 and monocyte chemoattractant protein-1 production. Cytokine production induced by invasive S. mutans via intracellular TLR2 and NOD2 in HAECs may be associated with inflammation in cardiovascular disease. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. The regulated expression, intracellular trafficking, and membrane recycling of the P2Y-like receptor GPR17 in Oli-neu oligodendroglial cells.

    Science.gov (United States)

    Fratangeli, Alessandra; Parmigiani, Elena; Fumagalli, Marta; Lecca, Davide; Benfante, Roberta; Passafaro, Maria; Buffo, Annalisa; Abbracchio, Maria P; Rosa, Patrizia

    2013-02-15

    GPR17 is a G-protein-coupled receptor that is activated by two classes of molecules: uracil-nucleotides and cysteinyl-leukotrienes. GPR17 is required for initiating the differentiation of oligodendrocyte precursors but has to be down-regulated to allow cells to undergo terminal maturation. Although a great deal has been learned about GPR17 expression and signaling, no information is currently available about the trafficking of native receptors after the exposure of differentiating oligodendrocytes to endogenous agonists. Here, we demonstrate that neuron-conditioned medium induces the transcriptionally mediated, time-regulated expression of GPR17 in Oli-neu, an oligodendrocyte precursor cell line, making these cells suitable for studying the endocytic traffic of the native receptor. Agonist-induced internalization, intracellular trafficking, and membrane recycling of GPR17 were analyzed by biochemical and immunofluorescence assays using an ad hoc-developed antibody against the extracellular N-terminal of GPR17. Both UDP-glucose and LTD(4) increased GPR17 internalization, although with different efficiency. At early time points, internalized GPR17 co-localized with transferrin receptor, whereas at later times it partially co-localized with the lysosomal marker Lamp1, suggesting that a portion of GPR17 is targeted to lysosomes upon ligand binding. An analysis of receptor recycling and degradation demonstrated that a significant aliquot of GPR17 is recycled to the cell surface. Furthermore, internalized GPR17 displayed a co-localization with the marker of the "short loop" recycling endosomes, Rab4, while showing very minor co-localization with the "long loop" recycling marker, Rab11. Our results provide the first data on the agonist-induced trafficking of native GPR17 in oligodendroglial cells and may have implications for both physiological and pathological myelination.

  10. Role of the ERC motif in the proximal part of the second intracellular loop and the C-terminal domain of the human prostaglandin F2alpha receptor (hFP-R) in G-protein coupling control.

    Science.gov (United States)

    Pathe-Neuschäfer-Rube, Andrea; Neuschäfer-Rube, Frank; Püschel, Gerhard P

    2005-05-15

    The human FP-R (F2alpha prostaglandin receptor) is a Gq-coupled heptahelical ectoreceptor, which is of significant medical interest, since it is a potential target for the treatment of glaucoma and preterm labour. On agonist exposure, it mediates an increase in intracellular inositol phosphate formation. Little is known about the structures that govern the agonist-dependent receptor activation. In other prostanoid receptors, the C-terminal domain has been inferred in the control of agonist-dependent receptor activation. A DRY motif at the beginning of the second intracellular loop is highly conserved throughout the G-protein-coupled receptor family and appears to be crucial for controlling agonist-dependent receptor activation. It is replaced by an ERC motif in the FP-R and no evidence for the relevance of this motif in ligand-dependent activation of prostanoid receptors has been provided so far. The aim of the present study was to elucidate the potential role of the C-terminal domain and the ERC motif in agonist-controlled intracellular signalling in FP-R mutants generated by site-directed mutagenesis. It was found that substitution of the acidic Glu(132) in the ERC motif by a threonine residue led to full constitutive activation, whereas truncation of the receptor's C-terminal domain led to partial constitutive activation of all three intracellular signal pathways that had previously been shown to be activated by the FP-R, i.e. inositol trisphosphate formation, focal adhesion kinase activation and T-cell factor signalling. Inositol trisphosphate formation and focal adhesion kinase phosphorylation were further enhanced by ligand binding in cells expressing the truncation mutant but not the E132T (Glu132-->Thr) mutant. Thus C-terminal truncation appeared to result in a receptor with partial constitutive activation, whereas substitution of Glu132 by threonine apparently resulted in a receptor with full constitutive activity.

  11. Cellular Quantitative Structure–Activity Relationship (Cell-QSAR): Conceptual Dissection of Receptor Binding and Intracellular Disposition in Antifilarial Activities of Selwood Antimycins

    Science.gov (United States)

    2012-01-01

    We present the cellular quantitative structure–activity relationship (cell-QSAR) concept that adapts ligand-based and receptor-based 3D-QSAR methods for use with cell-level activities. The unknown intracellular drug disposition is accounted for by the disposition function (DF), a model-based, nonlinear function of a drug’s lipophilicity, acidity, and other properties. We conceptually combined the DF with our multispecies, multimode version of the frequently used ligand-based comparative molecular field analysis (CoMFA) method, forming a single correlation function for fitting the cell-level activities. The resulting cell-QSAR model was applied to the Selwood data on filaricidal activities of antimycin analogues. Their molecules are flexible, ionize under physiologic conditions, form different intramolecular H-bonds for neutral and ionized species, and cross several membranes to reach unknown receptors. The calibrated cell-QSAR model is significantly more predictive than other models lacking the disposition part and provides valuable structure optimization clues by factorizing the cell-level activity of each compound into the contributions of the receptor binding and disposition. PMID:22468611

  12. Cellular quantitative structure-activity relationship (Cell-QSAR): conceptual dissection of receptor binding and intracellular disposition in antifilarial activities of Selwood antimycins.

    Science.gov (United States)

    Natesan, Senthil; Wang, Tiansheng; Lukacova, Viera; Bartus, Vladimir; Khandelwal, Akash; Subramaniam, Rajesh; Balaz, Stefan

    2012-04-26

    We present the cellular quantitative structure-activity relationship (cell-QSAR) concept that adapts ligand-based and receptor-based 3D-QSAR methods for use with cell-level activities. The unknown intracellular drug disposition is accounted for by the disposition function (DF), a model-based, nonlinear function of a drug's lipophilicity, acidity, and other properties. We conceptually combined the DF with our multispecies, multimode version of the frequently used ligand-based comparative molecular field analysis (CoMFA) method, forming a single correlation function for fitting the cell-level activities. The resulting cell-QSAR model was applied to the Selwood data on filaricidal activities of antimycin analogues. Their molecules are flexible, ionize under physiologic conditions, form different intramolecular H-bonds for neutral and ionized species, and cross several membranes to reach unknown receptors. The calibrated cell-QSAR model is significantly more predictive than other models lacking the disposition part and provides valuable structure optimization clues by factorizing the cell-level activity of each compound into the contributions of the receptor binding and disposition.

  13. Intracellular calcium measurements as a method in studies on activity of purinergic P2X receptor channels

    Czech Academy of Sciences Publication Activity Database

    He, M. L.; Zemková, Hana; Koshimizu, T.; Tomič, M.; Stojilkovic, S. S.

    2003-01-01

    Roč. 285, č. 2 (2003), s. C467-C479 ISSN 0363-6143 Institutional research plan: CEZ:AV0Z5011922 Keywords : P2X purinergic receptor * calcium signaling * membrane current Subject RIV: ED - Physiology Impact factor: 4.103, year: 2003

  14. Intracellular distribution of the vitamin D receptor in the brain: comparison with classic target tissues and redistribution with development.

    Science.gov (United States)

    Eyles, D W; Liu, P Y; Josh, P; Cui, X

    2014-05-30

    Apart from its role in regulating calcium there is growing evidence that vitamin D is a neuroactive steroid capable of regulating multiple pathways important for both brain development and mature brain function. Vitamin D induces its genomic effects through its nuclear receptor the vitamin D receptor (VDR). Although there is abundant evidence for this receptor's presence in the mammalian brain from studies employing immunohistochemistry, Western blot or quantitative RNA studies there remains some dispute regarding the validity of these studies. In this study we provide unambiguous confirmation for the VDR in adult rodent brain using proteomic techniques. However Western blot experiments show that compared to more classic target organs such as the gut and kidney, VDR expression is quantitatively lower in the brain. In addition we have examined VDR subcellular distribution in the gut, kidney and brain from both embryonic and adult tissues. We show that in all embryonic tissues VDR distribution is mostly nuclear, however by adulthood it appears that at least in the gut and kidney, VDR presence in the plasma membrane is more prominent perhaps reflecting some change in VDR function with the maturation of these tissues. Finally the subcellular distribution of VDR in the embryo did not appear to be altered by vitamin D deficiency indicating that perhaps there are other mechanisms at play in vivo to stabilize this receptor in the absence of its ligand. Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.

  15. The Contribution of Proteinase-Activated Receptors to Intracellular Signaling, Transcellular Transport and Autophagy in Alzheimer´s Disease

    Czech Academy of Sciences Publication Activity Database

    Matěj, R.; Rohan, Z.; Holada, K.; Olejár, Tomáš

    2015-01-01

    Roč. 12, č. 1 (2015), s. 2-12 ISSN 1567-2050 Institutional support: RVO:67985823 Keywords : Alzheimer ´s Disease * autophagy * proteinase-activated receptors Subject RIV: EA - Cell Biology Impact factor: 3.145, year: 2015

  16. Tris-hydroxymethyl-aminomethane enhances capsaicin-induced intracellular Ca2+ influx through transient receptor potential V1 (TRPV1 channels

    Directory of Open Access Journals (Sweden)

    Satoshi Murakami

    2016-02-01

    Full Text Available Non-selective transient receptor potential vanilloid (TRPV cation channels are activated by various insults, including exposure to heat, acidity, and the compound capsaicin, resulting in sensations of pain in the skin, visceral organs, and oral cavity. Recently, TRPV1 activation was also demonstrated in response to basic pH elicited by ammonia and intracellular alkalization. Tris-hydroxymethyl aminomethane (THAM is widely used as an alkalizing agent; however, the effects of THAM on TRPV1 channels have not been defined. In this study, we characterized the effects of THAM-induced TRPV1 channel activation in baby hamster kidney cells expressing human TRPV1 (hTRPV1 and the Ca2+-sensitive fluorescent sensor GCaMP2 by real-time confocal microscopy. Notably, both capsaicin (1 μM and pH 6.5 buffer elicited steep increases in the intracellular Ca2+ concentration ([Ca2+]i, while treatment with THAM (pH 8.5 alone had no effect. However, treatment with THAM (pH 8.5 following capsaicin application elicited a profound, long-lasting increase in [Ca2+]i that was completely inhibited by the TRPV1 antagonist capsazepine. Taken together, these results suggest that hTRPV1 pre-activation is required to provoke enhanced, THAM-induced [Ca2+]i increases, which could be a mechanism underlying pain induced by basic pH.

  17. Memantine, an antagonist of the NMDA glutamate receptor, affects cell proliferation, differentiation and the intracellular cycle and induces apoptosis in Trypanosoma cruzi.

    Directory of Open Access Journals (Sweden)

    Flávia Silva Damasceno

    2014-02-01

    Full Text Available Chagas' disease is caused by the protozoan parasite Trypanosoma cruzi and affects approximately 10 million people in endemic areas of Mexico and Central and South America. Currently available chemotherapies are limited to two compounds: Nifurtimox and Benznidazole. Both drugs reduce the symptoms of the disease and mortality among infected individuals when used during the acute phase, but their efficacy during the chronic phase (during which the majority of cases are diagnosed remains controversial. Moreover, these drugs have several side effects. The aim of this study was to evaluate the effect of Memantine, an antagonist of the glutamate receptor in the CNS of mammals, on the life cycle of T. cruzi. Memantine exhibited a trypanocidal effect, inhibiting the proliferation of epimastigotes (IC50 172.6 µM. Furthermore, this compound interfered with metacyclogenesis (approximately 30% reduction and affected the energy metabolism of the parasite. In addition, Memantine triggered mechanisms that led to the apoptosis-like cell death of epimastigotes, with extracellular exposure of phosphatidylserine, increased production of reactive oxygen species, decreased ATP levels, increased intracellular Ca(2+ and morphological changes. Moreover, Memantine interfered with the intracellular cycle of the parasite, specifically the amastigote stage (IC50 31 µM. Interestingly, the stages of the parasite life cycle that require more energy (epimastigote and amastigote were more affected as were the processes of differentiation and cell invasion.

  18. NMDA receptor blockade alters the intracellular distribution of neuronal nitric oxide synthase in the superficial layers of the rat superior colliculus

    Directory of Open Access Journals (Sweden)

    R.E. de Bittencourt-Navarrete

    2009-02-01

    Full Text Available Nitric oxide (NO is a molecular messenger involved in several events of synaptic plasticity in the central nervous system. Ca2+ influx through the N-methyl-D-aspartate receptor (NMDAR triggers the synthesis of NO by activating the enzyme neuronal nitric oxide synthase (nNOS in postsynaptic densities. Therefore, NMDAR and nNOS are part of the intricate scenario of postsynaptic densities. In the present study, we hypothesized that the intracellular distribution of nNOS in the neurons of superior colliculus (SC superficial layers is an NMDAR activity-dependent process. We used osmotic minipumps to promote chronic blockade of the receptors with the pharmacological agent MK-801 in the SC of 7 adult rats. The effective blockade of NMDAR was assessed by changes in the protein level of the immediate early gene NGFI-A, which is a well-known NMDAR activity-dependent expressing transcription factor. Upon chronic infusion of MK-801, a decrease of 47% in the number of cells expressing NGFI-A was observed in the SC of treated animals. Additionally, the filled dendritic extent by the histochemical product of nicotinamide adenine di-nucleotide phosphate diaphorase was reduced by 45% when compared to the contralateral SC of the same animals and by 64% when compared to the SC of control animals. We conclude that the proper intracellular localization of nNOS in the retinorecipient layers of SC depends on NMDAR activation. These results are consistent with the view that the participation of NO in the physiological and plastic events of the central nervous system might be closely related to an NMDAR activity-dependent function.

  19. The role of the class A scavenger receptors, SR-A and MARCO, in the immune system. Part 1. The structure of receptors, their ligand binding repertoires and ability to initiate intracellular signaling

    Directory of Open Access Journals (Sweden)

    Szczepan Józefowski

    2012-02-01

    Full Text Available  Recognition of pathogens by innate immune cells is mediated by pattern recognition receptors (PRR, which include scavenger receptors (SR. The class A SR, SR-A/CD204 and MARCO, are characterized by the presence of collagenous and SR cysteine-rich domains in their extracellular portions. Both receptors are expressed mainly on macrophages and dendritic cells. Thanks to their ability to bind to a wide range of polyanionic ligands, the class A SR may participate in numerous functions of these cells, such as endocytosis, and adhesion to extracellular matrix and to other cells. Among SR-A ligands are oxidized lipoproteins and β-amyloid fibrils, which link SR-A to the pathogenesis of arteriosclerosis and Alzheimer’s disease. Despite the demonstration of class A SR involvement in so many processes, the lack of selective ligands precluded reaching definite conclusions concerning their signaling abilities. Using specific receptor ligation with antibodies, we showed that SR-A and MARCO trigger intracellular signaling, modulating pro-inflammatory and microbicidal activities of macrophages. Surprisingly, despite similarities in structure and ligand binding repertoires, SR-A and MARCO exert opposite effects on interleukin-12 (IL-12 production in macrophages. SR-A ligation also stimulated H2O2 and IL-10 production, but had no effect on the release of several other cytokines. These limited effects of specific SR-A ligation contrast with generalized enhancement of immune responses observed in SR-A-deficient mice. Recent studies have revealed that many of these effects of SR-A deficiency may be caused by compensatory changes in the expression of other receptors and/or disinhibition of signal transduction from receptors belonging to the Toll/IL-1R family, rather than by the loss of the receptor function of SR-A.

  20. Intracellular cholesterol level regulates sensitivity of glioblastoma cells against temozolomide-induced cell death by modulation of caspase-8 activation via death receptor 5-accumulation and activation in the plasma membrane lipid raft.

    Science.gov (United States)

    Yamamoto, Yutaro; Tomiyama, Arata; Sasaki, Nobuyoshi; Yamaguchi, Hideki; Shirakihara, Takuya; Nakashima, Katsuhiko; Kumagai, Kosuke; Takeuchi, Satoru; Toyooka, Terushige; Otani, Naoki; Wada, Kojiro; Narita, Yoshitaka; Ichimura, Koichi; Sakai, Ryuichi; Namba, Hiroki; Mori, Kentaro

    2018-01-01

    Development of resistance against temozolomide (TMZ) in glioblastoma (GBM) after continuous treatment with TMZ is one of the critical problems in clinical GBM therapy. Intracellular cholesterol regulates cancer cell biology, but whether intracellular cholesterol is involved in TMZ resistance of GBM cells remains unclear. The involvement of intracellular cholesterol in acquired resistance against TMZ in GBM cells was investigated. Intracellular cholesterol levels were measured in human U251 MG cells with acquired TMZ resistance (U251-R cells) and TMZ-sensitive control U251 MG cells (U251-Con cells), and found that the intracellular cholesterol level was significantly lower in U251-R cells than in U251-Con cells. In addition, treatment by intracellular cholesterol remover, methyl-beta cyclodextrin (MβCD), or intracellular cholesterol inducer, soluble cholesterol (Chol), regulated TMZ-induced U251-Con cell death in line with changes in intracellular cholesterol level. Involvement of death receptor 5 (DR5), a death receptor localized in the plasma membrane, was evaluated. TMZ without or with MβCD and/or Chol caused accumulation of DR5 into the plasma membrane lipid raft and formed a complex with caspase-8, an extrinsic caspase cascade inducer, reflected in the induction of cell death. In addition, treatment with caspase-8 inhibitor or knockdown of DR5 dramatically suppressed U251-Con cell death induced by combination treatment with TMZ, MβCD, and Chol. Combined treatment of Chol with TMZ reversed the TMZ resistance of U251-R cells and another GBM cell model with acquired TMZ resistance, whereas clinical antihypercholesterolemia agents at physiological concentrations suppressed TMZ-induced cell death of U251-Con cells. These findings suggest that intracellular cholesterol level affects TMZ treatment of GBM mediated via a DR5-caspase-8 mechanism. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Growth hormone, interferon-gamma, and leukemia inhibitory factor utilize insulin receptor substrate-2 in intracellular signaling

    DEFF Research Database (Denmark)

    Argetsinger, L S; Norstedt, G; Billestrup, Nils

    1996-01-01

    In this report, we demonstrate that insulin receptor substrate-2 (IRS-2) is tyrosyl-phosphorylated following stimulation of 3T3-F442A fibroblasts with growth hormone (GH), leukemia inhibitory factor and interferon-gamma. In response to GH and leukemia inhibitory factor, IRS-2 is immediately...... for GH is further demonstrated by the finding that GH stimulates association of IRS-2 with the 85-kDa regulatory subunit of phosphatidylinositol 3'-kinase and with the protein-tyrosine phosphatase SHP2. These results are consistent with the possibility that IRS-2 is a downstream signaling partner...

  2. Inhibition of motor neuron death in vitro and in vivo by a p75 neurotrophin receptor intracellular domain fragment.

    Science.gov (United States)

    Matusica, Dusan; Alfonsi, Fabienne; Turner, Bradley J; Butler, Tim J; Shepheard, Stephanie R; Rogers, Mary-Louise; Skeldal, Sune; Underwood, Clare K; Mangelsdorf, Marie; Coulson, Elizabeth J

    2016-02-01

    The p75 neurotrophin receptor (p75(NTR); also known as NGFR) can mediate neuronal apoptosis in disease or following trauma, and facilitate survival through interactions with Trk receptors. Here we tested the ability of a p75(NTR)-derived trophic cell-permeable peptide, c29, to inhibit p75(NTR)-mediated motor neuron death. Acute c29 application to axotomized motor neuron axons decreased cell death, and systemic c29 treatment of SOD1(G93A) mice, a common model of amyotrophic lateral sclerosis, resulted in increased spinal motor neuron survival mid-disease as well as delayed disease onset. Coincident with this, c29 treatment of these mice reduced the production of p75(NTR) cleavage products. Although c29 treatment inhibited mature- and pro-nerve-growth-factor-induced death of cultured motor neurons, and these ligands induced the cleavage of p75(NTR) in motor-neuron-like NSC-34 cells, there was no direct effect of c29 on p75(NTR) cleavage. Rather, c29 promoted motor neuron survival in vitro by enhancing the activation of TrkB-dependent signaling pathways, provided that low levels of brain-derived neurotrophic factor (BDNF) were present, an effect that was replicated in vivo in SOD1(G93A) mice. We conclude that the c29 peptide facilitates BDNF-dependent survival of motor neurons in vitro and in vivo. © 2016. Published by The Company of Biologists Ltd.

  3. GABAAα1 and GABAAρ1 subunits are expressed in cultured human RPE cells and GABAA receptor agents modify the intracellular calcium concentration.

    Science.gov (United States)

    Cheng, Zhen-Ying; Wang, Xu-Ping; Schmid, Katrina L; Han, Xu-Guang; Song, Hui; Tang, Xin

    2015-01-01

    Gamma-aminobutyric acid A (GABAA) receptors (GABAARs), which are ionotropic receptors involving chloride channels, have been identified in various neural (e.g., mouse retinal ganglion cells) and nonneural cells (e.g., mouse lens epithelial cells) regulating the intracellular calcium concentration ([Ca(2+)]i). GABAAR β-subunit protein has been isolated in the cultured human and rat RPE, and GABAAα1 and GABAAρ1 mRNAs and proteins are present in the chick RPE. The purpose of this study was to investigate the expression of GABAAα1 and GABAAρ1, two important subunits in forming functional GABAARs, in the cultured human RPE, and further to explore whether altering receptor activation modifies [Ca(2+)]i. Human RPE cells were separately cultured from five donor eye cups. Real-time PCR, western blots, and immunofluorescence were used to test for GABAAα1 and GABAAρ1 mRNAs and proteins. The effects of the GABAAR agonist muscimol, antagonist picrotoxin, or the specific GABAAρ antagonist 1,2,5,6-tetrahydropyridin-4-yl) methylphosphinic acid (TPMPA) on [Ca(2+)]i in cultured human RPE were demonstrated using Fluo3-AM. Both GABAAα1 and GABAAρ1 mRNAs and proteins were identified in cultured human RPE cells; antibody staining was mainly localized to the cell membrane and was also present in the cytoplasm but not in the nucleus. Muscimol (100 μM) caused a transient increase of the [Ca(2+)]i in RPE cells regardless of whether Ca(2+) was added to the buffer. Muscimol-induced increases in the [Ca(2+)]i were inhibited by pretreatment with picrotoxin (300 μM) or TPMPA (500 μM). GABAAα1 and GABAAρ1 are expressed in cultured human RPE cells, and GABAA agents can modify [Ca(2+)]i.

  4. A Role of Endogenous Progesterone in Stroke Cerebroprotection Revealed by the Neural-Specific Deletion of Its Intracellular Receptors.

    Science.gov (United States)

    Zhu, Xiaoyan; Fréchou, Magalie; Liere, Philippe; Zhang, Shaodong; Pianos, Antoine; Fernandez, Neïké; Denier, Christian; Mattern, Claudia; Schumacher, Michael; Guennoun, Rachida

    2017-11-08

    Treatment with progesterone protects the male and female brain against damage after middle cerebral artery occlusion (MCAO). However, in both sexes, the brain contains significant amounts of endogenous progesterone. It is not known whether endogenously produced progesterone enhances the resistance of the brain to ischemic insult. Here, we used steroid profiling by gas chromatography-tandem mass spectrometry (GC-MS/MS) for exploring adaptive and sex-specific changes in brain levels of progesterone and its metabolites after MCAO. We show that, in the male mouse brain, progesterone is mainly metabolized via 5α-reduction leading to 5α-dihydroprogesterone (5α-DHP), also a progesterone receptor (PR) agonist ligand in neural cells, then to 3α,5α-tetrahydroprogesterone (3α,5α-THP). In the female mouse brain, levels of 5α-DHP and 3α,5α-THP are lower and levels of 20α-DHP are higher than in males. After MCAO, levels of progesterone and 5α-DHP are upregulated rapidly to pregnancy-like levels in the male but not in the female brain. To assess whether endogenous progesterone and 5α-DHP contribute to the resistance of neural cells to ischemic damage, we inactivated PR selectively in the CNS. Deletion of PR in the brain reduced its resistance to MCAO, resulting in increased infarct volumes and neurological deficits in both sexes. Importantly, endogenous PR ligands continue to protect the brain of aging mice. These results uncover the unexpected importance of endogenous progesterone and its metabolites in cerebroprotection. They also reveal that the female reproductive hormone progesterone is an endogenous cerebroprotective neurosteroid in both sexes. SIGNIFICANCE STATEMENT The brain responds to injury with protective signaling and has a remarkable capacity to protect itself. We show here that, in response to ischemic stroke, levels of progesterone and its neuroactive metabolite 5α-dihydroprogesterone are upregulated rapidly in the male mouse brain but not in the

  5. Distinct uptake mechanisms but similar intracellular processing of two different toll-like receptor ligand-peptide conjugates in dendritic cells.

    Science.gov (United States)

    Khan, Selina; Bijker, Martijn S; Weterings, Jimmy J; Tanke, Hans J; Adema, Gosse J; van Hall, Thorbald; Drijfhout, Jan W; Melief, Cornelis J M; Overkleeft, Hermen S; van der Marel, Gijsbert A; Filippov, Dmitri V; van der Burg, Sjoerd H; Ossendorp, Ferry

    2007-07-20

    Covalent conjugation of Toll-like receptor ligands (TLR-L) to synthetic antigenic peptides strongly improves antigen presentation in vitro and T lymphocyte priming in vivo. These molecularly well defined TLR-L-peptide conjugates, constitute an attractive vaccination modality, sharing the peptide antigen and a defined adjuvant in one single molecule. We have analyzed the intracellular trafficking and processing of two TLR-L conjugates in dendritic cells (DCs). Long synthetic peptides containing an ovalbumin cytotoxic T-cell epitope were chemically conjugated to two different TLR-Ls the TLR2 ligand, Pam(3)CysSK(4) (Pam) or the TLR9 ligand CpG. Rapid and enhanced uptake of both types of TLR-L-conjugated peptide occurred in DCs. Moreover, TLR-L conjugation greatly enhanced antigen presentation, a process that was dependent on endosomal acidification, proteasomal cleavage, and TAP translocation. The uptake of the CpG approximately conjugate was independent of endosomally-expressed TLR9 as reported previously. Unexpectedly, we found that Pam approximately conjugated peptides were likewise internalized independently of the expression of cell surface-expressed TLR2. Further characterization of the uptake mechanisms revealed that TLR2-L employed a different uptake route than TLR9-L. Inhibition of clathrin- or caveolin-dependent endocytosis greatly reduced uptake and antigen presentation of the Pam-conjugate. In contrast, internalization and antigen presentation of CpG approximately conjugates was independent of clathrin-coated pits but partly dependent on caveolae formation. Importantly, in contrast to the TLR-independent uptake of the conjugates, TLR expression and downstream TLR signaling was required for dendritic cell maturation and for priming of naïve CD8(+) T-cells. Together, our data show that targeting to two distinct TLRs requires distinct uptake mechanism but follows similar trafficking and intracellular processing pathways leading to optimal antigen

  6. Toll-like receptor 2 (TLR2) mediates intracellular signalling in human keratinocytes in response to Malassezia furfur.

    Science.gov (United States)

    Baroni, Adone; Orlando, Manuela; Donnarumma, Giovanna; Farro, Pietro; Iovene, Maria Rosaria; Tufano, Maria Antonietta; Buommino, Elisabetta

    2006-01-01

    Toll-like receptors (TLRs) are crucial players in the innate immune response to microbial invaders. The lipophilic yeast Malassezia furfur has been implicated in the triggering of scalp lesions in psoriasis. The aim of the present study was to assess the role of TLRs in the defence against M. furfur infection. The expression of the myeloid differentiation factor 88 (MyD88) gene, which is involved in the signalling pathway of many TLRs, was also analysed. In addition, a possible correlation of antimicrobial peptides of the beta-defensin family to TLRs was tested. Human keratinocytes infected with M. furfur and a variety of M. furfur-positive psoriatic skin biopsies were analysed by RT-PCR, for TLRs, MyD88, human beta-defensin 2 (HBD-2), HBD-3 and interleukin-8 (IL-8) mRNA expression. When keratinocytes were infected with M. furfur, an up-regulation for TLR2, MyD88, HBD-2, HBD-3 and IL-8 mRNA was demonstrated, compared to the untreated cells. The same results were obtained when psoriatic skin biopsies were analysed. The M. furfur-induced increase in HBD-2 and IL-8 gene expression is inhibited by anti-TLR2 neutralising antibodies, suggesting that TLR2 is involved in the M. furfur-induced expression of these molecules. These findings suggest the importance of TLRs in skin protection against fungi and the importance of keratinocytes as a component of innate immunity.

  7. The Retinoic acid receptor alpha (RARalpha) chimeric proteins PML-, PLZF-, NPM-, and NuMA-RARalpha have distinct intracellular localization patterns.

    Science.gov (United States)

    Hummel, Jeff L; Zhang, Tong; Wells, Richard A; Kamel-Reid, Suzanne

    2002-04-01

    Retinoic acid receptor alpha (RARalpha) gene rearrangement by reciprocal chromosome translocation is the molecular signature of acute promyelocytic leukemia (APL). Disruption of RARalpha function appears to be the likely cause of aberrant myelopoiesis observed in APL, because PML-RARalpha expression has been shown to deregulate the transcription of genes that control myelopoiesis. To target RARalpha chimeric proteins, we engineered epitope-tagged versions of PML-RARalpha, PLZF-RARalpha, NPM-RARalpha, and NuMA-RARalpha (X-RARalphaV5) and generated a panel of stable COS cell lines expressing X-RARalphaV5. Protein fractionation and Western analysis of these COS lines reveal that X-RARalpha proteins localize to both the cytoplasm and nucleus. NPM-RARalpha is predominantly nuclear whereas NuMA-RARalpha is predominantly cytoplasmic. Confocal immunofluorescent microscopy reveals that PML-RARalpha and PLZF-RARalpha share a primarily diffuse nuclear pattern that excludes the nucleolus. NPM-RARalpha is also diffuse in the nucleus but, in contrast to PML-RARalpha and PLZF-RARalpha, is strongly associated with the nucleolus. Strikingly, NuMA-RARalpha predominantly localizes throughout the cytoplasm in a microspeckled pattern. We further demonstrate that NPM and NuMA interact with NPM-RARalpha and NuMA-RARalpha, respectively. The distinct intracellular localization patterns and the shared ability of X-RARalpha to interact with their respective RARalpha partner proteins (X) further support the hypothesis that deregulation of these partners may play a role in APL pathogenesis.

  8. Intracellular β2-adrenergic receptor signaling specificity in mouse skeletal muscle in response to single-dose β2-agonist clenbuterol treatment and acute exercise.

    Science.gov (United States)

    Sato, Shogo; Shirato, Ken; Mitsuhashi, Ryosuke; Inoue, Daisuke; Kizaki, Takako; Ohno, Hideki; Tachiyashiki, Kaoru; Imaizumi, Kazuhiko

    2013-05-01

    The aim of this study was to clarify the intracellular β2-adrenergic receptor signaling specificity in mouse slow-twitch soleus and fast-twitch tibialis anterior (TA) muscles, resulting from single-dose β2-agonist clenbuterol treatment and acute exercise. At 1, 4, and 24 h after single-dose treatment with clenbuterol or after acute running exercise, the soleus and TA muscles were isolated and subjected to analysis. The phosphorylation of p38 mitogen-activated protein kinase (MAPK) increased after single-dose clenbuterol treatment and acute exercise in the soleus muscle but not in the TA muscle. Although there was no change in the phosphorylation of Akt after acute exercise in either muscle, phosphorylation of Akt in the soleus muscle increased after single-dose clenbuterol treatment, whereas that in the TA muscle remained unchanged. These results suggest that p38 MAPK and Akt pathways play a functional role in the adaptation to clenbuterol treatment and exercise, particularly in slow-twitch muscles.

  9. Evidence for CB2 receptor involvement in LPS-induced reduction of cAMP intracellular levels in uterine explants from pregnant mice: pathophysiological implications.

    Science.gov (United States)

    Salazar, Ana Inés; Carozzo, Alejandro; Correa, Fernando; Davio, Carlos; Franchi, Ana María

    2017-07-01

    What is the role of the endocannabinoid system (eCS) on the lipopolysaccharide (LPS) effects on uterine explants from 7-day pregnant mice in a murine model of endotoxin-induced miscarriage? We found evidence for cannabinoid receptor type2 (CB2) involvement in LPS-induced increased prostaglandin-F2α (PGF2α) synthesis and diminished cyclic adenosine monophosphate (cAMP) intracellular content in uterine explants from early pregnant mice. Genital tract infections by Gram-negative bacteria are a common complication of human pregnancy that results in an increased risk of pregnancy loss. LPS, the main component of the Gram-negative bacterial wall, elicits a strong maternal inflammatory response that results in embryotoxicity and embryo resorption in a murine model endotoxin-induced early pregnancy loss. We have previously shown that the eCS mediates the embryotoxic effects of LPS, mainly via CB1 receptor activation. An in vitro study of mice uterine explants was performed to investigate the eCS in mediating the effects of LPS on PGF2α production and cAMP intracellular content. Eight to 12-week-old virgin female BALB/c or CD1 (wild-type [WT] or CB1-knockout [CB1-KO]) mice were paired with 8- to 12-week-old BALB/c or CD1 (WT or CB1-KO) males, respectively. On day 7 of pregnancy, BALB/c, CD1 WT or CD1 CB1-KO mice were euthanized, the uteri were excised, implantation sites were removed and the uterine tissues were separated from decidual and embryo tissues. Uterine explants were cultured and exposed for an appropriate amount of time to different pharmacological treatments. The tissues were then collected for cAMP assay and PGF2α content determination by radioimmunoassay. In vitro treatment of uteri explants from 7-day pregnant BALB/c or CD1 (WT or CB1-KO) mice with LPS induced an increased production of PGF2α (P LPS-induced effects (P LPS-induced deleterious effects on reproductive tissues. Since our experimental design involves in vitro experiments of uterine explants

  10. Low density lipoprotein receptor-related protein 1 (LRP1) modulates N-methyl-D-aspartate (NMDA) receptor-dependent intracellular signaling and NMDA-induced regulation of postsynaptic protein complexes.

    Science.gov (United States)

    Nakajima, Chikako; Kulik, Akos; Frotscher, Michael; Herz, Joachim; Schäfer, Michael; Bock, Hans H; May, Petra

    2013-07-26

    The lipoprotein receptor LRP1 is essential in neurons of the central nervous system, as was revealed by the analysis of conditional Lrp1-deficient mouse models. The molecular basis of its neuronal functions, however, is still incompletely understood. Here we show by immunocytochemistry, electron microscopy, and postsynaptic density preparation that LRP1 is located postsynaptically. Basal and NMDA-induced phosphorylation of the transcription factor cAMP-response element-binding protein (CREB) as well as NMDA target gene transcription are reduced in LRP1-deficient neurons. In control neurons, NMDA promotes γ-secretase-dependent release of the LRP1 intracellular domain (LRP1-ICD). However, pull-down and chromatin immunoprecipitation (ChIP) assays showed no direct interaction between the LRP1-ICD and either CREB or target gene promoters. On the other hand, NMDA-induced degradation of the postsynaptic scaffold protein PSD-95 was impaired in the absence of LRP1, whereas its ubiquitination was increased, indicating that LRP1 influences the composition of postsynaptic protein complexes. Accordingly, NMDA-induced internalization of the AMPA receptor subunit GluA1 was impaired in LRP1-deficient neurons. These results show a role of LRP1 in the regulation and turnover of synaptic proteins, which may contribute to the reduced dendritic branching and to the neurological phenotype observed in the absence of LRP1.

  11. Low Density Lipoprotein Receptor-related Protein 1 (LRP1) Modulates N-Methyl-d-aspartate (NMDA) Receptor-dependent Intracellular Signaling and NMDA-induced Regulation of Postsynaptic Protein Complexes*

    Science.gov (United States)

    Nakajima, Chikako; Kulik, Akos; Frotscher, Michael; Herz, Joachim; Schäfer, Michael; Bock, Hans H.; May, Petra

    2013-01-01

    The lipoprotein receptor LRP1 is essential in neurons of the central nervous system, as was revealed by the analysis of conditional Lrp1-deficient mouse models. The molecular basis of its neuronal functions, however, is still incompletely understood. Here we show by immunocytochemistry, electron microscopy, and postsynaptic density preparation that LRP1 is located postsynaptically. Basal and NMDA-induced phosphorylation of the transcription factor cAMP-response element-binding protein (CREB) as well as NMDA target gene transcription are reduced in LRP1-deficient neurons. In control neurons, NMDA promotes γ-secretase-dependent release of the LRP1 intracellular domain (LRP1-ICD). However, pull-down and chromatin immunoprecipitation (ChIP) assays showed no direct interaction between the LRP1-ICD and either CREB or target gene promoters. On the other hand, NMDA-induced degradation of the postsynaptic scaffold protein PSD-95 was impaired in the absence of LRP1, whereas its ubiquitination was increased, indicating that LRP1 influences the composition of postsynaptic protein complexes. Accordingly, NMDA-induced internalization of the AMPA receptor subunit GluA1 was impaired in LRP1-deficient neurons. These results show a role of LRP1 in the regulation and turnover of synaptic proteins, which may contribute to the reduced dendritic branching and to the neurological phenotype observed in the absence of LRP1. PMID:23760271

  12. Nicotine Elevated Intracellular Ca2+ in Rat Airway Smooth Muscle Cells via Activating and Up-Regulating α7-Nicotinic Acetylcholine Receptor

    Directory of Open Access Journals (Sweden)

    Yongliang Jiang

    2014-02-01

    Full Text Available Background: Chronic obstructive pulmonary disease (COPD is characterized by airway remodeling with airway smooth muscle (ASM hypertrophy and hyperplasia. Since tobacco use is the key risk factor for the development of COPD and intracellular Ca2+ concentration ([Ca2+]i plays a major role in both cell proliferation and differentiation, we hypothesized that nicotinic acetylcholine receptor (nAChR activation plays a role in the elevation of [Ca2+]i in airway smooth muscle cells (ASMCs. Methods: We examined the expression of nAChR and characterized the functions of α7-nAChR in ASMCs. Results: RT-PCR analysis showed that α2-7, β2, and β3-nAChR subunits are expressed in rat ASMCs, with α7 being one of the most abundantly expressed subtypes. Chronic nicotine exposure increased α7-nAChR mRNA and protein expression, and elevated resting [Ca2+]i in cultured rat ASMCs. Acute application of nicotine evoked a rapid increase in [Ca2+]i in a concentration-dependent manner, and the response was significantly enhanced in ASMCs cultured with 1 µM nicotine for 48 hours. Nicotine-induced Ca2+ response was reversibly blocked by the α7-nAChR nicotinic antagonists, methyllycaconitine and α-bungarotoxin. Small interfering RNA suppression of α7-nAChR also substantially blunted the Ca2+ responses induced by nicotine. Conclusion: These observations suggest that nicotine elevates [Ca2+]i in ASMCs through α7-nAChR-mediated signals pathways, and highlight the possibility that α7-nAChR can be considered as a potential target for the treatment of airway remodeling.that nicotine elevates [Ca2+]i in ASMCs through α7-nAChR-mediated signals pathways, and highlight the possibility that α7-nAChR can be considered as a potential target for the treatment of airway remodeling.

  13. A Pepducin Derived from the Third Intracellular Loop of FPR2 Is a Partial Agonist for Direct Activation of This Receptor in Neutrophils But a Full Agonist for Cross-Talk Triggered Reactivation of FPR2

    DEFF Research Database (Denmark)

    Gabl, Michael; Winther, Malene; Skovbakke, Sarah Line

    2014-01-01

    We recently described a novel receptor cross-talk mechanism in neutrophils, unique in that the signals generated by the PAF receptor (PAFR) and the ATP receptor (P2Y2R) transfer formyl peptide receptor 1 (FPR1) from a desensitized (non-signaling) state back to an actively signaling state (Forsman H...... et al., PLoS One, 8:e60169, 2013; Önnheim K, et al., Exp Cell Res, 323:209, 2014). In addition to the G-protein coupled FPR1, neutrophils also express the closely related receptor FPR2. In this study we used an FPR2 specific pepducin, proposed to work as an allosteric modulator at the cytosolic...... signaling interface, to determine whether the cross-talk pathway is utilized also by FPR2. The pepducin used contains a fatty acid linked to a peptide sequence derived from the third intracellular loop of FPR2, and it activates as well as desensensitizes this receptor. We now show that neutrophils...

  14. Identification of tyrosine residues in the intracellular domain of the growth hormone receptor required for transcriptional signaling and Stat5 activation

    DEFF Research Database (Denmark)

    Hansen, L. H.; Wang, X.; Kopchick, J J

    1996-01-01

    The binding of growth hormone (GH) to its receptor results in its dimerization followed by activation of Jak2 kinase and tyrosine phosphorylation of the GH receptor itself, as well as Jak2 and the transcription factors Stat1, -3, and -5. In order to study the role of GH receptor tyrosine phosphor...

  15. Intracellular angiotensin II elicits Ca2+ increases in A7r5 vascular smooth muscle cells

    NARCIS (Netherlands)

    Filipeanu, CM; Brailoiu, E; Kok, JW; Henning, RH; De Zeeuw, D; Nelemans, SA

    2001-01-01

    Recent studies show that angiotensin II can act within the cell, possibly via intracellular receptors pharmacologically different from typical plasma membrane angiotensin II receptors. The signal transduction of intracellular angiotensin LI is unclear. Therefore. we investigated the effects of

  16. Assembly, intracellular processing, and expression at the cell surface of the human alpha beta T cell receptor/CD3 complex. Function of the CD3-zeta chain

    DEFF Research Database (Denmark)

    Geisler, C; Kuhlmann, J; Rubin, B

    1989-01-01

    complex, the role of the CD3 chains for the TCR/CD3 expression have not been experimentally addressed in human T cells. In this study the function of the CD3-zeta chain for the assembly, intracellular processing, and expression of the TCR/CD3 complex in the human leukemic T cell line Jurkat...

  17. Intracellular calcium elevation during plateau potentials mediated by extrasynaptic NMDA receptor activation in rat hippocampal CA1 pyramidal neurons is primarily due to calcium entry through voltage-gated calcium channels.

    Science.gov (United States)

    Oda, Yoshiaki; Kodama, Satoshi; Tsuchiya, Sadahiro; Inoue, Masashi; Miyakawa, Hiroyoshi

    2014-05-01

    We reported previously that plateau potentials mediated by extrasynaptic N-methyl-d-aspartate receptors (NMDARs) can be induced either by synaptic stimulation in the presence of glutamate transporter antagonist or by iontophoresis of NMDA in rat hippocampal CA1 pyramidal neurons. To examine whether the plateau potentials are accompanied by an elevation of intracellular Ca2+ and to determine the source of Ca2+ elevation, we performed Ca2+ imaging during the plateau potential. Neurons were loaded with Ca2+ indicator fluo-4, and the plateau potentials were generated either synaptically in the presence of glutamate transporter antagonist or by iontophoretically applying NMDA. We have found that a transient elevation in intracellular Ca2+ accompanies the plateau potential. The synaptically induced plateau potential and the Ca2+ elevation were blocked by 5,7-dichlorokynurenic acid (5,7-dCK), an antagonist for the glycine-binding sites of NMDAR. A mixture of Cd2+ and tetrodotoxin did not block NMDA-induced plateau potentials, but completely abolished the accompanying Ca2+ elevation in both the presence and absence of Mg2+ ions in the bathing solution. The NMDA-induced plateau potential was blocked by further adding 5,7-dCK. Our results show that the NMDAR-mediated plateau potential is accompanied by elevation of intracellular Ca2+ that is primarily caused by the influx of Ca2+ through voltage-gated Ca2+ channels. © 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  18. Identification of specific sites in the third intracellular loop and carboxyl terminus of the Bombyx mori PBAN receptor crucial for ligand-induced internalization

    Science.gov (United States)

    Sex pheromone production in most moths is mediated by the pheromone biosynthesis activating neuropeptide receptor (PBANR). Similar to other rhodopsin-like G protein-coupled receptors, the silkmoth Bombyx mori PBANR (BmPBANR) undergoes agonist-induced internalization. Despite interest in developing...

  19. 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

    Science.gov (United States)

    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

  20. A Stretch of Negatively Charged Amino Acids of Linker for Activation of T-Cell Adaptor Has a Dual Role in T-Cell Antigen Receptor Intracellular Signaling

    Directory of Open Access Journals (Sweden)

    Mikel M. Arbulo-Echevarria

    2018-02-01

    Full Text Available The adaptor protein linker for activation of T cells (LAT has an essential role transducing activatory intracellular signals coming from the TCR/CD3 complex. Previous reports have shown that upon T-cell activation, LAT interacts with the tyrosine kinase Lck, leading to the inhibition of its kinase activity. LAT–Lck interaction seemed to depend on a stretch of negatively charged amino acids in LAT. Here, we have substituted this segment of LAT between amino acids 113 and 126 with a non-charged segment and expressed the mutant LAT (LAT-NIL in J.CaM2 cells in order to analyze TCR signaling. Substitution of this segment in LAT prevented the activation-induced interaction with Lck. Moreover, cells expressing this mutant form of LAT showed a statistically significant increase of proximal intracellular signals such as phosphorylation of LAT in tyrosine residues 171 and 191, and also enhanced ZAP70 phosphorylation approaching borderline statistical significance (p = 0.051. Nevertheless, downstream signals such as Ca2+ influx or MAPK pathways were partially inhibited. Overall, our data reveal that LAT–Lck interaction constitutes a key element regulating proximal intracellular signals coming from the TCR/CD3 complex.

  1. P2Y receptor-mediated transient relaxation of rat longitudinal ileum preparations involves phospholipase C activation, intracellular Ca(2+) release and SK channel activation.

    Science.gov (United States)

    Mader, Felix; Krause, Ludwig; Tokay, Tursonjan; Hakenberg, Oliver W; Köhling, Rüdiger; Kirschstein, Timo

    2016-05-01

    Purinergic signaling plays a major role in the enteric nervous system, where it governs gut motility through a number of P2X and P2Y receptors. The aim of this study was to investigate the P2Y receptor-mediated motility in rat longitudinal ileum preparations. Ileum smooth muscle strips were prepared from rats, and fixed in an organ bath. Isometric contraction and relaxation responses of the muscle strips were measured with force transducers. Drugs were applied by adding of stock solutions to the organ bath to yield the individual final concentrations. Application of the non-hydrolyzable P2 receptor agonists α,β-Me-ATP or 2-Me-S-ADP (10, 100 μmol/L) dose-dependently elicited a transient relaxation response followed by a sustained contraction. The relaxation response was largely blocked by SK channel blockers apamin (500 nmol/L) and UCL1684 (10 μmol/L), PLC inhibitor U73122 (100 μmol/L), IP3 receptor blocker 2-APB (100 μmol/L) or sarcoendoplasmic Ca(2+) ATPase inhibitor thapsigargin (1 μmol/L), but not affected by atropine, NO synthase blocker L-NAME or tetrodotoxin. Furthermore, α,β-Me-ATP-induced relaxation was suppressed by P2Y1 receptor antagonist MRS2179 (50 μmol/L) or P2Y13 receptor antagonist MRS2211 (100 μmol/L), and was abolished by co-application of the two antagonists, whereas 2-Me-S-ADP-induced relaxation was abolished by P2Y6 receptor antagonist MRS2578 (50 μmol/L). In addition, P2Y1 receptor antagonist MRS2500 (1 μmol/L) not only abolished α,β-Me-ATP-induced relaxation, but also suppressed 2-Me-S-ADP-induced relaxation. P2Y receptor agonist-induced transient relaxation of rat ileum smooth muscle strips is mediated predominantly by P2Y1 receptor, but also by P2Y6 and P2Y13 receptors, and involves PLC, IP3, Ca(2+) release and SK channel activation, but is independent of acetylcholine and NO release.

  2. A pepducin derived from the third intracellular loop of FPR2 is a partial agonist for direct activation of this receptor in neutrophils but a full agonist for cross-talk triggered reactivation of FPR2.

    Directory of Open Access Journals (Sweden)

    Michael Gabl

    Full Text Available We recently described a novel receptor cross-talk mechanism in neutrophils, unique in that the signals generated by the PAF receptor (PAFR and the ATP receptor (P2Y2R transfer formyl peptide receptor 1 (FPR1 from a desensitized (non-signaling state back to an actively signaling state (Forsman H et al., PLoS One, 8:e60169, 2013; Önnheim K, et al., Exp Cell Res, 323∶209, 2014. In addition to the G-protein coupled FPR1, neutrophils also express the closely related receptor FPR2. In this study we used an FPR2 specific pepducin, proposed to work as an allosteric modulator at the cytosolic signaling interface, to determine whether the cross-talk pathway is utilized also by FPR2. The pepducin used contains a fatty acid linked to a peptide sequence derived from the third intracellular loop of FPR2, and it activates as well as desensensitizes this receptor. We now show that neutrophils desensitized with the FPR2-specific pepducin display increased cellular responses to stimulation with PAF or ATP. The secondary PAF/ATP induced response was sensitive to FPR2-specific inhibitors, disclosing a receptor cross-talk mechanism underlying FPR2 reactivation. The pepducin induced an activity in naïve cells similar to that of a conventional FPR2 agonist, but with lower potency (partial efficacy, meaning that the pepducin is a partial agonist. The PAF- or ATP-induced reactivation was, however, much more pronounced when neutrophils had been desensitized to the pepducin as compared to cells desensitized to conventional agonists. The pepducin should thus in this respect be classified as a full agonist. In summary, we demonstrate that desensitized FPR2 can be transferred back to an actively signaling state by receptor cross-talk signals generated through PAFR and P2Y2R, and the difference in agonist potency with respect to pepducin-induced direct receptor activation and cross-talk reactivation of FPR2 puts the concept of functional selectivity in focus.

  3. 17beta-estradiol rapidly mobilizes intracellular calcium from ryanodine-receptor-gated stores via a PKC-PKA-Erk-dependent pathway in the human eccrine sweat gland cell line NCL-SG3.

    LENUS (Irish Health Repository)

    Muchekehu, Ruth W

    2008-09-01

    We describe a novel rapid non-genomic effect of 17beta-estradiol (E2) on intracellular Ca2+ ([Ca2+]i) signalling in the eccrine sweat gland epithelial cell line NCL-SG3. E2 had no observable effect on basal [Ca2+]i, however exposure of cells to E2 in the presence of the microsomal Ca2+ ATPase pump inhibitor, thapsigargin, produced a secondary, sustained increase in [Ca2+]i compared to thapsigargin treatment alone, where cells responded with a transient single spike-like increase in [Ca2+]i. The E2-induced increase in [Ca2+]i was not dependent on the presence of extracellular calcium and was completely abolished by ryanodine (100 microM). The estrogen receptor antagonist ICI 182,780 (1 microM) prevented the E2-induced effects suggesting a role for the estrogen receptor in the release of [Ca2+]i from ryanodine-receptor-gated stores. The E2-induced effect on [Ca2+]i could also be prevented by the protein kinase C delta (PKCdelta)-specific inhibitor rottlerin (10 microM), the protein kinase A (PKA) inhibitor Rp-adenosine 3\\

  4. Plasma membrane cholesterol level and agonist-induced internalization of delta-opioid receptors; colocalization study with intracellular membrane markers of Rab family\

    Czech Academy of Sciences Publication Activity Database

    Brejchová, Jana; Vošahlíková, Miroslava; Roubalová, Lenka; Parenti, M.; Mauri, M.; Chernyavskiy, Oleksandr; Svoboda, Petr

    2016-01-01

    Roč. 48, č. 4 (2016), s. 375-396 ISSN 0145-479X R&D Projects: GA ČR(CZ) GAP207/12/0919 Institutional support: RVO:67985823 Keywords : cholesterol * plasma membrane * delta-opioid receptor * internalization * Rab proteins Subject RIV: CE - Biochemistry Impact factor: 2.576, year: 2016

  5. Multiple tyrosine residues in the intracellular domain of the common ß subunit of the interleukin 5 receptor are involved in activation of STAT5

    NARCIS (Netherlands)

    Dijk, Thamar B. van; Caldenhoven, Eric; Raaijmakers, J.A.M.; Lammers, J.W.J.; Koenderman, L.; Groot, Rolf P. de

    1997-01-01

    Abstract In contrast to the general model of cytokine-induced JAK/STAT signaling, tyrosine phosphorylation of the IL-5R ß chain seems to be dispensable for STAT activation in cells overexpressing exogenous STAT proteins. In this study we expressed IL-5 receptor mutants in 293 cells and studied

  6. In vivo experimental stroke and in vitro organ culture induce similar changes in vasoconstrictor receptors and intracellular calcium handling in rat cerebral arteries

    DEFF Research Database (Denmark)

    Povlsen, Gro Klitgaard; Waldsee, Roya; Ahnstedt, Hilda

    2012-01-01

    Cerebral arteries subjected to different types of experimental stroke upregulate their expression of certain G-protein-coupled vasoconstrictor receptors, a phenomenon that worsens the ischemic brain damage. Upregulation of contractile endothelin B (ET(B)) and 5-hydroxytryptamine 1B (5-HT(1B)) rec...

  7. The coronavirus transmissible gastroenteritis virus causes infection after receptor-mediated endocytosis and acid-dependent fusion with an intracellular compartment

    DEFF Research Database (Denmark)

    Hansen, Gert Helge; Delmas, B; Besnardeau, L

    1998-01-01

    Aminopeptidase N is a species-specific receptor for transmissible gastroenteritis virus (TGEV), which infects piglets, and for the 229E virus, which infects humans. It is not known whether these coronaviruses are endocytosed before fusion with a membrane of the target cell, causing a productive...

  8. Investigating Internalization and Intracellular Trafficking of GPCRs

    DEFF Research Database (Denmark)

    Foster, Simon R; Bräuner-Osborne, Hans

    2017-01-01

    for signal transduction. One of the major mechanisms for GPCR regulation involves their endocytic trafficking, which serves to internalize the receptors from the plasma membrane and thereby attenuate G protein-dependent signaling. However, there is accumulating evidence to suggest that GPCRs can signal...... independently of G proteins, as well as from intracellular compartments including endosomes. It is in this context that receptor internalization and intracellular trafficking have attracted renewed interest within the GPCR field. In this chapter, we will review the current understanding and methodologies...... that have been used to investigate internalization and intracellular signaling of GPCRs, with a particular focus on emerging real-time techniques. These recent developments have improved our understanding of the complexities of GPCR internalization and intracellular signaling and suggest that the broader...

  9. Switching of N-Methyl-d-aspartate (NMDA) Receptor-favorite Intracellular Signal Pathways from ERK1/2 Protein to p38 Mitogen-activated Protein Kinase Leads to Developmental Changes in NMDA Neurotoxicity*

    Science.gov (United States)

    Xiao, Lin; Hu, Chun; Feng, Chunzhi; Chen, Yizhang

    2011-01-01

    Excitotoxicity mediated by overactivation of N-methyl-d-aspartate receptors (NMDARs) has been implicated in a variety of neuropathological conditions in the central nervous system (CNS). It has been suggested that N-methyl-d-aspartate (NMDA) neurotoxicity is developmentally regulated, but the definite pattern of the regulation has been controversial, and the underlying mechanism remains largely unknown. Here, we show that NMDA treatment leads to significant cell death in mature (9 and 12 days in vitro) hippocampal neurons or hippocampi of young postnatal day 12 and adult rats but not in immature (3 and 6 days in vitro) neurons or embryonic day 18 and neonatal rat hippocampi. In contrast, NMDA promotes survival of immature neurons against tropic deprivation. Interestingly, it is found that NMDA preferentially activates p38 MAPK in mature neuron and adult rat hippocampus, but it favors ERK1/2 activation in immature neuron and postnatal day 0 rat hippocampus. Moreover, it is shown that NMDA neurotoxicity in mature neuron is mediated via p38 MAPK activation, and neuroprotection in immature neuron is mediated via ERK1/2 activation, whereas all these effects are NR2B-containing NMDAR-dependent, as well as Ca2+-dependent. We also revealed that mature and immature neurons showed no difference in the amplitude of NMDA-induced intracellular calcium ([Ca2+]i) increase. However, the basal level of [Ca2+]i is shown to elevate with the maturation of neuron, and this elevation is attributable to the changes in NMDA neurotoxicity but not to the switch of the NMDAR signaling pathway. Taken together, our results suggest that a switch of NMDA receptor-favorite intracellular signal pathways from ERK1/2 to p38 MAPK and the elevated basal level of [Ca2+]i with age might be critical for the developmental changes in NMDA neurotoxicity in the hippocampal neuron. PMID:21474451

  10. Switching of N-methyl-D-aspartate (NMDA) receptor-favorite intracellular signal pathways from ERK1/2 protein to p38 mitogen-activated protein kinase leads to developmental changes in NMDA neurotoxicity.

    Science.gov (United States)

    Xiao, Lin; Hu, Chun; Feng, Chunzhi; Chen, Yizhang

    2011-06-10

    Excitotoxicity mediated by overactivation of N-methyl-D-aspartate receptors (NMDARs) has been implicated in a variety of neuropathological conditions in the central nervous system (CNS). It has been suggested that N-methyl-D-aspartate (NMDA) neurotoxicity is developmentally regulated, but the definite pattern of the regulation has been controversial, and the underlying mechanism remains largely unknown. Here, we show that NMDA treatment leads to significant cell death in mature (9 and 12 days in vitro) hippocampal neurons or hippocampi of young postnatal day 12 and adult rats but not in immature (3 and 6 days in vitro) neurons or embryonic day 18 and neonatal rat hippocampi. In contrast, NMDA promotes survival of immature neurons against tropic deprivation. Interestingly, it is found that NMDA preferentially activates p38 MAPK in mature neuron and adult rat hippocampus, but it favors ERK1/2 activation in immature neuron and postnatal day 0 rat hippocampus. Moreover, it is shown that NMDA neurotoxicity in mature neuron is mediated via p38 MAPK activation, and neuroprotection in immature neuron is mediated via ERK1/2 activation, whereas all these effects are NR2B-containing NMDAR-dependent, as well as Ca(2+)-dependent. We also revealed that mature and immature neurons showed no difference in the amplitude of NMDA-induced intracellular calcium ([Ca(2+)](i)) increase. However, the basal level of [Ca(2+)](i) is shown to elevate with the maturation of neuron, and this elevation is attributable to the changes in NMDA neurotoxicity but not to the switch of the NMDAR signaling pathway. Taken together, our results suggest that a switch of NMDA receptor-favorite intracellular signal pathways from ERK1/2 to p38 MAPK and the elevated basal level of [Ca(2+)](i) with age might be critical for the developmental changes in NMDA neurotoxicity in the hippocampal neuron.

  11. Functional proteins involved in regulation of intracellular Ca(2+) for drug development: the extracellular calcium receptor and an innovative medical approach to control secondary hyperparathyroidism by calcimimetics.

    Science.gov (United States)

    Nagano, Nobuo; Nemeth, Edward F

    2005-03-01

    Circulating levels of calcium ion (Ca(2+)) are maintained within a narrow physiological range mainly by the action of parathyroid hormone (PTH) secreted from parathyroid cells. Parathyroid cells can sense small fluctuations in plasma Ca(2+) levels by virtue of a cell surface Ca(2+) receptor (CaR) that belongs to the superfamily of G-protein-coupled receptors. Calcimimetics are positive allosteric modulators that activate the CaR on parathyroid cells and thereby immediately suppress PTH secretion. Pre-clinical studies with NPS R-568, a first generation calcimimetic compound, have demonstrated that daily oral administration inhibits the elevation of plasma PTH levels and parathyroid gland hyperplasia and ameliorates impaired bone qualities in rats with chronic renal insufficiency. The results of clinical trials with cinacalcet hydrochloride, a second generation calcimimetic compound, have shown that calcimimetics possess lowering effects not only on serum PTH levels but also on serum calcium x phosphorus product levels, a hallmark of an increased risk for cardiovascular death in dialysis patients with end-stage renal disease (ESRD). Thus, calcimimetics have considerable potential as an innovative medical approach to manage secondary hyperparathyroidism associated with ESRD. Indeed, cinacalcet hydrochloride has been approved in several countries and is the first positive allosteric modulator of any G protein-coupled receptor to reach the market.

  12. The effects of 3,4-methylenedioxymethamphetamine (MDMA) on nicotinic receptors: Intracellular calcium increase, calpain/caspase 3 activation, and functional upregulation

    International Nuclear Information System (INIS)

    Garcia-Rates, Sara; Camarasa, Jordi; Sanchez-Garcia, Ana I.; Gandia, Luis; Escubedo, Elena; Pubill, David

    2010-01-01

    Previous work by our group demonstrated that homomeric α7 nicotinic acetylcholine receptors (nAChR) play a role in the neurotoxicity induced by 3,4-methylenedioxymethamphetamine (MDMA), as well as the binding affinity of this drug to these receptors. Here we studied the effect of MDMA on the activation of nAChR subtypes, the consequent calcium mobilization, and calpain/caspase 3 activation because prolonged Ca 2+ increase could contribute to cytotoxicity. As techniques, we used fluorimetry in Fluo-4-loaded PC12 cells and electrophysiology in Xenopus oocytes. MDMA produced a rapid and sustained increase in calcium without reaching the maximum effect induced by ACh. It also concentration-dependently inhibited the response induced by ACh, nicotine, and the specific α7 agonist PNU 282987 with IC 50 values in the low micromolar range. Similarly, MDMA induced inward currents in Xenopus oocytes transfected with human α7 but not with α4β2 nAChR and inhibited ACh-induced currents in both receptors in a concentration-dependent manner. The calcium response was inhibited by methyllycaconitine (MLA) and α-bungarotoxin but not by dihydro-β-erythroidine. These results therefore indicate that MDMA acts as a partial agonist on α7 nAChRs and as an antagonist on the heteromeric subtypes. Subsequently, calcium-induced Ca 2+ release from the endoplasmic reticulum and entry through voltage-operated calcium channels are also implicated as proved using specific antagonists. In addition, treatment with MDMA for 24 h significantly increased basal Ca 2+ levels and induced an increase in α-spectrin breakdown products, which indicates that calpain and caspase 3 were activated. These effects were inhibited by pretreatment with MLA. Moreover, pretreatment with MDMA induced functional upregulation of calcium responses to specific agonists of both heteromeric and α7 nAChR. Sustained calcium entry and calpain activation could favor the activation of Ca 2+ -dependent enzymes such as

  13. The coronavirus transmissible gastroenteritis virus causes infection after receptor-mediated endocytosis and acid-dependent fusion with an intracellular compartment

    DEFF Research Database (Denmark)

    Hansen, Gert Helge; Delmas, B; Besnardeau, L

    1998-01-01

    Aminopeptidase N is a species-specific receptor for transmissible gastroenteritis virus (TGEV), which infects piglets, and for the 229E virus, which infects humans. It is not known whether these coronaviruses are endocytosed before fusion with a membrane of the target cell, causing a productive...... infection, or whether they fuse directly with the plasma membrane. We have studied the interaction between TGEV and a cell line (MDCK) stably expressing recombinant pig aminopeptidase N (pAPN). By electron microscopy and flow cytometry, TGEV was found to be associated with the plasma membrane after...... adsorption to the pAPN-MDCK cells. TGEV was also observed in endocytic pits and apical vesicles after 3 to 10 min of incubation at 38 degrees C. The number of pits and apical vesicles was increased by the TGEV incubation, indicating an increase in endocytosis. After 10 min of incubation, a distinct TGEV...

  14. Plasma membrane cholesterol level and agonist-induced internalization of δ-opioid receptors; colocalization study with intracellular membrane markers of Rab family.

    Science.gov (United States)

    Brejchova, Jana; Vosahlikova, Miroslava; Roubalova, Lenka; Parenti, Marco; Mauri, Mario; Chernyavskiy, Oleksandr; Svoboda, Petr

    2016-08-01

    Decrease of cholesterol level in plasma membrane of living HEK293 cells transiently expressing FLAG-δ-OR by β-cyclodextrin (β-CDX) resulted in a slight internalization of δ-OR. Massive internalization of δ-OR induced by specific agonist DADLE was diminished in cholesterol-depleted cells. These results suggest that agonist-induced internalization of δ-OR, which has been traditionally attributed exclusively to clathrin-mediated pathway, proceeds at least partially via membrane domains. Identification of internalized pools of FLAG-δ-OR by colocalization studies with proteins of Rab family indicated the decreased presence of receptors in early endosomes (Rab5), late endosomes and lysosomes (Rab7) and fast recycling vesicles (Rab4). Slow type of recycling (Rab11) was unchanged by cholesterol depletion. As expected, agonist-induced internalization of oxytocin receptors was totally suppressed in β-CDX-treated cells. Determination of average fluorescence lifetime of TMA-DPH, the polar derivative of hydrophobic membrane probe diphenylhexatriene, in live cells by FLIM indicated a significant alteration of the overall PM structure which may be interpreted as an increased "water-accessible space" within PM area. Data obtained by studies of HEK293 cells transiently expressing FLAG-δ-OR by "antibody feeding" method were extended by analysis of the effect of cholesterol depletion on distribution of FLAG-δ-OR in sucrose density gradients prepared from HEK293 cells stably expressing FLAG-δ-OR. Major part of FLAG-δ-OR was co-localized with plasma membrane marker Na,K-ATPase and β-CDX treatment resulted in shift of PM fragments containing both FLAG-δ-OR and Na,K-ATPase to higher density. Thus, the decrease in content of the major lipid constituent of PM resulted in increased density of resulting PM fragments.

  15. Downmodulation of Vaccine-Induced Immunity and Protection against the Intracellular Bacterium Francisella tularensis by the Inhibitory Receptor FcγRIIB

    Directory of Open Access Journals (Sweden)

    Brian J. Franz

    2015-01-01

    Full Text Available Fc gamma receptor IIB (FcγRIIB is the only Fc gamma receptor (FcγR which negatively regulates the immune response, when engaged by antigen- (Ag- antibody (Ab complexes. Thus, the generation of Ag-specific IgG in response to infection or immunization has the potential to downmodulate immune protection against infection. Therefore, we sought to determine the impact of FcγRIIB on immune protection against Francisella tularensis (Ft, a Category A biothreat agent. We utilized inactivated Ft (iFt as an immunogen. Naïve and iFt-immunized FcγRIIB knockout (KO or wildtype (WT mice were challenged with Ft-live vaccine strain (LVS. While no significant difference in survival between naïve FcγRIIB KO versus WT mice was observed, iFt-immunized FcγRIIB KO mice were significantly better protected than iFt-immunized WT mice. Ft-specific IgA in serum and bronchial alveolar lavage, as well as IFN-γ, IL-10, and TNF-α production by splenocytes harvested from iFt-immunized FcγRIIB KO, were also significantly elevated. In addition, iFt-immunized FcγRIIB KO mice exhibited a reduction in proinflammatory cytokine levels in vivo at 5 days after challenge, which correlates with increased survival following Ft-LVS challenge in published studies. Thus, these studies demonstrate for the first time the ability of FcγRIIB to regulate vaccine-induced IgA production and downmodulate immunity and protection. The immune mechanisms behind the above observations and their potential impact on vaccine development are discussed.

  16. Stochastic models of intracellular transport

    KAUST Repository

    Bressloff, Paul C.

    2013-01-09

    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. © 2013 American Physical Society.

  17. The Conserved Arginine Cluster in the Insert of the Third Cytoplasmic Loop of the Long Form of the D2 Dopamine Receptor (D2L-R Acts as an Intracellular Retention Signal

    Directory of Open Access Journals (Sweden)

    Valentina Kubale

    2016-07-01

    Full Text Available This study examined whether the conserved arginine cluster present within the 29-amino acid insert of the long form of the D2 dopamine receptor (D2L-R confers its predominant intracellular localization. We hypothesized that the conserved arginine cluster (RRR located within the insert could act as an RXR-type endoplasmic reticulum (ER retention signal. Arginine residues (R within the cluster at positions 267, 268, and 269 were charge-reserved to glutamic acids (E, either individually or in clusters, thus generating single, double, and triple D2L-R mutants. Through analyses of cellular localization by confocal microscopy and enzyme-linked immunosorbent assay (ELISA, radioligand binding assay, bioluminescence resonance energy transfer (BRET2 β-arrestin 2 (βarr2 recruitment assay, and cAMP signaling, it was revealed that charge reversal of the R residues at all three positions within the motif impaired their colocalization with ER marker calnexin and led to significantly improved cell surface expression. Additionally, these data demonstrate that an R to glutamic acid (E substitution at position 2 within the RXR motif is not functionally permissible. Furthermore, all generated D2L-R mutants preserved their functional integrity regarding ligand binding, agonist-induced βarr2 recruitment and Gαi-mediated signaling. In summary, our results show that the conserved arginine cluster within the 29-amino acid insert of third cytoplasmic loop (IC3 of the D2L-R appears to be the ER retention signal.

  18. Intracellular ion channels and cancer

    Directory of Open Access Journals (Sweden)

    Luigi eLeanza

    2013-09-01

    Full Text Available Several types of channels play a role in the maintenance of ion homeostasis in subcellular organelles including endoplasmatic reticulum, nucleus, lysosome, endosome and mitochondria. Here we give a brief overview of the contribution of various mitochondrial and other organellar channels to cancer cell proliferation or death. Much attention is focused on channels involved in intracellular calcium signaling and on ion fluxes in the ATP-producing organelle mitochondria. Mitochondrial K+ channels (Ca2+-dependent BKCa and IKCa, ATP-dependent KATP, Kv1.3, two-pore TWIK-related Acid-Sensitive K+ channel-3 (TASK-3, Ca2+ uniporter MCU, Mg2+-permeable Mrs2, anion channels (voltage-dependent chloride channel VDAC, intracellular chloride channel CLIC and the Permeability Transition Pore (MPTP contribute importantly to the regulation of function in this organelle. Since mitochondria play a central role in apoptosis, modulation of their ion channels by pharmacological means may lead to death of cancer cells. The nuclear potassium channel Kv10.1 and the nuclear chloride channel CLIC4 as well as the endoplasmatic reticulum (ER-located inositol 1,4,5-trisphosphate (IP3 receptor, the ER-located Ca2+ depletion sensor STIM1 (stromal interaction molecule 1, a component of the store-operated Ca2+ channel and the ER-resident TRPM8 are also mentioned. Furthermore, pharmacological tools affecting organellar channels and modulating cancer cell survival are discussed. The channels described in this review are summarized on Figure 1. Overall, the view is emerging that intracellular ion channels may represent a promising target for cancer treatment.

  19. Micro- and nanotechnologies for intracellular delivery.

    Science.gov (United States)

    Yan, Li; Zhang, Jinfeng; Lee, Chun-Sing; Chen, Xianfeng

    2014-11-01

    The majority of drugs and biomolecules need to be delivered into cells to be effective. However, the cell membranes, a biological barrier, strictly resist drugs or biomolecules entering cells, resulting in significantly reduced intracellular delivery efficiency. To overcome this barrier, a variety of intracellular delivery approaches including chemical and physical ways have been developed in recent years. In this review, the focus is on summarizing the nanomaterial routes involved in making use of a collection of receptors for the targeted delivery of drugs and biomolecules and the physical ways of applying micro- and nanotechnologies for high-throughput intracellular delivery. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. receptores

    Directory of Open Access Journals (Sweden)

    Salete Regina Daronco Benetti

    2006-01-01

    Full Text Available Se trata de un estudio etnográfico, que tuvo lo objetivo de interpretar el sistema de conocimiento y del significado atribuidos a la sangre referente a la transfusión sanguínea por los donadores y receptores de un banco de sangre. Para la colecta de las informaciones se observaron los participantes y la entrevista etnográfica se realizó el análisis de dominio, taxonómicos y temáticos. Los dominios culturales fueron: la sangre es vida: fuente de vida y alimento valioso; creencias religiosas: fuentes simbólicas de apoyos; donación sanguínea: un gesto colaborador que exige cuidarse, gratifica y trae felicidad; donación sanguínea: fuente simbólica de inseguridad; estar enfermo es una condición para realizar transfusión sanguínea; transfusión sanguínea: esperanza de vida; Creencias populares: transfusión sanguínea como riesgo para la salud; donadores de sangre: personas benditas; donar y recibir sangre: como significado de felicidad. Temática: “líquido precioso que origina, sostiene, modifica la vida, provoca miedo e inseguridad”.

  1. Therapeutic Antibodies against Intracellular Tumor Antigens

    Directory of Open Access Journals (Sweden)

    Iva Trenevska

    2017-08-01

    Full Text Available Monoclonal antibodies are among the most clinically effective drugs used to treat cancer. However, their target repertoire is limited as there are relatively few tumor-specific or tumor-associated cell surface or soluble antigens. Intracellular molecules represent nearly half of the human proteome and provide an untapped reservoir of potential therapeutic targets. Antibodies have been developed to target externalized antigens, have also been engineered to enter into cells or may be expressed intracellularly with the aim of binding intracellular antigens. Furthermore, intracellular proteins can be degraded by the proteasome into short, commonly 8–10 amino acid long, peptides that are presented on the cell surface in the context of major histocompatibility complex class I (MHC-I molecules. These tumor-associated peptide–MHC-I complexes can then be targeted by antibodies known as T-cell receptor mimic (TCRm or T-cell receptor (TCR-like antibodies, which recognize epitopes comprising both the peptide and the MHC-I molecule, similar to the recognition of such complexes by the TCR on T cells. Advances in the production of TCRm antibodies have enabled the generation of multiple TCRm antibodies, which have been tested in vitro and in vivo, expanding our understanding of their mechanisms of action and the importance of target epitope selection and expression. This review will summarize multiple approaches to targeting intracellular antigens with therapeutic antibodies, in particular describing the production and characterization of TCRm antibodies, the factors influencing their target identification, their advantages and disadvantages in the context of TCR therapies, and the potential to advance TCRm-based therapies into the clinic.

  2. Intracellular calcium homeostasis and signaling.

    Science.gov (United States)

    Brini, Marisa; Calì, Tito; Ottolini, Denis; Carafoli, Ernesto

    2013-01-01

    Ca(2+) is a universal carrier of biological information: it controls cell life from its origin at fertilization to its end in the process of programmed cell death. Ca(2+) is a conventional diffusible second messenger released inside cells by the interaction of first messengers with plasma membrane receptors. However, it can also penetrate directly into cells to deliver information without the intermediation of first or second messengers. Even more distinctively, Ca(2+) can act as a first messenger, by interacting with a plasma membrane receptor to set in motion intracellular signaling pathways that involve Ca(2+) itself. Perhaps the most distinctive property of the Ca(2+) signal is its ambivalence: while essential to the correct functioning of cells, Ca(2+) becomes an agent that mediates cell distress, or even (toxic) cell death, if its concentration and movements inside cells are not carefully tuned. Ca(2+) is controlled by reversible complexation to specific proteins, which could be pure Ca(2+) buffers, or which, in addition to buffering Ca(2+), also decode its signal to pass it on to targets. The most important actors in the buffering of cell Ca(2+) are proteins that transport it across the plasma membrane and the membrane of the organelles: some have high Ca(2+) affinity and low transport capacity (e.g., Ca(2+) pumps), others have opposite properties (e.g., the Ca(2+) uptake system of mitochondria). Between the initial event of fertilization, and the terminal event of programmed cell death, the Ca(2+) signal regulates the most important activities of the cell, from the expression of genes, to heart and muscle contraction and other motility processes, to diverse metabolic pathways involved in the generation of cell fuels.

  3. Intracellular Polyamines Enhance Astrocytic Coupling

    Science.gov (United States)

    Benedikt, Jan; Inyushin, Mikhail; Kucheryavykh, Yuriy V.; Rivera, Yomarie; Kucheryavykh, Lilia Y.; Nichols, Colin G.; Eaton, Misty J.; Skatchkov, Serguei N.

    2013-01-01

    Spermine (SPM) and spermidine (SPD), endogenous polyamines (PA) with the ability to modulate various ion channels and receptors in the brain, exert neuroprotective, antidepressant, antioxidant and other effects in vivo such as increasing longevity. These PA are preferably accumulated in astrocytes, and we hypothesized that SPM increases glial intercellular communication by interacting with glial gap junctions. Results obtained in situ, using Lucifer yellow propagation in the astrocytic syncitium of 21–25 day old rat CA1 hippocampal slices, showed reduced coupling when astrocytes were dialyzed with standard intracellular solutions (ICS) without SPM. However, there was a robust increase in the spreading of Lucifer yellow via gap junctions to neighboring astrocytes when the cells were patched with ICS containing 1 mM SPM; a physiological concentration in glia. Lucifer yellow propagation was inhibited by gap junction blockers. Our findings show that the glial syncitium propagates SPM via gap junctions and further suggest a new role of polyamines in the regulation of the astroglial network in both normal and pathological conditions. PMID:23076119

  4. Cytokine-induced inside-out activation of FcaR (CD89) is mediated by a single serine residue (S263) in the intracellular domain of the receptor

    NARCIS (Netherlands)

    Bracke, M.S.G.M.; Lammers, J.W.J.; Coffer, P.J.; Koenderman, L.

    2001-01-01

    Fc receptors play an important role in leukocyte activation and the modulation of ligand binding ("activation") is a criti-cal point of regulation. Previous studies demonstrated that the Fc receptor for IgA (FcaRI/CD89) is regulated by cytokine stimulation, switching it to a

  5. FLIPR assays of intracellular calcium in GPCR drug discovery

    DEFF Research Database (Denmark)

    Hansen, Kasper Bø; Bräuner-Osborne, Hans

    2009-01-01

    Fluorescent dyes sensitive to changes in intracellular calcium have become increasingly popular in G protein-coupled receptor (GPCR) drug discovery for several reasons. First of all, the assays using the dyes are easy to perform and are of low cost compared to other assays. Second, most non...

  6. A bacteriophage endolysin that eliminates intracellular streptococci.

    Science.gov (United States)

    Shen, Yang; Barros, Marilia; Vennemann, Tarek; Gallagher, D Travis; Yin, Yizhou; Linden, Sara B; Heselpoth, Ryan D; Spencer, Dennis J; Donovan, David M; Moult, John; Fischetti, Vincent A; Heinrich, Frank; Lösche, Mathias; Nelson, Daniel C

    2016-03-15

    PlyC, a bacteriophage-encoded endolysin, lyses Streptococcus pyogenes (Spy) on contact. Here, we demonstrate that PlyC is a potent agent for controlling intracellular Spy that often underlies refractory infections. We show that the PlyC holoenzyme, mediated by its PlyCB subunit, crosses epithelial cell membranes and clears intracellular Spy in a dose-dependent manner. Quantitative studies using model membranes establish that PlyCB interacts strongly with phosphatidylserine (PS), whereas its interaction with other lipids is weak, suggesting specificity for PS as its cellular receptor. Neutron reflection further substantiates that PlyC penetrates bilayers above a PS threshold concentration. Crystallography and docking studies identify key residues that mediate PlyCB-PS interactions, which are validated by site-directed mutagenesis. This is the first report that a native endolysin can traverse epithelial membranes, thus substantiating the potential of PlyC as an antimicrobial for Spy in the extracellular and intracellular milieu and as a scaffold for engineering other functionalities.

  7. The intracellular pharmacokinetics of terminally capped peptides.

    NARCIS (Netherlands)

    Ruttekolk, I.R.R.; Witsenburg, J.J.; Glauner, H.B.; Bovee-Geurts, P.H.M.; Ferro, E.S.; Verdurmen, W.P.R.; Brock, R.E.

    2012-01-01

    With significant progress in delivery technologies, peptides and peptidomimetics are receiving increasing attention as potential therapeutics also for intracellular applications. However, analyses of the intracellular behavior of peptides are a challenge; therefore, knowledge on the intracellular

  8. Functional genomics of intracellular bacteria.

    Science.gov (United States)

    de Barsy, Marie; Greub, Gilbert

    2013-07-01

    During the genomic era, a large amount of whole-genome sequences accumulated, which identified many hypothetical proteins of unknown function. Rapidly, functional genomics, which is the research domain that assign a function to a given gene product, has thus been developed. Functional genomics of intracellular pathogenic bacteria exhibit specific peculiarities due to the fastidious growth of most of these intracellular micro-organisms, due to the close interaction with the host cell, due to the risk of contamination of experiments with host cell proteins and, for some strict intracellular bacteria such as Chlamydia, due to the absence of simple genetic system to manipulate the bacterial genome. To identify virulence factors of intracellular pathogenic bacteria, functional genomics often rely on bioinformatic analyses compared with model organisms such as Escherichia coli and Bacillus subtilis. The use of heterologous expression is another common approach. Given the intracellular lifestyle and the many effectors that are used by the intracellular bacteria to corrupt host cell functions, functional genomics is also often targeting the identification of new effectors such as those of the T4SS of Brucella and Legionella.

  9. Intracellular Signalling by C-Peptide

    Directory of Open Access Journals (Sweden)

    Claire E. Hills

    2008-01-01

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

  10. [Intracellular signaling mechanisms in thyroid cancer].

    Science.gov (United States)

    Mondragón-Terán, Paul; López-Hernández, Luz Berenice; Gutiérrez-Salinas, José; Suárez-Cuenca, Juan Antonio; Luna-Ceballos, Rosa Isela; Erazo Valle-Solís, Aura

    2016-01-01

    Thyroid cancer is the most common malignancy of the endocrine system, the papillary variant accounts for 80-90% of all diagnosed cases. In the development of papillary thyroid cancer, BRAF and RAS genes are mainly affected, resulting in a modification of the system of intracellular signaling proteins known as «protein kinase mitogen-activated» (MAPK) which consist of «modules» of internal signaling proteins (Receptor/Ras/Raf/MEK/ERK) from the cell membrane to the nucleus. In thyroid cancer, these signanling proteins regulate diverse cellular processes such as differentiation, growth, development and apoptosis. MAPK play an important role in the pathogenesis of thyroid cancer as they are used as molecular biomarkers for diagnostic, prognostic and as possible therapeutic molecular targets. Mutations in BRAF gene have been correlated with poor response to treatment with traditional chemotherapy and as an indicator of poor prognosis. To review the molecular mechanisms involved in intracellular signaling of BRAF and RAS genes in thyroid cancer. Molecular therapy research is in progress for this type of cancer as new molecules have been developed in order to inhibit any of the components of the signaling pathway (RET/PTC)/Ras/Raf/MEK/ERK; with special emphasis on the (RET/PTC)/Ras/Raf section, which is a major effector of ERK pathway. Copyright © 2016 Academia Mexicana de Cirugía A.C. Publicado por Masson Doyma México S.A. All rights reserved.

  11. Self-organization of intracellular gradients during mitosis

    Directory of Open Access Journals (Sweden)

    Fuller Brian G

    2010-01-01

    Full Text Available Abstract Gradients are used in a number of biological systems to transmit spatial information over a range of distances. The best studied are morphogen gradients where information is transmitted over many cell lengths. Smaller mitotic gradients reflect the need to organize several distinct events along the length of the mitotic spindle. The intracellular gradients that characterize mitosis are emerging as important regulatory paradigms. Intracellular gradients utilize intrinsic auto-regulatory feedback loops and diffusion to establish stable regions of activity within the mitotic cytosol. We review three recently described intracellular mitotic gradients. The Ran GTP gradient with its elaborate cascade of nuclear transport receptors and cargoes is the best characterized, yet the dynamics underlying the robust gradient of Ran-GTP have received little attention. Gradients of phosphorylation have been observed on Aurora B kinase substrates both before and after anaphase onset. In both instances the phosphorylation gradient appears to result from a soluble gradient of Aurora B kinase activity. Regulatory properties that support gradient formation are highlighted. Intracellular activity gradients that regulate localized mitotic events bare several hallmarks of self-organizing biologic systems that designate spatial information during pattern formation. Intracellular pattern formation represents a new paradigm in mitotic regulation.

  12. Repeated blockade of mineralocorticoid receptors, but not of glucocorticoid receptors impairs food rewarded spatial learning

    NARCIS (Netherlands)

    Douma, BRK; Korte, SM; Buwalda, B; la Fleur, SE; Bohus, B; Luiten, PGM

    Corticosteroids from the adrenal cortex influence a variety of behaviours including cognition, learning and memory. These hormones act via two intracellular receptors, the mineralo-corticoid receptor (MR) and the glucocorticoid receptor (GR). These two receptor types display a high concentration and

  13. Repeated blockade of mineralocorticoid receptors, but not of glucocorticoid receptors impairs food rewarded spatial learning

    NARCIS (Netherlands)

    Douma, B. R.; Korte, S. M.; Buwalda, B.; La Fleur, S. E.; Bohus, B.; Luiten, P. G.

    1998-01-01

    Corticosteroids from the adrenal cortex influence a variety of behaviours including cognition, learning and memory. These hormones act via two intracellular receptors, the mineralo-corticoid receptor (MR) and the glucocorticoid receptor (GR). These two receptor types display a high concentration and

  14. Mechanisms of Borrelia burgdorferi internalization and intracellular innate immune signaling

    Directory of Open Access Journals (Sweden)

    Tanja ePetnicki-Ocwieja

    2014-12-01

    Full Text Available Lyme disease is a long-term infection whose most severe pathology is characterized by inflammatory arthritis of the lower bearing joints, carditis and neuropathy. The inflammatory cascades are initiated through the early recognition of invading Borrelia burgdorferi spirochetes by cells of the innate immune response, such as neutrophils and macrophage. B. burgdorferi does not have an intracellular niche and thus much research has focused on immune pathways activated by pathogen recognition molecules at the cell surface, such as the Toll-like receptors (TLRs. However, in recent years, studies have shown that internalization of the bacterium by host cells is an important component of the defense machinery in response to B. burgdorferi. Upon internalization, B. burgdorferi is trafficked through an endo/lysosomal pathway resulting in the activation of a number of intracellular pathogen recognition receptors including TLRs and Nod-like receptors (NLRs. Here we will review the innate immune molecules that participate in both cell surface and intracellular immune activation by B. burgdorferi.

  15. NMDA receptor signaling: death or survival?

    OpenAIRE

    LUO, Tong; WU, Wei-Hua; CHEN, Bo-Shiun

    2011-01-01

    Glutamate-induced neuronal damage is mainly caused by overactivation of N-methyl-D-aspartate (NMDA) receptors. Conversely, normal physiological brain function and neuronal survival require adequate activation of NMDA receptors. Studies have revealed that NMDA receptor-induced neuronal death or survival is mediated through distinct subset of NMDA receptors triggering different intracellular signaling pathways. Here we discuss recent advances in the characterization of NMDA receptors in neurona...

  16. Eps15: a multifunctional adaptor protein regulating intracellular trafficking

    Directory of Open Access Journals (Sweden)

    van Bergen en Henegouwen Paul MP

    2009-10-01

    Full Text Available Abstract Over expression of receptor tyrosine kinases is responsible for the development of a wide variety of malignancies. Termination of growth factor signaling is primarily determined by the down regulation of active growth factor/receptor complexes. In recent years, considerable insight has been gained in the endocytosis and degradation of growth factor receptors. A crucial player in this process is the EGFR Protein tyrosine kinase Substrate #15, or Eps15. This protein functions as a scaffolding adaptor protein and is involved both in secretion and endocytosis. Eps15 has been shown to bind to AP-1 and AP-2 complexes, to bind to inositol lipids and to several other proteins involved in the regulation of intracellular trafficking. In addition, Eps15 has been detected in the nucleus of mammalian cells. Activation of growth factor receptors induces tyrosine phosphorylation and mono-ubiquitination of Eps15. The role of these post translational modifications of Eps15 is still a mystery. It is proposed that Eps15 and its family members Eps15R and Eps15b are involved in the regulation of membrane morphology, which is required for intracellular vesicle formation and trafficking.

  17. Host-directed antimicrobial drugs with broad-spectrum efficacy against intracellular bacterial pathogens.

    Science.gov (United States)

    Czyż, Daniel M; Potluri, Lakshmi-Prasad; Jain-Gupta, Neeta; Riley, Sean P; Martinez, Juan J; Steck, Theodore L; Crosson, Sean; Shuman, Howard A; Gabay, Joëlle E

    2014-07-29

    We sought a new approach to treating infections by intracellular bacteria, namely, by altering host cell functions that support their growth. We screened a library of 640 Food and Drug Administration (FDA)-approved compounds for agents that render THP-1 cells resistant to infection by four intracellular pathogens. We identified numerous drugs that are not antibiotics but were highly effective in inhibiting intracellular bacterial growth with limited toxicity to host cells. These compounds are likely to target three kinds of host functions: (i) G protein-coupled receptors, (ii) intracellular calcium signals, and (iii) membrane cholesterol distribution. The compounds that targeted G protein receptor signaling and calcium fluxes broadly inhibited Coxiella burnetii, Legionella pneumophila, Brucella abortus, and Rickettsia conorii, while those directed against cholesterol traffic strongly attenuated the intracellular growth of C. burnetii and L. pneumophila. These pathways probably support intracellular pathogen growth so that drugs that perturb them may be therapeutic candidates. Combining host- and pathogen-directed treatments is a strategy to decrease the emergence of drug-resistant intracellular bacterial pathogens. Importance: Although antibiotic treatment is often successful, it is becoming clear that alternatives to conventional pathogen-directed therapy must be developed in the face of increasing antibiotic resistance. Moreover, the costs and timing associated with the development of novel antimicrobials make repurposed FDA-approved drugs attractive host-targeted therapeutics. This paper describes a novel approach of identifying such host-targeted therapeutics against intracellular bacterial pathogens. We identified several FDA-approved drugs that inhibit the growth of intracellular bacteria, thereby implicating host intracellular pathways presumably utilized by bacteria during infection. Copyright © 2014 Czyż et al.

  18. Modulating cancer cell survival by targeting intracellular cholesterol transport.

    Science.gov (United States)

    Kuzu, Omer F; Gowda, Raghavendra; Noory, Mohammad A; Robertson, Gavin P

    2017-08-08

    Demand for cholesterol is high in certain cancers making them potentially sensitive to therapeutic strategies targeting cellular cholesterol homoeostasis. A potential approach involves disruption of intracellular cholesterol transport, which occurs in Niemann-Pick disease as a result of acid sphingomyelinase (ASM) deficiency. Hence, a class of lysosomotropic compounds that were identified as functional ASM inhibitors (FIASMAs) might exhibit chemotherapeutic activity by disrupting cancer cell cholesterol homoeostasis. Here, the chemotherapeutic utility of ASM inhibition was investigated. The effect of FIASMAs on intracellular cholesterol levels, cholesterol homoeostasis, cellular endocytosis and signalling cascades were investigated. The in vivo efficacy of ASM inhibition was demonstrated using melanoma xenografts and a nanoparticle formulation was developed to overcome dose-limiting CNS-associated side effects of certain FIASMAs. Functional ASM inhibitors inhibited intracellular cholesterol transport leading to disruption of autophagic flux, cellular endocytosis and receptor tyrosine kinase signalling. Consequently, major oncogenic signalling cascades on which cancer cells were reliant for survival were inhibited. Two tested ASM inhibitors, perphenazine and fluphenazine that are also clinically used as antipsychotics, were effective in inhibiting xenografted tumour growth. Nanoliposomal encapsulation of the perphenazine enhanced its chemotherapeutic efficacy while decreasing CNS-associated side effects. This study suggests that disruption of intracellular cholesterol transport by targeting ASM could be utilised as a potential chemotherapeutic approach for treating cancer.

  19. The role of TREM-2 in internalization and intracellular survival of Brucella abortus in murine macrophages.

    Science.gov (United States)

    Wei, Pan; Lu, Qiang; Cui, Guimei; Guan, Zhenhong; Yang, Li; Sun, Changjiang; Sun, Wanchun; Peng, Qisheng

    2015-02-15

    Triggering receptor expressed on myeloid cells-2 (TREM-2) is a cell surface receptor primarily expressed on macrophages and dendritic cells. TREM-2 functions as a phagocytic receptor for bacteria as well as an inhibitor of Toll like receptors (TLR) induced inflammatory cytokines. However, the role of TREM-2 in Brucella intracellular growth remains unknown. To investigate whether TREM-2 is involved in Brucella intracellular survival, we chose bone marrow derived macrophages (BMDMs), in which TREM-2 is stably expressed, as cell model. Colony formation Units (CFUs) assay suggests that TREM-2 is involved in the internalization of Brucella abortus (B. abortus) by macrophages, while silencing of TREM-2 decreases intracellular survival of B. abortus. To further study the underlying mechanisms of TREM-2-mediated bacterial intracellular survival, we examined the activation of B. abortus-infected macrophages through determining the kinetics of activation of the three MAPKs, including ERK, JNK and p38, and measuring TNFα production in response to lipopolysaccharide (LPS) of Brucella (BrLPS) or B. abortus stimulation. Our data show that TREM-2 deficiency promotes activation of Brucella-infected macrophages. Moreover, our data also demonstrate that macrophage activation promotes killing of Brucella by enhancing nitric oxygen (NO), but not reactive oxygen species (ROS) production, macrophage apoptosis or cellular death. Taken together, these findings provide a novel interpretation of Brucella intracellular growth through inhibition of NO production produced by TREM-2-mediated activated macrophages. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Evolutionary analysis of functional divergence among chemokine receptors, decoy receptors and viral receptors

    Directory of Open Access Journals (Sweden)

    Hiromi eDaiyasu

    2012-07-01

    Full Text Available Chemokine receptors (CKRs function in the inflammatory response and in vertebrate homeostasis. Decoy and viral receptors are two types of CKR homologues with modified functions from those of the typical CKRs. The decoy receptors are able to bind ligands without signaling. On the other hand, the viral receptors show constitutive signaling without ligands. We examined the sites related to the functional difference. At first, the decoy and viral receptors were each classified into five groups, based on the molecular phylogenetic analysis. A multiple amino acid sequence alignment between each group and the CKRs was then constructed. The difference in the amino acid composition between the group and the CKRs was evaluated as the Kullback-Leibler (KL information value at each alignment site. The KL information value is considered to reflect the difference in the functional constraints at the site. The sites with the top 5% of KL information values were selected and mapped on the structure of a CKR. The comparisons with decoy receptor groups revealed that the detected sites were biased on the intracellular side. In contrast, the sites detected from the comparisons with viral receptor groups were found on both the extracellular and intracellular sides. More sites were found in the ligand-binding pocket in the analyses of the viral receptor groups, as compared to the decoy receptor groups. Some of the detected sites were located in the GPCR motifs. For example, the DRY motif of the decoy receptors was often degraded, although the motif of the viral receptors was basically conserved. The observations for the viral receptor groups suggested that the constraints in the pocket region are loose and that the sites on the intracellular side are different from those for the decoy receptors, which may be related to the constitutive signaling activity of the viral receptors.

  1. Developmental regulation of intracellular calcium by N-methyl-D-aspartate and noradrenaline in rat visual cortex.

    Science.gov (United States)

    Kobayashi, M; Imamura, K; Kaub, P A; Nakadate, K; Watanabe, Y

    1999-01-01

    The effects of N-methyl-D-aspartate and noradrenaline on intracellular Ca2+ concentration in slices of rat visual cortex were studied using a fluorescent indicator, Fura-2. Bath application of N-methyl-D-aspartate (1-100 microM) increased intracellular Ca2+ concentration in a dose-dependent manner, especially in layers II/III. Noradrenaline (1-100 microM) also increased intracellular Ca2+ concentration in a dose-dependent manner, especially in layers I and IV. However, the maximum increase in intracellular Ca2+ concentration after 100 microM noradrenaline application was less than half of that after 100 microM N-methyl-D-aspartate application in slices obtained from animals in the sensitive period. The effect of noradrenaline was most prominent in slices of the sensitive period, whereas the N-methyl-D-aspartate-induced intracellular Ca2+ concentration response decreased with age. Additive effects from application of both N-methyl-D-aspartate and noradrenaline on intracellular Ca2+ concentration were found only in the neonatal stage. Pharmacological experiments showed that alpha1-adrenergic receptors play a major role in the noradrenaline-induced intracellular Ca2+ concentration response, although both alpha2- and beta-adrenergic receptors were also partially involved. The release of Ca2+ from intracellular storage underlay the early phase of the noradrenaline-induced intracellular Ca2+ concentration response, while extracellular Ca2+ influxes contributed to the sustained phase. Experiments using a gliotoxin, fluorocitric acid, suggested that the function of glial cells is involved in the noradrenaline-induced increase of intracellular Ca2+ concentration. The larger intracellular Ca2+ concentration response to noradrenaline during the sensitive period may modulate the increase in intracellular Ca2+ concentration by N-methyl-D-aspartate to maintain a higher level of cortical plasticity during this period.

  2. MR imaging of intracellular and extracellular deoxyhemoglobin

    International Nuclear Information System (INIS)

    Janick, P.A.; Grossman, R.I.; Asakura, T.

    1989-01-01

    MR imaging was performed on varying concentrations of intracellular and extracellular deoxyhemoglobin as well as varying proportions of deoxyhemoglobin and oxyhemoglobin in vitro at 1.5T with use of standard spin-echo and gradient-refocused spin sequences. This study indicates that susceptibility-induced T2 shortening occurs over a broad range of intracellular deoxyhemoglobin concentrations (maximal at hematocrits between 20% and 45%), reflecting diffusional effects at the cellular level. T2* gradient-echo imaging enhances the observed hypointensity in images of intracellular deoxyhemoglobin. The characteristic MR appearance of acute hemotomas can be modeled by the behavior of intracellular and extracellular deoxyhemoglobin and oxyhemoglobin

  3. Release of intracellular Calcium increase production of mitochondrial reactive oxygen species in renal distal epithelial cells

    DEFF Research Database (Denmark)

    Bjerregaard, Henning F.

    was inhibited by buffering of intracellular calcium with BAPTA, by the antioxidant N-acetylcysteine and by uncoupling of mitochondrial oxidative phosphorylation from respiration with CCCP. These results indicate that Cd generate a prompt initiation of ROS production from mitochondria due to an increase......Release of intracellular Calcium increase production of mitochondrial reactive oxygen species in renal distal epithelial cells. Henning F. Bjerregaard, Roskilde University, Department of Science, Systems and Models , 4000 Roskilde, Denmark. HFB@ RUC.DK Reactive oxygen species (ROS) like, hydrogen...... dynamics in living cells in response to hormonal signal events in the A6 cell culture. A6 cells have a divalent cation-sensing receptor (the extracellular calcium receptor) that can be stimulated with cadmium (Cd) and thereby induce a fast and transient liberation of calcium from intracellular stores, due...

  4. Vitamin D is positively associated with sperm motility and increases intracellular calcium in human spermatozoa

    DEFF Research Database (Denmark)

    Blomberg Jensen, Martin; Bjerrum, Poul J; Jessen, Torben E

    2011-01-01

    BACKGROUND The vitamin D receptor (VDR) is expressed in human spermatozoa, and VDR-knockout mice and vitamin D (VD) deficiency in rodents results in impaired fertility, low sperm counts and a low number of motile spermatozoa. We investigated the role of activated VD (1,25(OH)(2)D(3)) in human...... spermatozoa and whether VD serum levels are associated with semen quality. METHODS Cross-sectional association study of semen quality and VD serum level in 300 men from the general population, and in vitro studies on spermatozoa from 40 men to investigate the effects of VD on intracellular calcium, sperm......M). 1,25(OH)(2)D(3) increased intracellular calcium concentration in human spermatozoa through VDR-mediated calcium release from an intracellular calcium storage, increased sperm motility and induced the acrosome reaction in vitro. CONCLUSIONS 1,25(OH)(2)D(3) increased intracellular calcium...

  5. Intracellular antibody-mediated immunity and the role of TRIM21.

    Science.gov (United States)

    McEwan, William A; Mallery, Donna L; Rhodes, David A; Trowsdale, John; James, Leo C

    2011-11-01

    Protection against bacterial and viral pathogens by antibodies has always been thought to end at the cell surface. Once inside the cell, a pathogen was understood to be safe from humoral immunity. However, it has now been found that antibodies can routinely enter cells attached to viral particles and mediate an intracellular immune response. Antibody-coated virions are detected inside the cell by means of an intracellular antibody receptor, TRIM21, which directs their degradation by recruitment of the ubiquitin-proteasome system. In this article we assess how this discovery alters our view of the way in which antibodies neutralise viral infection. We also consider the antiviral function of TRIM21 in the context of its other reported roles in immune signalling and autoimmunity. Finally, we discuss the conceptual implications of intracellular antibody immunity and how it alters our view of the discrete separation of extracellular and intracellular environments. Copyright © 2011 WILEY Periodicals, Inc.

  6. Intracellular Angiotensin II and cell growth of vascular smooth muscle cells

    NARCIS (Netherlands)

    Filipeanu, CM; Henning, RH; de Zeeuw, D; Nelemans, A

    1 We recently demonstrated that intracellular application of Angiotensin II (Angiotensin IIintr) induces rat aorta contraction independent of plasma membrane Angiotensin II receptors. In this study we investigated the effects of Angiotensin IIintr on cell growth in A7r5 smooth muscle cells. 2

  7. The transduction channel TRPM5 is gated by intracellular calcium in taste cells.

    Science.gov (United States)

    Zhang, Zheng; Zhao, Zhen; Margolskee, Robert; Liman, Emily

    2007-05-23

    Bitter, sweet, and umami tastants are detected by G-protein-coupled receptors that signal through a common second-messenger cascade involving gustducin, phospholipase C beta2, and the transient receptor potential M5 (TRPM5) ion channel. The mechanism by which phosphoinositide signaling activates TRPM5 has been studied in heterologous cell types with contradictory results. To resolve this issue and understand the role of TRPM5 in taste signaling, we took advantage of mice in which the TRPM5 promoter drives expression of green fluorescent protein and mice that carry a targeted deletion of the TRPM5 gene to unequivocally identify TRPM5-dependent currents in taste receptor cells. Our results show that brief elevation of intracellular inositol trisphosphate or Ca2+ is sufficient to gate TRPM5-dependent currents in intact taste cells, but only intracellular Ca2+ is able to activate TRPM5-dependent currents in excised patches. Detailed study in excised patches showed that TRPM5 forms a nonselective cation channel that is half-activated by 8 microM Ca2+ and that desensitizes in response to prolonged exposure to intracellular Ca2+. In addition to channels encoded by the TRPM5 gene, we found that taste cells have a second type of Ca2+-activated nonselective cation channel that is less sensitive to intracellular Ca2+. These data constrain proposed models for taste transduction and suggest a link between receptor signaling and membrane potential in taste cells.

  8. Optochemokine Tandem for Light-Control of Intracellular Ca2.

    Directory of Open Access Journals (Sweden)

    Katrin Feldbauer

    Full Text Available An optochemokine tandem was developed to control the release of calcium from endosomes into the cytosol by light and to analyze the internalization kinetics of G-protein coupled receptors (GPCRs by electrophysiology. A previously constructed rhodopsin tandem was re-engineered to combine the light-gated Ca2+-permeable cation channel Channelrhodopsin-2(L132C, CatCh, with the chemokine receptor CXCR4 in a functional tandem protein tCXCR4/CatCh. The GPCR was used as a shuttle protein to displace CatCh from the plasma membrane into intracellular areas. As shown by patch-clamp measurements and confocal laser scanning microscopy, heterologously expressed tCXCR4/CatCh was internalized via the endocytic SDF1/CXCR4 signaling pathway. The kinetics of internalization could be followed electrophysiologically via the amplitude of the CatCh signal. The light-induced release of Ca2+ by tandem endosomes into the cytosol via CatCh was visualized using the Ca2+-sensitive dyes rhod2 and rhod2-AM showing an increase of intracellular Ca2+ in response to light.

  9. Receptors, G proteins, and their interactions

    NARCIS (Netherlands)

    Hollmann, Markus W.; Strumper, Danja; Herroeder, Susanne; Durieux, Marcel E.

    2005-01-01

    Membrane receptors coupling to intracellular G proteins (G protein-coupled receptors) form one of the major classes of membrane signaling proteins. They are of great importance to the practice of anesthesiology because they are involved in many systems of relevance to the specialty (cardiovascular

  10. Allopregnanolone-induced rise in intracellular calcium in embryonic hippocampal neurons parallels their proliferative potential

    Directory of Open Access Journals (Sweden)

    Brinton Roberta

    2008-12-01

    Full Text Available Abstract Background Factors that regulate intracellular calcium concentration are known to play a critical role in brain function and neural development, including neural plasticity and neurogenesis. We previously demonstrated that the neurosteroid allopregnanolone (APα; 5α-pregnan-3α-ol-20-one promotes neural progenitor proliferation in vitro in cultures of rodent hippocampal and human cortical neural progenitors, and in vivo in triple transgenic Alzheimer's disease mice dentate gyrus. We also found that APα-induced proliferation of neural progenitors is abolished by a calcium channel blocker, nifedipine, indicating a calcium dependent mechanism for the proliferation. Methods In the present study, we investigated the effect of APα on the regulation of intracellular calcium concentration in E18 rat hippocampal neurons using ratiometric Fura2-AM imaging. Results Results indicate that APα rapidly increased intracellular calcium concentration in a dose-dependent and developmentally regulated manner, with an EC50 of 110 ± 15 nM and a maximal response occurring at three days in vitro. The stereoisomers 3β-hydroxy-5α-hydroxy-pregnan-20-one, and 3β-hydroxy-5β-hydroxy-pregnan-20-one, as well as progesterone, were without significant effect. APα-induced intracellular calcium concentration increase was not observed in calcium depleted medium and was blocked in the presence of the broad spectrum calcium channel blocker La3+, or the L-type calcium channel blocker nifedipine. Furthermore, the GABAA receptor blockers bicuculline and picrotoxin abolished APα-induced intracellular calcium concentration rise. Conclusion Collectively, these data indicate that APα promotes a rapid, dose-dependent, stereo-specific, and developmentally regulated increase of intracellular calcium concentration in rat embryonic hippocampal neurons via a mechanism that requires both the GABAA receptor and L-type calcium channel. These data suggest that AP

  11. Structure-Function Studies on the Prolactin Receptor

    DEFF Research Database (Denmark)

    Haxholm, Gitte Wolfsberg

    information on the intracellular domains (ICDs) of these receptors. The overall aim of this study was to obtain an improved understanding of cytokine receptor signaling through structure-function studies on the prolactin receptor (PRLR). The primary focus of this thesis was to structurally characterize...

  12. Nanoparticles for intracellular-targeted drug delivery

    International Nuclear Information System (INIS)

    Paulo, Cristiana S O; Pires das Neves, Ricardo; Ferreira, Lino S

    2011-01-01

    Nanoparticles (NPs) are very promising for the intracellular delivery of anticancer and immunomodulatory drugs, stem cell differentiation biomolecules and cell activity modulators. Although initial studies in the area of intracellular drug delivery have been performed in the delivery of DNA, there is an increasing interest in the use of other molecules to modulate cell activity. Herein, we review the latest advances in the intracellular-targeted delivery of short interference RNA, proteins and small molecules using NPs. In most cases, the drugs act at different cellular organelles and therefore the drug-containing NPs should be directed to precise locations within the cell. This will lead to the desired magnitude and duration of the drug effects. The spatial control in the intracellular delivery might open new avenues to modulate cell activity while avoiding side-effects.

  13. Intracellular insulin processing is altered in monocytes from patients with type II diabetes mellitus

    International Nuclear Information System (INIS)

    Trischitta, V.; Benzi, L.; Brunetti, A.; Cecchetti, P.; Marchetti, P.; Vigneri, R.; Navalesi, R.

    1987-01-01

    We studied total cell-associated A14-[ 125 I]insulin radioactivity (including surface-bound and internalized radioactivity), insulin internalization, and its intracellular degradation at 37 C in monocytes from nonobese type II untreated diabetic patients (n = 9) and normal subjects (n = 7). Total cell-associated radioactivity was decreased in diabetic patients [2.65 +/- 1.21% (+/- SD) vs. 4.47 +/- 1.04% of total radioactivity. Insulin internalization was also reduced in diabetic patients (34.0 +/- 6.8% vs. 59.0 +/- 11.3% of cell-associated radioactivity. Using high performance liquid chromatography six intracellular forms of radioactivity derived from A14-[ 125 I] insulin were identified; 10-20% of intracellular radioactivity had approximately 300,000 mol wt and was identified as radioactivity bound to the insulin receptor, and the remaining intracellular radioactivity included intact A14-[ 125 I]insulin, [ 125 I]iodide, or [ 125 I]tyrosine, and three intermediate compounds. A progressive reduction of intact insulin and a corresponding increase in iodine were found when the incubation time was prolonged. Intracellular insulin degradation was reduced in monocytes from diabetic patients; intracellular intact insulin was 65.6 +/- 18.1% vs. 37.4 +/- 18.0% of intracellular radioactivity after 2 min and 23.6 +/- 22.3% vs. 3.9 +/- 2.3% after 60 min in diabetic patients vs. normal subjects, respectively. In conclusion, 1) human monocytes internalize and degrade insulin in the intracellular compartment in a stepwise time-dependent manner; and 2) in monocytes from type II diabetic patients total cell-associated radioactivity, insulin internalization, and insulin degradation are significantly reduced. These defects may be related to the cellular insulin resistance present in these patients

  14. Purinergic receptors expressed in human skeletal muscle fibres

    DEFF Research Database (Denmark)

    Bornø, A; Ploug, Thorkil; Bune, L T

    2012-01-01

    in immunolabelled transverse sections of muscle biopsies. The receptors P2Y(4), P2Y(11) and likely P2X(1) were present intracellularly or in the plasma membrane of muscle fibres and were thus selected for further detailed morphological analysis. P2X(1) receptors were expressed in intracellular vesicles...... of purinergic receptors in skeletal muscle fibres in patients with type 2 diabetes and age-matched controls. Muscle biopsies from vastus lateralis were obtained from six type 2 diabetic patients and seven age-matched controls. Purinergic receptors were analysed using light and confocal microscopy...... and sarcolemma. P2Y(4) receptors were present in sarcolemma. P2Y(11) receptors were abundantly and diffusely expressed intracellularly and were more explicitly expressed in type I than in type II fibres, whereas P2X(1) and P2Y(4) showed no fibre-type specificity. Both diabetic patients and healthy controls...

  15. Intracellular Signaling Mediators in the Circulatory and Ventilatory Systems

    CERN Document Server

    Thiriet, Marc

    2013-01-01

    The volumes in this authoritative series present a multidisciplinary approach to modeling and simulation of flows in the cardiovascular and ventilatory systems, especially multiscale modeling and coupled simulations. The cardiovascular and respiratory systems are tightly coupled, as their primary function is to supply oxygen to and remove carbon dioxide from the body's cells. Because physiological conduits have deformable and reactive walls, macroscopic flow behavior and prediction must be coupled to phenomenological models of nano- and microscopic events in a corrector scheme of regulated mechanisms when the vessel lumen caliber varies markedly. Therefore, investigation of flows of blood and air in physiological conduits requires an understanding of the biology, chemistry, and physics of these systems together with the mathematical tools to describe their functioning. Volume 4 is devoted to major sets of intracellular mediators that transmit signals upon stimulation of cell-surface receptors.  Activation of...

  16. The intracellular cholesterol landscape: dynamic integrator of the immune response

    Science.gov (United States)

    Fessler, Michael B.

    2016-01-01

    Cholesterol has typically been considered an exogenous, disease-related factor in immunity; however, recent literature suggests that a paradigm shift is in order. Sterols are now recognized to ligate several immune receptors. Altered flux through the mevalonic acid synthesis pathway also appears to be a required event in the antiviral interferon response of macrophages and in the activation, proliferation, and differentiation of T cells. In this review, evidence is discussed that suggests an intrinsic, ‘professional’ role for sterols and oxysterols in macrophage and T cell immunity. Host defense may have been the original selection pressure behind the development of mechanisms for intracellular cholesterol homeostasis. Functional coupling between sterol metabolism and immunity has fundamental implications for health and disease. PMID:27692616

  17. Bacteriomimetic invasin-functionalized nanocarriers for intracellular delivery.

    Science.gov (United States)

    Labouta, Hagar Ibrahim; Menina, Sara; Kochut, Annika; Gordon, Sarah; Geyer, Rebecca; Dersch, Petra; Lehr, Claus-Michael

    2015-12-28

    Intracellular bacteria invade mammalian cells to establish an infectious niche. The current work models adhesion and subsequent internalization strategy of pathogenic bacteria into mammalian cells to design a bacteriomimetic bioinvasive delivery system. We report on the surface functionalization of liposomes with a C-terminal fragment of invasin (InvA497), an invasion factor in the outer membrane of Yersinia pseudotuberculosis. InvA497-functionalized liposomes adhere to mammalian epithelial HEp-2 cell line at different infection stages with a significantly higher efficiency than liposomes functionalized with bovine serum albumin. Covalent attachment of InvA497 results in higher cellular adhesion than liposomes with physically adsorbed InvA497 with non-specific surface protein alignment. Uptake studies in HEp-2 cells indicate active internalization of InvA497-functionalized liposomes via β1-integrin receptor-mediated uptake mechanism mimicking the natural invasion strategy of Y. pseudotuberculosis. Uptake studies in Caco-2 cells at different polarization states demonstrate specific targeting of the InvA497-functionalized liposomes to less polarized cells reflecting the status of inflamed cells. Moreover, when loaded with the anti-infective agent gentamicin and applied to HEp-2 cells infected with Y. pseudotuberculosis, InvA497-functionalized liposomes are able to significantly reduce the infection load relative to non-functionalized drug-loaded liposomes. This indicates a promising application of such a bacteriomimetic system for drug delivery to intracellular compartments. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Human β-Cell Proliferation and Intracellular Signaling: Part 3

    Science.gov (United States)

    Hussain, Mehboob A.; García-Ocaña, Adolfo; Vasavada, Rupangi C.; Bhushan, Anil; Bernal-Mizrachi, Ernesto

    2015-01-01

    This is the third in a series of Perspectives on intracellular signaling pathways coupled to proliferation in pancreatic β-cells. We contrast the large knowledge base in rodent β-cells with the more limited human database. With the increasing incidence of type 1 diabetes and the recognition that type 2 diabetes is also due in part to a deficiency of functioning β-cells, there is great urgency to identify therapeutic approaches to expand human β-cell numbers. Therapeutic approaches might include stem cell differentiation, transdifferentiation, or expansion of cadaver islets or residual endogenous β-cells. In these Perspectives, we focus on β-cell proliferation. Past Perspectives reviewed fundamental cell cycle regulation and its upstream regulation by insulin/IGF signaling via phosphatidylinositol-3 kinase/mammalian target of rapamycin signaling, glucose, glycogen synthase kinase-3 and liver kinase B1, protein kinase Cζ, calcium-calcineurin–nuclear factor of activated T cells, epidermal growth factor/platelet-derived growth factor family members, Wnt/β-catenin, leptin, and estrogen and progesterone. Here, we emphasize Janus kinase/signal transducers and activators of transcription, Ras/Raf/extracellular signal–related kinase, cadherins and integrins, G-protein–coupled receptors, and transforming growth factor β signaling. We hope these three Perspectives will serve to introduce these pathways to new researchers and will encourage additional investigators to focus on understanding how to harness key intracellular signaling pathways for therapeutic human β-cell regeneration for diabetes. PMID:25999530

  19. Inflammatory intracellular pathways activated by electronegative LDL in monocytes.

    Science.gov (United States)

    Estruch, Montserrat; Sanchez-Quesada, Jose Luis; Ordoñez-Llanos, Jordi; Benitez, Sonia

    2016-09-01

    Electronegative LDL (LDL(-)) is a plasma LDL subfraction that induces cytokine release in monocytes through toll-like receptor 4 (TLR4) activation. However, the intracellular pathways induced by LDL(-) downstream TLR4 activation are unknown. We aimed to identify the pathways activated by LDL(-) leading to cytokine release in monocytes. We determined LDL(-)-induced activation of several intracellular kinases in protein extracts from monocytes using a multikinase ELISA array. LDL(-) induced higher p38 mitogen-activated protein kinase (MAPK) phosphorylation than native LDL. This was corroborated by a specific cell-based assay and it was dependent on TLR4 and phosphoinositide 3-kinase (PI3k)/Akt pathway. P38 MAPK activation was involved in cytokine release promoted by LDL(-). A specific ELISA showed that LDL(-) activated cAMP response-element binding (CREB) in a p38 MAPK dependent manner. P38 MAPK was also involved in the nuclear factor kappa-B (NF-kB) and activating protein-1 (AP-1) activation by LDL(-). We found that NF-kB, AP-1 and CREB inhibitors decreased LDL(-)-induced cytokine release, mainly on MCP1, IL6 and IL10 release, respectively. LDL(-) promotes p38 MAPK phosphorylation through TLR4 and PI3k/Akt pathways. Phosphorylation of p38 MAPK is involved in NF-kB, AP-1 and CREB activation, leading to LDL(-)-induced cytokine release in monocytes. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Insulin receptors

    International Nuclear Information System (INIS)

    Kahn, C.R.; Harrison, L.C.

    1988-01-01

    This book contains the proceedings on insulin receptors. Part A: Methods for the study of structure and function. Topics covered include: Method for purification and labeling of insulin receptors, the insulin receptor kinase, and insulin receptors on special tissues

  1. Thymosin β4 promotes the migration of endothelial cells without intracellular Ca2+ elevation

    International Nuclear Information System (INIS)

    Selmi, Anna; Malinowski, Mariusz; Brutkowski, Wojciech; Bednarek, Radoslaw; Cierniewski, Czeslaw S.

    2012-01-01

    Numerous studies have demonstrated the effects of Tβ4 on cell migration, proliferation, apoptosis and inflammation after exogenous treatment, but the mechanism by which Tβ4 functions is still unclear. Previously, we demonstrated that incubation of endothelial cells with Tβ4 induced synthesis and secretion of various proteins, including plasminogen activator inhibitor type 1 and matrix metaloproteinases. We also showed that Tβ4 interacts with Ku80, which may operate as a novel receptor for Tβ4 and mediates its intracellular activity. In this paper, we provide evidence that Tβ4 induces cellular processes without changes in the intracellular Ca 2+ concentration. External treatment of HUVECs with Tβ4 and its mutants deprived of the N-terminal tetrapeptide AcSDKP (Tβ4 AcSDKPT/4A ) or the actin-binding sequence KLKKTET (Tβ4 KLKKTET/7A ) resulted in enhanced cell migration and formation of tubular structures in Matrigel. Surprisingly, the increased cell motility caused by Tβ4 was not associated with the intracellular Ca 2+ elevation monitored with Fluo-4 NW or Fura-2 AM. Therefore, it is unlikely that externally added Tβ4 induces HUVEC migration via the surface membrane receptors known to generate Ca 2+ influx. Our data confirm the concept that externally added Tβ4 must be internalized to induce intracellular mechanisms supporting endothelial cell migration.

  2. Intracellular transport: from physics to ... biology.

    Science.gov (United States)

    Roux, Aurélien; Cuvelier, Damien; Bassereau, Patricia; Goud, Bruno

    2008-03-01

    Considerable effort over the past three decades has allowed the identification of the protein families that control the cellular machinery responsible for intracellular transport within eukaryotic cells. These proteins are estimated to represent about 10-20% of the human "proteome." The complexity of intracellular transport makes useful the development of model membranes. We describe here experimental systems based on lipid giant unilamellar vesicles (GUVs), which are attached to kinesin molecules. These systems give rise to thin membrane tubes and to complex tubular networks when incubated in vitro with microtubules and ATP. This type of assay, which mimics key events occurring during intracellular transport, allows physicists and biologists to understand how the unique mechanical properties of lipid membranes could be involved in the budding process, the sorting of cargo proteins and lipids, and the separation of the buds from a donor membrane.

  3. Fluorescent nanothermometers for intracellular thermal sensing.

    Science.gov (United States)

    Jaque, Daniel; Rosal, Blanca Del; Rodríguez, Emma Martín; Maestro, Laura Martínez; Haro-González, Patricia; Solé, José García

    2014-05-01

    The importance of high-resolution intracellular thermal sensing and imaging in the field of modern biomedicine has boosted the development of novel nanosized fluorescent systems (fluorescent nanothermometers) as the next generation of probes for intracellular thermal sensing and imaging. This thermal mapping requires fluorescent nanothermometers with good biocompatibility and high thermal sensitivity in order to obtain submicrometric and subdegree spatial and thermal resolutions, respectively. This review describes the different nanosized systems used up to now for intracellular thermal sensing and imaging. We also include the later advances in molecular systems based on fluorescent proteins for thermal mapping. A critical overview of the state of the art and the future perspective is also included.

  4. Macrophage defense mechanisms against intracellular bacteria.

    Science.gov (United States)

    Weiss, Günter; Schaible, Ulrich E

    2015-03-01

    Macrophages and neutrophils play a decisive role in host responses to intracellular bacteria including the agent of tuberculosis (TB), Mycobacterium tuberculosis as they represent the forefront of innate immune defense against bacterial invaders. At the same time, these phagocytes are also primary targets of intracellular bacteria to be abused as host cells. Their efficacy to contain and eliminate intracellular M. tuberculosis decides whether a patient initially becomes infected or not. However, when the infection becomes chronic or even latent (as in the case of TB) despite development of specific immune activation, phagocytes have also important effector functions. Macrophages have evolved a myriad of defense strategies to combat infection with intracellular bacteria such as M. tuberculosis. These include induction of toxic anti-microbial effectors such as nitric oxide and reactive oxygen intermediates, the stimulation of microbe intoxication mechanisms via acidification or metal accumulation in the phagolysosome, the restriction of the microbe's access to essential nutrients such as iron, fatty acids, or amino acids, the production of anti-microbial peptides and cytokines, along with induction of autophagy and efferocytosis to eliminate the pathogen. On the other hand, M. tuberculosis, as a prime example of a well-adapted facultative intracellular bacterium, has learned during evolution to counter-balance the host's immune defense strategies to secure survival or multiplication within this otherwise hostile environment. This review provides an overview of innate immune defense of macrophages directed against intracellular bacteria with a focus on M. tuberculosis. Gaining more insights and knowledge into this complex network of host-pathogen interaction will identify novel target sites of intervention to successfully clear infection at a time of rapidly emerging multi-resistance of M. tuberculosis against conventional antibiotics. © 2015 The Authors

  5. Macrophage defense mechanisms against intracellular bacteria

    Science.gov (United States)

    Weiss, Günter; Schaible, Ulrich E

    2015-01-01

    Macrophages and neutrophils play a decisive role in host responses to intracellular bacteria including the agent of tuberculosis (TB), Mycobacterium tuberculosis as they represent the forefront of innate immune defense against bacterial invaders. At the same time, these phagocytes are also primary targets of intracellular bacteria to be abused as host cells. Their efficacy to contain and eliminate intracellular M. tuberculosis decides whether a patient initially becomes infected or not. However, when the infection becomes chronic or even latent (as in the case of TB) despite development of specific immune activation, phagocytes have also important effector functions. Macrophages have evolved a myriad of defense strategies to combat infection with intracellular bacteria such as M. tuberculosis. These include induction of toxic anti-microbial effectors such as nitric oxide and reactive oxygen intermediates, the stimulation of microbe intoxication mechanisms via acidification or metal accumulation in the phagolysosome, the restriction of the microbe's access to essential nutrients such as iron, fatty acids, or amino acids, the production of anti-microbial peptides and cytokines, along with induction of autophagy and efferocytosis to eliminate the pathogen. On the other hand, M. tuberculosis, as a prime example of a well-adapted facultative intracellular bacterium, has learned during evolution to counter-balance the host's immune defense strategies to secure survival or multiplication within this otherwise hostile environment. This review provides an overview of innate immune defense of macrophages directed against intracellular bacteria with a focus on M. tuberculosis. Gaining more insights and knowledge into this complex network of host-pathogen interaction will identify novel target sites of intervention to successfully clear infection at a time of rapidly emerging multi-resistance of M. tuberculosis against conventional antibiotics. PMID:25703560

  6. Neural cell adhesion molecule induces intracellular signaling via multiple mechanisms of Ca2+ homeostasis

    DEFF Research Database (Denmark)

    Kiryushko, Darya; Korshunova, Irina; Berezin, Vladimir

    2006-01-01

    The neural cell adhesion molecule (NCAM) plays a pivotal role in the development of the nervous system, promoting neuronal differentiation via homophilic (NCAM-NCAM) as well as heterophilic (NCAM-fibroblast growth factor receptor [FGFR]) interactions. NCAM-induced intracellular signaling has been...... with the Src-family kinases, were also involved in neuritogenesis induced by physiological, homophilic NCAM interactions. Thus, unanticipated mechanisms of Ca2+ homeostasis are shown to be activated by NCAM and to contribute to neuronal differentiation....

  7. Intracellular Ca2+ and the phospholipid PIP2 regulate the taste transduction ion channel TRPM5

    OpenAIRE

    Liu, Dan; Liman, Emily R.

    2003-01-01

    The transduction of taste is a fundamental process that allows animals to discriminate nutritious from noxious substances. Three taste modalities, bitter, sweet, and amino acid, are mediated by G protein-coupled receptors that signal through a common transduction cascade: activation of phospholipase C β2, leading to a breakdown of phosphatidylinositol-4,5-bisphosphate (PIP2) into diacylglycerol and inositol 1,4,5-trisphosphate, which causes release of Ca2+ from intracellular stores. The ion c...

  8. Role of intracellular infections in premature childbirth.

    Science.gov (United States)

    Zurabishvili, S; Mamamtavrishvili, I; Apridonidze, K; Shanidze, L

    2005-09-01

    Vaginal Smear taken by sterile Folkman spoon from 15 women with premature birth was studied. The study was performed by the direct immune fluorescence method with the luminescence microscope. We aimed to study the effect of intracellular infections: ureaplasma urealitikum, mycoplasma hominis, Chlamydia trachomatis, herpes simplex virus of I and II type and cytomegalovirus. Intracellular infections were detected in at about 82% of cases, which included mono infections with cytomegalovirus and in 9 cases in the form of bi-component associations. The obtained results may be interesting from the etiologic point of view of premature births in Georgian population.

  9. The effect of intracellular calcium oscillations on fluid secretion in airway epithelium.

    Science.gov (United States)

    Warren, N J; Tawhai, M H; Crampin, E J

    2010-08-07

    Airway epithelium has been shown to elicit fluid secretion after a rise in intracellular calcium. This rise in intracellular calcium has been shown to display complex oscillations in many species after the binding of particular agonists to extracellular receptors. Fluid secreted by the airway epithelium is used to maintain the depth of the periciliary liquid (PCL) above the apical membrane of the epithelial cells lining the bronchial airways. Previous mathematical models have been published which separately consider the electrophysiology involved in regulating periciliary liquid depth, and the transmission of intracellular calcium waves in airway epithelial tissue. In this paper we present a mathematical model that combines these previous models and allows the effect of oscillations in intracellular calcium on fluid secretion by airway epithelial cells to be investigated. We show that an oscillatory calcium response produces different fluid secretion properties to that elicited by a tonic rise in intracellular calcium. These differences are shown to be due to saturation of the Ca(2+) activated ion channels. Copyright 2010 Elsevier Ltd. All rights reserved.

  10. Differential inhibitory response to telcagepant on αCGRP induced vasorelaxation and intracellular Ca(2+) levels in the perfused and non-perfused isolated rat middle cerebral artery

    DEFF Research Database (Denmark)

    Erdling, André; Sheykhzade, Majid; Edvinsson, Lars

    2017-01-01

    and tension in rat middle cerebral arteries (MCA) by pressurized arteriography, FURA-2/wire myography and immunohistochemistry. METHODS: A pressurized arteriograph system was used to evaluate changes in MCA tension when subjected to CGRP and/or telcagepant. Intracellular calcium levels were evaluated using......, while abluminal telcagepant inhibited the relaxation (10(-6) M). Using the FURA-2 method in combination with wire myography we observed that αCGRP reduced intracellular calcium levels and in parallel the vascular tone. Telcagepant (10(-6) M) inhibited both vasorelaxation and drop in intracellular...... calcium levels. Both functional components of the CGRP receptor, CLR (calcitonin receptor-like receptor) and RAMP1 (receptor activity modifying peptide 1) were found in the smooth muscle cells but not in the endothelial cells of the cerebral vasculature. CONCLUSIONS: This study thus demonstrates...

  11. Expanding the neuron's calcium signaling repertoire: intracellular calcium release via voltage-induced PLC and IP3R activation.

    Directory of Open Access Journals (Sweden)

    Stefanie Ryglewski

    2007-04-01

    Full Text Available Neuronal calcium acts as a charge carrier during information processing and as a ubiquitous intracellular messenger. Calcium signals are fundamental to numerous aspects of neuronal development and plasticity. Specific and independent regulation of these vital cellular processes is achieved by a rich bouquet of different calcium signaling mechanisms within the neuron, which either can operate independently or may act in concert. This study demonstrates the existence of a novel calcium signaling mechanism by simultaneous patch clamping and calcium imaging from acutely isolated central neurons. These neurons possess a membrane voltage sensor that, independent of calcium influx, causes G-protein activation, which subsequently leads to calcium release from intracellular stores via phospholipase C and inositol 1,4,5-trisphosphate receptor activation. This allows neurons to monitor activity by intracellular calcium release without relying on calcium as the input signal and opens up new insights into intracellular signaling, developmental regulation, and information processing in neuronal compartments lacking calcium channels.

  12. Viral evasion of intracellular DNA and RNA sensing

    Science.gov (United States)

    Chan, Ying Kai; Gack, Michaela U.

    2016-01-01

    The co-evolution of viruses with their hosts has led to the emergence of viral pathogens that are adept at evading or actively suppressing host immunity. Pattern recognition receptors (PRRs) are key components of antiviral immunity that detect conserved molecular features of viral pathogens and initiate signalling that results in the expression of antiviral genes. In this Review, we discuss the strategies that viruses use to escape immune surveillance by key intracellular sensors of viral RNA or DNA, with a focus on RIG-I-like receptors (RLRs), cyclic GMP–AMP synthase (cGAS) and interferon-γ (IFNγ)-inducible protein 16 (IFI16). Such viral strategies include the sequestration or modification of viral nucleic acids, interference with specific post-translational modifications of PRRs or their adaptor proteins, the degradation or cleavage of PRRs or their adaptors, and the sequestration or relocalization of PRRs. An understanding of viral immune-evasion mechanisms at the molecular level may guide the development of vaccines and antivirals. PMID:27174148

  13. Viral evasion of intracellular DNA and RNA sensing.

    Science.gov (United States)

    Chan, Ying Kai; Gack, Michaela U

    2016-06-01

    The co-evolution of viruses with their hosts has led to the emergence of viral pathogens that are adept at evading or actively suppressing host immunity. Pattern recognition receptors (PRRs) are key components of antiviral immunity that detect conserved molecular features of viral pathogens and initiate signalling that results in the expression of antiviral genes. In this Review, we discuss the strategies that viruses use to escape immune surveillance by key intracellular sensors of viral RNA or DNA, with a focus on RIG-I-like receptors (RLRs), cyclic GMP-AMP synthase (cGAS) and interferon-γ (IFNγ)-inducible protein 16 (IFI16). Such viral strategies include the sequestration or modification of viral nucleic acids, interference with specific post-translational modifications of PRRs or their adaptor proteins, the degradation or cleavage of PRRs or their adaptors, and the sequestration or relocalization of PRRs. An understanding of viral immune-evasion mechanisms at the molecular level may guide the development of vaccines and antivirals.

  14. Hepatitis C virus intracellular host interactions

    NARCIS (Netherlands)

    Liefhebber, Johanna Maaike Pieternella

    2010-01-01

    Hepatitis C virus (HCV) infects about 170 million people worldwide causing a major healthcare problem. The virus lifecycle is greatly dependent on the host-cell for effective replication. In this thesis, the intracellular interactions of the non-structural HCV proteins with the host-cell were

  15. Enhanced production of intracellular dextran dextrinase from ...

    African Journals Online (AJOL)

    Enhanced production of intracellular dextran dextrinase from Gluconobacter oxydans using statistical experimental methods. ... the Plackett-Burman screening. A four-factor five-level central composite design (CCD) was chosen to explain the combined effects of the four medium constituents. The optimum medium consisted ...

  16. Biological synthesis and characterization of intracellular gold ...

    Indian Academy of Sciences (India)

    ... nontoxic, safe, biocompatible and environmentally acceptable. In the present study, Aspergillus fumigatus was used for the intracellular synthesis of gold nanoparticles. Stable nanoparticles were produced when an aqueous solution of chloroauric acid (HAuCl4) was reduced by A. fumigatus biomass as the reducing agent ...

  17. Efficient intracellular delivery of native proteins

    NARCIS (Netherlands)

    D'Astolfo, Diego S; Pagliero, Romina J; Pras, Anita; Karthaus, Wouter R; Clevers, Hans; Prasad, Vikram; Lebbink, Robert Jan; Rehmann, Holger; Geijsen, Niels

    2015-01-01

    Modulation of protein function is used to intervene in cellular processes but is often done indirectly by means of introducing DNA or mRNA encoding the effector protein. Thus far, direct intracellular delivery of proteins has remained challenging. We developed a method termed iTOP, for induced

  18. Temporal protein expression pattern in intracellular signalling ...

    Indian Academy of Sciences (India)

    2015-09-28

    Sep 28, 2015 ... [Ganguli P, Chowdhury S, Bhowmick R and Sarkar RR 2015 Temporal protein expression pattern in intracellular signalling cascade during T-cell activation: A ... cells and tissues by studying different signalling pathways, such as Hedgehog ...... Murray JD 2003 On the mechanochemical theory of biological.

  19. Optimizing Nanoelectrode Arrays for Scalable Intracellular Electrophysiology.

    Science.gov (United States)

    Abbott, Jeffrey; Ye, Tianyang; Ham, Donhee; Park, Hongkun

    2018-03-20

    Electrode technology for electrophysiology has a long history of innovation, with some decisive steps including the development of the voltage-clamp measurement technique by Hodgkin and Huxley in the 1940s and the invention of the patch clamp electrode by Neher and Sakmann in the 1970s. The high-precision intracellular recording enabled by the patch clamp electrode has since been a gold standard in studying the fundamental cellular processes underlying the electrical activities of neurons and other excitable cells. One logical next step would then be to parallelize these intracellular electrodes, since simultaneous intracellular recording from a large number of cells will benefit the study of complex neuronal networks and will increase the throughput of electrophysiological screening from basic neurobiology laboratories to the pharmaceutical industry. Patch clamp electrodes, however, are not built for parallelization; as for now, only ∼10 patch measurements in parallel are possible. It has long been envisioned that nanoscale electrodes may help meet this challenge. First, nanoscale electrodes were shown to enable intracellular access. Second, because their size scale is within the normal reach of the standard top-down fabrication, the nanoelectrodes can be scaled into a large array for parallelization. Third, such a nanoelectrode array can be monolithically integrated with complementary metal-oxide semiconductor (CMOS) electronics to facilitate the large array operation and the recording of the signals from a massive number of cells. These are some of the central ideas that have motivated the research activity into nanoelectrode electrophysiology, and these past years have seen fruitful developments. This Account aims to synthesize these findings so as to provide a useful reference. Summing up from the recent studies, we will first elucidate the morphology and associated electrical properties of the interface between a nanoelectrode and a cellular membrane

  20. Donepezil suppresses intracellular Ca2+mobilization through the PI3K pathway in rodent microglia.

    Science.gov (United States)

    Haraguchi, Yoshinori; Mizoguchi, Yoshito; Ohgidani, Masahiro; Imamura, Yoshiomi; Murakawa-Hirachi, Toru; Nabeta, Hiromi; Tateishi, Hiroshi; Kato, Takahiro A; Monji, Akira

    2017-12-22

    Microglia are resident innate immune cells which release many factors including proinflammatory cytokines or nitric oxide (NO) when they are activated in response to immunological stimuli. Pathophysiology of Alzheimer's disease (AD) is related to the inflammatory responses mediated by microglia. Intracellular Ca 2+ signaling is important for microglial functions such as release of NO and cytokines. In addition, alteration of intracellular Ca 2+ signaling underlies the pathophysiology of AD, while it remains unclear how donepezil, an acetylcholinesterase inhibitor, affects intracellular Ca 2+ mobilization in microglial cells. We examined whether pretreatment with donepezil affects the intracellular Ca 2+ mobilization using fura-2 imaging and tested the effects of donepezil on phagocytic activity by phagocytosis assay in rodent microglial cells. In this study, we observed that pretreatment with donepezil suppressed the TNFα-induced sustained intracellular Ca 2+ elevation in both rat HAPI and mouse primary microglial cells. On the other hand, pretreatment with donepezil did not suppress the mRNA expression of both TNFR1 and TNFR2 in rodent microglia we used. Pretreatment with acetylcholine but not donepezil suppressed the TNFα-induced intracellular Ca 2+ elevation through the nicotinic α7 receptors. In addition, sigma 1 receptors were not involved in the donepezil-induced suppression of the TNFα-mediated intracellular Ca 2+ elevation. Pretreatment with donepezil suppressed the TNFα-induced intracellular Ca 2+ elevation through the PI3K pathway in rodent microglial cells. Using DAF-2 imaging, we also found that pretreatment with donepezil suppressed the production of NO induced by TNFα treatment and the PI3K pathway could be important for the donepezil-induced suppression of NO production in rodent microglial cells. Finally, phagocytosis assay showed that pretreatment with donepezil promoted phagocytic activity of rodent microglial cells through the PI3K but not

  1. Biochemical and ultrastructural studies suggest that the effects of thapsigargin on human platelets are mediated by changes in intracellular calcium but not by intracellular histamine

    DEFF Research Database (Denmark)

    Saxena, S P; McNicol, A; Becker, A B

    1992-01-01

    was observed at 1 microM Tg. Preincubation of platelets with inhibitors of histamine metabolizing enzymes had little effect on intracellular histamine levels in platelets stimulated by 0.5 microM Tg. In addition, the inhibitors of histidine decarboxylase (HDC), alpha-methyl histidine (alpha-MH) and alpha......-fluoromethyl histidine (alpha-FMH) failed to inhibit Tg-induced aggregation. The intracellular histamine receptor antagonist, N,N-diethyl-2-[4-(phenylmethyl)phenoxy] ethanamine. HCl (DPPE), inhibited Tg-induced aggregation but with IC50 values dependent on the concentration of agonist used. The inhibitory effects...... of DPPE on Tg-induced aggregation were not reversed by the addition of histamine to saponin-permeabilized platelets suggesting non-histamine mediated effects of DPPE on Tg-induced aggregation. Tg stimulated an increase in the cytosolic free calcium concentration which was unaffected by DPPE indicating...

  2. Intracellular Modifiers of Integrin Alpha 6p Production in Aggressive Prostate and Breast Cancer Cell Lines

    Science.gov (United States)

    Kacsinta, Apollo D.; Rubenstein, Cynthia S.; Sroka, Isis C.; Pawar, Sangita; Gard, Jaime M.; Nagle, Raymond B.; Cress, Anne E.

    2014-01-01

    Cancer metastasis is a multi-step process in which tumor cells gain the ability to invade beyond the primary tumor and colonize distant sites. The mechanisms regulating the metastatic process confer changes to cell adhesion receptors including the integrin family of receptors. Our group previously discovered that the α6 integrin (ITGA6/CD49f) is post translationally modified by urokinase plasminogen activator (uPA) and its receptor, urokinase plasminogen activator receptor (uPAR), to form the variant ITGA6p. This variant of ITGA6 is a cleaved form of the receptor that lacks the ligand-binding domain. Although it is established that the uPA/uPAR axis drives ITGA6 cleavage, the mechanisms regulating cleavage have not been defined. Intracellular integrin dependent “inside-out” signaling is a major regulator of integrin function and the uPA/uPAR axis. We hypothesized that intracellular signaling molecules play a role in formation of ITGA6p to promote cell migration during cancer metastasis. In order to test our hypothesis, DU145 and PC3B1 prostate cancer and MDA-MB-231 breast cancer cell lines were treated with small interfering RNA targeting actin and the intracellular signaling regulators focal adhesion kinase (FAK), integrin linked kinase (ILK), and paxillin. The results demonstrated that inhibition of actin, FAK, and ILK expression resulted in significantly increased uPAR expression and ITGA6p production. Inhibition of actin increased ITGA6p, although inhibition of paxillin did not affect ITGA6p formation. Taken together, these results suggest that FAK and ILK dependent “inside-out” signaling, and actin dynamics regulate extracellular production of ITGA6p and the aggressive phenotype. PMID:25450398

  3. Role of protein dynamics in transmembrane receptor signalling

    DEFF Research Database (Denmark)

    Wang, Yong; Bugge, Katrine Østergaard; Kragelund, Birthe Brandt

    2018-01-01

    Cells are dependent on transmembrane receptors to communicate and transform chemical and physical signals into intracellular responses. Because receptors transport 'information', conformational changes and protein dynamics play a key mechanistic role. We here review examples where experiment...... and computation have been used to study receptor dynamics. Recent studies on three distinct classes of receptors (G-protein coupled receptors, ligand-gated ion-channels and single-pass receptors) are highlighted to show that conformational changes across a range of time-scales and length-scales are central...

  4. Bioreducible Lipid-like Nanoparticles for Intracellular Protein Delivery

    Science.gov (United States)

    Arellano, Carlos Luis

    Protein-based therapy is one of the most direct ways to manipulate cell function and treat human disease. Although protein therapeutics has made its way to clinical practice, with five of the top fifteen global pharmaceuticals being peptide or protein-based drugs, one common limitation is that the effects of protein therapy are only achieved through the targeting of cell surface receptors and intracellular domains. Due to the impermeability of the cell membrane to most foreign materials, entire classes of potentially therapeutic proteins cannot thoroughly be studied without a safe and efficient method of transporting proteins into the cytosol. We report the use of a combinatorially-designed bioreducible lipid-like material (termed "lipidoid") - based protein delivery platform for the transfection of human cancer cell lines. Lipidoid nanoparticles are synthesized through a thin film dispersion method. The degradation of the bioreducible nanoparticles was observed when exposed to glutathione, a highly reductive compound present in the cytosol. We demonstrate that the nanoparticles are capable of transfecting a dose-dependent concentration of our model protein, beta-galactosidase into HeLa cells. Furthermore, formulations of the lipidoid containing the cytotoxic proteins saporin and RNase-A are both capable of inhibiting tumor cell proliferation as observed in in vitro treatment of different human cancer cell lines. There was no observed loss in protein activity after lyophilization and long--term storage, indicating the potential of pre-clinical applications. Overall, we demonstrate an effective approach to protein formulation and intracellular delivery. We believe that our formulations will lead to the study of a whole class of previously untapped therapeutics that may generate new solutions for previously untreatable diseases.

  5. Axotomy depletes intracellular calcium stores in primary sensory neurons.

    Science.gov (United States)

    Rigaud, Marcel; Gemes, Geza; Weyker, Paul D; Cruikshank, James M; Kawano, Takashi; Wu, Hsiang-En; Hogan, Quinn H

    2009-08-01

    The cellular mechanisms of neuropathic pain are inadequately understood. Previous investigations have revealed disrupted Ca signaling in primary sensory neurons after injury. The authors examined the effect of injury on intracellular Ca stores of the endoplasmic reticulum, which critically regulate the Ca signal and neuronal function. Intracellular Ca levels were measured with Fura-2 or mag-Fura-2 microfluorometry in axotomized fifth lumbar (L5) dorsal root ganglion neurons and adjacent L4 neurons isolated from hyperalgesic rats after L5 spinal nerve ligation, compared to neurons from control animals. Endoplasmic reticulum Ca stores released by the ryanodine-receptor agonist caffeine decreased by 46% in axotomized small neurons. This effect persisted in Ca-free bath solution, which removes the contribution of store-operated membrane Ca channels, and after blockade of the mitochondrial, sarco-endoplasmic Ca-ATPase and the plasma membrane Ca ATPase pathways. Ca released by the sarco-endoplasmic Ca-ATPase blocker thapsigargin and by the Ca-ionophore ionomycin was also diminished by 25% and 41%, respectively. In contrast to control neurons, Ca stores in axotomized neurons were not expanded by neuronal activation by K depolarization, and the proportionate rate of refilling by sarco-endoplasmic Ca-ATPase was normal. Luminal Ca concentration was also reduced by 38% in axotomized neurons in permeabilized neurons. The adjacent neurons of the L4 dorsal root ganglia showed modest and inconsistent changes after L5 spinal nerve ligation. Painful nerve injury leads to diminished releasable endoplasmic reticulum Ca stores and a reduced luminal Ca concentration. Depletion of Ca stores may contribute to the pathogenesis of neuropathic pain.

  6. Reduction of intracellular glutathione content and radiosensitivity

    International Nuclear Information System (INIS)

    Vos, O.; Schans, G.P. van der; Roos-Verheij, W.S.D.

    1986-01-01

    The intracellular glutathione (GSH) content of HeLa, CHO and V79 cells was reduced by incubating the cells in growth medium containing buthionine sulphoximine or diethyl maleate (DEM). Clonogenicity, single-strand DNA breaks (ssb) and double-strand DNA breaks (dsb) were used as criteria for radiation-induced damage after X- or γ-irradiation. In survival experiments, DEM gave a slightly larger sensitization although it gave a smaller reduction of the intracellular GSH. In general, sensitization was larger for dsb than for ssb, also the reduction of the o.e.r. was generally larger for dsb than for ssb. This may be due to the higher dose rate in case of dsb experiments resulting in a higher rate of radiochemical oxygen consumption. In general, no effect was found on post-irradiation repair of ssb and dsb. (author)

  7. Intracellular mechanisms of solar water disinfection

    Science.gov (United States)

    Castro-Alférez, María; Polo-López, María Inmaculada; Fernández-Ibáñez, Pilar

    2016-12-01

    Solar water disinfection (SODIS) is a zero-cost intervention measure to disinfect drinking water in areas of poor access to improved water sources, used by more than 6 million people in the world. The bactericidal action of solar radiation in water has been widely proven, nevertheless the causes for this remain still unclear. Scientific literature points out that generation of reactive oxygen species (ROS) inside microorganisms promoted by solar light absorption is the main reason. For the first time, this work reports on the experimental measurement of accumulated intracellular ROS in E. coli during solar irradiation. For this experimental achievement, a modified protocol based on the fluorescent probe dichlorodihydrofluorescein diacetate (DCFH-DA), widely used for oxidative stress in eukaryotic cells, has been tested and validated for E. coli. Our results demonstrate that ROS and their accumulated oxidative damages at intracellular level are key in solar water disinfection.

  8. Reduction of intracellular glutathione content and radiosensitivity

    International Nuclear Information System (INIS)

    Vos, O.; Schans, G.P. van der; Roos-Verheij, W.S.D.

    1986-05-01

    The intracellular glutathione (GSH) content in HeLa, CHO and V79 cells was reduced by incubating the cells in growth medium containing buthionine sulfoximine (BSO) or diethyl maleate (DEM). Clonogenicity, single strand DNA breaks (ssb) and double strand DNA breaks (dsb) were used as criteria for radiation induced damage after X- or γ irradiation. In survival experiments DEM gave a slightly larger sensitization although it gave a smaller reduction of the intracellular GSH. In general, sensitization was larger for dsb than for ssb, also the reduction of the OER was generally larger for dsb than for ssb. This may be due to the higher dose rate in case of dsb experiments resulting in a higher rate of radiochemical oxygen consumption. In general, no effect was found on post-irradiation repair of ssb and dsb. (Auth.)

  9. Intracellular Protein Delivery for Treating Breast Cancer

    Science.gov (United States)

    2014-08-01

    Intracellular delivery of such proteins, including human tumor suppressors (such as p53) (Brown et al., 2009) and exogenous tumor-killing proteins...vivo systems. Nature materials 11, 1038-1043. Chorny, M., Hood, E., Levy, R.J., and Muzykantov, V.R. (2010). Endothelial delivery of antioxidant ...for the ntracellular delivery of such proteins, including human umor suppressors [7] and exogenous tumor-killing proteins 8—10]), is attractive as a

  10. Fluorescent nanoparticles for intracellular sensing: A review

    International Nuclear Information System (INIS)

    Ruedas-Rama, Maria J.; Walters, Jamie D.; Orte, Angel; Hall, Elizabeth A.H.

    2012-01-01

    Highlights: ► Analytical applications of fluorescent nanoparticles (NPs) in intracellular sensing. ► Critical review on performance of QDots, metal NPs, silica NPs, and polymer NPs. ► Highlighted potential of fluorescence lifetime imaging microscopy (FLIM). - Abstract: Fluorescent nanoparticles (NPs), including semiconductor NPs (Quantum Dots), metal NPs, silica NPs, polymer NPs, etc., have been a major focus of research and development during the past decade. The fluorescent nanoparticles show unique chemical and optical properties, such as brighter fluorescence, higher photostability and higher biocompatibility, compared to classical fluorescent organic dyes. Moreover, the nanoparticles can also act as multivalent scaffolds for the realization of supramolecular assemblies, since their high surface to volume ratio allow distinct spatial domains to be functionalized, which can provide a versatile synthetic platform for the implementation of different sensing schemes. Their excellent properties make them one of the most useful tools that chemistry has supplied to biomedical research, enabling the intracellular monitoring of many different species for medical and biological purposes. In this review, we focus on the developments and analytical applications of fluorescent nanoparticles in chemical and biological sensing within the intracellular environment. The review also points out the great potential of fluorescent NPs for fluorescence lifetime imaging microscopy (FLIM). Finally, we also give an overview of the current methods for delivering of fluorescent NPs into cells, where critically examine the benefits and liabilities of each strategy.

  11. Fluorescent nanoparticles for intracellular sensing: A review

    Energy Technology Data Exchange (ETDEWEB)

    Ruedas-Rama, Maria J., E-mail: mjruedas@ugr.esmailto [Department of Physical Chemistry, Faculty of Pharmacy, University of Granada, Campus Cartuja, 18071, Granada (Spain); Walters, Jamie D. [Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, UK CB2 1QT (United Kingdom); Orte, Angel [Department of Physical Chemistry, Faculty of Pharmacy, University of Granada, Campus Cartuja, 18071, Granada (Spain); Hall, Elizabeth A.H., E-mail: lisa.hall@biotech.cam.ac.uk [Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QT (United Kingdom)

    2012-11-02

    Highlights: Black-Right-Pointing-Pointer Analytical applications of fluorescent nanoparticles (NPs) in intracellular sensing. Black-Right-Pointing-Pointer Critical review on performance of QDots, metal NPs, silica NPs, and polymer NPs. Black-Right-Pointing-Pointer Highlighted potential of fluorescence lifetime imaging microscopy (FLIM). - Abstract: Fluorescent nanoparticles (NPs), including semiconductor NPs (Quantum Dots), metal NPs, silica NPs, polymer NPs, etc., have been a major focus of research and development during the past decade. The fluorescent nanoparticles show unique chemical and optical properties, such as brighter fluorescence, higher photostability and higher biocompatibility, compared to classical fluorescent organic dyes. Moreover, the nanoparticles can also act as multivalent scaffolds for the realization of supramolecular assemblies, since their high surface to volume ratio allow distinct spatial domains to be functionalized, which can provide a versatile synthetic platform for the implementation of different sensing schemes. Their excellent properties make them one of the most useful tools that chemistry has supplied to biomedical research, enabling the intracellular monitoring of many different species for medical and biological purposes. In this review, we focus on the developments and analytical applications of fluorescent nanoparticles in chemical and biological sensing within the intracellular environment. The review also points out the great potential of fluorescent NPs for fluorescence lifetime imaging microscopy (FLIM). Finally, we also give an overview of the current methods for delivering of fluorescent NPs into cells, where critically examine the benefits and liabilities of each strategy.

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

    Science.gov (United States)

    Osadchiĭ, O E

    2006-01-01

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

  13. Effects of inhibitors of protein synthesis and intracellular transport on the gamma-aminobutyric acid agonist-induced functional differentiation of cultured cerebellar granule cells

    DEFF Research Database (Denmark)

    Belhage, B; Hansen, Gert Helge; Meier, E

    1990-01-01

    an intracellular and a plasma membrane localization of the receptors. In all experiments cultures treated with THIP alone served as controls. The inhibitors of protein synthesis totally abolished the ability of THIP to induce low-affinity GABA receptors. In contrast, the inhibitors of intracellular transport......The effect of inhibitors of protein synthesis (actinomycin D, cycloheximide), proteases (leupeptin), and intracellular transport (colchicine, monensin) on the gamma-aminobutyric acid (GABA) agonist [4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP)]-induced changes in morphological...... as well as the protease inhibitor did not affect this parameter. However, studies of effects of GABA on transmitter release from monensin-treated cultures showed that transmitter release could not be inhibited by GABA in these cells in spite of the presence of low-affinity GABA receptors in the membrane...

  14. Rapid and transient stimulation of intracellular reactive oxygen species by melatonin in normal and tumor leukocytes

    International Nuclear Information System (INIS)

    Radogna, Flavia; Paternoster, Laura; De Nicola, Milena; Cerella, Claudia; Ammendola, Sergio; Bedini, Annalida; Tarzia, Giorgio; Aquilano, Katia; Ciriolo, Maria; Ghibelli, Lina

    2009-01-01

    Melatonin is a modified tryptophan with potent biological activity, exerted by stimulation of specific plasma membrane (MT1/MT2) receptors, by lower affinity intracellular enzymatic targets (quinone reductase, calmodulin), or through its strong anti-oxidant ability. Scattered studies also report a perplexing pro-oxidant activity, showing that melatonin is able to stimulate production of intracellular reactive oxygen species (ROS). Here we show that on U937 human monocytes melatonin promotes intracellular ROS in a fast (< 1 min) and transient (up to 5-6 h) way. Melatonin equally elicits its pro-radical effect on a set of normal or tumor leukocytes; intriguingly, ROS production does not lead to oxidative stress, as shown by absence of protein carbonylation, maintenance of free thiols, preservation of viability and regular proliferation rate. ROS production is independent from MT1/MT2 receptor interaction, since a) requires micromolar (as opposed to nanomolar) doses of melatonin; b) is not contrasted by the specific MT1/MT2 antagonist luzindole; c) is not mimicked by a set of MT1/MT2 high affinity melatonin analogues. Instead, chlorpromazine, the calmodulin inhibitor shown to prevent melatonin-calmodulin interaction, also prevents melatonin pro-radical effect, suggesting that the low affinity binding to calmodulin (in the micromolar range) may promote ROS production.

  15. The first intracellular loop of GLUT4 contains a retention motif.

    Science.gov (United States)

    Talantikite, Maya; Berenguer, Marion; Gonzalez, Teresa; Alessi, Marie Christine; Poggi, Marjorie; Peiretti, Franck; Govers, Roland

    2016-06-01

    Glucose transporter GLUT4 (also known as SLC2A4) plays a major role in glucose homeostasis and is efficiently retained intracellularly in adipocytes and myocytes. To simplify the analysis of its retention, here, various intracellular GLUT4 domains were fused individually to reporter molecules. Of the four short cytoplasmic loops of GLUT4, only the first nine-residue-long loop conferred intracellular retention of truncated forms of the transferrin receptor and CD4 in adipocytes. In contrast, the same loop of GLUT1 was without effect. The reporter molecules to which the first loop of GLUT4 was fused localized, unlike GLUT4, to the trans-Golgi network (TGN), possibly explaining why these molecules did not respond to insulin. The retention induced by the GLUT4 loop was specific to adipocytes as it did not induce retention in preadipocytes. Of the SQWLGRKRA sequence that constitutes this loop, mutation of either the tryptophan or lysine residue abrogated reporter retention. Mutation of these residues individually into alanine residues in the full-length GLUT4 molecule resulted in a decreased retention for GLUT4-W105A. We conclude that the first intracellular loop of GLUT4 contains the retention motif WLGRK, in which W105 plays a prominent role. © 2016. Published by The Company of Biologists Ltd.

  16. Use of magnetic nanobeads to study intracellular antigen processing

    Energy Technology Data Exchange (ETDEWEB)

    Perrin-Cocon, Laure A.; Chesne, Serge; Pignot-Paintrand, Isabelle; Marche, Patrice N.; Villiers, Christian L. E-mail: christian.villiers@cea.fr

    2001-07-01

    Magnetic nanobeads were covalently linked to antigens and used as a tool to simultaneously follow their intracellular transport into the cells and specifically purify the intracellular compartments implicated in antigen processing. The protein content of these vesicles was analysed by 2D-electrophoresis. Furthermore, nanobeads allowed intracellular localisation of the antigen in electron and fluorescence microscopy.

  17. Use of magnetic nanobeads to study intracellular antigen processing

    International Nuclear Information System (INIS)

    Perrin-Cocon, Laure A.; Chesne, Serge; Pignot-Paintrand, Isabelle; Marche, Patrice N.; Villiers, Christian L.

    2001-01-01

    Magnetic nanobeads were covalently linked to antigens and used as a tool to simultaneously follow their intracellular transport into the cells and specifically purify the intracellular compartments implicated in antigen processing. The protein content of these vesicles was analysed by 2D-electrophoresis. Furthermore, nanobeads allowed intracellular localisation of the antigen in electron and fluorescence microscopy

  18. BDNF-Induced Potentiation of Spontaneous Twitching in Innervated Myocytes Requires Calcium Release From Intracellular Stores

    Science.gov (United States)

    KLEIMAN, ROBIN J.; TIAN, NING; KRIZAJ, DAVID; HWANG, THOMAS N.; COPENHAGEN, DAVID R.; REICHARDT, LOUIS F.

    2009-01-01

    Brain-derived neurotrophic factor (BDNF) can potentiate synaptic release at newly developed frog neuromuscular junctions. Although this potentiation depends on extracellular Ca2+ and reflects changes in acetylcholine release, little is known about the intracellular transduction or calcium signaling pathways. We have developed a video assay for neurotrophin-induced potentiation of myocyte twitching as a measure of potentiation of synaptic activity. We use this assay to show that BDNF-induced synaptic potentiation is not blocked by cadmium, indicating that Ca2+ influx through voltage-gated Ca2+ channels is not required. TrkB autophosphorylation is not blocked in Ca2+-free conditions, indicating that TrkB activity is not Ca2+ dependent. Additionally, an inhibitor of phospholipase C interferes with BDNF-induced potentiation. These results suggest that activation of the TrkB receptor activates phospholipase C to initiate intracellular Ca2+ release from stores which subsequently potentiates transmitter release. PMID:10899220

  19. Comparison of the intracellular trafficking itinerary of ctla-4 orthologues.

    Directory of Open Access Journals (Sweden)

    Satdip Kaur

    Full Text Available CTLA-4 is an essential inhibitor of T cell immune responses. At steady state, most CTLA-4 resides in intracellular compartments due to constitutive internalisation mediated via a tyrosine based endocytic motif (YVKM within the cytoplasmic domain. This domain is highly conserved in mammals suggesting strong selective pressure. In contrast, the C-terminal domain varies considerably in non-mammals such as fish, xenopus and birds. We compared the ability of the C-terminus of these species to direct the trafficking of CTLA-4 with human CTLA-4. Using a chimeric approach, endocytosis was found to be conserved between human, xenopus and chicken CTLA-4 but was reduced substantially in trout CTLA-4, which lacks the conserved YXXM motif. Nevertheless, we identified an alternative YXXF motif in trout CTLA-4 that permitted limited endocytosis. Post-internalisation, CTLA-4 was either recycled or targeted for degradation. Human and chicken CTLA-4, which contain a YVKM motif, showed efficient recycling compared to xenopus CTLA-4 which contains a less efficient YEKM motif. Specific mutation of this motif in human CTLA-4 reduced receptor recycling. These findings suggest evolutionary development in the endocytic and recycling potential of CTLA-4, which may facilitate more refined functions of CTLA-4 within the mammalian immune system.

  20. Ketamine Metabolites Enantioselectively Decrease Intracellular D-Serine Concentrations in PC-12 Cells.

    Directory of Open Access Journals (Sweden)

    Nagendra S Singh

    Full Text Available D-Serine is an endogenous NMDA receptor co-agonist that activates synaptic NMDA receptors modulating neuronal networks in the cerebral cortex and plays a key role in long-term potentiation of synaptic transmission. D-serine is associated with NMDA receptor neurotoxicity and neurodegeneration and elevated D-serine concentrations have been associated with Alzheimer's and Parkinsons' diseases and amyotrophic lateral sclerosis. Previous studies have demonstrated that the ketamine metabolites (rac-dehydronorketamine and (2S,6S-hydroxynorketamine decrease intracellular D-serine concentrations in a concentration dependent manner in PC-12 cells. In the current study, PC-12 cells were incubated with a series of ketamine metabolites and the IC50 values associated with attenuated intracellular D-serine concentrations were determined. The results demonstrate that structural and stereochemical features of the studied compounds contribute to the magnitude of the inhibitory effect with (2S,6S-hydroxynorketamine and (2R,6R-hydroxynorketamine displaying the most potent inhibition with IC50 values of 0.18 ± 0.04 nM and 0.68 ± 0.09 nM. The data was utilized to construct a preliminary 3D-QSAR/pharmacophore model for use in the design of new and more efficient modulators of D-serine.

  1. Dynamic mobility of functional GABAA receptors at inhibitory synapses.

    Science.gov (United States)

    Thomas, Philip; Mortensen, Martin; Hosie, Alastair M; Smart, Trevor G

    2005-07-01

    Importing functional GABAA receptors into synapses is fundamental for establishing and maintaining inhibitory transmission and for controlling neuronal excitability. By introducing a binding site for an irreversible inhibitor into the GABAA receptor alpha1 subunit channel lining region that can be accessed only when the receptor is activated, we have determined the dynamics of receptor mobility between synaptic and extrasynaptic locations in hippocampal pyramidal neurons. We demonstrate that the cell surface GABAA receptor population shows no fast recovery after irreversible inhibition. In contrast, after selective inhibition, the synaptic receptor population rapidly recovers by the import of new functional entities within minutes. The trafficking pathways that promote rapid importation of synaptic receptors do not involve insertion from intracellular pools, but reflect receptor diffusion within the plane of the membrane. This process offers the synapse a rapid mechanism to replenish functional GABAA receptors at inhibitory synapses and a means to control synaptic efficacy.

  2. Cytoskeletal Network Morphology Regulates Intracellular Transport Dynamics

    Science.gov (United States)

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

    2015-01-01

    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. PMID:26488648

  3. Intracellular bacteria: the origin of dinoflagellate toxicity.

    Science.gov (United States)

    Silva, E S

    1990-01-01

    Dinoflagellate blooms of the same species have been registered either as toxic or nontoxic and, in the latter case, toxicity may be of different types. A hypothesis has been formulated according to which the bacteria having in some way taken part in the toxin formation are either inside the dinoflagellate cell or in the nutritive liquid. The presence of intracellular bacteria in those microorganisms has been studied mainly in material from cultures, a few from the sea, and several strains were isolated from different species. Experiments with crossed inoculations have shown that the bacterial strain from Gonyaulax tamarensis caused the cells of some other species to become toxic. From nontoxic clonal cultures of Prorocentrum balticum, Glenodinium foliaceum, and Gyrodinium instriatum, after inoculation of that bacterial strain, cultures were obtained whose cell extracts showed the same kind of toxicity as G. tamarensis. No toxic action could be found in the extracts of the bacterial cells form the assayed strains. The interference of intracellular bacteria in the metabolism of dinoflagellates must be the main cause of their toxicity.

  4. Cytoskeletal Network Morphology Regulates Intracellular Transport Dynamics.

    Science.gov (United States)

    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. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  5. Intracellular accumulation of norfloxacin in Mycobacterium smegmatis.

    Science.gov (United States)

    Corti, S; Chevalier, J; Cremieux, A

    1995-01-01

    To evaluate the intracellular accumulation of norfloxacin in mycobacteria, two methods were used with Mycobacterium smegmatis. A radiometric method (K. V. Cundy, C. E. Fasching, K. E. Willard, and L. R. Peterson, J. Antimicrob. Chemother. 28:491-497, 1991) was used without great modification, but the fluorometric method (P. G. S. Mortimer and L. J. V. Piddock, J. Antimicrob. Chemother. 28:639-653, 1991) was changed considerably. Indeed, adsorption of the quinolone to the bacterial surface was characterized by measuring the level of accumulation of 0 degree C. Taking into account the adsorption, the pH of the washing buffer was increased from 7.0 to 9.0 to improve the desorption of norfloxacin from the cell surface. Both the fluorometric method, with the technical improvement, and the radiometric method could be used to estimate the intracellular accumulation of norfloxacin, which resulted from the difference between the whole uptake measured at 37 degrees C and the adsorption measured at 0 degrees C. A total of 35 ng of norfloxacin per mg of cells (dry weight) penetrated into the M. smegmatis cell, and the steady state was achieved in 5 min. Use of inhibitors of the proton motive force revealed that transport of norfloxacin was energy independent. Thus, the same mechanisms of quinolone accumulation that occur in eubacteria seem to occur in mycobacteria, at least in M. smegmatis. PMID:8585727

  6. Fluorescent nanoparticles for intracellular sensing: a review.

    Science.gov (United States)

    Ruedas-Rama, Maria J; Walters, Jamie D; Orte, Angel; Hall, Elizabeth A H

    2012-11-02

    Fluorescent nanoparticles (NPs), including semiconductor NPs (Quantum Dots), metal NPs, silica NPs, polymer NPs, etc., have been a major focus of research and development during the past decade. The fluorescent nanoparticles show unique chemical and optical properties, such as brighter fluorescence, higher photostability and higher biocompatibility, compared to classical fluorescent organic dyes. Moreover, the nanoparticles can also act as multivalent scaffolds for the realization of supramolecular assemblies, since their high surface to volume ratio allow distinct spatial domains to be functionalized, which can provide a versatile synthetic platform for the implementation of different sensing schemes. Their excellent properties make them one of the most useful tools that chemistry has supplied to biomedical research, enabling the intracellular monitoring of many different species for medical and biological purposes. In this review, we focus on the developments and analytical applications of fluorescent nanoparticles in chemical and biological sensing within the intracellular environment. The review also points out the great potential of fluorescent NPs for fluorescence lifetime imaging microscopy (FLIM). Finally, we also give an overview of the current methods for delivering of fluorescent NPs into cells, where critically examine the benefits and liabilities of each strategy. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Stochastic models of intracellular calcium signals

    Energy Technology Data Exchange (ETDEWEB)

    Rüdiger, Sten, E-mail: sten.ruediger@physik.hu-berlin.de

    2014-01-10

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

  8. Cell-Surface Receptors Transactivation Mediated by G Protein-Coupled Receptors

    Science.gov (United States)

    Cattaneo, Fabio; Guerra, Germano; Parisi, Melania; De Marinis, Marta; Tafuri, Domenico; Cinelli, Mariapia; Ammendola, Rosario

    2014-01-01

    G protein-coupled receptors (GPCRs) are seven transmembrane-spanning proteins belonging to a large family of cell-surface receptors involved in many intracellular signaling cascades. Despite GPCRs lack intrinsic tyrosine kinase activity, tyrosine phosphorylation of a tyrosine kinase receptor (RTK) occurs in response to binding of specific agonists of several such receptors, triggering intracellular mitogenic cascades. This suggests that the notion that GPCRs are associated with the regulation of post-mitotic cell functions is no longer believable. Crosstalk between GPCR and RTK may occur by different molecular mechanism such as the activation of metalloproteases, which can induce the metalloprotease-dependent release of RTK ligands, or in a ligand-independent manner involving membrane associated non-receptor tyrosine kinases, such as c-Src. Reactive oxygen species (ROS) are also implicated as signaling intermediates in RTKs transactivation. Intracellular concentration of ROS increases transiently in cells stimulated with GPCR agonists and their deliberated and regulated generation is mainly catalyzed by enzymes that belong to nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family. Oxidation and/or reduction of cysteine sulfhydryl groups of phosphatases tightly controls the activity of RTKs and ROS-mediated inhibition of cellular phosphatases results in an equilibrium shift from the non-phosphorylated to the phosphorylated state of RTKs. Many GPCR agonists activate phospholipase C, which catalyze the hydrolysis of phosphatidylinositol 4,5-bis-phosphate to produce inositol 1,4,5-triphosphate and diacylglicerol. The consequent mobilization of Ca2+ from endoplasmic reticulum leads to the activation of protein kinase C (PKC) isoforms. PKCα mediates feedback inhibition of RTK transactivation during GPCR stimulation. Recent data have expanded the coverage of transactivation to include Serine/Threonine kinase receptors and Toll-like receptors. Herein, we

  9. Intracellular signaling by diffusion: can waves of hydrogen peroxide transmit intracellular information in plant cells?

    DEFF Research Database (Denmark)

    Vestergaard, Christian L.; Flyvbjerg, Henrik; Møller, Ian Max

    2012-01-01

    Amplitude- and frequency-modulated waves of Ca(2+) ions transmit information inside cells. Reactive Oxygen Species (ROS), specifically hydrogen peroxide, have been proposed to have a similar role in plant cells. We consider the feasibility of such an intracellular communication system in view...

  10. The calcium feedback loop and T cell activation: how cytoskeleton networks control intracellular calcium flux.

    Science.gov (United States)

    Joseph, Noah; Reicher, Barak; Barda-Saad, Mira

    2014-02-01

    During T cell activation, the engagement of a T cell with an antigen-presenting cell (APC) results in rapid cytoskeletal rearrangements and a dramatic increase of intracellular calcium (Ca(2+)) concentration, downstream to T cell antigen receptor (TCR) ligation. These events facilitate the organization of an immunological synapse (IS), which supports the redistribution of receptors, signaling molecules and organelles towards the T cell-APC interface to induce downstream signaling events, ultimately supporting T cell effector functions. Thus, Ca(2+) signaling and cytoskeleton rearrangements are essential for T cell activation and T cell-dependent immune response. Rapid release of Ca(2+) from intracellular stores, e.g. the endoplasmic reticulum (ER), triggers the opening of Ca(2+) release-activated Ca(2+) (CRAC) channels, residing in the plasma membrane. These channels facilitate a sustained influx of extracellular Ca(2+) across the plasma membrane in a process termed store-operated Ca(2+) entry (SOCE). Because CRAC channels are themselves inhibited by Ca(2+) ions, additional factors are suggested to enable the sustained Ca(2+) influx required for T cell function. Among these factors, we focus here on the contribution of the actin and microtubule cytoskeleton. The TCR-mediated increase in intracellular Ca(2+) evokes a rapid cytoskeleton-dependent polarization, which involves actin cytoskeleton rearrangements and microtubule-organizing center (MTOC) reorientation. Here, we review the molecular mechanisms of Ca(2+) flux and cytoskeletal rearrangements, and further describe the way by which the cytoskeletal networks feedback to Ca(2+) signaling by controlling the spatial and temporal distribution of Ca(2+) sources and sinks, modulating TCR-dependent Ca(2+) signals, which are required for an appropriate T cell response. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters

  11. Somatostatin receptors

    DEFF Research Database (Denmark)

    Møller, Lars Neisig; Stidsen, Carsten Enggaard; Hartmann, Bolette

    2003-01-01

    therefore been acknowledged to be a third endogenous ligand at SRIF receptors. This review goes through mechanisms of signal transduction, pharmacology, and anatomical distribution of SRIF receptors. Structurally, SRIF receptors belong to the superfamily of G protein-coupled (GPC) receptors, sharing....... The generation of knock-out (KO) mice, intended as a means to define the contributions made by individual receptor subtypes, necessarily marks but an approximation. Furthermore, we must now take into account the stunning complexity of receptor co-operation indicated by the observation of receptor homo......-peptides, receptor agonists and antagonists. Relatively long half lives, as compared to those of the endogenous ligands, have been paramount from the outset. Motivated by theoretical puzzles or the shortcomings of present-day diagnostics and therapy, investigators have also aimed to produce subtype...

  12. Temporal protein expression pattern in intracellular signalling ...

    Indian Academy of Sciences (India)

    2016-08-26

    Aug 26, 2016 ... Various T-cell co-receptor molecules and calcium channel CRAC play a pivotal role in the maintenance of cell's functional responses by regulating the production of effector molecules (mostly cytokines) that aids in immune clearance and also maintaining the cell in a functionally active state. Any defect in ...

  13. Innate host defense against intracellular pathogens

    NARCIS (Netherlands)

    Vaart, Michiel van der

    2013-01-01

    This thesis focuses on the recognition of pathogenic bacteria and the defense mechanisms that are activated during the innate immune response to infection. Detection of pathogens, such as bacteria, viruses, and parasites, depends on receptors that bind to evolutionary conserved structures on their

  14. Porphyromonas gingivalis evasion of autophagy and intracellular killing by human myeloid dendritic cells involves DC-SIGN-TLR2 crosstalk.

    Directory of Open Access Journals (Sweden)

    Ahmed R El-Awady

    2015-02-01

    Full Text Available Signaling via pattern recognition receptors (PRRs expressed on professional antigen presenting cells, such as dendritic cells (DCs, is crucial to the fate of engulfed microbes. Among the many PRRs expressed by DCs are Toll-like receptors (TLRs and C-type lectins such as DC-SIGN. DC-SIGN is targeted by several major human pathogens for immune-evasion, although its role in intracellular routing of pathogens to autophagosomes is poorly understood. Here we examined the role of DC-SIGN and TLRs in evasion of autophagy and survival of Porphyromonas gingivalis in human monocyte-derived DCs (MoDCs. We employed a panel of P. gingivalis isogenic fimbriae deficient strains with defined defects in Mfa-1 fimbriae, a DC-SIGN ligand, and FimA fimbriae, a TLR2 agonist. Our results show that DC-SIGN dependent uptake of Mfa1+P. gingivalis strains by MoDCs resulted in lower intracellular killing and higher intracellular content of P. gingivalis. Moreover, Mfa1+P. gingivalis was mostly contained within single membrane vesicles, where it survived intracellularly. Survival was decreased by activation of TLR2 and/or autophagy. Mfa1+P. gingivalis strain did not induce significant levels of Rab5, LC3-II, and LAMP1. In contrast, P. gingivalis uptake through a DC-SIGN independent manner was associated with early endosomal routing through Rab5, increased LC3-II and LAMP-1, as well as the formation of double membrane intracellular phagophores, a characteristic feature of autophagy. These results suggest that selective engagement of DC-SIGN by Mfa-1+P. gingivalis promotes evasion of antibacterial autophagy and lysosome fusion, resulting in intracellular persistence in myeloid DCs; however TLR2 activation can overcome autophagy evasion and pathogen persistence in DCs.

  15. Porphyromonas gingivalis evasion of autophagy and intracellular killing by human myeloid dendritic cells involves DC-SIGN-TLR2 crosstalk.

    Science.gov (United States)

    El-Awady, Ahmed R; Miles, Brodie; Scisci, Elizabeth; Kurago, Zoya B; Palani, Chithra D; Arce, Roger M; Waller, Jennifer L; Genco, Caroline A; Slocum, Connie; Manning, Matthew; Schoenlein, Patricia V; Cutler, Christopher W

    2015-02-01

    Signaling via pattern recognition receptors (PRRs) expressed on professional antigen presenting cells, such as dendritic cells (DCs), is crucial to the fate of engulfed microbes. Among the many PRRs expressed by DCs are Toll-like receptors (TLRs) and C-type lectins such as DC-SIGN. DC-SIGN is targeted by several major human pathogens for immune-evasion, although its role in intracellular routing of pathogens to autophagosomes is poorly understood. Here we examined the role of DC-SIGN and TLRs in evasion of autophagy and survival of Porphyromonas gingivalis in human monocyte-derived DCs (MoDCs). We employed a panel of P. gingivalis isogenic fimbriae deficient strains with defined defects in Mfa-1 fimbriae, a DC-SIGN ligand, and FimA fimbriae, a TLR2 agonist. Our results show that DC-SIGN dependent uptake of Mfa1+P. gingivalis strains by MoDCs resulted in lower intracellular killing and higher intracellular content of P. gingivalis. Moreover, Mfa1+P. gingivalis was mostly contained within single membrane vesicles, where it survived intracellularly. Survival was decreased by activation of TLR2 and/or autophagy. Mfa1+P. gingivalis strain did not induce significant levels of Rab5, LC3-II, and LAMP1. In contrast, P. gingivalis uptake through a DC-SIGN independent manner was associated with early endosomal routing through Rab5, increased LC3-II and LAMP-1, as well as the formation of double membrane intracellular phagophores, a characteristic feature of autophagy. These results suggest that selective engagement of DC-SIGN by Mfa-1+P. gingivalis promotes evasion of antibacterial autophagy and lysosome fusion, resulting in intracellular persistence in myeloid DCs; however TLR2 activation can overcome autophagy evasion and pathogen persistence in DCs.

  16. Intracellular Calcium Plays a Critical Role in the Microcystin-LR-Elicited Neurotoxicity Through PLC/IP3 Pathway.

    Science.gov (United States)

    Cai, Fei; Liu, Jue; Li, Cairong; Wang, Jianghua

    2015-01-01

    Neurotoxicity of microcystin-leucine-arginine (MCLR) has been widely reported. However, the mechanism is not fully understood. Using primary hippocampal neurons, we tested the hypothesis that MCLR-triggered activation in intracellular free calcium concentration ([Ca(2+)](i)) induces the death of neurons. Microcystin-leucine-arginine inhibited cell viability at a range of 0.1 to 30 μmol/L and caused a dose-dependent increase in [Ca(2+)](i). This increase in [Ca(2+)](i) was observed in Ca(2+)-free media and blocked by an endoplasmic reticulum Ca(2+) pump inhibitor, suggesting intracellular Ca(2+) release. Moreover, pretreatment of hippocampal neurons with intracellular Ca(2+) chelator (O,O'-bis (2-aminophenyl) ethyleneglycol-N,N,N',N'-tetraacetic acid, tetraacetoxy-methyl ester) and inositol 1,4,5-trisphosphate receptor antagonist (2-aminoethoxydiphenyl borate) could block both the Ca(2+) mobilization and the neuronal death following MCLR exposure. In contrast, the ryanodine receptor inhibitor (dantrolene) did not ameliorate the effect of MCLR. In conclusion, MCLR disrupts [Ca(2+)](i) homeostasis in neurons by releasing Ca(2+) from intracellular stores, and this increase in [Ca(2+)](i) may be a key determinant in the mechanism underlying MCLR-induced neurotoxicity. © The Author(s) 2015.

  17. Intracellular pH in sperm physiology.

    Science.gov (United States)

    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. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Drosophila VAMP7 regulates Wingless intracellular trafficking.

    Science.gov (United States)

    Gao, Han; He, Fang; Lin, Xinhua; Wu, Yihui

    2017-01-01

    Drosophila Wingless (Wg) is a morphogen that determines cell fate during development. Previous studies have shown that endocytic pathways regulate Wg trafficking and signaling. Here, we showed that loss of vamp7, a gene required for vesicle fusion, dramatically increased Wg levels and decreased Wg signaling. Interestingly, we found that levels of Dally-like (Dlp), a glypican that can interact with Wg to suppress Wg signaling at the dorsoventral boundary of the Drosophila wing, were also increased in vamp7 mutant cells. Moreover, Wg puncta in Rab4-dependent recycling endosomes were Dlp positive. We hypothesize that VAMP7 is required for Wg intracellular trafficking and the accumulation of Wg in Rab4-dependent recycling endosomes might affect Wg signaling.

  19. Intracellular Na⁺ and cardiac metabolism.

    Science.gov (United States)

    Bay, Johannes; Kohlhaas, Michael; Maack, Christoph

    2013-08-01

    In heart failure, alterations of excitation-contraction underlie contractile dysfunction. One important defect is an elevation of the intracellular Na(+) concentration in cardiac myocytes ([Na(+)]i), which has an important impact on cytosolic and mitochondrial Ca(2+) homeostasis. While elevated [Na(+)]i is thought to compensate for decreased Ca(2+) load of the sarcoplasmic reticulum (SR), it yet negatively affects energy supply-and-demand matching and can even induce mitochondrial oxidative stress. Here, we review the mechanisms underlying these pathophysiological changes. The chain of events may constitute a vicious cycle of ion dysregulation, oxidative stress and energetic deficit, resembling characteristic cellular deficits that are considered key hallmarks of the failing heart. This article is part of a Special Issue entitled "Na(+) Regulation in Cardiac Myocytes". Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. An intracellular anion channel critical for pigmentation.

    Science.gov (United States)

    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.

  1. Nanobodies: Chemical Functionalization Strategies and Intracellular Applications

    Science.gov (United States)

    Schumacher, Dominik; Helma, Jonas; Schneider, Anselm F. L.; Leonhardt, Heinrich

    2018-01-01

    Abstract Nanobodies can be seen as next‐generation tools for the recognition and modulation of antigens that are inaccessible to conventional antibodies. Due to their compact structure and high stability, nanobodies see frequent usage in basic research, and their chemical functionalization opens the way towards promising diagnostic and therapeutic applications. In this Review, central aspects of nanobody functionalization are presented, together with selected applications. While early conjugation strategies relied on the random modification of natural amino acids, more recent studies have focused on the site‐specific attachment of functional moieties. Such techniques include chemoenzymatic approaches, expressed protein ligation, and amber suppression in combination with bioorthogonal modification strategies. Recent applications range from sophisticated imaging and mass spectrometry to the delivery of nanobodies into living cells for the visualization and manipulation of intracellular antigens. PMID:28913971

  2. Decreased intracellular [Ca2+ ] coincides with reduced expression of Dhprα1s, RyR1, and diaphragmatic dysfunction in a rat model of sepsis.

    Science.gov (United States)

    Wang, Meng-Meng; Hao, Li-Ying; Guo, Feng; Zhong, Bin; Zhong, Xiao-Mei; Yuan, Jing; Hao, Yi-Fei; Zhao, Shuang; Sun, Xue-Fei; Lei, Ming; Jiao, Guang-Yu

    2017-12-01

    Sepsis can cause decreased diaphragmatic contractility. Intracellular calcium as a second messenger is central to diaphragmatic contractility. However, changes in intracellular calcium concentration ([Ca 2+ ]) and the distribution and co-localization of relevant calcium channels [dihydropyridine receptors, (DHPRα1s) and ryanodine receptors (RyR1)] remain unclear during sepsis. In this study we investigated the effect of changed intracellular [Ca 2+ ] and expression and distribution of DHPRα1s and RyR1 on diaphragm function during sepsis. We measured diaphragm contractility and isolated diaphragm muscle cells in a rat model of sepsis. The distribution and co-localization of DHPRα1s and RyR1 were determined using immunohistochemistry and immunofluorescence, whereas intracellular [Ca 2+ ] was measured by confocal microscopy and fluorescence spectrophotometry. Septic rat diaphragm contractility, expression of DHPRα1s and RyR1, and intracellular [Ca 2+ ] were significantly decreased in the rat sepsis model compared with controls. Decreased intracellular [Ca 2+ ] coincides with diaphragmatic contractility and decreased expression of DHPRα1s and RyR1 in sepsis. Muscle Nerve 56: 1128-1136, 2017. © 2017 Wiley Periodicals, Inc.

  3. The Role of Autophagy in Intracellular Pathogen Nutrient Acquisition

    Directory of Open Access Journals (Sweden)

    Shaun eSteele

    2015-06-01

    Full Text Available Following entry into host cells intracellular pathogens must simultaneously evade innate host defense mechanisms and acquire energy and anabolic substrates from the nutrient-limited intracellular environment. Most of the potential intracellular nutrient sources are stored within complex macromolecules that are not immediately accessible by intracellular pathogens. To obtain nutrients for proliferation, intracellular pathogens must compete with the host cell for newly-imported simple nutrients or degrade host nutrient storage structures into their constituent components (fatty acids, carbohydrates and amino acids. It is becoming increasingly evident that intracellular pathogens have evolved a wide variety of strategies to accomplish this task. One recurrent microbial strategy is to exploit host degradative processes that break down host macromolecules into simple nutrients that the microbe can use. Herein we focus on how a subset of bacterial, viral and eukaryotic pathogens leverage the host process of autophagy to acquire nutrients that support their growth within infected cells

  4. A novel intracellular pool of LFA-1 is critical for asymmetric CD8+ T cell activation and differentiation.

    Science.gov (United States)

    Capece, Tara; Walling, Brandon L; Lim, Kihong; Kim, Kyun-Do; Bae, Seyeon; Chung, Hung-Li; Topham, David J; Kim, Minsoo

    2017-11-06

    The integrin lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18) is a key T cell adhesion receptor that mediates stable interactions with antigen-presenting cell (APC), as well as chemokine-mediated migration. Using our newly generated CD11a-mYFP knock-in mice, we discovered that naive CD8 + T cells reserve a significant intracellular pool of LFA-1 in the uropod during migration. Intracellular LFA-1 quickly translocated to the cell surface with antigenic stimulus. Importantly, the redistribution of intracellular LFA-1 at the contact with APC was maintained during cell division and led to an unequal inheritance of LFA-1 in divided T cells. The daughter CD8 + T cells with disparate LFA-1 expression showed different patterns of migration on ICAM-1, APC interactions, and tissue retention, as well as altered effector functions. In addition, we identified Rab27 as an important regulator of the intracellular LFA-1 translocation. Collectively, our data demonstrate that an intracellular pool of LFA-1 in naive CD8 + T cells plays a key role in T cell activation and differentiation. © 2017 Capece et al.

  5. The decompensated detrusor I: the effects of bladder outlet obstruction on the use of intracellular calcium stores.

    Science.gov (United States)

    Rohrmann, D; Levin, R M; Duckett, J W; Zderic, S A

    1996-08-01

    As in other smooth muscle groups, extracellular calcium influx as well as the release of calcium from intracellular storage sites or sarcoplasmic reticulum occur in response to receptor stimulation. The relative participation of extracellular influx versus intracellular release has recently been shown to be influenced by developmental stage and obstruction. Partial bladder outlet obstruction results in marked hypertrophy of the bladder and produces alterations in contractile function. To understand better how this contractile dysfunction after outlet obstruction is influenced by intracellular calcium handling we tested the effects of 2 drugs with known effects on the sarcoplasmic reticulum. We evaluated ryanodine, which blocks the release of calcium from the sarcoplasmic reticulum, and thapsigargin, which blocks the ability of the sarcoplasmic reticulum to pump cytosolic calcium back into the storage sites. Rabbit bladders were obstructed for different periods, after which detrusor muscle strips were harvested and contractile performance was evaluated in the absence and presence of ryanodine and thapsigargin. In the early phases of outlet obstruction the release of intracellular calcium increased significantly. With prolonged obstruction and detrusor decompensation the intracellular storage sites lost the ability to contribute to the generation of contractile force. Alterations in the calcium handling ability of the smooth muscle cell appear to have an important role in the process of decompensation of bladder function in infravesical obstruction.

  6. Somatostatin receptors

    DEFF Research Database (Denmark)

    Møller, Lars Neisig; Stidsen, Carsten Enggaard; Hartmann, Bolette

    2003-01-01

    therefore been acknowledged to be a third endogenous ligand at SRIF receptors. This review goes through mechanisms of signal transduction, pharmacology, and anatomical distribution of SRIF receptors. Structurally, SRIF receptors belong to the superfamily of G protein-coupled (GPC) receptors, sharing......- and heterodimerisation, let alone oligomerisation. Theoretically, this phenomenon adds a novel series of functional megareceptors/super-receptors, with varied pharmacological profiles, to the catalogue of monomeric receptor subtypes isolated and cloned in the past. SRIF analogues include both peptides and non......-peptides, receptor agonists and antagonists. Relatively long half lives, as compared to those of the endogenous ligands, have been paramount from the outset. Motivated by theoretical puzzles or the shortcomings of present-day diagnostics and therapy, investigators have also aimed to produce subtype...

  7. Intracellular Uropathogenic E. coli Exploits Host Rab35 for Iron Acquisition and Survival within Urinary Bladder Cells.

    Science.gov (United States)

    Dikshit, Neha; Bist, Pradeep; Fenlon, Shannon N; Pulloor, Niyas Kudukkil; Chua, Christelle En Lin; Scidmore, Marci A; Carlyon, Jason A; Tang, Bor Luen; Chen, Swaine L; Sukumaran, Bindu

    2015-08-01

    Recurrent urinary tract infections (UTIs) caused by uropathogenic E. coli (UPEC) are common and morbid infections with limited therapeutic options. Previous studies have demonstrated that persistent intracellular infection of bladder epithelial cells (BEC) by UPEC contributes to recurrent UTI in mouse models of infection. However, the mechanisms employed by UPEC to survive within BEC are incompletely understood. In this study we aimed to understand the role of host vesicular trafficking proteins in the intracellular survival of UPEC. Using a cell culture model of intracellular UPEC infection, we found that the small GTPase Rab35 facilitates UPEC survival in UPEC-containing vacuoles (UCV) within BEC. Rab35 plays a role in endosomal recycling of transferrin receptor (TfR), the key protein responsible for transferrin-mediated cellular iron uptake. UPEC enhance the expression of both Rab35 and TfR and recruit these proteins to the UCV, thereby supplying UPEC with the essential nutrient iron. Accordingly, Rab35 or TfR depleted cells showed significantly lower intracellular iron levels and reduced ability to support UPEC survival. In the absence of Rab35, UPEC are preferentially trafficked to degradative lysosomes and killed. Furthermore, in an in vivo murine model of persistent intracellular infection, Rab35 also colocalizes with intracellular UPEC. We propose a model in which UPEC subverts two different vesicular trafficking pathways (endosomal recycling and degradative lysosomal fusion) by modulating Rab35, thereby simultaneously enhancing iron acquisition and avoiding lysosomal degradation of the UCV within bladder epithelial cells. Our findings reveal a novel survival mechanism of intracellular UPEC and suggest a potential avenue for therapeutic intervention against recurrent UTI.

  8. Weakened Intracellular Zn2+-Buffering in the Aged Dentate Gyrus and Its Involvement in Erasure of Maintained LTP.

    Science.gov (United States)

    Takeda, Atsushi; Tamano, Haruna; Murakami, Taku; Nakada, Hiroyuki; Minamino, Tatsuya; Koike, Yuta

    2018-05-01

    Memory is lost by the increased influx of extracellular Zn 2+ into neurons. It is possible that intracellular Zn 2+ dynamics is modified even at non-zincergic medial perforant pathway-dentate granule cell synapses along with aging and that vulnerability to the modification is linked to age-related cognitive decline. To examine these possibilities, vulnerability of long-term potentiation (LTP) maintenance, which underlies memory retention, to modification of synaptic Zn 2+ dynamics was compared between young and aged rats. The influx of extracellular Zn 2+ into dentate granule cells was increased in aged rats after injection of high K + into the dentate gyrus, but not in young rats. This increase impaired maintained LTP in aged rats. However, the impairment was rescued by co-injection of CaEDTA, an extracellular Zn 2+ chelator, or CNQX, an AMPA receptor antagonist, which suppressed the Zn 2+ influx. Maintained LTP was also impaired in aged rats after injection of ZnAF-2DA into the dentate gyrus that chelates intracellular Zn 2+ , but not in young rats. Interestingly, the capacity of chelating intracellular Zn 2+ with intracellular ZnAF-2 was almost lost in the aged dentate gyrus 2 h after injection of ZnAF-2DA into the dentate gyrus, suggesting that intracellular Zn 2+ -buffering is weakened in the aged dentate gyrus, compared to the young dentate gyrus. In the dentate gyrus of aged rats, maintained LTP is more vulnerable to modification of intracellular Zn 2+ dynamics than in young rats, probably due to weakened intracellular Zn 2+ -buffering.

  9. Strategies of Intracellular Pathogens for Obtaining Iron from the Environment

    Directory of Open Access Journals (Sweden)

    Nidia Leon-Sicairos

    2015-01-01

    Full Text Available Most microorganisms are destroyed by the host tissues through processes that usually involve phagocytosis and lysosomal disruption. However, some organisms, called intracellular pathogens, are capable of avoiding destruction by growing inside macrophages or other cells. During infection with intracellular pathogenic microorganisms, the element iron is required by both the host cell and the pathogen that inhabits the host cell. This minireview focuses on how intracellular pathogens use multiple strategies to obtain nutritional iron from the intracellular environment in order to use this element for replication. Additionally, the implications of these mechanisms for iron acquisition in the pathogen-host relationship are discussed.

  10. Ryanodine receptor channelopathies

    Science.gov (United States)

    Betzenhauser, Matthew J.

    2010-01-01

    Ryanodine receptors (RyR) are intracellular Ca2+-permeable channels that provide the sarcoplasmic reticulum Ca2+ release required for skeletal and cardiac muscle contractions. RyR1 underlies skeletal muscle contraction, and RyR2 fulfills this role in cardiac muscle. Over the past 20 years, numerous mutations in both RyR isoforms have been identified and linked to skeletal and cardiac diseases. Malignant hyperthermia, central core disease, and catecholaminergic polymorphic ventricular tachycardia have been genetically linked to mutations in either RyR1 or RyR2. Thus, RyR channelopathies are both of interest because they cause significant human diseases and provide model systems that can be studied to elucidate important structure–function relationships of these ion channels. PMID:20179962

  11. Activation of a Neospora caninum EGFR-Like Kinase Facilitates Intracellular Parasite Proliferation

    Directory of Open Access Journals (Sweden)

    Xiaoxia Jin

    2017-10-01

    Full Text Available The Apicomplexan parasite Neospora caninum, an obligate intracellular protozoan, causes serious diseases in a number of mammalian species, especially in cattle. Infection with N. caninum is associated with abortions in both dairy and beef cattle worldwide which have a major economic impact on the cattle industry. However, the mechanism by which N. caninum proliferates within host cells is poorly understood. Epidermal growth factor receptor (EGFR is a protein kinase ubiquitously expressed, present on cell surfaces in numerous species, which has been confirmed to be essential in signal transduction involved in cell growth, proliferation, survival, and many other intracellular processes. However, the presence of EGFR in N. caninum and its role in N. caninum proliferation remain unclear. In the present study, we identified a putative EGFR-like kinase in N. caninum, which could be activated in tachyzoites by infection or treatment with rNcMIC3 [containing four epidermal growth factor (EGF domains] or human EGF. Blockade of EGFR-like in tachyzoites by AG1478 significantly reduced parasite proliferation in host cells. Our data suggested that the activation of tachyzoite EGFR-like might facilitate the intracellular proliferation of N. caninum.

  12. 3-Methylcholanthrene inhibits lymphocyte proliferation and increases intracellular calcium levels in common carp (Cyprinus carpio L)

    International Nuclear Information System (INIS)

    Reynaud, S.; Duchiron, C.; Deschaux, P.

    2003-01-01

    Polycyclic aromatic hydrocarbons (PAHs) are an important class of environmental pollutants that are known to be carcinogenic and immunotoxic. Many authors have focused on macrophage activities in fish exposed to PAHs. However, fewer studies have reported decrease in specific immunity in such fish. We investigated the intracellular mechanisms by which the 3-methylcholanthrene (3-MC) decreased lymphocyte proliferation in carp. T- and B-lymphocyte proliferation induced by Concanavalin A (Con A) and lipopolysaccharide (LPS) were inhibited by 3-MC (0.5-50 μM). 3-MC also produced a rapid and a sustained increase in intracellular calcium concentration ([Ca 2+ ] i ) (2 h minimum). However, the cytochrome P450 1A and Ah receptor inhibitor, α-naphtoflavone (a-NF), also inhibited lymphocyte proliferation and did not reverse the effects of 3-MC. Moreover, since a-NF and 3-MC increased [Ca 2+ ] i and inhibited lymphocyte proliferation it was possible that calcium release played a role in 3-MC-inhibited lymphocyte proliferation. The rise in [Ca 2+ ] i induced by 3-MC was potentiated by the inhibitor of the endoplasmic reticulum calcium ATPases, thapsigargin. Treating cells with 3-MC decreased calcium mobilization caused by thapsigargin. These results suggest that 3-MC acts on the endoplasmic reticulum, perhaps directly on calcium ATPases, to increase intracellular calcium levels in carp leucocytes

  13. Intracellular Ca2+ and the phospholipid PIP2 regulate the taste transduction ion channel TRPM5.

    Science.gov (United States)

    Liu, Dan; Liman, Emily R

    2003-12-09

    The transduction of taste is a fundamental process that allows animals to discriminate nutritious from noxious substances. Three taste modalities, bitter, sweet, and amino acid, are mediated by G protein-coupled receptors that signal through a common transduction cascade: activation of phospholipase C beta2, leading to a breakdown of phosphatidylinositol-4,5-bisphosphate (PIP2) into diacylglycerol and inositol 1,4,5-trisphosphate, which causes release of Ca2+ from intracellular stores. The ion channel, TRPM5, is an essential component of this cascade; however, the mechanism by which it is activated is not known. Here we show that heterologously expressed TRPM5 forms a cation channel that is directly activated by micromolar concentrations of intracellular Ca2+ (K1/2 = 21 microM). Sustained exposure to Ca2+ desensitizes TRPM5 channels, but PIP2 reverses desensitization, partially restoring channel activity. Whole-cell TRPM5 currents can be activated by intracellular Ca2+ and show strong outward rectification because of voltage-sensitive gating of the channels. TRPM5 channels are nonselective among monovalent cations and not detectably permeable to divalent cations. We propose that the regulation of TRPM5 by Ca2+ mediates sensory activation in the taste system.

  14. Mutations in G protein-coupled receptors that impact receptor trafficking and reproductive function.

    Science.gov (United States)

    Ulloa-Aguirre, Alfredo; Zariñán, Teresa; Dias, James A; Conn, P Michael

    2014-01-25

    G protein coupled receptors (GPCRs) are a large superfamily of integral cell surface plasma membrane proteins that play key roles in transducing extracellular signals, including sensory stimuli, hormones, neurotransmitters, or paracrine factors into the intracellular environment through the activation of one or more heterotrimeric G proteins. Structural alterations provoked by mutations or variations in the genes coding for GPCRs may lead to misfolding, altered plasma membrane expression of the receptor protein and frequently to disease. A number of GPCRs regulate reproductive function at different levels; these receptors include the gonadotropin-releasing hormone receptor (GnRHR) and the gonadotropin receptors (follicle-stimulating hormone receptor and luteinizing hormone receptor), which regulate the function of the pituitary-gonadal axis. Loss-of-function mutations in these receptors may lead to hypogonadotropic or hypergonadotropic hypogonadism, which encompass a broad spectrum of clinical phenotypes. In this review we describe mutations that provoke misfolding and failure of these receptors to traffick from the endoplasmic reticulum to the plasma membrane. We also discuss some aspects related to the therapeutic potential of some target-specific drugs that selectively bind to and rescue function of misfolded mutant GnRHR and gonadotropin receptors, and that represent potentially valuable strategies to treat diseases caused by inactivating mutations of these receptors. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  15. Real-Time G-Protein-Coupled Receptor Imaging to Understand and Quantify Receptor Dynamics

    Directory of Open Access Journals (Sweden)

    María S. Aymerich

    2011-01-01

    Full Text Available Understanding the trafficking of G-protein-coupled receptors (GPCRs and their regulation by agonists and antagonists is fundamental to develop more effective drugs. Optical methods using fluorescent-tagged receptors and spinning disk confocal microscopy are useful tools to investigate membrane receptor dynamics in living cells. The aim of this study was to develop a method to characterize receptor dynamics using this system which offers the advantage of very fast image acquisition with minimal cell perturbation. However, in short-term assays photobleaching was still a problem. Thus, we developed a procedure to perform a photobleaching-corrected image analysis. A study of short-term dynamics of the long isoform of the dopamine type 2 receptor revealed an agonist-induced increase in the mobile fraction of receptors with a rate of movement of 0.08 μm/s For long-term assays, the ratio between the relative fluorescence intensity at the cell surface versus that in the intracellular compartment indicated that receptor internalization only occurred in cells co-expressing G protein-coupled receptor kinase 2. These results indicate that the lateral movement of receptors and receptor internalization are not directly coupled. Thus, we believe that live imaging of GPCRs using spinning disk confocal image analysis constitutes a powerful tool to study of receptor dynamics.

  16. Intracellular CXCR4+ cell targeting with T22-empowered protein-only nanoparticles

    Science.gov (United States)

    Unzueta, Ugutz; Céspedes, María Virtudes; Ferrer-Miralles, Neus; Casanova, Isolda; Cedano, Juan; Corchero, José Luis; Domingo-Espín, Joan; Villaverde, Antonio; Mangues, Ramón; Vázquez, Esther

    2012-01-01

    Background Cell-targeting peptides or proteins are appealing tools in nanomedicine and innovative medicines because they increase the local drug concentration and reduce potential side effects. CXC chemokine receptor 4 (CXCR4) is a cell surface marker associated with several severe human pathologies, including colorectal cancer, for which intracellular targeting agents are currently missing. Results Four different peptides that bind CXCR4 were tested for their ability to internalize a green fluorescent protein-based reporter nanoparticle into CXCR4+ cells. Among them, only the 18 mer peptide T22, an engineered segment derivative of polyphemusin II from the horseshoe crab, efficiently penetrated target cells via a rapid, receptor-specific endosomal route. This resulted in accumulation of the reporter nanoparticle in a fully fluorescent and stable form in the perinuclear region of the target cells, without toxicity either in cell culture or in an in vivo model of metastatic colorectal cancer. Conclusion Given the urgent demand for targeting agents in the research, diagnosis, and treatment of CXCR4-linked diseases, including colorectal cancer and human immunodeficiency virus infection, T22 appears to be a promising tag for the intracellular delivery of protein drugs, nanoparticles, and imaging agents. PMID:22923991

  17. Nongenomic actions of estrogens and xenoestrogens by binding at a plasma membrane receptor unrelated to estrogen receptor α and estrogen receptor β

    Science.gov (United States)

    Nadal, Angel; Ropero, Ana B.; Laribi, Ouahiba; Maillet, Marjorie; Fuentes, Esther; Soria, Bernat

    2000-01-01

    The molecular mechanism used by environmental chemicals to exert their hormone-like actions is still only partially resolved. Although it generally is accepted that xenoestrogens act at the genomic level by binding to intracellular estrogen receptors, we have shown here that they trigger nongenomic effects in pancreatic β cells. Both xenoestrogens and the circulating hormone, 17β-estradiol, bind with high affinity to a common membrane binding site unrelated to the intracellular estrogen receptors ERα and ERβ. This binding site is shared by dopamine, epinephrine, and norepinephrine and has the pharmacological profile of the γadrenergic receptor. This study provides an outline of the membrane receptor involved in rapid xenoestrogen actions. PMID:11027358

  18. Intracellular Shuttle: The Lactate Aerobic Metabolism

    Directory of Open Access Journals (Sweden)

    Rogério Santos de Oliveira Cruz

    2012-01-01

    Full Text Available Lactate is a highly dynamic metabolite that can be used as a fuel by several cells of the human body, particularly during physical exercise. Traditionally, it has been believed that the first step of lactate oxidation occurs in cytosol; however, this idea was recently challenged. A new hypothesis has been presented based on the fact that lactate-to-pyruvate conversion cannot occur in cytosol, because the LDH enzyme characteristics and cytosolic environment do not allow the reaction in this way. Instead, the Intracellular Lactate Shuttle hypothesis states that lactate first enters in mitochondria and only then is metabolized. In several tissues of the human body this idea is well accepted but is quite resistant in skeletal muscle. In this paper, we will present not only the studies which are protagonists in this discussion, but the potential mechanism by which this oxidation occurs and also a link between lactate and mitochondrial proliferation. This new perspective brings some implications and comes to change our understanding of the interaction between the energy systems, because the product of one serves as a substrate for the other.

  19. Intracellular sphingosine releases calcium from lysosomes

    Science.gov (United States)

    Höglinger, Doris; Haberkant, Per; Aguilera-Romero, Auxiliadora; Riezman, Howard; Porter, Forbes D; Platt, Frances M; Galione, Antony; Schultz, Carsten

    2015-01-01

    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. DOI: http://dx.doi.org/10.7554/eLife.10616.001 PMID:26613410

  20. An intracellular anion channel critical for pigmentation

    Science.gov (United States)

    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

  1. LIPID SYNTHESIS, INTRACELLULAR TRANSPORT, AND SECRETION

    Science.gov (United States)

    Stein, Olga; Stein, Yechezkiel

    1967-01-01

    In the mammary glands of lactating albino mice injected intravenously with 9, 10-oleic acid-3H or 9, 10-palmitic acid-3H, it has been shown that the labeled fatty acids are incorporated into mammary gland glycerides. The labeled lipid in the mammary gland 1 min after injection was in esterified form (> 95%), and the radioautographic reaction was seen over the rough endoplasmic reticulum and over lipid droplets, both intracellular and intraluminal. At 10–60 min after injection, the silver grains were concentrated predominantly over lipid droplets. There was no concentration of radioactivity over the granules in the Golgi apparatus, at any time interval studied. These findings were interpreted to indicate that after esterification of the fatty acid into glycerides in the rough endoplasmic reticulum an in situ aggregation of lipid occurs, with acquisition of droplet form. The release of the lipid into the lumen proceeds directly and not through the Golgi apparatus, in contradistinction to the mode of secretion of casein in the mammary gland or of lipoprotein in the liver. The presence of strands of endoplasmic reticulum attached to intraluminal lipid droplets provides a structural counterpart to the milk microsomes described in ruminant milk. PMID:6033535

  2. On the Computing Potential of Intracellular Vesicles.

    Science.gov (United States)

    Mayne, Richard; Adamatzky, Andrew

    2015-01-01

    Collision-based computing (CBC) is a form of unconventional computing in which travelling localisations represent data and conditional routing of signals determines the output state; collisions between localisations represent logical operations. We investigated patterns of Ca2+-containing vesicle distribution within a live organism, slime mould Physarum polycephalum, with confocal microscopy and observed them colliding regularly. Vesicles travel down cytoskeletal 'circuitry' and their collisions may result in reflection, fusion or annihilation. We demonstrate through experimental observations that naturally-occurring vesicle dynamics may be characterised as a computationally-universal set of Boolean logical operations and present a 'vesicle modification' of the archetypal CBC 'billiard ball model' of computation. We proceed to discuss the viability of intracellular vesicles as an unconventional computing substrate in which we delineate practical considerations for reliable vesicle 'programming' in both in vivo and in vitro vesicle computing architectures and present optimised designs for both single logical gates and combinatorial logic circuits based on cytoskeletal network conformations. The results presented here demonstrate the first characterisation of intracelluar phenomena as collision-based computing and hence the viability of biological substrates for computing.

  3. Modeling HIV-1 intracellular replication: two simulation approaches

    NARCIS (Netherlands)

    Zarrabi, N.; Mancini, E.; Tay, J.; Shahand, S.; Sloot, P.M.A.

    2010-01-01

    Many mathematical and computational models have been developed to investigate the complexity of HIV dynamics, immune response and drug therapy. However, there are not many models which consider the dynamics of virus intracellular replication at a single level. We propose a model of HIV intracellular

  4. Pico gauges for minimally invasive intracellular hydrostatic pressure measurements

    DEFF Research Database (Denmark)

    Knoblauch, Jan; Mullendore, Daniel L.; Jensen, Kaare Hartvig

    2014-01-01

    Intracellular pressure has a multitude of functions in cells surrounded by a cell wall or similar matrix in all kingdoms of life. The functions include cell growth, nastic movements, and penetration of tissue by parasites. The precise measurement of intracellular pressure in the majority of cells...

  5. Development of bacterial cell-based system for intracellular ...

    African Journals Online (AJOL)

    Development of bacterial cell-based system for intracellular antioxidant activity screening assay using green fluorescence protein (GFP) reporter. ... Both strains demonstrated that quercetin and α- tocopherol exhibited the most potent and significant antioxidant activity with more than 60% reduction of intracellular superoxide ...

  6. Determination of Six Transmembrane Protein of Prostate 2 Gene Expression and Intracellular Localization in Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Bora İrer

    2017-12-01

    Full Text Available Objective: In this study, we aimed to determine the relationship between the RNA and protein expression profile of six transmembrane protein of prostate 2 (STAMP2 gene and androgen and the intracellular localization of STAMP2. Materials and Methods: RNA and protein were obtained from androgen treated lymph node carcinoma of the prostate (LNCaP cells, untreated LNCaP cells, DU145 cells with no androgen receptor, and STAMP2 transfected COS-7 cells. The expression profile of STAMP2 gene and the effect of androgenes on the expression was shown in RNA and protein levels by using Northern and Western blotting methods. In addition, intracellular localization of the naturally synthesized STAMP2 protein and the transfected STAMP2 protein in COS-7 cells after androgen administration in both LNCaP cells was determined by immunofluorescence microscopy. Results: We found that the RNA and protein expression of STAMP2 gene in LNCaP cells are regulated by androgenes, the power of expression is increased with the duration of androgen treatment and there is no STAMP2 expression in DU145 cells which has no androgen receptor. As a result of the immunofluorescence microscopy study we observed that STAMP2 protein was localized at golgi complex and cell membrane. Conclusion: In conclusion, we have demonstrated that STAMP2 may play an important role in the pathogenesis of the prostate cancer and in the androgen-dependent androgen-independent staging of prostate cancer. In addition, STAMP2 protein, which is localized in the intracellular golgi complex and cell membrane, may be a new target molecule for prostate cancer diagnosis and treatment.

  7. TRPM2 contributes to LPC-induced intracellular Ca2+influx and microglial activation.

    Science.gov (United States)

    Jeong, Heejin; Kim, Yong Ho; Lee, Yunsin; Jung, Sung Jun; Oh, Seog Bae

    2017-04-01

    Microglia are the resident immune cells which become activated in some pathological conditions in central nervous system (CNS). Lysophosphatidylcholine (LPC), an endogenous inflammatory phospholipid, is implicated in immunomodulatory function of glial cells in the CNS. Although several studies uncovered that LPC induces intracellular Ca 2+ influx and morphologic change in microglia, there is still no direct evidence showing change of phosphorylation of mitogen-activated protein kinase (MAPK) p38 (p-p38), a widely used microglia activation marker, by LPC. Furthermore, the cellular mechanism of LPC-induced microglia activation remains unknown. In this study, we found that LPC induced intracellular Ca 2+ increase in primary cultured microglia, which was blocked in the presence of Gd 3+ , non-selective transient receptor potential (TRP) channel blocker. RT-PCR and whole cell patch clamp recordings revealed molecular and functional expression of TRP melastatin 2 (TRPM2) in microglia. Using western blotting, we also observed that LPC increased phosphorylation of p38 MAPK, and the increase of p-p38 expression is also reversed in TRPM2-knockout (KO) microglia. Moreover, LPC induced membrane trafficking of TRPM2 and intrathecal injection of LPC increased Iba-1 immunoreactivity in the spinal cord, which were significantly reduced in KO mice. In addition, LPC-induced intracellular Ca 2+ increase and inward currents were abolished in TRPM2-KO microglia. Taken together, our results suggest that LPC induces intracellular Ca 2+ influx and increases phosphorylation of p38 MAPK via TRPM2, which in turn activates microglia. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Folate mediated self-assembled phytosterol-alginate nanoparticles for targeted intracellular anticancer drug delivery.

    Science.gov (United States)

    Wang, Jianting; Wang, Ming; Zheng, Mingming; Guo, Qiong; Wang, Yafan; Wang, Heqing; Xie, Xiangrong; Huang, Fenghong; Gong, Renmin

    2015-05-01

    Self-assembled core/shell nanoparticles (NPs) were synthesized from water-soluble alginate substituted by hydrophobic phytosterols. Folate, a cancer-cell-specific ligand, was conjugated to the phytosterol-alginate (PA) NPs for targeting folate-receptor-overexpressing cancer cells. The physicochemical properties of folate-phytosterol-alginate (FPA) NPs were characterized by nuclear magnetic resonance, transmission electron microscopy, dynamic light scattering, electrophoretic light scattering, and fluorescence spectroscopy. Doxorubicin (DOX), an anticancer drug, was entrapped inside prepared NPs by dialysis method. The identification of prepared FPA NPs to folate-receptor-overexpressing cancer cells (KB cells) was confirmed by cytotoxicity and folate competition assays. Compared to the pure DOX and DOX/PA NPs, the DOX/FPA NPs had lower IC50 value to KB cells because of folate-receptor-mediated endocytosis process and the cytotoxicity of DOX/FPA NPs to KB cells could be competitively inhibited by free folate. The cellular uptake and internalization of pure DOX and DOX/FPA NPs was confirmed by confocal laser scanning microscopy image and the higher intracellular uptake of drug for DOX/FPA NPs over pure DOX was observed. The FPA NPs had the potential as a promising carrier to target drugs to cancer cells overexpressing folate receptors and avoid cytotoxicity to normal tissues. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Analysis of Intracellular Metabolites from Microorganisms: Quenching and Extraction Protocols.

    Science.gov (United States)

    Pinu, Farhana R; Villas-Boas, Silas G; Aggio, Raphael

    2017-10-23

    Sample preparation is one of the most important steps in metabolome analysis. The challenges of determining microbial metabolome have been well discussed within the research community and many improvements have already been achieved in last decade. The analysis of intracellular metabolites is particularly challenging. Environmental perturbations may considerably affect microbial metabolism, which results in intracellular metabolites being rapidly degraded or metabolized by enzymatic reactions. Therefore, quenching or the complete stop of cell metabolism is a pre-requisite for accurate intracellular metabolite analysis. After quenching, metabolites need to be extracted from the intracellular compartment. The choice of the most suitable metabolite extraction method/s is another crucial step. The literature indicates that specific classes of metabolites are better extracted by different extraction protocols. In this review, we discuss the technical aspects and advancements of quenching and extraction of intracellular metabolite analysis from microbial cells.

  10. Biochemical and ultrastructural studies suggest that the effects of thapsigargin on human platelets are mediated by changes in intracellular calcium but not by intracellular histamine

    DEFF Research Database (Denmark)

    Saxena, S P; McNicol, A; Becker, A B

    1992-01-01

    -fluoromethyl histidine (alpha-FMH) failed to inhibit Tg-induced aggregation. The intracellular histamine receptor antagonist, N,N-diethyl-2-[4-(phenylmethyl)phenoxy] ethanamine. HCl (DPPE), inhibited Tg-induced aggregation but with IC50 values dependent on the concentration of agonist used. The inhibitory effects...... of DPPE on Tg-induced aggregation were not reversed by the addition of histamine to saponin-permeabilized platelets suggesting non-histamine mediated effects of DPPE on Tg-induced aggregation. Tg stimulated an increase in the cytosolic free calcium concentration which was unaffected by DPPE indicating...... that the effects of DPPE are also not due to the inhibition of mobilization of cytosolic calcium. The ultrastructural studies suggest that the major Tg-induced changes (pseudopod formation and granule centralization) are consistent with a primary role for Tg to mobilize calcium; DPPE had very little effect...

  11. Regulation of Notch1 signaling by the APP intracellular domain facilitates degradation of the Notch1 intracellular domain and RBP-Jk.

    Science.gov (United States)

    Kim, Mi-Yeon; Mo, Jung-Soon; Ann, Eun-Jung; Yoon, Ji-Hye; Jung, Jane; Choi, Yun-Hee; Kim, Su-Man; Kim, Hwa-Young; Ahn, Ji-Seon; Kim, Hangun; Kim, Kwonseop; Hoe, Hyang-Sook; Park, Hee-Sae

    2011-06-01

    The Notch1 receptor is a crucial controller of cell fate decisions, and is also a key regulator of cell growth and differentiation in a variety of contexts. In this study, we have demonstrated that the APP intracellular domain (AICD) attenuates Notch1 signaling by accelerated degradation of the Notch1 intracellular domain (Notch1-IC) and RBP-Jk, through different degradation pathways. AICD suppresses Notch1 transcriptional activity by the dissociation of the Notch1-IC-RBP-Jk complex after processing by γ-secretase. Notch1-IC is capable of forming a trimeric complex with Fbw7 and AICD, and AICD enhances the protein degradation of Notch1-IC through an Fbw7-dependent proteasomal pathway. AICD downregulates the levels of RBP-Jk protein through the lysosomal pathway. AICD-mediated degradation is involved in the preferential degradation of non-phosphorylated RBP-Jk. Collectively, our results demonstrate that AICD functions as a negative regulator in Notch1 signaling through the promotion of Notch1-IC and RBP-Jk protein degradation.

  12. Harmonization of the intracellular cytokine staining assay.

    Science.gov (United States)

    Welters, Marij J P; Gouttefangeas, Cécile; Ramwadhdoebe, Tamara H; Letsch, Anne; Ottensmeier, Christian H; Britten, Cedrik M; van der Burg, Sjoerd H

    2012-07-01

    Active immunotherapy for cancer is an accepted treatment modality aiming to reinforce the T-cell response to cancer. T-cell reactivity is measured by various assays and used to guide the clinical development of immunotherapeutics. However, data obtained across different institutions may vary substantially making comparative conclusions difficult. The Cancer Immunotherapy Immunoguiding Program organizes proficiency panels to identify key parameters influencing the outcome of commonly used T-cell assays followed by harmonization. Our successes with IFNγ-ELISPOT and peptide HLA multimer analysis have led to the current study on intracellular cytokine staining (ICS). We report the results of three successive panels evaluating this assay. At the beginning, 3 out of 9 participants (33 %) were able to detect >6 out of 8 known virus-specific T-cell responses in peripheral blood of healthy individuals. This increased to 50 % of the laboratories in the second phase. The reported percentages of cytokine-producing T cells by the different laboratories were highly variable with coefficients of variation well over 60 %. Variability could partially be explained by protocol-related differences in background cytokine production leading to sub-optimal signal-to-noise ratios. The large number of protocol variables prohibited identification of prime guidelines to harmonize the assays. In addition, the gating strategy used to identify reactive T cells had a major impact on assay outcome. Subsequent harmonization of the gating strategy considerably reduced the variability within the group of participants. In conclusion, we propose that first basic guidelines should be applied for gating in ICS experiments before harmonizing assay protocol variables.

  13. Dimerization of nuclear receptors.

    Science.gov (United States)

    Germain, Pierre; Bourguet, William

    2013-01-01

    Multicellular organisms require specific intercellular communication to properly organize the complex body plan during embryogenesis and maintain its properties and functions during the entire life. While growth factors, neurotransmitters, and peptide hormones bind to membrane receptors, thereby inducing the activity of intracellular kinase cascades or the JAK-STAT signaling pathways, other small signaling compounds such as steroid hormones, certain vitamins, and metabolic intermediates enter, or are generated, within the target cells and bind to members of a large family of nuclear receptors (NRs). NRs are ligand-inducible transcription factors that control a plethora of biological phenomena, thus orchestrating complex events like development, organ homeostasis, immune function, and reproduction. NR-NR interactions are of major importance in these regulatory processes, as NRs regulate their target genes by binding to cognate DNA response elements essentially as homo- or heterodimers. A number of structural and functional studies have provided significant insights as to how combinatorial NRs rely on protein-protein contacts that discriminate geometric features of their DNA response elements, thereby allowing both binding site diversity and physiological specificity. Here, we will review our current understanding of NR-NR interactions and provide protocols for a number of experimental approaches that are useful for their study. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Sweet Taste Receptor Signaling Network: Possible Implication for Cognitive Functioning

    Directory of Open Access Journals (Sweden)

    Menizibeya O. Welcome

    2015-01-01

    Full Text Available Sweet taste receptors are transmembrane protein network specialized in the transmission of information from special “sweet” molecules into the intracellular domain. These receptors can sense the taste of a range of molecules and transmit the information downstream to several acceptors, modulate cell specific functions and metabolism, and mediate cell-to-cell coupling through paracrine mechanism. Recent reports indicate that sweet taste receptors are widely distributed in the body and serves specific function relative to their localization. Due to their pleiotropic signaling properties and multisubstrate ligand affinity, sweet taste receptors are able to cooperatively bind multiple substances and mediate signaling by other receptors. Based on increasing evidence about the role of these receptors in the initiation and control of absorption and metabolism, and the pivotal role of metabolic (glucose regulation in the central nervous system functioning, we propose a possible implication of sweet taste receptor signaling in modulating cognitive functioning.

  15. Functional conservation study of polarity protein Crumbs intracellular domain.

    Science.gov (United States)

    Shi, Qi-ping; Cao, Hao-wei; Xu, Rui; Zhang, Dan-dan; Huang, Juan

    2017-01-20

    The transmembrane protein Crumbs (Crb) plays key roles in the establishing and maintaining cell apical-basal polarity in epithelial cells by determining the apical plasma membrane identity. Although its intracellular domain contains only 37 amino acids, it is absolutely essential for its function. In Drosophila, mutations in this intracellular domain result in severe defects in epithelial polarity and abnormal embryonic development. The intracellular domain of Crb shows high homology across species from Drosophila to Mus musculus and Homo sapiens. However, the intracellular domains of the two Crb proteins in C. elegans are rather divergent from those of Drosophila and mammals, raising the question on whether the function of the intracellular domain of the Crb protein is conserved in C. elegans. Using genomic engineering approach, we replaced the intracellular domain of the Drosophila Crb with that of C. elegans Crb2 (CeCrb2), which has extremely low homology with those from the Crb proteins of Drosophila and mammals. Surprisingly, substituting the intracellular domain of Drosophila Crb with that of CeCrb2 did not cause any abnormalities in development of the Drosophila embryo, in terms of expression and localization of Crb and other polarity proteins and apical-basal polarity in embryonic epithelial cells. Our results support the notion that despite their extensive sequence variations, all functionally critical amino acid residues and motifs of the intercellular domain of Crb proteins are fully conserved between Drosophila and C. elegans.

  16. Elementary Steps in T Cell Receptor Triggering

    OpenAIRE

    Dushek, Omer

    2012-01-01

    The mechanism by which antigen binding to the T cell antigen receptor (TCR) generates intracellular signaling, a process termed TCR triggering, is incompletely understood. A large body of experimental evidence has implicated multiple biophysical/biochemical effects and multiple molecules in the process of TCR triggering, which likely reflect the uniquely demanding role of the TCR in recognizing diverse antigenic ligands. In this perspective, I propose that breaking down the process of TCR tri...

  17. Cerebrovascular endothelin receptor upregulation in cerebral ischemia

    DEFF Research Database (Denmark)

    Edvinsson, Lars

    2009-01-01

    either by an embolus or by local thrombosis. Several studies have shown an involvement of the endothelin system in ischemic stroke. This review aims to examine the alterations of vascular endothelin receptor expression in ischemic stroke. Furthermore, studies of the intracellular signalling pathways...... leading to the enhanced expression of vascular endothelin receptors show that both protein kinase C (PKC) and mitogen activating protein kinase (MAPK) play important roles. The results from this work provide new perspectives on the pathophysiology of ischemic stroke, and give a possible explanation...

  18. Identification of Target Genes of the Steroid Hormone Receptor Co-Activator AIB1 (Amplified in Breast Cancer 1)

    National Research Council Canada - National Science Library

    List, Heinz-Joachim

    2002-01-01

    Steroid hormones play pivotal roles in the control of breast cancer proliferation and differentiation, The biological effects of these hormones are mediated through intracellular receptor proteins, e.g...

  19. New perspective in the assessment of total intracellular magnesium

    Directory of Open Access Journals (Sweden)

    Azzurra Sargenti

    2014-01-01

    Full Text Available Magnesium (Mg is essential for biological processes, but its cellular homeostasis has not been thoroughly elucidated, mainly because of the inadequacy of the available techniques to map intracellular Mg distribution. Recently, particular interest has been raised by a new family of fluorescent probes, diaza-18-crown-hydroxyquinoline (DCHQ, that shows remarkably high affinity and specificity for Mg, thus permitting the detection of the total intracellular Mg. The data obtained by fluori- metric and cytofluorimetric assays performed with DCHQ5 are in good agreement with atomic absorption spectroscopy, confirming that DCHQ5 probe allows both qualitative and quantitative determination of total intracellular Mg.

  20. EVIDENCE FOR THE MACROPHAGE INDUCING GENE IN MYCOBACTERIUM INTRACELLULARE

    Science.gov (United States)

    Background: The Mycobacterium avium Complex (MAC) includes the species M. avium (MA), M. intracellulare (MI), and possibly others. Organisms belonging to the MAC are phylogenetically closely related, opportunistic pathogens. The macrophage inducing gene (mig) is the only well-des...

  1. Spatial Cell Biology : Dissecting and directing intracellular transport mechanisms

    NARCIS (Netherlands)

    Adrian, M.

    2017-01-01

    Cellular compartmentalization and intracellular transport mechanisms are important to establish and maintain the spatial organisation of proteins and organelles needed to ensure proper cellular functioning. Especially in polarized cells like neurons, the proper distribution of proteins into the

  2. Intracellular Renin Disrupts Chemical Communication between Heart Cells. Pathophysiological Implications.

    Science.gov (United States)

    De Mello, Walmor C

    2014-01-01

    HighlightsIntracellular renin disrupts chemical communication in the heartAngiotensinogen enhances the effect of reninIntracellular enalaprilat reduces significantly the effect of reninIntracellular renin increases the inward calcium currentHarmful versus beneficial effect during myocardial infarction The influence of intracellular renin on the process of chemical communication between cardiac cells was investigated in cell pairs isolated from the left ventricle of adult Wistar Kyoto rats. The enzyme together with Lucifer yellow CH was dialyzed into one cell of the pair using the whole cell clamp technique. The diffusion of the dye in the dialyzed and in non-dialyzed cell was followed by measuring the intensity of fluorescence in both cells as a function of time. The results indicated that; (1) under normal conditions, Lucifer Yellow flows from cell to cell through gap junctions; (2) the intracellular dialysis of renin (100 nM) disrupts chemical communication - an effect enhanced by simultaneous administration of angiotensinogen (100 nM); (3) enalaprilat (10(-9) M) administered to the cytosol together with renin reduced drastically the uncoupling action of the enzyme; (4) aliskiren (10(-8) M) inhibited the effect of renin on chemical communication; (5) the possible role of intracellular renin independently of angiotensin II (Ang II) was evaluated including the increase of the inward calcium current elicited by the enzyme and the possible role of oxidative stress on the disruption of cell communication; (6) the possible harmful versus the beneficial effect of intracellular renin during myocardial infarction was discussed; (7) the present results indicate that intracellular renin due to internalization or in situ synthesis causes a severe impairment of chemical communication in the heart resulting in derangement of metabolic cooperation with serious consequences for heart function.

  3. Western Analysis of Intracellular Interleukin-8 in Human Mononuclear Leukocytes

    OpenAIRE

    Miskolci, Veronika; Hodgson, Louis; Cox, Dianne; Vancurova, Ivana

    2014-01-01

    Most cytokines are stored in the cytoplasm until their release into the extracellular environment; however, some cytokines have been reported to localize in the nucleus. Traditional whole cell extract preparation does not provide information about the intracellular localization of cytokines. Here, we describe how to prepare cytoplasmic and nuclear extracts that can be analyzed by immunoblotting. While in this chapter we use this method to analyze intracellular localization of interleukin-8 (I...

  4. Acquisition of an animal gene by microsporidian intracellular parasites

    OpenAIRE

    Selman, Mohammed; Pombert, Jean-François; Solter, Leellen; Farinelli, Laurent; Weiss, Louis M.; Keeling, Patrick; Corradi, Nicolas

    2011-01-01

    Parasites have adapted to their specialised way of life by a number of means, including the acquisition of genes by horizontal gene transfer. These newly acquired genes seem to come from a variety of sources, but seldom from the host, even in the most intimate associations between obligate intracellular parasite and host [1]. Microsporidian intracellular parasites have acquired a handful of genes, mostly from bacteria, that help them take energy from their hosts or protect them from the envir...

  5. Hypoxia Induces Internalization of κ-Opioid Receptor.

    Science.gov (United States)

    Xi, Chunhua; Liang, Xuan; Chen, Chunhua; Babazada, Hasan; Li, Tianzuo; Liu, Renyu

    2017-05-01

    It has been demonstrated that κ-opioid receptor agonists can reduce hypoxia-ischemia brain injury in animal models. However, it is unclear how the κ-opioid receptor responds to hypoxia-ischemia. In the current study, the authors used an in vitro model of oxygen-glucose deprivation and reoxygenation to explore how κ-opioid receptors respond to hypoxia and reoxygenation. Mouse neuroblastoma Neuro2A cells were stably transfected with mouse κ-opioid receptor-tdTomato fusion protein or Flag-tagged mouse κ-opioid receptor, divided into several groups (n = 6 to 12), and used to investigate the κ-opioid receptor movement. Observations were performed under normal oxygen, at 30 min to 1 h after oxygen-glucose deprivation and at 1 h after reoxygenation using high-resolution imaging techniques including immunoelectronmicroscopy in the presence and absence of κ-opioid receptor antagonist, dynamin inhibitors, potassium channel blockers, and dopamine receptor inhibitor. Hypoxic conditions caused the κ-opioid receptor to be internalized into the cells. Inhibition of dynamin by Dyngo-4a prevented the receptor internalization. Interestingly, a specific κ-opioid receptor antagonist norbinaltorphimine blocked internalization, suggesting the involvement of activation of a specific κ-opioid receptor. κ-Opioid receptor internalization appears to be reversed by reoxygenation. Quantities of intracellular κ-opioid receptor-associated gold particles as demonstrated by immunoelectron microscopy were increased from 37 to 85% (P internalization. Hypoxia induces reversible κ-opioid receptor internalization, which was inhibited by selective κ-opioid receptor antagonists or dynamin inhibitor, and can be reversed by reoxygenation in neuroblastoma cells, indicating the modulating effects between κ-opioid receptor and hypoxia via κ-opioid receptor activation and the dynamin-dependent mechanism.

  6. Cloning and expression of a widely expressed receptor tyrosine phosphatase

    DEFF Research Database (Denmark)

    Sap, J; D'Eustachio, P; Givol, D

    1990-01-01

    antigen yielded cDNA clones coding for a 794-amino acid transmembrane protein [hereafter referred to as receptor protein tyrosine phosphatase alpha (R-PTP-alpha)] with an intracellular domain displaying clear homology to the catalytic domains of CD45 and LAR (45% and 53%, respectively). The 142-amino acid...

  7. Immune regulation of Rab proteins expression and intracellular transport.

    Science.gov (United States)

    Pei, Gang; Bronietzki, Marc; Gutierrez, Maximiliano Gabriel

    2012-07-01

    Compartmentalization in cells of the immune system, the focus of this review, facilitates the spatiotemporal organization of cellular responses essential for specialized immune functions. In this process of compartment maintenance, Rab proteins are central regulators of protein-mediated transport and fusion of intracellular structures. It is widely believed that the intracellular concentration of proteins that regulate intracellular transport, including Rab proteins, is constitutively mantained. However, there is a growing body of evidence indicating that transcriptional rates of Rab proteins can be modified. This process is especially evident during immune activation and argues that after activation, these cells require higher levels of Rab proteins. The aim of this review is to discuss evidence showing the increasing links between Rab protein expression and intracellular transport, particularly in monocytes and macrophages. We highlight here biological processes in which the expression of Rab GTPases is selectively regulated, leading to the activation of specific intracellular routes. Further, we focus on the immune regulation of intracellular transport after cytokine activation and microbial infection, with an emphasis in mycobacterial infection.

  8. Muscarinic signalling affects intracellular calcium concentration during the first cell cycle of sea urchin embryos.

    Science.gov (United States)

    Harrison, P K; Falugi, C; Angelini, C; Whitaker, M J

    2002-06-01

    The existence of a response to acetylcholine (ACh) and cholinomimetic drugs in sea urchin eggs and zygotes was investigated in two sea urchin species: Paracentrotus lividus and Lytechinus pictus. The calcium sensitive fluorescent probe, Fura-2 dextran, was employed to investigate the regulation of cytosolic free calcium concentration ([Ca(2+)](i)) by cholinomimetic drugs in unfertilised and fertilised eggs of both the sea urchin species. Exposure to cholinomimetic agonists/antagonists, either extracellularly or intracellularly, had no effect either on resting [Ca(2+)](i) levels in the unfertilised sea urchin egg, or on the transient [Ca(2+)](i) increase at fertilisation. However, following fertilisation, extracellular application of ACh receptors agonists, such as ACh and carbachol, predominantly muscarinic agonist, but not nicotine, induced a significant increase in [Ca(2+)](i), which was partially inhibited by atropine. As a consequence of exposure after fertilisation to the agonists of ACh receptors, chromatin structure was transiently affected. The hypothesis is proposed that muscarinic receptors may be involved in the (presumably Ca(2+)-dependent) modulation of the nuclear status during the first cell cycles.

  9. Intracellular sequestration of the NKG2D ligand ULBP3 by human cytomegalovirus.

    Science.gov (United States)

    Bennett, Neil J; Ashiru, Omodele; Morgan, Fiona J E; Pang, Yin; Okecha, Georgina; Eagle, Rob A; Trowsdale, John; Sissons, J G Patrick; Wills, Mark R

    2010-07-15

    Human CMV (HCMV) encodes multiple genes that control NK cell activation and cytotoxicity. Some of these HCMV-encoded gene products modulate NK cell activity as ligands expressed at the cell surface that engage inhibitory NK cell receptors, whereas others prevent the infected cell from upregulating ligands that bind to activating NK cell receptors. A major activating NKR is the homodimeric NKG2D receptor, which has eight distinct natural ligands in humans. It was shown that HCMV is able to prevent the surface expression of five of these ligands (MIC A/B and ULBP1, 2, and 6). In this article, we show that the HCMV gene product UL142 can prevent cell surface expression of ULBP3 during infection. We further show that UL142 interacts with ULBP3 and mediates its intracellular retention in a compartment that colocalizes with markers of the cis-Golgi complex. In doing so, UL142 prevents ULBP3 trafficking to the surface and protects transfected cells from NK-mediated cytotoxicity. This is the first description of a viral gene able to mediate downregulation of ULBP3.

  10. Intrinsically disordered cytoplasmic domains of two cytokine receptors mediate conserved interactions with membranes

    DEFF Research Database (Denmark)

    Haxholm, Gitte Wolfsberg; Nikolajsen, Louise Fletcher; Olsen, Johan Gotthardt

    2015-01-01

    Class 1 cytokine receptors regulate essential biological processes through complex intracellular signaling networks. However, the structural platform for understanding their functions is currently incomplete as structure-function studies of the intracellular domains (ICDs) are critically lacking...... of the inner plasma membrane leaflet through conserved motifs resembling immuno T-cell receptor activation motifs(ITAMs). However, contrary to the observations made for ITAMs, lipid association of the prolactin and growth hormone receptor ICDs was shown to be unaccompanied by changes in transient secondary...... structure and independent of tyrosine phosphorylation. The data presented here provides a new structural platform for studying class 1 cytokine receptors and may implicate the membrane as an active component regulating intracellular signaling....

  11. Receptor activation and 2 distinct COOH-terminal motifs control G-CSF receptor distribution and internalization kinetics

    NARCIS (Netherlands)

    L.H.J. Aarts (Bart); O. Roovers (Onno); A.C. Ward (Alister); I.P. Touw (Ivo)

    2004-01-01

    textabstractWe have studied the intracellular distribution and internalization kinetics of the granulocyte colony-stimulating factor receptor (G-CSF-R) in living cells using fusion constructs of wild-type or mutant G-CSF-R and enhanced green fluorescent protein (EGFP). Under

  12. Quantitative imaging of intracellular signaling for personalized pancreatic cancer therapy in an in vivo avatar (Conference Presentation)

    Science.gov (United States)

    Samkoe, Kimberley S.; Schultz, Emily; Park, Yeonjae; Fischer, Dawn; Pogue, Brian W.; Smith, Kerrington; Tichauer, Kenneth M.; Gibbs, Summer L.

    2017-02-01

    Pancreatic ductal adenocarcinomas (PDAC) are notoriously difficult to treat and in general, molecular targeted therapies have failed even when the targeted protein is overexpressed in the tumor tissue. Genetic mutations in extracellular receptors and downstream signaling proteins (i.e., RAS signaling pathway) and convoluted intracellular cross-talk between cell signaling pathways are likely reasons that these promising therapies fail. Monitoring the complex relationship between intracellular protein signaling is difficult and to-date, standard techniques that are used (Western blot, flow cytometry, immunohistochemistry, etc.) are invasive, static and do not accurately represent in vivo structure-function relationships. Here, we describe the development of an in ovo avatar using patient derived tumors grown on the chicken chorioallantoic membrane (CAM) and the novel fluorescence-based Quantitative Protein Expression Tracking (QUIET) methodology to bridge the gap between oncology, genomics and patient outcomes. Previously developed paired-agent imaging, was extended to a three-compartment model system in QUIET, which utilizes three types of imaging agents: novel fluorophore conjugated cell permeable targeted and untargeted small molecule paired-agents, in addition to a tumor perfusion agent that is not cell membrane permeable. We have demonstrated the ability to quantify the intracellular binding domain of a trans-membrane protein in vitro using cell permeable fluorescent agents (erlotinib-TRITC and control isotype-BODIPY FL). In addition, we have demonstrated imaging protocols to simultaneously image up to 6 spectrally distinct organic fluorophores in in ovo avatars using the Nuance EX (Perkin Elmer) and established proof-of-principle intracellular and extracellular protein concentrations of epidermal growth factor receptor using QUIET and traditional paired-agent imaging.

  13. Astroglial expression of the P-glycoprotein is controlled by intracellular CNTF

    Directory of Open Access Journals (Sweden)

    Guillet Catherine

    2002-07-01

    Full Text Available Abstract Background The P-glycoprotein (P-gp, an ATP binding cassette transmembrane transporter, is expressed by astrocytes in the adult brain, and is positively modulated during astrogliosis. In a search for factors involved in this modulation, P-gp overexpression was studied in long-term in vitro astroglial cultures. Results Surprisingly, most factors that are known to induce astroglial activation in astroglial cultures failed to increase P-gp expression. The only effective proteins were IFNγ and those belonging to the IL-6 family of cytokines (IL-6, LIF, CT-1 and CNTF. As well as P-gp expression, the IL-6 type cytokines - but not IFNγ - stimulated the expression of endogenous CNTF in astrocytes. In order to see whether an increased intracellular level of CNTF was necessary for induction of P-gp overexpression by IL-6 type cytokines, by the same cytokines analysis was carried out on astrocytes obtained from CNTF knockout mice. In these conditions, IFNγ produced increased P-gp expression, but no overexpression of P-gp was observed with either IL-6, LIF or CT-1, pointing to a role of CNTF in the intracellular signalling pathway leading to P-gp overexpression. In agreement with this suggestion, application of exogenous CNTF -which is internalised with its receptor - produced an overexpression of P-gp in CNTF-deficient astrocytes. Conclusions These results reveal two different pathways regulating P-gp expression and activity in reactive astrocytes, one of which depends upon the intracellular concentration of CNTF. This regulation of P-gp may be one of the long searched for physiological roles of CNTF.

  14. Modulation of intracellular calcium waves and triggered activities by mitochondrial ca flux in mouse cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Zhenghang Zhao

    Full Text Available Recent studies have suggested that mitochondria may play important roles in the Ca(2+ homeostasis of cardiac myocytes. However, it is still unclear if mitochondrial Ca(2+ flux can regulate the generation of Ca(2+ waves (CaWs and triggered activities in cardiac myocytes. In the present study, intracellular/cytosolic Ca(2+ (Cai (2+ was imaged in Fluo-4-AM loaded mouse ventricular myocytes. Spontaneous sarcoplasmic reticulum (SR Ca(2+ release and CaWs were induced in the presence of high (4 mM external Ca(2+ (Cao (2+. The protonophore carbonyl cyanide p-(trifluoromethoxyphenylhydrazone (FCCP reversibly raised basal Cai (2+ levels even after depletion of SR Ca(2+ in the absence of Cao (2+ , suggesting Ca(2+ release from mitochondria. FCCP at 0.01 - 0.1 µM partially depolarized the mitochondrial membrane potential (Δψ m and increased the frequency and amplitude of CaWs in a dose-dependent manner. Simultaneous recording of cell membrane potentials showed the augmentation of delayed afterdepolarization amplitudes and frequencies, and induction of triggered action potentials. The effect of FCCP on CaWs was mimicked by antimycin A (an electron transport chain inhibitor disrupting Δψ m or Ru360 (a mitochondrial Ca(2+ uniporter inhibitor, but not by oligomycin (an ATP synthase inhibitor or iodoacetic acid (a glycolytic inhibitor, excluding the contribution of intracellular ATP levels. The effects of FCCP on CaWs were counteracted by the mitochondrial permeability transition pore blocker cyclosporine A, or the mitochondrial Ca(2+ uniporter activator kaempferol. Our results suggest that mitochondrial Ca(2+ release and uptake exquisitely control the local Ca(2+ level in the micro-domain near SR ryanodine receptors and play an important role in regulation of intracellular CaWs and arrhythmogenesis.

  15. Dehydroepiandrosterone inhibits intracellular calcium release in beta-cells by a plasma membrane-dependent mechanism.

    Science.gov (United States)

    Liu, Dongmin; Ren, Min; Bing, Xinyu; Stotts, Corey; Deorah, Sundeep; Love-Homan, Laurie; Dillon, Joseph S

    2006-08-01

    Both dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS) affect glucose stimulated insulin secretion, though their cellular mechanisms of action are not well characterized. We tested the hypothesis that human physiological concentrations of DHEA alter insulin secretion by an action initiated at the plasma membrane of beta-cells. DHEA alone had no effect on intracellular calcium concentration ([Ca(2+)](i)) in a rat beta-cell line (INS-1). However, it caused an immediate and dose-dependent inhibition of carbachol-induced Ca(2+) release from intracellular stores, with a 25% inhibition at zero. One nanometer DHEA. DHEA also inhibited the Ca(2+) mobilizing effect of bombesin (29% decrease), but did not inhibit the influx of extracellular Ca(2+) evoked by glyburide (100 microM) or glucose (15 mM). The steroids (androstenedione, 17-alpha-hydroxypregnenolone, and DHEAS) had no inhibitory effect on carbachol-induced intracellular Ca(2+) release. The action of DHEA depended on a signal initiated at the plasma membrane, since membrane impermeant DHEA-BSA complexes also inhibited the carbachol effect on [Ca(2+)](i) (39% decrease). The inhibition of carbachol-induced Ca(2+) release by DHEA was blocked by pertussis toxin (PTX). DHEA also inhibited the carbachol induction of phosphoinositide generation, with a maximal inhibition at 0.1 nM DHEA. Furthermore, DHEA inhibited insulin secretion induced by carbachol in INS-1 cells by 25%, and in human pancreatic islets by 53%. Taken together, this is the first report showing that human physiological concentrations of DHEA decrease agonist-induced Ca(2+) release by a rapid, non-genomic mechanism in INS-1 cells. Furthermore, these data provide evidence consistent with the existence of a specific plasma membrane DHEA receptor, mediating this signal transduction pathway by pertussis toxin-sensitive G-proteins.

  16. Wave failure at strong coupling in intracellular C a2 + signaling system with clustered channels

    Science.gov (United States)

    Li, Xiang; Wu, Yuning; Gao, Xuejuan; Cai, Meichun; Shuai, Jianwei

    2018-01-01

    As an important intracellular signal, C a2 + ions control diverse cellular functions. In this paper, we discuss the C a2 + signaling with a two-dimensional model in which the inositol 1,4,5-trisphosphate (I P3 ) receptor channels are distributed in clusters on the endoplasmic reticulum membrane. The wave failure at large C a2 + diffusion coupling is discussed in detail in the model. We show that with varying model parameters the wave failure is a robust behavior with either deterministic or stochastic channel dynamics. We suggest that the wave failure should be a general behavior in inhomogeneous diffusing systems with clustered excitable regions and may occur in biological C a2 + signaling systems.

  17. The GARP Complex Is Involved in Intracellular Cholesterol Transport via Targeting NPC2 to Lysosomes.

    Science.gov (United States)

    Wei, Jian; Zhang, Ying-Yu; Luo, Jie; Wang, Ju-Qiong; Zhou, Yu-Xia; Miao, Hong-Hua; Shi, Xiong-Jie; Qu, Yu-Xiu; Xu, Jie; Li, Bo-Liang; Song, Bao-Liang

    2017-06-27

    Proper intracellular cholesterol trafficking is critical for cellular function. Two lysosome-resident proteins, NPC1 and NPC2, mediate the egress of low-density lipoprotein-derived cholesterol from lysosomes. However, other proteins involved in this process remain largely unknown. Through amphotericin B-based selection, we isolated two cholesterol transport-defective cell lines. Subsequent whole-transcriptome-sequencing analysis revealed two cell lines bearing the same mutation in the vacuolar protein sorting 53 (Vps53) gene. Depletion of VPS53 or other subunits of the Golgi-associated retrograde protein (GARP) complex impaired NPC2 sorting to lysosomes and caused cholesterol accumulation. GARP deficiency blocked the retrieval of the cation-independent mannose 6-phosphate receptor (CI-MPR) to the trans-Golgi network. Further, Vps54 mutant mice displayed reduced cellular NPC2 protein levels and increased cholesterol accumulation, underscoring the physiological role of the GARP complex in cholesterol transport. We conclude that the GARP complex contributes to intracellular cholesterol transport by targeting NPC2 to lysosomes in a CI-MPR-dependent manner. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  18. Mesoporous silica nanorods toward efficient loading and intracellular delivery of siRNA

    Science.gov (United States)

    Chen, Lijue; She, Xiaodong; Wang, Tao; Shigdar, Sarah; Duan, Wei; Kong, Lingxue

    2018-02-01

    The technology of RNA interference (RNAi) that uses small interfering RNA (siRNA) to silence the gene expression with complementary messenger RNA (mRNA) sequence has great potential for the treatment of cancer in which certain genes were usually found overexpressed. However, the carry and delivery of siRNA to the target site in the human body can be challenging for this technology to be used clinically to silence the cancer-related gene expression. In this work, rod shaped mesoporous silica nanoparticles (MSNs) were developed as siRNA delivery system for specific intracellular delivery. The rod MSNs with an aspect ratio of 1.5 had a high surface area of 934.28 m2/g and achieved a siRNA loading of more than 80 mg/g. With the epidermal growth factor (EGF) grafted on the surface of the MSNs, siRNA can be delivered to the epidermal growth factor receptor (EGFR) overexpressed colorectal cancer cells with high intracellular concentration compared to MSNs without EGF and lead to survivin gene knocking down to less than 30%.

  19. Tissue architecture and function: dynamic reciprocity via extra- and intra-cellular matrices

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Ren; Boudreau, Aaron; Bissell, Mina J

    2008-12-23

    Mammary gland development, functional differentiation, and homeostasis are orchestrated and sustained by a balance of biochemical and biophysical cues from the organ's microenvironment. The three-dimensional microenvironment of the mammary gland, predominantly 'encoded' by a collaboration between the extracellular matrix (ECM), hormones, and growth factors, sends signals from ECM receptors through the cytoskeletal intracellular matrix to nuclear and chromatin structures resulting in gene expression; the ECM in turn is regulated and remodeled by signals from the nucleus. In this chapter, we discuss how coordinated ECM deposition and remodeling is necessary for mammary gland development, how the ECM provides structural and biochemical cues necessary for tissue-specific function, and the role of the cytoskeleton in mediating the extra - to intracellular dialogue occurring between the nucleus and the microenvironment. When operating normally, the cytoskeletal-mediated dynamic and reciprocal integration of tissue architecture and function directs mammary gland development, tissue polarity, and ultimately, tissue-specific gene expression. Cancer occurs when these dynamic interactions go awry for an extended time.

  20. Bisindoylmaleimide I suppresses adipocyte differentiation through stabilization of intracellular β-catenin protein

    International Nuclear Information System (INIS)

    Cho, Munju; Park, Seoyoung; Gwak, Jungsug; Kim, Dong-Eun; Yea, Sung Su; Shin, Jae-Gook; Oh, Sangtaek

    2008-01-01

    The Wnt/β-catenin signaling pathway plays important roles in cell differentiation. Activation of this pathway, likely by Wnt-10b, has been shown to inhibit adipogenesis in cultured 3T3-L1 preadipocytes and mice. Here we revealed that bisindoylmaleimide I (BIM), which is widely used as a specific inhibitor of protein kinase C (PKC), inhibits adipocyte differentiation through activation of the Wnt/β-catenin signaling pathway. BIM increased β-catenin responsive transcription (CRT) and up-regulated intracellular β-catenin levels in HEK293 cells and 3T3-L1 preadipocytes. BIM significantly decreased intracellular lipid accumulation and reduced expression of important adipocyte marker genes including peroxisome-proliferator-activated receptor γ (PPARγ) and CAATT enhancer-binding protein α (C/EBPα) in 3T3-L1 preadipocytes. Taken together, our findings indicate that BIM inhibits adipogenesis by increasing the stability of β-catenin protein in 3T3-L1 preadipocyte cells

  1. Receptor structure-based discovery of non-metabolite agonists for the succinate receptor GPR91

    DEFF Research Database (Denmark)

    Trauelsen, Mette; Rexen Ulven, Elisabeth; Hjorth, Siv A

    2017-01-01

    OBJECTIVE: Besides functioning as an intracellular metabolite, succinate acts as a stress-induced extracellular signal through activation of GPR91 (SUCNR1) for which we lack suitable pharmacological tools. METHODS AND RESULTS: Here we first determined that the cis conformation of the succinate...... backbone is preferred and that certain backbone modifications are allowed for GPR91 activation. Through receptor modeling over the X-ray structure of the closely related P2Y1 receptor, we discovered that the binding pocket is partly occupied by a segment of an extracellular loop and that succinate...

  2. Intracellular disposition of chitosan nanoparticles in macrophages: intracellular uptake, exocytosis, and intercellular transport

    Directory of Open Access Journals (Sweden)

    Jiang LQ

    2017-08-01

    Full Text Available Li Qun Jiang,1 Ting Yu Wang,1 Thomas J Webster,2 Hua-Jian Duan,1 Jing Ying Qiu,1 Zi Ming Zhao,1 Xiao Xing Yin,1,* Chun Li Zheng3,* 1Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, School of Pharmacy, Xuzhou Medical University, Xuzhou, People’s Republic of China; 2Department of Chemical Engineering, Northeastern University, Boston, MA, USA; 3School of Pharmacy, China Pharmaceutical University, Nanjing, People’s Republic of China *These authors contributed equally to this work Abstract: Biodegradable nanomaterials have been widely used in numerous medical fields. To further improve such efforts, this study focused on the intracellular disposition of chitosan nanoparticles (CsNPs in macrophages, a primary cell of the mononuclear phagocyte system (MPS. Such interactions with the MPS determine the nanoparticle retention time in the body and consequently play a significant role in their own clinical safety. In this study, various dye-labeled CsNPs (about 250 nm were prepared, and a murine macrophage cell line (RAW 264.7 was selected as a model macrophage. The results showed two mechanisms of macrophage incorporation of CsNPs, ie, a clathrin-mediated endocytosis pathway (the primary and phagocytosis. Following internalization, the particles partly dissociated in the cells, indicating cellular digestion of the nanoparticles. It was proved that, after intracellular uptake, a large proportion of CsNPs were exocytosed within 24 h; this excretion induced a decrease in fluorescence intensity in cells by 69%, with the remaining particles possessing difficulty being cleared. Exocytosis could be inhibited by both wortmannin and vacuolin-1, indicating that CsNP uptake was mediated by lysosomal and multivesicular body pathways, and after exocytosis, the reuptake of CsNPs by neighboring cells was verified by further experiments. This study, thus, elucidated the fate of CsNPs in macrophages as well as identified cellular disposition

  3. Cellular Internalization Mechanism and Intracellular Trafficking of Filamentous M13 Phages Displaying a Cell-Penetrating Transbody and TAT Peptide

    Science.gov (United States)

    Shin, Seung-Min; Pham, Chuong D.; Choi, Dong-Ki; Kwon, Myung-Hee; Kim, Yong-Sung

    2012-01-01

    Cellular internalization of bacteriophage by surface-displayed cell penetrating peptides has been reported, though the underlying mechanism remains elusive. Here we describe in detail the internalization mechanism and intracellular trafficking and stability of filamentous M13 phages, the cellular entry of which is mediated by surface-displayed cell-penetrating light chain variable domain 3D8 VL transbody (3D8 VL-M13) or TAT peptide (TAT-M13). Recombinant 3D8 VL-M13 and TAT-M13 phages were efficiently internalized into living mammalian cells via physiologically relevant, energy-dependent endocytosis and were recovered from the cells in their infective form with the yield of 3D8 VL-M13 being higher (0.005∼0.01%) than that of TAT-M13 (0.001∼0.005%). Biochemical and genetic studies revealed that 3D8 VL-M13 was internalized principally by caveolae-mediated endocytosis via interaction with heparan sulfate proteoglycans as cell surface receptors, whereas TAT-M13 was internalized by clathrin- and caveolae-mediated endocytosis utilizing chondroitin sulfate proteoglycans as cell surface receptors, suggesting that phage internalization occurs by physiological endocytotic mechanism through specific cell surface receptors rather than non-specific transcytotic pathways. Internalized 3D8 VL-M13 phages routed to the cytosol and remained stable for more than 18 h without further trafficking to other subcellular compartments, whereas TAT-M13 phages routed to several subcellular compartments before being degraded in lysosomes even after 2 h of internalization. Our results suggest that the internalizing mechanism and intracellular trafficking of filamentous M13 bacteriophages largely follow the attributes of the displayed cell-penetrating moiety. Efficient internalization and cytosolic localization of 3D8 VL transbody-displayed phages will provide a useful tool for intracellular delivery of polar macromolecules such as proteins, peptides, and siRNAs. PMID:23251631

  4. Cellular internalization mechanism and intracellular trafficking of filamentous M13 phages displaying a cell-penetrating transbody and TAT peptide.

    Science.gov (United States)

    Kim, Aeyung; Shin, Tae-Hwan; Shin, Seung-Min; Pham, Chuong D; Choi, Dong-Ki; Kwon, Myung-Hee; Kim, Yong-Sung

    2012-01-01

    Cellular internalization of bacteriophage by surface-displayed cell penetrating peptides has been reported, though the underlying mechanism remains elusive. Here we describe in detail the internalization mechanism and intracellular trafficking and stability of filamentous M13 phages, the cellular entry of which is mediated by surface-displayed cell-penetrating light chain variable domain 3D8 VL transbody (3D8 VL-M13) or TAT peptide (TAT-M13). Recombinant 3D8 VL-M13 and TAT-M13 phages were efficiently internalized into living mammalian cells via physiologically relevant, energy-dependent endocytosis and were recovered from the cells in their infective form with the yield of 3D8 VL-M13 being higher (0.005 ≈ 0.01%) than that of TAT-M13 (0.001 ≈ 0.005%). Biochemical and genetic studies revealed that 3D8 VL-M13 was internalized principally by caveolae-mediated endocytosis via interaction with heparan sulfate proteoglycans as cell surface receptors, whereas TAT-M13 was internalized by clathrin- and caveolae-mediated endocytosis utilizing chondroitin sulfate proteoglycans as cell surface receptors, suggesting that phage internalization occurs by physiological endocytotic mechanism through specific cell surface receptors rather than non-specific transcytotic pathways. Internalized 3D8 VL-M13 phages routed to the cytosol and remained stable for more than 18 h without further trafficking to other subcellular compartments, whereas TAT-M13 phages routed to several subcellular compartments before being degraded in lysosomes even after 2 h of internalization. Our results suggest that the internalizing mechanism and intracellular trafficking of filamentous M13 bacteriophages largely follow the attributes of the displayed cell-penetrating moiety. Efficient internalization and cytosolic localization of 3D8 VL transbody-displayed phages will provide a useful tool for intracellular delivery of polar macromolecules such as proteins, peptides, and siRNAs.

  5. Cellular internalization mechanism and intracellular trafficking of filamentous M13 phages displaying a cell-penetrating transbody and TAT peptide.

    Directory of Open Access Journals (Sweden)

    Aeyung Kim

    Full Text Available Cellular internalization of bacteriophage by surface-displayed cell penetrating peptides has been reported, though the underlying mechanism remains elusive. Here we describe in detail the internalization mechanism and intracellular trafficking and stability of filamentous M13 phages, the cellular entry of which is mediated by surface-displayed cell-penetrating light chain variable domain 3D8 VL transbody (3D8 VL-M13 or TAT peptide (TAT-M13. Recombinant 3D8 VL-M13 and TAT-M13 phages were efficiently internalized into living mammalian cells via physiologically relevant, energy-dependent endocytosis and were recovered from the cells in their infective form with the yield of 3D8 VL-M13 being higher (0.005 ≈ 0.01% than that of TAT-M13 (0.001 ≈ 0.005%. Biochemical and genetic studies revealed that 3D8 VL-M13 was internalized principally by caveolae-mediated endocytosis via interaction with heparan sulfate proteoglycans as cell surface receptors, whereas TAT-M13 was internalized by clathrin- and caveolae-mediated endocytosis utilizing chondroitin sulfate proteoglycans as cell surface receptors, suggesting that phage internalization occurs by physiological endocytotic mechanism through specific cell surface receptors rather than non-specific transcytotic pathways. Internalized 3D8 VL-M13 phages routed to the cytosol and remained stable for more than 18 h without further trafficking to other subcellular compartments, whereas TAT-M13 phages routed to several subcellular compartments before being degraded in lysosomes even after 2 h of internalization. Our results suggest that the internalizing mechanism and intracellular trafficking of filamentous M13 bacteriophages largely follow the attributes of the displayed cell-penetrating moiety. Efficient internalization and cytosolic localization of 3D8 VL transbody-displayed phages will provide a useful tool for intracellular delivery of polar macromolecules such as proteins, peptides, and siRNAs.

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

    Science.gov (United States)

    Kanoski, S E; Ong, Z Y; Fortin, S M; Schlessinger, E S; Grill, H J

    2015-03-01

    To investigate the behavioural and intracellular mechanisms by which the glucagon like peptide-1 (GLP-1) receptor agonist, liraglutide, and leptin in combination enhance the food intake inhibitory and weight loss effects of either treatment alone. We examined the effects of liraglutide (a long-acting GLP-1 analogue) and leptin co-treatment, delivered in low or moderate doses subcutaneously (s.c.) or to the third ventricle, respectively, on cumulative intake, meal patterns and hypothalamic expression of intracellular signalling proteins [phosphorylated signal transducer and activator of transcription-3 (pSTAT3) and protein tyrosine phosphatase-1B (PTP1B)] in lean rats. A low-dose combination of liraglutide (25 µg/kg) and leptin (0.75 µg) additively reduced cumulative food intake and body weight, a result mediated predominantly through a significant reduction in meal frequency that was not present with either drug alone. Liraglutide treatment alone also reduced meal size; an effect not enhanced with leptin co-administration. Moderate doses of liraglutide (75 µg/kg) and leptin (4 µg), examined separately, each reduced meal frequency, cumulative food intake and body weight; only liraglutide reduced meal size. In combination these doses did not further enhance the anorexigenic effects of either treatment alone. Ex vivo immunoblot analysis showed elevated pSTAT3 in the hypothalamic tissue after liraglutide-leptin co-treatment, an effect which was greater than that of leptin treatment alone. In addition, s.c. liraglutide reduced the expression of PTP1B (a negative regulator of leptin receptor signalling), revealing a potential mechanism for the enhanced pSTAT3 response after liraglutide-leptin co-administration. Collectively, these results show novel behavioural and molecular mechanisms underlying the additive reduction in food intake and body weight after liraglutide-leptin combination treatment. © 2014 John Wiley & Sons Ltd.

  7. Des-Arg9-bradykinin increases intracellular Ca2+ in bronchoalveolar eosinophils from ovalbumin-sensitized and -challenged mice.

    Science.gov (United States)

    Eric, Jadranka; Bkaily, Ghassan; Bkaily, Ghassan B; Volkov, Leonid; Gabra, Bichoy H; Sirois, Pierre

    2003-08-15

    The effects of the selective bradykinin B1 receptor agonist, des-Arg9-bradykinin and the bradykinin B2 receptor agonist, bradykinin were studied on the intracellular free Ca2+ concentration ([Ca2+]i) in murine bronchoalveolar lavage cells from control and ovalbumin-sensitized mice using fura-2 microfluorimetry. The bronchoalveolar lavage cells of control mice, which were predominantly alveolar macrophages, showed an increase in [Ca2+]i in response to bradykinin (1 microM) but not to des-Arg9-bradykinin (1 microM), indicating the presence of functional bradykinin B2 receptors and the absence of B1 receptors. Such elevation in [Ca2+]i induced by bradykinin was totally inhibited by the selective bradykinin B2 receptor antagonist, D-Arg0-Hyp3-Thi5-D-Tic7-Oic8-bradykinin (HOE-140; 10 microM). In contrast, bronchoalveolar lavage cells from ovalbumin-sensitized and -challenged mice significantly responded to both bradykinin and des-Arg9-bradykinin, indicating the presence of both functional bradykinin B1 and B2 receptors. Eosinophils exhibited higher response to des-Arg9-bradykinin (1 microM; 485% increase in [Ca2+]i) compared to bradykinin (1 microM; 163% increase in [Ca2+]i). This des-Arg9-bradykinin-induced [Ca2+]i increase was markedly inhibited by the selective bradykinin B1 receptor antagonist, Ac-Lys-[D-betaNal7, Ile8]des-Arg9-bradykinin (R-715; 10 microM). Des-Arg9-bradykinin neither modified the basal [Ca2+]i in lymphocytes nor in mononuclear cells from ovalbumin-sensitized and challenged mice, while bradykinin produced a [Ca2+]i increase in both cell types. Our results further support the implication of the inducible bradykinin B1 receptors in airway inflammatory response in ovalbumin-sensitized and challenged mice.

  8. Analysis of intracellular expressed proteins of Mycobacterium tuberculosis clinical isolates

    Directory of Open Access Journals (Sweden)

    Singhal Neelja

    2012-03-01

    Full Text Available Abstract Background Tuberculosis (TB is the most threatening infectious disease globally. Although progress has been made to reduce global incidence of TB, emergence of multidrug resistant (MDR TB threatens to undermine these advances. To combat the disease, novel intervention strategies effective against drug resistant and sensitive subpopulations of M. tuberculosis are urgently required as adducts in the present treatment regimen. Using THP-1 cells we have analyzed and compared the global protein expression profile of broth-cultured and intraphagosomally grown drug resistant and sensitive M.tuberculosis clinical isolates. Results On comparing the two dimensional (2-DE gels, many proteins were found to be upregulated/expressed during intracellular state which were identified by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS. Four proteins (adenosylhomocysteinase, aspartate carbomyltransferase, putatitive thiosulfate sulfurtransferase and universal stress protein were present in both intracellular MDR and sensitive isolates and three of these belonged to intermediary metabolism and respiration category. Two proteins (alanine dehydrogenase and adenosine kinase of intracellular MDR isolate and two (glucose-6-phosphate isomerase and ATP synthase epsilon chain of intracellular sensitive isolate belonged to intermediary metabolism and respiration category. One protein (Peroxidase/Catalase of intracellular MDR and three (HSPX, 14 kDa antigen and 10 kDa chaperonin of sensitive isolate belonged to virulence, detoxification and adaptation category. ESAT-6 of intracellular MDR belonged to cell wall and cell processes category. Two proteins (Antigen 85-C and Antigen 85-A of intracellular sensitive isolate were involved in lipid metabolism while probable peptidyl-prolyl cis-trans isomerase A was involved in information pathways. Four (Rv0635, Rv1827, Rv0036c and Rv2032 of intracellular MDR and two proteins (Rv2896c and Rv2558c of

  9. Intracellular renin disrupts chemical communication between heart cells. Pathophysiological implications

    Directory of Open Access Journals (Sweden)

    Walmor eDe Mello

    2015-01-01

    Full Text Available The influence of intracellular renin on the process of chemical communication between cardiac cells was investigated in cell pairs isolated from the left ventricle of adult Wistar Kyoto rats. The enzyme together with Lucifer yellow CH was dialyzed into one cell of the pair using the whole cell clamp technique. The diffusion of the dye in the dialyzed and in non-dialyzed cell was followed by measuring the intensity of fluorescence in both cells as a function of time. The results indicated that; 1 under normal conditions, Lucifer Yellow flows from cell-to-cell through gap junctions; 2 the intracellular dialysis of renin (100nM disrupts chemical communication-an effect enhanced by simultaneous administration of angiotensinogen (100nM; 3 enalaprilat (10-9M administered to the cytosol together with renin reduced drastically the uncoupling action of the enzyme; 4 aliskiren (10-8M inhibited the effect of renin on chemical communication;5 the possible role of intracellular renin independently of angiotensin II (Ang II was evaluated including the increase of the inward calcium current elicited by the enzyme and the possible role of oxidative stress on the disruption of cell communication; 6 the possible harmful versus the beneficial effect of intracellular renin during myocardial infarction was discussed;7 the present results indicate that intracellular renin due to internalization or in situ synthesis, causes a severe impairment of chemical communication in the heart resulting in derangement of metabolic cooperation with serious consequences for heart function.

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

    International Nuclear Information System (INIS)

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

    2014-01-01

    Highlights: • High intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import. • The effect of Ca 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

  11. Intracellular transport of fat-soluble vitamins A and E.

    Science.gov (United States)

    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. © 2014 The Authors. Traffic published by John Wiley & Sons Ltd.

  12. Advances in genetic manipulation of obligate intracellular bacterial pathogens

    Directory of Open Access Journals (Sweden)

    Paul eBeare

    2011-05-01

    Full Text Available Infections by obligate intracellular bacterial pathogens result in significant morbidity and mortality worldwide. These bacteria include Chlamydia spp., which causes millions of cases of sexually transmitted disease and blinding trachoma annually, and members of the α-proteobacterial genera Anaplasma, Ehrlichia, Orientia and Rickettsia, agents of serious human illnesses including epidemic typhus. Coxiella burnetii, the agent of human Q fever, has also been considered a prototypical obligate intracellular bacterium, but recent host cell-free (axenic growth has rescued it from obligatism. The historic genetic intractability of obligate intracellular bacteria has severely limited molecular dissection of their unique lifestyles and virulence factors involved in pathogenesis. Host cell restricted growth is a significant barrier to genetic transformation that can make simple procedures for free-living bacteria, such as cloning, exceedingly difficult. Low transformation efficiency requiring long term culture in host cells to expand small transformant populations is another obstacle. Despite numerous technical limitations, the last decade has witnessed significant gains in genetic manipulation of obligate intracellular bacteria including allelic exchange. Continued development of genetic tools should soon enable routine mutation and complementation strategies for virulence factor discovery and stimulate renewed interest in these refractory pathogens. In this review, we discuss the technical challenges associated with genetic transformation of obligate intracellular bacteria and highlight advances made with individual genera.

  13. The orphan receptor GPR17 identified as a new dual uracil nucleotides/cysteinyl-leukotrienes receptor.

    Science.gov (United States)

    Ciana, Paolo; Fumagalli, Marta; Trincavelli, Maria Letizia; Verderio, Claudia; Rosa, Patrizia; Lecca, Davide; Ferrario, Silvia; Parravicini, Chiara; Capra, Valérie; Gelosa, Paolo; Guerrini, Uliano; Belcredito, Silvia; Cimino, Mauro; Sironi, Luigi; Tremoli, Elena; Rovati, G Enrico; Martini, Claudia; Abbracchio, Maria P

    2006-10-04

    Nucleotides and cysteinyl-leukotrienes (CysLTs) are unrelated signaling molecules inducing multiple effects through separate G-protein-coupled receptors: the P2Y and the CysLT receptors. Here we show that GPR17, a Gi-coupled orphan receptor at intermediate phylogenetic position between P2Y and CysLT receptors, is specifically activated by both families of endogenous ligands, leading to both adenylyl cyclase inhibition and intracellular calcium increases. Agonist-response profile, as determined by [(35)S]GTPgammaS binding, was different from that of already known CysLT and P2Y receptors, with EC(50) values in the nanomolar and micromolar range, for CysLTs and uracil nucleotides, respectively. Both rat and human receptors are highly expressed in the organs typically undergoing ischemic damage, that is, brain, heart and kidney. In vivo inhibition of GPR17 by either CysLT/P2Y receptor antagonists or antisense technology dramatically reduced ischemic damage in a rat focal ischemia model, suggesting GPR17 as the common molecular target mediating brain damage by nucleotides and CysLTs. In conclusion, the deorphanization of GPR17 revealed a dualistic receptor for two endogenous unrelated ligand families. These findings may lead to dualistic drugs of previously unexplored therapeutic potential.

  14. A Dual-Sensing Receptor Confers Robust Cellular Homeostasis

    Directory of Open Access Journals (Sweden)

    Hannah Schramke

    2016-06-01

    Full Text Available Cells have evolved diverse mechanisms that maintain intracellular homeostasis in fluctuating environments. In bacteria, control is often exerted by bifunctional receptors acting as both kinase and phosphatase to regulate gene expression, a design known to provide robustness against noise. Yet how such antagonistic enzymatic activities are balanced as a function of environmental change remains poorly understood. We find that the bifunctional receptor that regulates K+ uptake in Escherichia coli is a dual sensor, which modulates its autokinase and phosphatase activities in response to both extracellular and intracellular K+ concentration. Using mathematical modeling, we show that dual sensing is a superior strategy for ensuring homeostasis when both the supply of and demand for a limiting resource fluctuate. By engineering standards, this molecular control system displays a strikingly high degree of functional integration, providing a reference for the vast numbers of receptors for which the sensing strategy remains elusive.

  15. A model for the biosynthesis and transport of plasma membrane-associated signaling receptors to the cell surface

    Directory of Open Access Journals (Sweden)

    Sorina Claudia Popescu

    2012-04-01

    Full Text Available Intracellular protein transport is emerging as critical in determining the outcome of receptor-activated signal transduction pathways. In plants, relatively little is known about the nature of the molecular components and mechanisms involved in coordinating receptor synthesis and transport to the cell surface. Recent advances in this field indicate that signaling pathways and intracellular transport machinery converge and coordinate to render receptors competent for signaling at their plasma membrane activity sites. The biogenesis and transport to the cell surface of signaling receptors appears to require both general trafficking and receptor-specific factors. Several molecular determinants, residing or associated with compartments of the secretory pathway and known to influence aspects in receptor biogenesis, are discussed and integrated into a predictive cooperative model for the functional expression of signaling receptors at the plasma membrane.

  16. Caveolae facilitate muscarinic receptor-mediated intracellular Ca2+ mobilization and contraction in airway smooth muscle

    NARCIS (Netherlands)

    Gosens, Reinoud; Stelmack, Gerald L.; Dueck, Gordon; Mutawe, Mark M.; Hinton, Martha; McNeill, Karol D.; Paulson, Angela; Dakshinamurti, Shyamala; Gerthoffer, William T.; Thliveris, James A.; Unruh, Helmut; Zaagsma, Johan; Halayko, Andrew J.

    2007-01-01

    Contractile responses of airway smooth muscle ( ASM) determine airway resistance in health and disease. Caveolae microdomains in the plasma membrane are marked by caveolin proteins and are abundant in contractile smooth muscle in association with nanospaces involved in Ca2+ homeostasis. Caveolin-1

  17. DMPD: Intracellular NOD-like receptors in host defense and disease. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available ) Open .csml file with CIOPlayer Open .csml file with CIOPlayer - ※CIO Playerのご利用上の注意 Open .csml file with CIO Open .csml file with CIO - ※CIOのご利用上の注意 ...

  18. Intracellular Delivery of a Planar DNA Origami Structure by the Transferrin-Receptor Internalization Pathway

    DEFF Research Database (Denmark)

    Schaffert, David Henning; Okholm, Anders Hauge; Sørensen, Rasmus Schøler

    2016-01-01

    DNA origami provides rapid access to easily functionalized, nanometer-sized structures making it an intriguing platform for the development of defined drug delivery and sensor systems. Low cellular uptake of DNA nanostructures is a major obstacle in the development of DNA-based delivery platforms....... Herein, significant strong increase in cellular uptake in an established cancer cell line by modifying a planar DNA origami structure with the iron transport protein transferrin (Tf) is demonstrated. A variable number of Tf molecules are coupled to the origami structure using a DNA-directed, site...... on the origami surface....

  19. Purine Biosynthesis Metabolically Constrains Intracellular Survival of Uropathogenic Escherichia coli

    Science.gov (United States)

    Shaffer, Carrie L.; Zhang, Ellisa W.; Dudley, Anne G.; Dixon, Beverly R. E. A.; Guckes, Kirsten R.; Breland, Erin J.; Floyd, Kyle A.; Casella, Daniel P.; Algood, Holly M. Scott; Clayton, Douglass B.

    2016-01-01

    ABSTRACT The ability to de novo synthesize purines has been associated with the intracellular survival of multiple bacterial pathogens. Uropathogenic Escherichia coli (UPEC), the predominant cause of urinary tract infections, undergoes a transient intracellular lifestyle during which bacteria clonally expand into multicellular bacterial communities within the cytoplasm of bladder epithelial cells. Here, we characterized the contribution of the conserved de novo purine biosynthesis-associated locus cvpA-purF to UPEC pathogenesis. Deletion of cvpA-purF, or of purF alone, abolished de novo purine biosynthesis but did not impact bacterial adherence properties in vitro or in the bladder lumen. However, upon internalization by bladder epithelial cells, UPEC deficient in de novo purine biosynthesis was unable to expand into intracytoplasmic bacterial communities over time, unless it was extrachromosomally complemented. These findings indicate that UPEC is deprived of purine nucleotides within the intracellular niche and relies on de novo purine synthesis to meet this metabolic requirement. PMID:27795353

  20. EFFECT OF TETRACYCLINES ON THE INTRACELLULAR AMINO ACIDS OF MOLDS.

    Science.gov (United States)

    FREEMAN, B A; CIRCO, R

    1963-07-01

    Freeman, Bob A. (University of Chicago, Chicago, Ill.) and Richard Circo. Effect of tetracyclines on the intracellular amino acids of molds. J. Bacteriol. 86:38-44. 1963.-The tetracycline antibiotics were shown to alter the amino acid metabolism of molds whose growth is not markedly affected. Eight molds were grown in the presence of these antiobiotics; four exhibited a general reduction in the concentration of the intracellular amino acids, except for glutamic acid and alanine. In most of these four cultures, the tetracyclines also caused the complete disappearance of arginine, lysine, proline, phenylalanine, and tyrosine from the intracellular amino acid pool. The significance of these observations and the usefulness of the method in the study of the mechanisms of antibiotic action are discussed.

  1. Quantifying intracellular hydrogen peroxide perturbations in terms of concentration

    Science.gov (United States)

    Huang, Beijing K.; Sikes, Hadley D.

    2014-01-01

    Molecular level, mechanistic understanding of the roles of reactive oxygen species (ROS) in a variety of pathological conditions is hindered by the difficulties associated with determining the concentration of various ROS species. Here, we present an approach that converts fold-change in the signal from an intracellular sensor of hydrogen peroxide into changes in absolute concentration. The method uses extracellular additions of peroxide and an improved biochemical measurement of the gradient between extracellular and intracellular peroxide concentrations to calibrate the intracellular sensor. By measuring peroxiredoxin activity, we found that this gradient is 650-fold rather than the 7–10-fold that is widely cited. The resulting calibration is important for understanding the mass-action kinetics of complex networks of redox reactions, and it enables meaningful characterization and comparison of outputs from endogenous peroxide generating tools and therapeutics across studies. PMID:25460730

  2. Glutamate receptors

    DEFF Research Database (Denmark)

    Kristensen, Anders S; Geballe, Matthew T; Snyder, James P

    2006-01-01

    Fast excitatory synaptic transmission in the CNS relies almost entirely on the neurotransmitter glutamate and its family of ion channel receptors. An appreciation of the coupling between agonist binding and channel opening has advanced rapidly during the past five years, largely as a result of ne...

  3. Intracellular CXCR4+ cell targeting with T22-empowered protein-only nanoparticles

    Directory of Open Access Journals (Sweden)

    Unzueta U

    2012-08-01

    Full Text Available Ugutz Unzueta,1–3 María Virtudes Céspedes,3,4 Neus Ferrer-Miralles,1–3 Isolda Casanova,3,4 Juan Cedano,5 José Luis Corchero,1–3 Joan Domingo-Espín,1–3 Antonio Villaverde,1–3 Ramón Mangues,3,4 Esther Vázquez1–31Institut de Biotecnologia i de Biomedicina, 2Departamento de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, 3CIBER en Bioingeniería, Biomateriales y Nanomedicina, Bellaterra, Barcelona, 4Oncogenesis and Antitumor Drug Group, Biomedical Research Institute Sant Pau, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; 5Laboratory of Immunology, Regional Norte, Universidad de la Republica, Salto, UruguayBackground: Cell-targeting peptides or proteins are appealing tools in nanomedicine and innovative medicines because they increase the local drug concentration and reduce potential side effects. CXC chemokine receptor 4 (CXCR4 is a cell surface marker associated with several severe human pathologies, including colorectal cancer, for which intracellular targeting agents are currently missing.Results: Four different peptides that bind CXCR4 were tested for their ability to internalize a green fluorescent protein-based reporter nanoparticle into CXCR4+ cells. Among them, only the 18 mer peptide T22, an engineered segment derivative of polyphemusin II from the horseshoe crab, efficiently penetrated target cells via a rapid, receptor-specific endosomal route. This resulted in accumulation of the reporter nanoparticle in a fully fluorescent and stable form in the perinuclear region of the target cells, without toxicity either in cell culture or in an in vivo model of metastatic colorectal cancer.Conclusion: Given the urgent demand for targeting agents in the research, diagnosis, and treatment of CXCR4-linked diseases, including colorectal cancer and human immunodeficiency virus infection, T22 appears to be a promising tag for the intracellular delivery of protein drugs, nanoparticles

  4. Cadmium Induces Transcription Independently of Intracellular Calcium Mobilization

    Science.gov (United States)

    Tvermoes, Brooke E.; Bird, Gary S.; Freedman, Jonathan H.

    2011-01-01

    Background Exposure to cadmium is associated with human pathologies and altered gene expression. The molecular mechanisms by which cadmium affects transcription remain unclear. It has been proposed that cadmium activates transcription by altering intracellular calcium concentration ([Ca2+]i) and disrupting calcium-mediated intracellular signaling processes. This hypothesis is based on several studies that may be technically problematic; including the use of BAPTA chelators, BAPTA-based fluorescent sensors, and cytotoxic concentrations of metal. Methodology/Principal Finding In the present report, the effects of cadmium on [Ca2+]i under non-cytotoxic and cytotoxic conditions was monitored using the protein-based calcium sensor yellow cameleon (YC3.60), which was stably expressed in HEK293 cells. In HEK293 constitutively expressing YC3.60, this calcium sensor was found to be insensitive to cadmium. Exposing HEK293::YC3.60 cells to non-cytotoxic cadmium concentrations was sufficient to induce transcription of cadmium-responsive genes but did not affect [Ca2+]i mobilization or increase steady-state mRNA levels of calcium-responsive genes. In contrast, exposure to cytotoxic concentrations of cadmium significantly reduced intracellular calcium stores and altered calcium-responsive gene expression. Conclusions/Significance These data indicate that at low levels, cadmium induces transcription independently of intracellular calcium mobilization. The results also support a model whereby cytotoxic levels of cadmium activate calcium-responsive transcription as a general response to metal-induced intracellular damage and not via a specific mechanism. Thus, the modulation of intracellular calcium may not be a primary mechanism by which cadmium regulates transcription. PMID:21694771

  5. Identification of Human P2X1 Receptor-interacting Proteins Reveals a Role of the Cytoskeleton in Receptor Regulation*

    Science.gov (United States)

    Lalo, Ulyana; Roberts, Jonathan A.; Evans, Richard J.

    2011-01-01

    P2X1 receptors are ATP-gated ion channels expressed by smooth muscle and blood cells. Carboxyl-terminally His-FLAG-tagged human P2X1 receptors were stably expressed in HEK293 cells and co-purified with cytoskeletal proteins including actin. Disruption of the actin cytoskeleton with cytochalasin D inhibited P2X1 receptor currents with no effect on the time course of the response or surface expression of the receptor. Stabilization of the cytoskeleton with jasplakinolide had no effect on P2X1 receptor currents but decreased receptor mobility. P2X2 receptor currents were unaffected by cytochalasin, and P2X1/2 receptor chimeras were used to identify the molecular basis of actin sensitivity. These studies showed that the intracellular amino terminus accounts for the inhibitory effects of cytoskeletal disruption similar to that shown for lipid raft/cholesterol sensitivity. Stabilization of the cytoskeleton with jasplakinolide abolished the inhibitory effects of cholesterol depletion on P2X1 receptor currents, suggesting that lipid rafts may regulate the receptor through stabilization of the cytoskeleton. These studies show that the cytoskeleton plays an important role in P2X1 receptor regulation. PMID:21757694

  6. The role of Toll-like receptors in skin diseases

    Directory of Open Access Journals (Sweden)

    Joanna Bacharewicz

    2014-09-01

    Full Text Available The innate immune system has the ability to recognize pathogens through Toll-like receptors (TLRs, which are transmembrane glycoproteins on the cell surface. These receptors present on the surface of immunological cells – macrophages, dendritic cells, mast cells and some populations of lymphocytes – play an important role in the defense against bacterial, viral and fungal infections. The connection of a Toll-like receptor with the microbial cell component known as pathogen associated molecular pattern (PAMP induces intracellular mechanisms leading to the synthesis of proinflammatory cytokines. Depending on the kind of the recognized ligand, TLRs are classified into subfamilies. So far, 13 TLRs have been described in mice and 11 in humans. These receptors may be expressed extracellularly (TLRs 1, 2, 4, 5, 6, 10, 11 or intracellularly, located in endosomes (TLRs 3, 7, 8, 9. Recent studies also indicate their role in the development of many dermatoses. Occurrence of these receptors has been found on the surface of epidermal and dermal cells: keratinocytes, Langerhans cells, fibroblasts, endo-thelial cells, melanocytes and adipocytes. This paper presents the structure and function of Toll-like receptors and their role in the pathogenesis of some infectious skin diseases, autoimmune and allergic dermatoses as well as skin neoplasms. The knowledge about the role of Toll-like receptors in the development of skin diseases creates the possibility to use them in treatment in the future.

  7. Ligand-directed trafficking of receptor stimulus.

    Science.gov (United States)

    Chilmonczyk, Zdzisław; Bojarski, Andrzej J; Sylte, Ingebrigt

    2014-12-01

    GPCRs are seven transmembrane-spanning receptors that convey specific extracellular stimuli to intracellular signalling. They represent the largest family of cell surface proteins that are therapeutically targeted. According to the traditional two-state model of receptor theory, GPCRs were considered as operating in equilibrium between two functional conformations, an active (R*) and inactive (R) state. Thus, it was assumed that a GPCR can exist either in an "off" or "on" conformation causing either no activation or equal activation of all its signalling pathways. Over the past several years it has become evident that this model is too simple and that GPCR signalling is far more complex. Different studies have presented a multistate model of receptor activation in which ligand-specific receptor conformations are able to differentiate between distinct signalling partners. Recent data show that beside G proteins numerous other proteins, such as β-arrestins and kinases, may interact with GPCRs and activate intracellular signalling pathways. GPCR activation may therefore involve receptor desensitization, coupling to multiple G proteins, Gα or Gβγ signalling, and pathway activation that is independent of G proteins. This latter effect leads to agonist "functional selectivity" (also called ligand-directed receptor trafficking, stimulus trafficking, biased agonism, biased signalling), and agonist intervention with functional selectivity may improve the therapy. Many commercially available drugs with beneficial efficacy also show various undesirable side effects. Further studies of biased signalling might facilitate our understanding of the side effects of current drugs and take us to new avenues to efficiently design pathway-specific medications. Copyright © 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  8. Autophagic clearance of bacterial pathogens: molecular recognition of intracellular microorganisms.

    Science.gov (United States)

    Pareja, Maria Eugenia Mansilla; Colombo, Maria I

    2013-01-01

    Autophagy is involved in several physiological and pathological processes. One of the key roles of the autophagic pathway is to participate in the first line of defense against the invasion of pathogens, as part of the innate immune response. Targeting of intracellular bacteria by the autophagic machinery, either in the cytoplasm or within vacuolar compartments, helps to control bacterial proliferation in the host cell, controlling also the spreading of the infection. In this review we will describe the means used by diverse bacterial pathogens to survive intracellularly and how they are recognized by the autophagic molecular machinery, as well as the mechanisms used to avoid autophagic clearance.

  9. ROR-Family Receptor Tyrosine Kinases.

    Science.gov (United States)

    Stricker, Sigmar; Rauschenberger, Verena; Schambony, Alexandra

    2017-01-01

    ROR-family receptor tyrosine kinases form a small subfamily of receptor tyrosine kinases (RTKs), characterized by a conserved, unique domain architecture. ROR RTKs are evolutionary conserved throughout the animal kingdom and act as alternative receptors and coreceptors of WNT ligands. The intracellular signaling cascades activated downstream of ROR receptors are diverse, including but not limited to ROR-Frizzled-mediated activation of planar cell polarity signaling, RTK-like signaling, and antagonistic regulation of WNT/β-Catenin signaling. In line with their diverse repertoire of signaling functions, ROR receptors are involved in the regulation of multiple processes in embryonic development such as development of the axial and paraxial mesoderm, the nervous system and the neural crest, the axial and appendicular skeleton, and the kidney. In humans, mutations in the ROR2 gene cause two distinct developmental syndromes, recessive Robinow syndrome (RRS; MIM 268310) and dominant brachydactyly type B1 (BDB1; MIM 113000). In Robinow syndrome patients and animal models, the development of multiple organs is affected, whereas BDB1 results only in shortening of the distal phalanges of fingers and toes, reflecting the diversity of functions and signaling activities of ROR-family RTKs. In this chapter, we give an overview on ROR receptor structure and function. We discuss their signaling functions and role in vertebrate embryonic development with a focus on those developmental processes that are affected by mutations in the ROR2 gene in human patients. © 2017 Elsevier Inc. All rights reserved.

  10. Antibody-induced activation of the epidermal growth factor receptor tyrosine kinase requires the presence of detergent

    NARCIS (Netherlands)

    Spaargaren, M.; Defize, L. H.; de Laat, S. W.; Boonstra, J.

    1990-01-01

    Activation of the epidermal growth factor receptor (EGF-R) tyrosine kinase was investigated in membrane preparations as well as intact A431 cells, using anti-EGF-R antibodies directed against extra- and intracellular receptor domains. In vitro assay conditions were mimicked on whole cells by a mild

  11. The Dkk3 gene encodes a vital intracellular regulator of cell proliferation.

    Directory of Open Access Journals (Sweden)

    Jack L Leonard

    Full Text Available Members of the Dickkopf (Dkk family of Wnt antagonists interrupt Wnt-induced receptor assembly and participate in axial patterning and cell fate determination. One family member, DKK3, does not block Wnt receptor activation. Loss of Dkk3 expression in cancer is associated with hyperproliferation and dysregulated ß-catenin signaling, and ectopic expression of Dkk3 halts cancer growth. The molecular events mediating the DKK3-dependent arrest of ß-catenin-driven cell proliferation in cancer cells are unknown. Here we report the identification of a new intracellular gene product originating from the Dkk3 locus. This Dkk3b transcript originates from a second transcriptional start site located in intron 2 of the Dkk3 gene. It is essential for early mouse development and is a newly recognized regulator of ß-catenin signaling and cell proliferation. Dkk3b interrupts nuclear translocation ß-catenin by capturing cytoplasmic, unphosphorylated ß-catenin in an extra-nuclear complex with ß-TrCP. These data reveal a new regulator of one of the most studied signal transduction pathways in metazoans and provides a novel, completely untapped therapeutic target for silencing the aberrant ß-catenin signaling that drives hyperproliferation in many cancers.

  12. Anabolic Androgenic Steroids and Intracellular Calcium Signaling: A Mini Review on Mechanisms and Physiological Implications

    Science.gov (United States)

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

    2015-01-01

    Increasing evidence suggests that nongenomic effects of testosterone and anabolic androgenic steroids (AAS) operate concertedly with genomic effects. Classically, these responses have been viewed as separate and independent processes, primarily because nongenomic responses are faster and appear to be mediated by membrane androgen receptors, whereas long-term genomic effects are mediated through cytosolic androgen receptors regulating transcriptional activity. Numerous studies have demonstrated increases in intracellular Ca2+ in response to AAS. These Ca2+ mediated responses have been seen in a diversity of cell types, including osteoblasts, platelets, skeletal muscle cells, cardiac myocytes and neurons. The versatility of Ca2+ as a second messenger provides these responses with a vast number of pathophysiological implications. In cardiac cells, testosterone elicits voltage-dependent Ca2+ oscillations and IP3R-mediated Ca2+ release from internal stores, leading to activation of MAPK and mTOR signaling that promotes cardiac hypertrophy. In neurons, depending upon concentration, testosterone can provoke either physiological Ca2+ oscillations, essential for synaptic plasticity, or sustained, pathological Ca2+ transients that lead to neuronal apoptosis. We propose therefore, that Ca2+ acts as an important point of crosstalk between nongenomic and genomic AAS signaling, representing a central regulator that bridges these previously thought to be divergent responses. PMID:21443511

  13. Synergistically enhanced selective intracellular uptake of anticancer drug carrier comprising folic acid-conjugated hydrogels containing magnetite nanoparticles

    Science.gov (United States)

    Kim, Haneul; Jo, Ara; Baek, Seulgi; Lim, Daeun; Park, Soon-Yong; Cho, Soo Kyung; Chung, Jin Woong; Yoon, Jinhwan

    2017-01-01

    Targeted drug delivery has long been extensively researched since drug delivery and release at the diseased site with minimum dosage realizes the effective therapy without adverse side effects. In this work, to achieve enhanced intracellular uptake of anticancer drug carriers for efficient chemo-therapy, we have designed targeted multifunctional anticancer drug carrier hydrogels. Temperature-responsive poly(N-isopropylacrylamide) (PNIPAm) hydrogel core containing superparamagnetic magnetite nanoparticles (MNP) were prepared using precipitation polymerization, and further polymerized with amine-functionalized copolymer shell to facilitate the conjugation of targeting ligand. Then, folic acid, specific targeting ligand for cervical cancer cell line (HeLa), was conjugated on the hydrogel surface, yielding the ligand conjugated hybrid hydrogels. We revealed that enhanced intracellular uptake by HeLa cells in vitro was enabled by both magnetic attraction and receptor-mediated endocytosis, which were contributed by MNP and folic acid, respectively. Furthermore, site-specific uptake of the developed carrier was confirmed by incubating with several other cell lines. Based on synergistically enhanced intracellular uptake, efficient cytotoxicity and apoptotic activity of HeLa cells incubated with anticancer drug loaded hybrid hydrogels were successfully achieved. The developed dual-targeted hybrid hydrogels are expected to provide a platform for the next generation intelligent drug delivery systems.

  14. The effect of intracellular trafficking of CD1d on the formation of TCR repertoire of NKT cells.

    Science.gov (United States)

    Shin, Jung Hoon; Park, Se-Ho

    2014-05-01

    CD1 molecules belong to non-polymorphic MHC class I-like proteins and present lipid antigens to T cells. Five different CD1 genes (CD1a-e) have been identified and classified into two groups. Group 1 include CD1a-c and present pathogenic lipid antigens to αβ T cells reminiscence of peptide antigen presentation by MHC-I molecules. CD1d is the only member of Group 2 and presents foreign and self lipid antigens to a specialized subset of αβ T cells, NKT cells. NKT cells are involved in diverse immune responses through prompt and massive production of cytokines. CD1d-dependent NKT cells are categorized upon the usage of their T cell receptors. A major subtype of NKT cells (type I) is invariant NKT cells which utilize invariant Vα14-Jα18 TCR alpha chain in mouse. The remaining NKT cells (type II) utilize diverse TCR alpha chains. Engineered CD1d molecules with modified intracellular trafficking produce either type I or type II NKT cell-defects suggesting the lipid antigens for each subtypes of NKT cells are processed/generated in different intracellular compartments. Since the usage of TCR by a T cell is the result of antigen-driven selection, the intracellular metabolic pathways of lipid antigen are a key in forming the functional NKT cell repertoire.

  15. Detection of ubiquitinated huntingtin species in intracellular aggregates

    NARCIS (Netherlands)

    Juenemann, Katrin; Wiemhoefer, Anne; Reits, Eric A.

    2015-01-01

    Protein conformation diseases, including polyglutamine (polyQ) diseases, result from the accumulation and aggregation of misfolded proteins. Huntington's disease (HD) is one of nine diseases caused by an expanded polyQ repeat within the affected protein and is hallmarked by intracellular inclusion

  16. Facilitating Intracellular Drug Delivery by Ultrasound-Activated Microbubbles

    NARCIS (Netherlands)

    Lammertink, BHA

    2017-01-01

    The goal of this thesis was to investigate the combination of ultrasound and microbubbles (USMB) for intracellular delivery of (model) drugs in vitro. We have focused on clinically approved drugs, i.e. cisplatin, and microbubbles, i.e. SonoVue™, to facilitate clinical translation. In addition, model

  17. Chitosan conjugation enables intracellular bacteria susceptible to aminoglycoside antibiotic.

    Science.gov (United States)

    Mu, Haibo; Niu, Hong; Wang, Dongdong; Sun, Feifei; Sun, Yuelin; Duan, Jinyou

    2016-11-01

    Most chronic infections are difficult to eradicate because bacteria capable of surviving in host-infected cells may be protected from the killing actions of antibiotics, leading to therapy failures and disease relapses. Here we demonstrated that covalent-coupling chitosan to streptomycin significantly improved intracellular bactericidal capacity towards multiple organisms within phagocytic or nonphagocytic cells. Structure-activity relationship investigations indicated that antibiotic contents, molecular size and positive charges of the conjugate were the key to retain this intracellular bactericidal activity. Mechanistic insight demonstrated the conjugate was capable to target and eliminate endocytic or endosomal escaped bacteria through facilitating the direct contact between the antibiotic and intracellular organism. In vivo acute infection models indicated that compared to equal dose of the antibiotic, chitosan-streptomycin (C-S) conjugate and especially the human serum album binding chitosan-streptomycin conjugate (HCS) complex formed by human serum album and C-S conjugate greatly decreased the bacteria burden in the spleen and liver in both wild type and immuno-suppressive mice. Furthermore, the HCS complex remarkably reduced mortality of infected TLR2 deficient mice, mimicking immune-compromised persons who were more susceptible to bacterial infections. These findings might open up a new avenue to combat intracellular bacterial infection by aminoglycosides antibiotics at a lower effective dose. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  18. Intracellular localization of Na + /H + antiporter from Malus zumi ...

    African Journals Online (AJOL)

    In this study, we examined the intracellular localization of the product of Na+/H+ antiporter gene (MzNHX1) cloned from Malus zumi. Analysis using yeast cells expressing a fusion protein of MzNHX1 and green fluorescent protein confirmed the localization of MzNHX1 on the tonoplast.

  19. Intracellular pH in rat pancreatic ducts

    DEFF Research Database (Denmark)

    Novak, I; Hug, M; Greger, R

    1997-01-01

    In order to study the mechanism of H+ and HCO3- transport in a HCO3- secreting epithelium, pancreatic ducts, we have measured the intracellular pH (pHi) in this tissue using the pH sensitive probe BCECF. We found that exposures of ducts to solutions containing acetate/acetic acid or NH4+/NH3 buff...

  20. Bioinspired Nanocarriers Designed to Enhance Intracellular Delivery of Biotherapeutics

    Science.gov (United States)

    2001-10-25

    therapeutic and vaccine development. Keywords - gene therapy, vaccine, bioinspired, biotherapeutic I. INTRODUCTION The efficacy of many protein and DNA...DNA, RNA and proteins . While these therapeutics have tremendous potential, effectively formulating and delivering them has also been a widely...intracellular trafficking that is inspired by biological polymers, i.e. proteins , that are involved in controlling vesicular trafficking pathways. For

  1. Comparing mannose binding lectin genetic diversity in intracellular ...

    African Journals Online (AJOL)

    SERVER

    2007-09-05

    Sep 5, 2007 ... binding lectin of Escherichia coli (Kawasaki et al., 1989) and Salmonella (Ihara et al., 1991). However some reports could not find any effect of mannose binding lectin on complement activation upon extracellular infec- tion of Staphylococcus aureus (Cunion et al., 2001). In intracellular infections, there is ...

  2. Association between VDAC1 mRNA expression and intracellular ...

    African Journals Online (AJOL)

    One way in which xenobiotics induce apoptotic cell death is to alter the selective permeability of the intracellular voltage-dependent anion channel (VDAC1) in the mitochondrial membrane. In this study, we explored the association between VDAC1 mRNA expression and mitochondrial function during hexavalent chromium ...

  3. Monitoring intracellular oxidative events using dynamic spectral unmixing microscopy

    Science.gov (United States)

    There is increasing interest in using live-cell imaging to monitor not just individual intracellular endpoints, but to investigate the interplay between multiple molecular events as they unfold in real time within the cell. A major impediment to simultaneous acquisition of multip...

  4. CONTRIBUTIONS OF INTRACELLULAR IONS TO Kv CHANNEL VOLTAGE SENSOR DYNAMICS.

    Directory of Open Access Journals (Sweden)

    Samuel eGoodchild

    2012-06-01

    Full Text Available Voltage sensing domains of Kv channels control ionic conductance through coupling of the movement of charged residues in the S4 segment to conformational changes at the cytoplasmic region of the pore domain, that allow K+ ions to flow. Conformational transitions within the voltage sensing domain caused by changes in the applied voltage across the membrane field are coupled to the conducting pore region and the gating of ionic conductance. However, several other factors not directly linked to the voltage dependent movement of charged residues within the voltage sensor impact the dynamics of the voltage sensor, such as inactivation, ionic conductance, intracellular ion identity and block of the channel by intracellular ligands. The effect of intracellular ions on voltage sensor dynamics is of importance in the interpretation of gating current measurements and the physiology of pore/voltage sensor coupling. There is a significant amount of variability in the reported kinetics of voltage sensor deactivation kinetics of Kv channels attributed to different mechanisms such as open state stabilization, immobilization and relaxation processes of the voltage sensor. Here we separate these factors and focus on the causal role that intracellular ions can play in allosterically modulating the dynamics of Kv voltage sensor deactivation kinetics. These considerations are of critical importance in understanding the molecular determinants of the complete channel gating cycle from activation to deactivation.

  5. Dihydroceramide biology - Structure-specific metabolism and intracellular localization

    NARCIS (Netherlands)

    Kok, JW; NikolovaKarakashian, M; Klappe, K; Alexander, C; Merrill, AH

    1997-01-01

    This study utilized fluorescent analogs to characterize the intracellular transport and metabolism of dihydroceramide (DN-Cer), an intermediate in de novo sphingolipid biosynthesis, When 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]hexanoyl-DH-Cer (C-6-NBD-DH-Cer) was incubated with HT29, NRK, BHK,

  6. Galectin-3 guides intracellular trafficking of some human serotransferrin glycoforms

    DEFF Research Database (Denmark)

    Carlsson, Carl Michael; Bengtson, Per; Cucak, Helena

    2013-01-01

    these transferrin glycoforms differently after preloading with exogenously added galectin-3. In all, this study provides the first evidence of a functional role for transferrin glycans, in intracellular trafficking after uptake. Moreover, the galectin-3 bound glycoform increased in cancer, suggesting...

  7. Cytoplasmic tail of coronavirus spike protein has intracellular ...

    Indian Academy of Sciences (India)

    2017-04-18

    Apr 18, 2017 ... if the histidine residue is protonated. Lontok et al., in their chimeric S protein studies used C terminal 11 amino acids of SARS-S protein attached to the plasma membrane re- porter protein VSV-G to show KXHXX motif is an intra- cellular localization signal for SARS, and the intracellular distribution closely ...

  8. Biomineralization Patterns of Intracellular Carbonatogenesis in Cyanobacteria: Molecular Hypotheses

    Directory of Open Access Journals (Sweden)

    Jinhua Li

    2016-02-01

    Full Text Available The recent discovery of intracellular carbonatogenesis in several cyanobacteria species has challenged the traditional view that this process was extracellular and not controlled. However, a detailed analysis of the size distribution, chemical composition and 3-D-arrangement of carbonates in these cyanobacteria is lacking. Here, we characterized these features in Candidatus Gloeomargarita lithophora C7 and Candidatus Synechococcus calcipolaris G9 by conventional transmission electron microscopy, tomography, ultramicrotomy, and scanning transmission X-ray microscopy (STXM. Both Ca. G. lithophora C7 and Ca. S. calcipolaris G9 formed numerous polyphosphate granules adjacent or engulfing Ca-carbonate inclusions when grown in phosphate-rich solutions. Ca-carbonates were scattered within Ca. G. lithophora C7 cells under these conditions, but sometimes arranged in one or several chains. In contrast, Ca-carbonates formed at cell septa in Ca. S. calcipolaris G9 and were segregated equally between daughter cells after cell division, arranging as distorted disks at cell poles. The size distribution of carbonates evolved from a positively to a negatively skewed distribution as particles grew. Conventional ultramicrotomy did not preserve Ca-carbonates explaining partly why intracellular calcification has been overlooked in the past. All these new observations allow discussing with unprecedented insight some nucleation and growth processes occurring in intracellularly calcifying cyanobacteria with a particular emphasis on the possible involvement of intracellular compartments and cytoskeleton.

  9. Deciphering the Intracellular Fate of Propionibacterium acnes in Macrophages

    Science.gov (United States)

    Fischer, Natalie; Mak, Tim N.; Shinohara, Debika Biswal; Sfanos, Karen S.; Meyer, Thomas F.

    2013-01-01

    Propionibacterium acnes is a Gram-positive bacterium that colonizes various niches of the human body, particularly the sebaceous follicles of the skin. Over the last years a role of this common skin bacterium as an opportunistic pathogen has been explored. Persistence of P. acnes in host tissue has been associated with chronic inflammation and disease development, for example, in prostate pathologies. This study investigated the intracellular fate of P. acnes in macrophages after phagocytosis. In a mouse model of P. acnes-induced chronic prostatic inflammation, the bacterium could be detected in prostate-infiltrating macrophages at 2 weeks postinfection. Further studies performed in the human macrophage cell line THP-1 revealed intracellular survival and persistence of P. acnes but no intracellular replication or escape from the host cell. Confocal analyses of phagosome acidification and maturation were performed. Acidification of P. acnes-containing phagosomes was observed at 6 h postinfection but then lost again, indicative of cytosolic escape of P. acnes or intraphagosomal pH neutralization. No colocalization with the lysosomal markers LAMP1 and cathepsin D was observed, implying that the P. acnes-containing phagosome does not fuse with lysosomes. Our findings give first insights into the intracellular fate of P. acnes; its persistency is likely to be important for the development of P. acnes-associated inflammatory diseases. PMID:23862148

  10. Cell-penetrating antimicrobial peptides - prospectives for targeting intracellular infections

    DEFF Research Database (Denmark)

    Bahnsen, Jesper S; Franzyk, Henrik; Sayers, Edward J

    2015-01-01

    . TPk showed the highest antibacterial activity. SA-3 exhibited selective disruption of liposomes mimicking Gram-positive and Gram-negative membranes. CONCLUSION: PK-12-KKP is an unlikely candidate for targeting intracellular bacteria, as the eukaryotic cell-penetrating ability is poor. SA-3, affected...

  11. Purification of an Intracellular Fibrinolytic Protease from Ganoderma ...

    African Journals Online (AJOL)

    Erah

    Method: The intracellular fibrinolytic protease produced by Ganoderma lucidum VK12 was isolated from the mycelia grown in MCDBF broth ... The inhibitory effect of different metal ions and commercial protease inhibitors on enzyme activity was studied. ... sodium hydroxide and 2.9 %w/v sodium carbonate in glass-distilled ...

  12. Cytoplasmic tail of coronavirus spike protein has intracellular

    Indian Academy of Sciences (India)

    Intracellular trafficking and localization studies of spike protein from SARS and OC43 showed that SARS spikeprotein is localized in the ER or ERGIC compartment and OC43 spike protein is predominantly localized in thelysosome. Differential localization can be explained by signal sequence. The sequence alignment ...

  13. Regulation of β2-adrenergic receptor function by conformationally selective single-domain intrabodies

    DEFF Research Database (Denmark)

    Staus, Dean P; Wingler, Laura M; Strachan, Ryan T

    2014-01-01

    . However, a monomeric single-domain antibody (nanobody) from the Camelid family was recently found to allosterically bind and stabilize an active conformation of the β2-adrenergic receptor (β2AR). Here, we set out to study the functional interaction of 18 related nanobodies with the β2AR to investigate...... their roles as novel tools for studying GPCR biology. Our studies revealed several sequence-related nanobody families with preferences for active (agonist-occupied) or inactive (antagonist-occupied) receptors. Flow cytometry analysis indicates that all nanobodies bind to epitopes displayed...... on the intracellular receptor surface; therefore, we transiently expressed them intracellularly as "intrabodies" to test their effects on β2AR-dependent signaling. Conformational specificity was preserved after intrabody conversion as demonstrated by the ability for the intracellularly expressed nanobodies...

  14. Membrane depolarization regulates intracellular RANKL transport in non-excitable osteoblasts.

    Science.gov (United States)

    Notomi, Takuya; Kuno, Miyuki; Hiyama, Akiko; Ezura, Yoichi; Honma, Masashi; Ishizuka, Toru; Ohura, Kiyoshi; Yawo, Hiromu; Noda, Masaki

    2015-12-01

    Parathyroid hormone (PTH) and 1α,25-dihydroxyvitamin D3 (VD3) are important factors in Ca(2+) homeostasis, and promote osteoclastogenesis by modulating receptor activator of nuclear factor kappa-B ligand (RANKL) mRNA expression. However, their contribution to RANKL intracellular transport (RANKLiT), including the trigger for RANKL lysosomal vesicle (RANKL-lv) fusion to the cell membrane, is unclear. In neurons, depolarization of membrane potential increases the intracellular Ca(2+) level ([Ca(2+)]i) and promotes neurotransmitter release via fusion of the synaptic vesicles to the cell membrane. To determine whether membrane depolarization also regulates cellular processes such as RANKLiT in MC3T3-E1 osteoblasts (OBs), we generated a light-sensitive OB cell line and developed a system for altering their membrane potential via delivery of a blue light stimulus. In the membrane fraction of RANKL-overexpressing OBs, PTH and VD3 increased the membrane-bound RANKL (mbRANKL) level at 10 min after application without affecting the mRNA expression level, and depolarized the cell membrane while transiently increasing [Ca(2+)]i. In our novel OB line stably expressing the channelrhodopsin-wide receiver, blue light-induced depolarization increased the mbRANKL level, which was reversed by treatment of blockers for L-type voltage-gated Ca(2+) channels and Ca(2+) release from the endoplasmic reticulum. In co-cultures of osteoclast precursor-like RAW264.7 cells and light-sensitive OBs overexpressing RANKL, light stimulation induced an increase in tartrate-resistant acid phosphatase activity and promoted osteoclast differentiation. These results indicate that depolarization of the cell membrane is a trigger for RANKL-lv fusion to the membrane and that membrane potential contributes to the function of OBs. In addition, the non-genomic action of VD3-induced RANKL-lv fusion included the membrane-bound VD3 receptor (1,25D3-MARRS receptor). Elucidating the mechanism of RANKLiT regulation

  15. Posttranslational Modification Biology of Glutamate Receptors and Drug Addiction

    Directory of Open Access Journals (Sweden)

    Li-Min eMao

    2011-03-01

    Full Text Available Posttranslational covalent modifications of glutamate receptors remain a hot topic. Early studies have established that this family of receptors, including almost all ionotropic and metabotropic glutamate receptor subtypes, undergoes active phosphorylation at serine, threonine, or tyrosine residues on their intracellular domains. Recent evidence identifies several glutamate receptor subtypes to be direct substrates for palmitoylation at cysteine residues. Other modifications such as ubiquitination and sumoylation at lysine residues also occur to certain glutamate receptors. These modifications are dynamic and reversible in nature and are regulatable by changing synaptic inputs. The regulated modifications significantly impact the receptor in many ways, including interrelated changes in biochemistry (synthesis, subunit assembling and protein-protein interactions, subcellular redistribution (trafficking, endocytosis, synaptic delivery and clustering, and physiology, usually associated with changes in synaptic plasticity. Glutamate receptors are enriched in the striatum and cooperate closely with dopamine to regulate striatal signaling. Emerging evidence shows that modification processes of striatal glutamate receptors are sensitive to addictive drugs, such as psychostimulants (cocaine and amphetamines. Altered modifications are believed to be directly linked to enduring receptor/synaptic plasticity and drug-seeking. This review summarizes several major types of modifications of glutamate receptors and analyzes the role of these modifications in striatal signaling and in the pathogenesis of psychostimulant addiction.

  16. Microsporidia are natural intracellular parasites of the nematode Caenorhabditis elegans.

    Science.gov (United States)

    Troemel, Emily R; Félix, Marie-Anne; Whiteman, Noah K; Barrière, Antoine; Ausubel, Frederick M

    2008-12-09

    For decades the soil nematode Caenorhabditis elegans has been an important model system for biology, but little is known about its natural ecology. Recently, C. elegans has become the focus of studies of innate immunity and several pathogens have been shown to cause lethal intestinal infections in C. elegans. However none of these pathogens has been shown to invade nematode intestinal cells, and no pathogen has been isolated from wild-caught C. elegans. Here we describe an intracellular pathogen isolated from wild-caught C. elegans that we show is a new species of microsporidia. Microsporidia comprise a large class of eukaryotic intracellular parasites that are medically and agriculturally important, but poorly understood. We show that microsporidian infection of the C. elegans intestine proceeds through distinct stages and is transmitted horizontally. Disruption of a conserved cytoskeletal structure in the intestine called the terminal web correlates with the release of microsporidian spores from infected cells, and appears to be part of a novel mechanism by which intracellular pathogens exit from infected cells. Unlike in bacterial intestinal infections, the p38 MAPK and insulin/insulin-like growth factor (IGF) signaling pathways do not appear to play substantial roles in resistance to microsporidian infection in C. elegans. We found microsporidia in multiple wild-caught isolates of Caenorhabditis nematodes from diverse geographic locations. These results indicate that microsporidia are common parasites of C. elegans in the wild. In addition, the interaction between C. elegans and its natural microsporidian parasites provides a system in which to dissect intracellular intestinal infection in vivo and insight into the diversity of pathogenic mechanisms used by intracellular microbes.

  17. Legionella pneumophila transcriptome during intracellular multiplication in human macrophages

    Directory of Open Access Journals (Sweden)

    Sebastien P Faucher

    2011-04-01

    Full Text Available Legionella pneumophila is the causative agent of Legionnaires’ disease, an acute pulmonary infection. L. pneumophila is able to infect and multiply in both phagocytic protozoa, such as Acanthamoeba castellanii, and mammalian professional phagocytes. The best-known L. pneumophila virulence determinant is the Icm/Dot Type IVB secretion system (TFBSS, which is used to translocate more than 150 effector proteins to host cells. While the transcriptional response of Legionella to the intracellular environment of A. castellanii has been investigated, much less is known about the Legionella transcriptional response inside human macrophages. In this study, the transcriptome of L. pneumophila was monitored during exponential and post-exponential phase in rich AYE broth as well as during infection of human cultured macrophages. This was accomplished with microarrays and an RNA amplification procedure called SCOTS to detect small amounts of mRNA from low numbers of intracellular bacteria. Among the genes induced intracellularly are those involved in amino acid biosynthetic pathways leading to L-arginine, L-histidine and L-proline as well as many transport systems involved in amino acid and iron uptake. Gene involved in catabolism of glycerol is also induced during intracellular growth and could be used as a carbon source. The genes encoding the Icm/Dot system are not differentially expressed inside cells compared to control bacteria grown in rich broth, but the genes encoding several translocated effectors are strongly induced. Moreover, we used the transcriptome data to predict previously unrecognized Icm/Dot effector genes based on their expression pattern and confirmed translocation for three candidates. This study provides a comprehensive view of how L. pneumophila responds to the human macrophage intracellular environment.

  18. Activation of the Innate Immune Receptors: Guardians of the Micro Galaxy : Activation and Functions of the Innate Immune Receptors.

    Science.gov (United States)

    De Nardo, Dominic

    2017-01-01

    The families of innate immune receptors are the frontline responders to danger. These superheroes of the host immune systems populate innate immune cells, surveying the extracellular environment and the intracellular endolysosomal compartments and cytosol for exogenous and endogenous danger signals. As a collective the innate immune receptors recognise a wide array of stimuli, and in response they initiate specific signalling pathways leading to activation of transcriptional or proteolytic pathways and the production of inflammatory molecules to destroy foreign pathogens and/or resolve tissue injury. In this review, I will give an overview of the innate immune system and the activation and effector functions of the families of receptors it comprises. Current key concepts will be described throughout, including innate immune memory, formation of innate immune receptor signalosomes, inflammasome formation and pyroptosis, methods of extrinsic cell communication and examples of receptor cooperation. Finally, several open questions and future directions in the field of innate immunity will be presented and discussed.

  19. NCS-1 associates with adenosine A2A receptors and modulates receptor function

    Directory of Open Access Journals (Sweden)

    Gemma eNavarro

    2012-04-01

    Full Text Available Modulation of G protein-coupled receptor (GPCR signalling by local changes in intracellular calcium concentration is an established function of Calmodulin which is known to interact with many GPCRs. Less is known about the functional role of the closely related neuronal EF-hand Ca2+-sensor proteins that frequently associate with calmodulin targets with different functional outcome. In the present study we aimed to investigate if a target of calmodulin – the A2A adenosine receptor, is able to associate with two other neuronal calcium binding proteins, namely NCS-1 and caldendrin. Using bioluminescence resonance energy transfer and co-immunoprecipitation experiments we show the existence of A2A - NCS-1 complexes in living cells whereas caldendrin did not associate with A2A receptors under the conditions tested. Interestingly, NCS-1 binding modulated downstream A2A receptor intracellular signalling in a Ca2+-dependent manner. Taken together this study provides further evidence that neuronal Ca2+-sensor proteins play an important role in modulation of GPCR signalling.

  20. Signal transduction through the IL-4 and insulin receptor families.

    Science.gov (United States)

    Wang, L M; Keegan, A; Frankel, M; Paul, W E; Pierce, J H

    1995-07-01

    Activation of tyrosine kinase-containing receptors and intracellular tyrosine kinases by ligand stimulation is known to be crucial for mediating initial and subsequent events involved in mitogenic signal transduction. Receptors for insulin and insulin-like growth factor 1 (IGF-1) contain cytoplasmic tyrosine kinase domains that undergo autophosphorylation upon ligand stimulation. Activation of these receptors also leads to pronounced and rapid tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) in cells of connective tissue origin. A related substrate, designated 4PS, is similarly phosphorylated by insulin and IGF-1 stimulation in many hematopoietic cell types. IRS-1 and 4PS possess a number of tyrosine phosphorylation sites that are within motifs that bind specific SH2-containing molecules known to be involved in mitogenic signaling such as PI-3 kinase, SHPTP-2 (Syp) and Grb-2. Thus, they appear to act as docking substrates for a variety of signaling molecules. The majority of hematopoietic cytokines bind to receptors that do not possess intrinsic kinase activity, and these receptors have been collectively termed as members of the hematopoietin receptor superfamily. Despite their lack of tyrosine kinase domains, stimulation of these receptors has been demonstrated to activate intracellular kinases leading to tyrosine phosphorylation of multiple substrates. Recent evidence has demonstrated that activation of different members of the Janus family of tyrosine kinases is involved in mediating tyrosine phosphorylation events by specific cytokines. Stimulation of the interleukin 4 (IL-4) receptor, a member of the hematopoietin receptor superfamily, is thought to result in activation of Jak1, Jak3, and/or Fes tyrosine kinases.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. Valproic Acid Influences MTNR1A Intracellular Trafficking and Signaling in a β-Arrestin 2-Dependent Manner.

    Science.gov (United States)

    Hong, Ling-juan; Jiang, Quan; Long, Sen; Wang, Huan; Zhang, Ling-di; Tian, Yun; Wang, Cheng-kun; Cao, Jing-jing; Tao, Rong-rong; Huang, Ji-yun; Liao, Mei-hua; Lu, Ying-mei; Fukunaga, Kohji; Zhou, Nai-ming; Han, Feng

    2016-03-01

    Valproate exposure is associated with increased risks of autism spectrum disorder. To date, the mechanistic details of disturbance of melatonin receptor subtype 1 (MTNR1A) internalization upon valproate exposure remain elusive. By expressing epitope-tagged receptors (MTNR1A-EGFP) in HEK-293 and Neuro-2a cells, we recorded the dynamic changes of MTNR1A intracellular trafficking after melatonin treatment. Using time-lapse confocal microscopy, we showed in living cells that valproic acid interfered with the internalization kinetics of MTNR1A in the presence of melatonin. This attenuating effect was associated with a decrease in the phosphorylation of PKA (Thr197) and ERK (Thr202/Tyr204). VPA treatment did not alter the whole-cell currents of cells with or without melatonin. Furthermore, fluorescence resonance energy transfer imaging data demonstrated that valproic acid reduced the melatonin-initiated association between YFP-labeled β-arrestin 2 and CFP-labeled MTNR1A. Together, we suggest that valproic acid influences MTNR1A intracellular trafficking and signaling in a β-arrestin 2-dependent manner.

  2. Internalized Listeria monocytogenes modulates intracellular trafficking and delays maturation of the phagosome.

    Science.gov (United States)

    Alvarez-Dominguez, C; Roberts, R; Stahl, P D

    1997-03-01

    Previous studies have shown that early phagosome-endosome fusion events following phagocytosis of Listeria monocytogenes are modulated by the live organism. In the present study, we have characterized more fully the intracellular pathway of dead and live Listeria phagosomes. To examine access of endosomal and lysosomal markers to phagosomes containing live and dead Listeria, quantitative electron microscopy was carried out with intact cells using internalized BSA-gold as a marker to quantify transfer of solute from endosomal and lysosomal compartments to phagosomes. To monitor the protein composition of phagosomal membranes and to quantify transfer of HRP from endosomes and lysosomes to phagosomes, highly enriched phagosomes containing live and dead Listeria were isolated. Enriched phagosomal membranes were used for western blotting experiments with endosomal and lysosomal markers. In this study, we used a listeriolysin-deficient mutant, Listeria(hly-), that is retained within the phagosome following phagocytosis. Western blotting experiments indicate that early endosomal markers (mannose receptor, transferrin receptor) and key fusion factors necessary for early events (NSF, alpha/beta-SNAP) but not late endosomal markers (cation dependent mannose 6-phosphate receptor) or lysosomal proteins (cathepsin D or lamp-1) accumulate on the live-Listeria phagosomal membranes. On the contrary, phagosomes containing dead-Listeria are readily accessible by both endocytic and lysosomal markers. Studies with radiolabeled dead- and live-Listeria(hly-) indicate that, following phagocytosis, degradation of the live microorganism is substantially delayed. These findings indicate that dead-Listeria containing phagosomes rapidly mature to a phagolysosomal stage whereas live-Listeria(hly-) prevents maturation, in part, by avoiding fusion with lysosomes. The data suggest that by delaying phagosome maturation and subsequent degradation, Listeria prolongs survival inside the phagosome

  3. Activation of Adenylyl Cyclase Causes Stimulation of Adenosine Receptors

    Directory of Open Access Journals (Sweden)

    Thomas Pleli

    2018-03-01

    Full Text Available Background/Aims: Signaling of Gs protein-coupled receptors (GsPCRs is accomplished by stimulation of adenylyl cyclase, causing an increase of the intracellular cAMP concentration, activation of the intracellular cAMP effectors protein kinase A (PKA and Epac, and an efflux of cAMP, the function of which is still unclear. Methods: Activation of adenylyl cyclase by GsPCR agonists or cholera toxin was monitored by measurement of the intracellular cAMP concentration by ELISA, anti-phospho-PKA substrate motif phosphorylation by immunoblotting, and an Epac-FRET assay in the presence and absence of adenosine receptor antagonists or ecto-nucleotide phosphodiesterase/pyrophosphatase2 (eNPP2 inhibitors. The production of AMP from cAMP by recombinant eNPP2 was measured by HPLC. Extracellular adenosine was determined by LC-MS/MS, extracellular ATP by luciferase and LC-MS/MS. The expression of eNPP isoenzymes 1-3 was examined by RT-PCR. The expression of multidrug resistance protein 4 was suppressed by siRNA. Results: Here we show that the activation of GsPCRs and the GsPCRs-independent activation of Gs proteins and adenylyl cyclase by cholera toxin induce stimulation of cell surface adenosine receptors (A2A or A2B adenosine receptors. In PC12 cells stimulation of adenylyl cyclase by GsPCR or cholera toxin caused activation of A2A adenosine receptors by an autocrine signaling pathway involving cAMP efflux through multidrug resistance protein 4 and hydrolysis of released cAMP to AMP by eNPP2. In contrast, in PC3 cells cholera toxin- and GsPCR-induced stimulation of adenylyl cyclase resulted in the activation of A2B adenosine receptors. Conclusion: Our findings show that stimulation of adenylyl cyclase causes a remarkable activation of cell surface adenosine receptors.

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

    Directory of Open Access Journals (Sweden)

    Taslima T. Lina

    2016-07-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

  6. A Dual Role for the Nonreceptor Tyrosine Kinase Pyk2 during the Intracellular Trafficking of Human Papillomavirus 16.

    Science.gov (United States)

    Gottschalk, Elinor Y; Meneses, Patricio I

    2015-09-01

    The infectious process of human papillomaviruses (HPVs) has been studied considerably, and many cellular components required for viral entry and trafficking continue to be revealed. In this study, we investigated the role of the nonreceptor tyrosine kinase Pyk2 during HPV16 pseudovirion infection of human keratinocytes. We found that Pyk2 is necessary for infection and appears to be involved in the intracellular trafficking of the virus. Small interfering RNA-mediated reduction of Pyk2 resulted in a significant decrease in infection but did not prevent viral entry at the plasma membrane. Pyk2 depletion resulted in altered endolysosomal trafficking of HPV16 and accelerated unfolding of the viral capsid. Furthermore, we observed retention of the HPV16 pseudogenome in the trans-Golgi network (TGN) in Pyk2-depleted cells, suggesting that the kinase could be required for the viral DNA to exit the TGN. While Pyk2 has previously been shown to function during the entry of enveloped viruses at the plasma membrane, the kinase has not yet been implicated in the intracellular trafficking of a nonenveloped virus such as HPV. Additionally, these data enrich the current literature on Pyk2's function in human keratinocytes. In this study, we investigated the role of the nonreceptor tyrosine kinase Pyk2 during human papillomavirus (HPV) infection of human skin cells. Infections with high-risk types of HPV such as HPV16 are the leading cause of cervical cancer and a major cause of genital and oropharyngeal cancer. As a nonenveloped virus, HPV enters cells by interacting with cellular receptors and established cellular trafficking routes to ensure that the viral DNA reaches the nucleus for productive infection. This study identified Pyk2 as a cellular component required for the intracellular trafficking of HPV16 during infection. Understanding the infectious pathways of HPVs is critical for developing additional preventive therapies. Furthermore, this study advances our knowledge of

  7. Topological and functional characterization of an insect gustatory receptor.

    Directory of Open Access Journals (Sweden)

    Hui-Jie Zhang

    Full Text Available Insect gustatory receptors are predicted to have a seven-transmembrane structure and are distantly related to insect olfactory receptors, which have an inverted topology compared with G-protein coupled receptors, including mammalian olfactory receptors. In contrast, the topology of insect gustatory receptors remains unknown. Except for a few examples from Drosophila, the specificity of individual insect gustatory receptors is also unknown. In this study, the total number of identified gustatory receptors in Bombyx mori was expanded from 65 to 69. BmGr8, a silkmoth gustatory receptor from the sugar receptor subfamily, was expressed in insect cells. Membrane topology studies on BmGr8 indicate that, like insect olfactory receptors, it has an inverted topology relative to G protein-coupled receptors. An orphan GR from the bitter receptor family, BmGr53, yielded similar results. We infer, from the finding that two distantly related BmGrs have an intracellular N-terminus and an odd number of transmembrane spans, that this is likely to be a general topology for all insect gustatory receptors. We also show that BmGr8 functions independently in Sf9 cells and responds in a concentration-dependent manner to the polyalcohols myo-inositol and epi-inositol but not to a range of mono- and di-saccharides. BmGr8 is the first chemoreceptor shown to respond specifically to inositol, an important or essential nutrient for some Lepidoptera. The selectivity of BmGr8 responses is consistent with the known responses of one of the gustatory receptor neurons in the lateral styloconic sensilla of B. mori, which responds to myo-inositol and epi-inositol but not to allo-inositol.

  8. Topological and functional characterization of an insect gustatory receptor.

    Science.gov (United States)

    Zhang, Hui-Jie; Anderson, Alisha R; Trowell, Stephen C; Luo, A-Rong; Xiang, Zhong-Huai; Xia, Qing-You

    2011-01-01

    Insect gustatory receptors are predicted to have a seven-transmembrane structure and are distantly related to insect olfactory receptors, which have an inverted topology compared with G-protein coupled receptors, including mammalian olfactory receptors. In contrast, the topology of insect gustatory receptors remains unknown. Except for a few examples from Drosophila, the specificity of individual insect gustatory receptors is also unknown. In this study, the total number of identified gustatory receptors in Bombyx mori was expanded from 65 to 69. BmGr8, a silkmoth gustatory receptor from the sugar receptor subfamily, was expressed in insect cells. Membrane topology studies on BmGr8 indicate that, like insect olfactory receptors, it has an inverted topology relative to G protein-coupled receptors. An orphan GR from the bitter receptor family, BmGr53, yielded similar results. We infer, from the finding that two distantly related BmGrs have an intracellular N-terminus and an odd number of transmembrane spans, that this is likely to be a general topology for all insect gustatory receptors. We also show that BmGr8 functions independently in Sf9 cells and responds in a concentration-dependent manner to the polyalcohols myo-inositol and epi-inositol but not to a range of mono- and di-saccharides. BmGr8 is the first chemoreceptor shown to respond specifically to inositol, an important or essential nutrient for some Lepidoptera. The selectivity of BmGr8 responses is consistent with the known responses of one of the gustatory receptor neurons in the lateral styloconic sensilla of B. mori, which responds to myo-inositol and epi-inositol but not to allo-inositol.

  9. Insulin-like growth factor-II receptors in cultured rat hepatocytes: regulation by cell density

    International Nuclear Information System (INIS)

    Scott, C.D.; Baxter, R.C.

    1987-01-01

    Insulin-like growth factor-II (IGF-II) receptors in primary cultures of adult rat hepatocytes were characterized and their regulation by cell density examined. In hepatocytes cultured at 5 X 10(5) cells per 3.8 cm2 plate [ 125 I]IGF-II bound to specific, high affinity receptors (Ka = 4.4 +/- 0.5 X 10(9) l/mol). Less than 1% cross-reactivity by IGF-I and no cross-reactivity by insulin were observed. IGF-II binding increased when cells were permeabilized with 0.01% digitonin, suggesting the presence of an intracellular receptor pool. Determined by Scatchard analysis and by polyacrylamide gel electrophoresis after affinity labeling, the higher binding was due solely to an increase in binding sites present on 220 kDa type II IGF receptors. In hepatocytes cultured at low densities, the number of cell surface receptors increased markedly, from 10-20,000 receptors per cell at a culture density of 6 X 10(5) cells/well to 70-80,000 receptors per cell at 0.38 X 10(5) cells/well. The increase was not due simply to the exposure of receptors from the intracellular pool, as a density-related increase in receptors was also seen in cells permeabilized with digitonin. There was no evidence that IGF binding proteins, either secreted by hepatocytes or present in fetal calf serum, had any effect on the measurement of receptor concentration or affinity. We conclude that rat hepatocytes in primary culture contain specific IGF-II receptors and that both cell surface and intracellular receptors are regulated by cell density

  10. Does GRK-β arrestin machinery work as a "switch on" for GPR17-mediated activation of intracellular signaling pathways?

    Science.gov (United States)

    Daniele, Simona; Trincavelli, Maria Letizia; Fumagalli, Marta; Zappelli, Elisa; Lecca, Davide; Bonfanti, Elisabetta; Campiglia, Pietro; Abbracchio, Maria P; Martini, Claudia

    2014-06-01

    During oligodendrocyte-precursor cell (OPC) differentiation program, an impairment in the regulatory mechanisms controlling GPR17 spatio-temporal expression and functional activity has been suggested to contribute to defective OPC maturation, a crucial event in the pathogenesis of multiple sclerosis. GRK-β arrestin machinery is the primary actor in the control of G-protein coupled receptor (GPCR) functional responses and changes in these regulatory protein activities have been demonstrated in several immune/inflammatory diseases. Herein, in order to shed light on the molecular mechanisms controlling GPR17 regulatory events during cell differentiation, the role of GRK/β-arrestin machinery in receptor desensitization and signal transduction was investigated, in transfected cells and primary OPC. Following cell treatment with the two classes of purinergic and cysteinyl-leukotriene (cysLT) ligands, different GRK isoforms were recruited to regulate GPR17 functional responses. CysLT-mediated receptor desensitization mainly involved GRK2; this kinase, via a G protein-dependent mechanism, promoted a transient binding of the receptor to β-arrestins, rapid ERK phosphorylation and sustained nuclear CREB activation. Furthermore, GRK2, whose expression parallels that of the receptor during differentiation process, appeared to be crucial to induce cysLT-mediated maturation of OPCs. On the other hand, purinergic ligand exclusively recruited the GRK5 subtype, and induced, via a G protein-independent/β-arrestin-dependent mechanism, a receptor/β-arrestin stable association, slower and sustained ERK stimulation and marginal CREB activation. These results show that purinergic and cysLT ligands, through the recruitment of specific GRK isoforms, address distinct intracellular pathways, most likely reinforcing the same final response. The identification of these mechanisms and players controlling GPR17 responses during OPC differentiation could be useful to identify new targets in

  11. ERK1/2 mediates sperm acrosome reaction through elevation of intracellular calcium concentration.

    Science.gov (United States)

    Jaldety, Yael; Breitbart, Haim

    2015-10-01

    Mammalian sperm acquire fertilization capacity after residing in the female reproductive tract for a few hours in a process called capacitation. Only capacitated sperm can bind the zona pellucida (ZP) of the egg and undergo the acrosome reaction, a process that allows penetration and fertilization. Extracellular signal regulated kinase (ERK1/2) mediates signalling in many cell types, however its role in sperm function is largely unknown. Here we show that ERK1/2 is highly phosphorylated/activated after a short incubation of mouse sperm under capacitation conditions and that this phosphorylation is reduced after longer incubation. Further phosphorylation was observed upon addition of crude extract of egg ZP or epidermal growth factor (EGF). The mitogen-activated ERK-kinase (MEK) inhibitor U0126 abolished ERK1/2 phosphorylation, in vitro fertilization rate and the acrosome reaction induced by ZP or EGF but not by the Ca2+-ionophore A23187. Moreover, inhibition of ERK1/2 along the capacitation process diminished almost completely the sperm's ability to go through the acrosome reaction, while inhibition at the end of capacitation attenuated the acrosome reaction rate by only 45%. The fact that the acrosome reaction, induced by the Ca2+ -ionophore A23187, was not inhibited by U0126 suggests that ERK1/2 mediates the acrosome reaction by activating Ca2+ transport into the cell. Direct determination of intracellular [Ca2+] revealed that Ca2+ influx induced by EGF or ZP was completely blocked by U0126. Thus, it has been established that the increase in ERK1/2 phosphorylation/activation in response to ZP or by activation of the EGF receptor (EGFR) by EGF, is a key event for intracellular Ca2+ elevation and the subsequent occurrence of the acrosome reaction.

  12. Diffusive spatio-temporal noise in a first-passage time model for intracellular calcium release

    KAUST Repository

    Flegg, Mark B.

    2013-01-01

    The intracellular release of calcium from the endoplasmic reticulum is controlled by ion channels. The resulting calcium signals exhibit a rich spatio-temporal signature, which originates at least partly from microscopic fluctuations. While stochasticity in the gating transition of ion channels has been incorporated into many models, the distribution of calcium is usually described by deterministic reaction-diffusion equations. Here we test the validity of the latter modeling approach by using two different models to calculate the frequency of localized calcium signals (calcium puffs) from clustered IP3 receptor channels. The complexity of the full calcium system is here limited to the basic opening mechanism of the ion channels and, in the mathematical reduction simplifies to the calculation of a first passage time. Two models are then studied: (i) a hybrid model, where channel gating is treated stochastically, while calcium concentration is deterministic and (ii) a fully stochastic model with noisy channel gating and Brownian calcium ion motion. The second model utilises the recently developed two-regime method [M. B. Flegg, S. J. Chapman, and R. Erban, "The two-regime method for optimizing stochastic reaction-diffusion simulations," J. R. Soc., Interface 9, 859-868 (2012)] in order to simulate a large domain with precision required only near the Ca2+ absorbing channels. The expected time for a first channel opening that results in a calcium puff event is calculated. It is found that for a large diffusion constant, predictions of the interpuff time are significantly overestimated using the model (i) with a deterministic non-spatial calcium variable. It is thus demonstrated that the presence of diffusive noise in local concentrations of intracellular Ca2+ ions can substantially influence the occurrence of calcium signals. The presented approach and results may also be relevant for other cell-physiological first-passage time problems with small ligand concentration

  13. Refrigerated storage of platelets initiates changes in platelet surface marker expression and localization of intracellular proteins.

    Science.gov (United States)

    Wood, Ben; Padula, Matthew P; Marks, Denese C; Johnson, Lacey

    2016-10-01

    Platelets (PLTs) are currently stored at room temperature (22°C), which limits their shelf life, primarily due to the risk of bacterial growth. Alternatives to room temperature storage include PLT refrigeration (2-6°C), which inhibits bacterial growth, thus potentially allowing an extension of shelf life. Additionally, refrigerated PLTs appear more hemostatically active than conventional PLTs, which may be beneficial in certain clinical situations. However, the mechanisms responsible for this hemostatic function are not well characterized. The aim of this study was to assess the protein profile of refrigerated PLTs in an effort to understand these functional consequences. Buffy coat PLTs were pooled, split, and stored either at room temperature (20-24°C) or under refrigerated (2-6°C) conditions (n = 8 in each group). PLTs were assessed for changes in external receptor expression and actin filamentation using flow cytometry. Intracellular proteomic changes were assessed using two-dimensional gel electrophoresis and Western blotting. PLT refrigeration significantly reduced the abundance of glycoproteins (GPIb, GPIX, GPIIb, and GPIV) on the external membrane. However, refrigeration resulted in the increased expression of high-affinity integrins (αIIbβ3 and β1) and activation and apoptosis markers (CD62P, CD63, and phosphatidylserine). PLT refrigeration substantially altered the abundance and localization of several cytoskeletal proteins and resulted in an increase in actin filamentation, as measured by phalloidin staining. Refrigerated storage of PLTs induces significant changes in the expression and localization of both surface-expressed and intracellular proteins. Understanding these proteomic changes may help to identify the mechanisms resulting in the refrigeration-associated alterations in PLT function and clearance. © 2016 AABB.

  14. Activation of protein kinase C alters the intracellular distribution and mobility of cardiac Na+ channels.

    Science.gov (United States)

    Hallaq, Haifa; Wang, Dao W; Kunic, Jennifer D; George, Alfred L; Wells, K Sam; Murray, Katherine T

    2012-02-01

    Na(+) current derived from expression of the cardiac isoform SCN5A is reduced by receptor-mediated or direct activation of protein kinase C (PKC). Previous work has suggested a possible role for loss of Na(+) channels at the plasma membrane in this effect, but the results are controversial. In this study, we tested the hypothesis that PKC activation acutely modulates the intracellular distribution of SCN5A channels and that this effect can be visualized in living cells. In human embryonic kidney cells that stably expressed SCN5A with green fluorescent protein (GFP) fused to the channel COOH-terminus (SCN5A-GFP), Na(+) currents were suppressed by an exposure to PKC activation. Using confocal microscopy, colocalization of SCN5A-GFP channels with the plasma membrane under control and stimulated conditions was quantified. A separate population of SCN5A channels containing an extracellular epitope was immunolabeled to permit temporally stable labeling of the plasma membrane. Our results demonstrated that Na(+) channels were preferentially trafficked away from the plasma membrane by PKC activation, with a major contribution by Ca(2+)-sensitive or conventional PKC isoforms, whereas stimulation of protein kinase A (PKA) had the opposite effect. Removal of the conserved PKC site Ser(1503) or exposure to the NADPH oxidase inhibitor apocynin eliminated the PKC-mediated effect to alter channel trafficking, indicating that both channel phosphorylation and ROS were required. Experiments using fluorescence recovery after photobleaching demonstrated that both PKC and PKA also modified channel mobility in a manner consistent with the dynamics of channel distribution. These results demonstrate that the activation of protein kinases can acutely regulate the intracellular distribution and molecular mobility of cardiac Na(+) channels in living cells.

  15. Venus Kinase Receptors: prospects in signalling and biological functions of these invertebrate receptors

    Directory of Open Access Journals (Sweden)

    Colette eDissous

    2014-05-01

    Full Text Available Venus Kinase Receptors (VKRs form a family of invertebrate receptor tyrosine kinases (RTKs initially discovered in the parasitic platyhelminth Schistosoma mansoni. VKRs are single transmembrane receptors which contain an extracellular Venus Flytrap (VFT structure similar to the ligand binding domain of G Protein Coupled Receptors of class C, and an intracellular Tyrosine Kinase domain close to that of Insulin Receptors. VKRs are found in a large variety of invertebrates from cnidarians to echinoderms, and are highly expressed in larval stages and in gonads, suggesting a role of these proteins in embryonic and larval development as well as in reproduction. Vkr gene silencing could demonstrate the function of these receptors in oogenesis as well as in spermatogenesis in Schistosoma .mansoni. VKRs are activated by amino-acids, and highly responsive to arginine. As many other RTKs, they form dimers when activated by ligands and induce intracellular pathways involved in protein synthesis and cellular growth, such as MAPK and PI3K/Akt/S6K pathways. VKRs are not present in vertebrates, nor in some invertebrate species. Questions remain open about the origin of this little-known RTK family in evolution and its role in emergence and specialization of Metazoa. What is the meaning of maintenance or loss of VKR in some phyla or species in terms of development and physiological functions? The presence of VKRs in invertebrates of economical and medical importance, such as pests, vectors of pathogens and platyhelminth parasites, and the implication of these RTKs in gametogenesis and reproduction processes are valuable reasons to consider VKRs as interesting targets in new programs for eradication/ control of pests and infectious diseases, with the main advantage in the case of parasite targeting that VKR counterparts are absent from the vertebrate host kinase panel.

  16. Responses to microbial challenges by SLAMF receptors

    Directory of Open Access Journals (Sweden)

    Boaz Job Van Driel

    2016-01-01

    Full Text Available The SLAMF Family (SLAMF of cell surface glycoproteins is comprised of nine glycoproteins and whilst SLAMF1, 3, 5, 6, 7, 8, 9 are self-ligand receptors, SLAMF2 and SLAMF4 interact with each other. Their interactions induce signal transduction networks in trans, thereby shaping immune cell-cell communications. Collectively, these receptors modulate a wide range of functions, such as myeloid cell and lymphocyte development and, T and B cell responses to microbes and parasites. In addition, several SLAMF receptors serve as microbial sensors, which either positively or negatively modulate the function of macrophages, dendritic cells, neutrophils and NK cells in response to microbial challenges. The SLAMF receptor-microbe interactions contribute both to intracellular microbicidal activity as well as to migration of phagocytes to the site of inflammation. In this review, we describe the current knowledge on how the SLAMF receptors and their specific adapters SAP and EAT-2 regulate innate and adaptive immune responses to microbes.

  17. A Miniature Graphene-based Biosensor for Intracellular Glucose Measurements

    International Nuclear Information System (INIS)

    Hasan, Kamran ul; Asif, Muhammad H.; Hassan, Muhammad Umair; Sandberg, Mats O.; Nur, O.; Willander, M.; Fagerholm, Siri; Strålfors, Peter

    2015-01-01

    We report on a small and simple graphene-based potentiometric sensor for the measurement of intracellular glucose concentration. A fine borosilicate glass capillary coated with graphene and subsequently immobilized with glucose oxidase (GOD) enzyme is inserted into the intracellular environment of a single human cell. The functional groups on the edge plane of graphene assist the attachment with the free amine terminals of GOD enzyme, resulting in a better immobilization. The sensor exhibits a glucose-dependent electrochemical potential against an Ag/AgCl reference microelectrode which is linear across the whole concentration range of interest (10 – 1000 μM). Glucose concentration in human fat cell measured by our graphene-based sensor is in good agreement with nuclear magnetic resonance (NMR) spectroscopy

  18. Enzyme encapsulated hollow silica nanospheres for intracellular biocatalysis.

    Science.gov (United States)

    Chang, Feng-Peng; Hung, Yann; Chang, Jen-Hsuan; Lin, Chen-Han; Mou, Chung-Yuan

    2014-05-14

    Hollow silica nanospheres (HSN) with low densities, large interior spaces and permeable silica shells are suitable for loading enzymes in the cavity to carry out intracellular biocatalysis. The porous shell can protect the encapsulated enzymes against proteolysis and attenuate immunological response. We developed a microemulsion-templating method for confining horseradish peroxidase (HRP) in the cavity of HSN. This simple one-pot enzyme encapsulation method allows entrapping of the enzyme, which retains high catalytic activity. Compared with HRP supported on solid silica spheres, HRP@HSN with thin porous silica shells displayed better enzyme activity. The small HRP@HSN (∼50 nm in diameter), giving satisfactory catalytic activity, can act as an intracellular catalyst for the oxidation of the prodrug indole-3-acetic acid to produce toxic free radicals for killing cancer cells. We envision this kind of hollow nanosystem could encapsulate multiple enzymes or other synergistic drugs and function as therapeutic nanoreactors.

  19. Siderocalin inhibits the intracellular replication of Mycobacterium tuberculosis in macrophages

    DEFF Research Database (Denmark)

    Johnson, Erin E; Srikanth, Chittur V; Sandgren, Andreas

    2010-01-01

    that siderocalin expression is upregulated following M.tb infection of mouse macrophage cell lines and primary murine alveolar macrophages. Furthermore, siderocalin added exogenously as a recombinant protein or overexpressed in the RAW264.7 macrophage cell line inhibited the intracellular growth of the pathogen......Siderocalin is a secreted protein that binds to siderophores to prevent bacterial iron acquisition. While it has been shown to inhibit the growth of Mycobacterium tuberculosis (M.tb) in extracellular cultures, its effect on this pathogen within macrophages is not clear. Here, we show....... A variant form of siderocalin, which is expressed only in the macrophage cytosol, inhibited intracellular M.tb growth as effectively as the normal, secreted form, an observation that provides mechanistic insight into how siderocalin might influence iron acquisition by the bacteria in the phagosome. Our...

  20. Intracellular transport and compartmentation of phosphate in plants.

    Science.gov (United States)

    Versaw, Wayne K; Garcia, L Rene

    2017-10-01

    Phosphate (Pi) is an essential macronutrient with structural and metabolic roles within every compartment of the plant cell. Intracellular Pi transporters direct Pi to each organelle and also control its exchange between subcellular compartments thereby providing the means to coordinate compartmented metabolic processes, including glycolysis, photosynthesis, and respiration. In this review we summarize recent advances in the identification and functional analysis of Pi transporters that localize to vacuoles, chloroplasts, non-photosynthetic plastids, mitochondria, and the Golgi apparatus. Electrical potentials across intracellular membranes and the pH of subcellular environments will also be highlighted as key factors influencing the energetics of Pi transport, and therefore pose limits for Pi compartmentation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Regulation of dopamine transporter trafficking by intracellular amphetamine

    DEFF Research Database (Denmark)

    Kahlig, Kristopher M; Lute, Brandon J; Wei, Yuqiang

    2006-01-01

    -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...

  2. Intracellular transport proteins: classification, structure and function of kinesins

    Directory of Open Access Journals (Sweden)

    Agnieszka Chudy

    2011-09-01

    Full Text Available Correct cell functioning, division and morphogenesis rely on efficient intracellular transport. Apart from dyneins and myosins, kinesins are the main proteins responsible for intracellular movement. Kinesins are a large, diverse group of motor proteins, which based on phylogenetic similarity were classified into fourteen families. Among these families, due to the location of their motor domains, three groups have been characterized: N-, C- and M-kinesin. As molecular motors, kinesins transport various molecules and vesicles mainly towards the microtubule plus end (from the cell body participating in anterograde transport, although there are also kinesins involved in retrograde transport (C-kinesins. Kinesins are also involved in spindle formation, chromosome segregation, and spermatogenesis. Because of their great importance for the correct functioning of cells, mutations in kinesin coding genes may lead to such neurodegenerative diseases as dominant hereditary spastic paraplegia or Charcot-Marie-Tooth disease.

  3. Mycobacterium avium-intracellulare: a rare cause of subacromial bursitis.

    Science.gov (United States)

    Sinha, Raj; Tuckett, John; Hide, Geoff; Dildey, Petra; Karsandas, Alvin

    2015-01-01

    Septic subacromial bursitis is an uncommon disorder with only a few reported cases in the literature. The most common causative organism is Staphylococcus aureus. We report the case of a 61-year-old female with a septic subacromial bursitis where the causative organism was found to be Mycobacterium avium-intracellulare (MAI). The diagnosis was only made following a biopsy, and we use this case to highlight the importance of recognising the need to consider a biopsy and aspiration in atypical situations.

  4. Evaluation of two novel methods for assessing intracellular oxygen

    International Nuclear Information System (INIS)

    Williams, Catrin F; Lloyd, David; Kombrabail, M; Vijayalakshmi, K; Krishnamoorthy, G; White, Nick

    2012-01-01

    The ability to resolve the spatio-temporal complexity of intracellular O 2 distribution is the ‘Holy Grail’ of cellular physiology. In an effort to obtain a non-invasive approach of mapping intracellular O 2 tensions, two methods of phosphorescent lifetime imaging microscopy were examined in the current study. These were picosecond time-resolved epiphosphorescence microscopy (single 0.5 µm focused spot) and two-photon confocal laser scanning microscopy with pinhole shifting. Both methods utilized nanoparticle-embedded Ru complex (45 nm diameter) as the phosphorescent probe, excited using pulsed outputs of Ti–sapphire Tsunami lasers (710–1050 nm). The former method used a 1 ps pulse width excitation beam with vertical polarization via a dichroic mirror (610 nm, XF43) and a 20× objective (NA 0.55, Nikon). Transmitted luminescence (1–2 × 10 4 counts s −1 ) was collected and time-correlated single photon counted decay times measured. Alternatively, an unmodified Zeiss LSM510 Confocal NLO microscope with 40× objective (NA 1.3) used successively shifted pinhole positions to collect image data from the lagging trail of the raster scan. Images obtained from two-photon excitation of a yeast (Schizosaccharomyces pombe) and a flagellate fish parasite (Spironucleus vortens), electroporated with Ru complex, indicated the intracellular location and magnitude of O 2 gradients, thus confirming the feasibility of optical mapping under different external O 2 concentrations. Both methods gave similar lifetimes for Ru complex phosphorescence under aerobic and anaerobic gas phases. Estimation of O 2 tensions within individual fibroblasts (human dermal fibroblast (HDF)) and mammary adenocarcinoma (MCF-7) cells was possible using epiphosphorescence microscopy. MCF-7 cells showed lower intracellular O 2 concentrations than HDF cells, possibly due to higher metabolic rates in the former. Future work should involve construction of higher resolution 3D maps of Ru coordinate

  5. Evaluation of two novel methods for assessing intracellular oxygen

    Science.gov (United States)

    Williams, Catrin F.; Kombrabail, M.; Vijayalakshmi, K.; White, Nick; Krishnamoorthy, G.; Lloyd, David

    2012-08-01

    The ability to resolve the spatio-temporal complexity of intracellular O2 distribution is the ‘Holy Grail’ of cellular physiology. In an effort to obtain a non-invasive approach of mapping intracellular O2 tensions, two methods of phosphorescent lifetime imaging microscopy were examined in the current study. These were picosecond time-resolved epiphosphorescence microscopy (single 0.5 µm focused spot) and two-photon confocal laser scanning microscopy with pinhole shifting. Both methods utilized nanoparticle-embedded Ru complex (45 nm diameter) as the phosphorescent probe, excited using pulsed outputs of Ti-sapphire Tsunami lasers (710-1050 nm). The former method used a 1 ps pulse width excitation beam with vertical polarization via a dichroic mirror (610 nm, XF43) and a 20× objective (NA 0.55, Nikon). Transmitted luminescence (1-2 × 104 counts s-1) was collected and time-correlated single photon counted decay times measured. Alternatively, an unmodified Zeiss LSM510 Confocal NLO microscope with 40× objective (NA 1.3) used successively shifted pinhole positions to collect image data from the lagging trail of the raster scan. Images obtained from two-photon excitation of a yeast (Schizosaccharomyces pombe) and a flagellate fish parasite (Spironucleus vortens), electroporated with Ru complex, indicated the intracellular location and magnitude of O2 gradients, thus confirming the feasibility of optical mapping under different external O2 concentrations. Both methods gave similar lifetimes for Ru complex phosphorescence under aerobic and anaerobic gas phases. Estimation of O2 tensions within individual fibroblasts (human dermal fibroblast (HDF)) and mammary adenocarcinoma (MCF-7) cells was possible using epiphosphorescence microscopy. MCF-7 cells showed lower intracellular O2 concentrations than HDF cells, possibly due to higher metabolic rates in the former. Future work should involve construction of higher resolution 3D maps of Ru coordinate complex lifetime

  6. Control of intracellular heme levels: Heme transporters and heme oxygenases

    OpenAIRE

    Khan, Anwar A.; Quigley, John G.

    2011-01-01

    Heme serves as a co-factor in proteins involved in fundamental biological processes including oxidative metabolism, oxygen storage and transport, signal transduction and drug metabolism. In addition, heme is important for systemic iron homeostasis in mammals. Heme has important regulatory roles in cell biology, yet excessive levels of intracellular heme are toxic; thus, mechanisms have evolved to control the acquisition, synthesis, catabolism and expulsion of cellular heme. Recently, a number...

  7. Intracellular Detection of Viral Transcription and Replication Using RNA FISH

    Science.gov (United States)

    2016-05-26

    Chapter 14. Intracellular detection of viral transcription and replication using RNA FISH i. Summary/Abstract Many hemorrhagic fever viruses...resolution. However, viral RNA tends to cluster in specific subcellular sites (e.g. viral replication factories). Thus while true single-molecule...assays [4]. Detection of viral RNA allows for in depth interrogation of the subcellular sites of viral replication and such experiments will help further

  8. NAD+-Glycohydrolase Promotes Intracellular Survival of Group A Streptococcus.

    Directory of Open Access Journals (Sweden)

    Onkar Sharma

    2016-03-01

    Full Text Available A global increase in invasive infections due to group A Streptococcus (S. pyogenes or GAS has been observed since the 1980s, associated with emergence of a clonal group of strains of the M1T1 serotype. Among other virulence attributes, the M1T1 clone secretes NAD+-glycohydrolase (NADase. When GAS binds to epithelial cells in vitro, NADase is translocated into the cytosol in a process mediated by streptolysin O (SLO, and expression of these two toxins is associated with enhanced GAS intracellular survival. Because SLO is required for NADase translocation, it has been difficult to distinguish pathogenic effects of NADase from those of SLO. To resolve the effects of the two proteins, we made use of anthrax toxin as an alternative means to deliver NADase to host cells, independently of SLO. We developed a novel method for purification of enzymatically active NADase fused to an amino-terminal fragment of anthrax toxin lethal factor (LFn-NADase that exploits the avid, reversible binding of NADase to its endogenous inhibitor. LFn-NADase was translocated across a synthetic lipid bilayer in vitro in the presence of anthrax toxin protective antigen in a pH-dependent manner. Exposure of human oropharyngeal keratinocytes to LFn-NADase in the presence of protective antigen resulted in cytosolic delivery of NADase activity, inhibition of protein synthesis, and cell death, whereas a similar construct of an enzymatically inactive point mutant had no effect. Anthrax toxin-mediated delivery of NADase in an amount comparable to that observed during in vitro infection with live GAS rescued the defective intracellular survival of NADase-deficient GAS and increased the survival of SLO-deficient GAS. Confocal microscopy demonstrated that delivery of LFn-NADase prevented intracellular trafficking of NADase-deficient GAS to lysosomes. We conclude that NADase mediates cytotoxicity and promotes intracellular survival of GAS in host cells.

  9. Potent Antibacterial Nanoparticles against Biofilm and Intracellular Bacteria

    OpenAIRE

    Mu, Haibo; Tang, Jiangjiang; Liu, Qianjin; Sun, Chunli; Wang, Tingting; Duan, Jinyou

    2016-01-01

    The chronic infections related to biofilm and intracellular bacteria are always hard to be cured because of their inherent resistance to both antimicrobial agents and host defenses. Herein we develop a facile approach to overcome the above conundrum through phosphatidylcholine-decorated Au nanoparticles loaded with gentamicin (GPA NPs). The nanoparticles were characterized by scanning electron microscopy (SEM), dynamic light scattering (DLS) and ultraviolet?visible (UV?vis) absorption spectra...

  10. Molecular evolution, intracellular organization, and the quinary structure of proteins.

    OpenAIRE

    McConkey, E H

    1982-01-01

    High-resolution two-dimensional polyacrylamide gel electrophoresis shows that at least half of 370 denatured polypeptides from hamster cells and human cells are indistinguishable in terms of isoelectric points and molecular weights. Molecular evolution may have been more conservative for this set of proteins than sequence studies on soluble proteins have implied. This may be a consequence of complexities of intracellular organization and the numerous macromolecular interactions in which most ...

  11. Sticholysin II-mediated cytotoxicity involves the activation of regulated intracellular responses that anticipates cell death.

    Science.gov (United States)

    Soto, Carmen; Bergado, Gretchen; Blanco, Rancés; Griñán, Tania; Rodríguez, Hermis; Ros, Uris; Pazos, Fabiola; Lanio, María Eliana; Hernández, Ana María; Álvarez, Carlos

    2018-02-13

    Sticholysin II (StII) is a pore-forming toxin of biomedical interest that belongs to the actinoporin protein family. Sticholysins are currently under examination as an active immunomodulating component of a vaccinal platform against tumoral cells and as a key element of a nucleic acids delivery system to cell cytosol. These proteins form pores in the plasma membrane leading to ion imbalance and cell lysis. However, the intracellular mechanisms triggered by actinoporins upon binding to membranes and its consequences for cell death are barely understood. Here, we have examined the cytotoxicity and intracellular responses induced by StII upon binding to human B-cell lymphoma Raji in vitro. StII cytotoxicity involves a functional actin cytoskeleton, induces cellular swelling, lysis and the concomitant release of cytosol content. In addition, StII induces calcium release mainly from the Endoplasmic Reticulum, activates Mitogen-Activated Protein Kinase ERK and impairs mitochondrial membrane potential. Furthermore, StII stimulates the expression of receptor interacting protein kinase 1 (RIP1), normally related to different forms of regulated cell death such as apoptosis and necroptosis. In correspondence, necrostatin-1, an inhibitor of this kinase, reduces StII cytotoxicity. However, the mechanism of cell death activated by StII does not involve caspases activation, typical molecular features of apoptosis and pyroptosis. Our results suggest that, beyond pore-formation and cell lysis, StII-induced cytotoxicity could involve other regulated intracellular mechanisms connected to RIP1-MEK1/2 -ERK1/2- pathways. This opens new perspectives and challenges the general point of view that these toxins induce a completely unregulated mechanism of necrotic cell death. This study contributes to a better understanding of the molecular mechanisms involved in toxin-cell interaction and the implications for cell functioning, with connotation for the exploitations of these toxins in

  12. Muscarinic acetylcholine receptor subtypes in cerebral cortex and hippocampus.

    Science.gov (United States)

    Volpicelli, Laura A; Levey, Allan I

    2004-01-01

    The M1, M2 and M4 subtypes of mAChRs are the predominant receptors in the CNS. These receptors activate a multitude of signaling pathways important for modulating neuronal excitability, synaptic plasticity and feedback regulation of ACh release. In addition, novel functions mediated by mAChRs are currently being discovered. These studies are greatly facilitated by the recent development of subtype selective toxins and mice lacking individual mAChR genes. Studies in cell culture and the rodent brain demonstrate that mAChR internalization and intracellular trafficking is an important component of mAChR regulation. Characterizing mAChR intracellular trafficking could help facilitate the development of selective mAChR ligands. For example, a selective M1 agonist would cause a shift in the distribution of M1 from the cell surface to an intracellular distribution, while M2 and M4 would remain on the cell surface. Characterizing mAChR intracellular trafficking is also important for understanding the cellular mechanisms that regulate mAChR cell surface expression and signaling. Furthermore, intracellular trafficking has recently been demonstrated to play a role in the development of tolerance to drugs (Whistler et al., 1999; He et al., 2002). Because individual mAChR subtypes are novel targets for treatments of diseases such as Alzheimer's disease and schizophrenia, understanding the mechanisms that regulate mAChR signaling and intracellular trafficking following acute and chronic stimulation might lead to the development of rational strategies.

  13. Forced resurgence and targeting of intracellular uropathogenic Escherichia coli reservoirs.

    Directory of Open Access Journals (Sweden)

    Matthew G Blango

    Full Text Available Intracellular quiescent reservoirs of uropathogenic Escherichia coli (UPEC, which can seed the bladder mucosa during the acute phase of a urinary tract infection (UTI, are protected from antibiotic treatments and are extremely difficult to eliminate. These reservoirs are a potential source for recurrent UTIs that affect millions annually. Here, using murine infection models and the bladder cell exfoliant chitosan, we demonstrate that intracellular UPEC populations shift within the stratified layers of the urothelium during the course of a UTI. Following invasion of the terminally differentiated superficial layer of epithelial cells that line the bladder lumen, UPEC can multiply and disseminate, eventually establishing reservoirs within underlying immature host cells. If given access, UPEC can invade the superficial and immature bladder cells equally well. As infected immature host cells differentiate and migrate towards the apical surface of the bladder, UPEC can reinitiate growth and discharge into the bladder lumen. By inducing the exfoliation of the superficial layers of the urothelium, chitosan stimulates rapid regenerative processes and the reactivation and efflux of quiescent intracellular UPEC reservoirs. When combined with antibiotics, chitosan treatment significantly reduces bacterial loads within the bladder and may therefore be of therapeutic value to individuals with chronic, recurrent UTIs.

  14. Purine Biosynthesis Metabolically Constrains Intracellular Survival of Uropathogenic Escherichia coli.

    Science.gov (United States)

    Shaffer, Carrie L; Zhang, Ellisa W; Dudley, Anne G; Dixon, Beverly R E A; Guckes, Kirsten R; Breland, Erin J; Floyd, Kyle A; Casella, Daniel P; Algood, Holly M Scott; Clayton, Douglass B; Hadjifrangiskou, Maria

    2017-01-01

    The ability to de novo synthesize purines has been associated with the intracellular survival of multiple bacterial pathogens. Uropathogenic Escherichia coli (UPEC), the predominant cause of urinary tract infections, undergoes a transient intracellular lifestyle during which bacteria clonally expand into multicellular bacterial communities within the cytoplasm of bladder epithelial cells. Here, we characterized the contribution of the conserved de novo purine biosynthesis-associated locus cvpA-purF to UPEC pathogenesis. Deletion of cvpA-purF, or of purF alone, abolished de novo purine biosynthesis but did not impact bacterial adherence properties in vitro or in the bladder lumen. However, upon internalization by bladder epithelial cells, UPEC deficient in de novo purine biosynthesis was unable to expand into intracytoplasmic bacterial communities over time, unless it was extrachromosomally complemented. These findings indicate that UPEC is deprived of purine nucleotides within the intracellular niche and relies on de novo purine synthesis to meet this metabolic requirement. Copyright © 2016 American Society for Microbiology.

  15. Correlation between intracellular accumulation of peptidoglycan precursors and streptomycin formation

    International Nuclear Information System (INIS)

    Nimi, Osamu; Kawashima, Hiroki; Sugiyama, Masanori; Nomi, Ryosaku

    1984-01-01

    When the mycelium of Streptomyces HUT 6037 was suspended in 0.5% NaCl solution containing 14 C-glucosamine, peptidoglycan precursors accumulated in the cells. While UDP-N-acetylglucosamine accumulated in the largest amount among the precursors, extracellularly added and intracellularly accumulated UDP-N-acetylglucosamine were not used to synthesize streptomycin and were probably used for peptidoglycan formation. On the other hand, correlation was recognized between accumulation of glucosamine-6-phosphate (GlcN-6P) and streptomycin formation. Addition of an inhibitor of peptidoglycan synthesis such as enduracidin, vancomycin or cycloserine to a mycelium-suspended culture changed the ratio of accumulated peptidoglycan precursors. When streptomycin formation was stimulated by addition of enduracidin or vancomycin, intracellular GlcN-6P remarkably increased and then decreased rapidly. On the contrary, when cycloserine was added to the culture, no increase of GlcN-6P was observed and streptomycin formation was not stimulated. These results suggest that an increase in the intracellular concentration of GlcN-6P is required for activation or induction of the system for utilizing GlcN-6P for streptomycin formation. (author)

  16. New intracellular activities of matrix metalloproteinases shine in the moonlight.

    Science.gov (United States)

    Jobin, Parker G; Butler, Georgina S; Overall, Christopher M

    2017-11-01

    Adaption of a single protein to perform multiple independent functions facilitates functional plasticity of the proteome allowing a limited number of protein-coding genes to perform a multitude of cellular processes. Multifunctionality is achievable by post-translational modifications and by modulating subcellular localization. Matrix metalloproteinases (MMPs), classically viewed as degraders of the extracellular matrix (ECM) responsible for matrix protein turnover, are more recently recognized as regulators of a range of extracellular bioactive molecules including chemokines, cytokines, and their binders. However, growing evidence has convincingly identified select MMPs in intracellular compartments with unexpected physiological and pathological roles. Intracellular MMPs have both proteolytic and non-proteolytic functions, including signal transduction and transcription factor activity thereby challenging their traditional designation as extracellular proteases. This review highlights current knowledge of subcellular location and activity of these "moonlighting" MMPs. Intracellular roles herald a new era of MMP research, rejuvenating interest in targeting these proteases in therapeutic strategies. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Confocal microscopy for intracellular co-localization of proteins.

    Science.gov (United States)

    Miyashita, Toshiyuki

    2015-01-01

    Confocal laser scanning microscopy is the best method to visualize intracellular co-localization of proteins in intact cells. Because of the point scan/pinhole detection system, light contribution from the neighborhood of the scanning spot in the specimen can be eliminated, allowing high Z-axis resolution. Fluorescence detection by sensitive photomultiplier tubes allows the usage of filters with a narrow bandpath, resulting in minimal cross-talk (overlap) between two spectra. This is particularly important in demonstrating co-localization of proteins with multicolor labeling. Here, the methods outlining the detection of transiently expressed tagged proteins and the detection of endogenous proteins are described. Ideally, the intracellular co-localization of two endogenous proteins should be demonstrated. However, when antibodies raised against the protein of interest are unavailable for immunofluorescence or the available cell lines do not express the protein of interest sufficiently enough for immunofluorescence, an alternative method is to transfect cells with expression plasmids that encode tagged proteins and stain the cells with anti-tag antibodies. However, it should be noted that the tagging of proteins of interest or their overexpression could potentially alter the intracellular localization or the function of the target protein.

  18. A first step toward liposome-mediated intracellular bacteriophage therapy.

    Science.gov (United States)

    Nieth, Anita; Verseux, Cyprien; Barnert, Sabine; Süss, Regine; Römer, Winfried

    2015-01-01

    The emergence of antibiotic-resistant bacteria presents a severe challenge to medicine and public health. While bacteriophage therapy is a promising alternative to traditional antibiotics, the general inability of bacteriophages to penetrate eukaryotic cells limits their use against resistant bacteria, causing intracellular diseases like tuberculosis. Bacterial vectors show some promise in carrying therapeutic bacteriophages into cells, but also bring a number of risks like an overload of bacterial antigens or the acquisition of virulence genes from the pathogen. As a first step in the development of a non-bacterial vector for bacteriophage delivery into pathogen-infected cells, we attempted to encapsulate bacteriophages into liposomes. Here we report effective encapsulation of the model bacteriophage λeyfp and the mycobacteriophage TM4 into giant liposomes. Furthermore, we show that liposome-associated bacteriophages are taken up into eukaryotic cells more efficiently than free bacteriophages. These are important milestones in the development of an intracellular bacteriophage therapy that might be useful in the fight against multi-drug-resistant intracellular pathogens like Mycobacterium tuberculosis.

  19. Potent Antibacterial Nanoparticles against Biofilm and Intracellular Bacteria

    Science.gov (United States)

    Mu, Haibo; Tang, Jiangjiang; Liu, Qianjin; Sun, Chunli; Wang, Tingting; Duan, Jinyou

    2016-01-01

    The chronic infections related to biofilm and intracellular bacteria are always hard to be cured because of their inherent resistance to both antimicrobial agents and host defenses. Herein we develop a facile approach to overcome the above conundrum through phosphatidylcholine-decorated Au nanoparticles loaded with gentamicin (GPA NPs). The nanoparticles were characterized by scanning electron microscopy (SEM), dynamic light scattering (DLS) and ultraviolet-visible (UV-vis) absorption spectra which demonstrated that GPA NPs with a diameter of approximately 180 nm were uniform. The loading manner and release behaviors were also investigated. The generated GPA NPs maintained their antibiotic activities against planktonic bacteria, but more effective to damage established biofilms and inhibited biofilm formation of pathogens including Gram-positive and Gram-negative bacteria. In addition, GPA NPs were observed to be nontoxic to RAW 264.7 cells and readily engulfed by the macrophages, which facilitated the killing of intracellular bacteria in infected macrophages. These results suggested GPA NPs might be a promising antibacterial agent for effective treatment of chronic infections due to microbial biofilm and intracellular bacteria.

  20. Intracellular Hg(0) Oxidation in Desulfovibrio desulfuricans ND132.

    Science.gov (United States)

    Wang, Yuwei; Schaefer, Jeffra K; Mishra, Bhoopesh; Yee, Nathan

    2016-10-03

    The disposal of elemental mercury (Hg(0)) wastes in mining and manufacturing areas has caused serious soil and groundwater contamination issues. Under anoxic conditions, certain anaerobic bacteria can oxidize dissolved elemental mercury and convert the oxidized Hg to neurotoxic methylmercury. In this study, we conducted experiments with the Hg-methylating bacterium Desulfovibrio desulfuricans ND132 to elucidate the role of cellular thiols in anaerobic Hg(0) oxidation. The concentrations of cell-surface and intracellular thiols were measured, and specific fractions of D. desulfuricans ND132 were examined for Hg(0) oxidation activity and analyzed with extended X-ray absorption fine structure (EXAFS) spectroscopy. The experimental data indicate that intracellular thiol concentrations are approximately six times higher than those of the cell wall. Cells reacted with a thiol-blocking reagent were severely impaired in Hg(0) oxidation activity. Spheroplasts lacking cell walls rapidly oxidized Hg(0) to Hg(II), while cell wall fragments exhibited low reactivity toward Hg(0). EXAFS analysis of spheroplast samples revealed that multiple different forms of Hg-thiols are produced by the Hg(0) oxidation reaction and that the local coordination environment of the oxidized Hg changes with reaction time. The results of this study indicate that Hg(0) oxidation in D. desulfuricans ND132 is an intracellular process that occurs by reaction with thiol-containing molecules.

  1. The association of metabotropic glutamate receptor type 5 with the neuronal Ca2+-binding protein 2 modulates receptor function.

    Science.gov (United States)

    Canela, Laia; Fernández-Dueñas, Víctor; Albergaria, Catarina; Watanabe, Masahiko; Lluís, Carme; Mallol, Josefa; Canela, Enric I; Franco, Rafael; Luján, Rafael; Ciruela, Francisco

    2009-10-01

    Metabotropic glutamate (mGlu) receptors mediate in part the CNS effects of glutamate. These receptors interact with a large array of intracellular proteins in which the final role is to regulate receptor function. Here, using co-immunoprecipitation and pull-down experiments we showed a close and specific interaction between mGlu(5) receptor and NECAB2 in both transfected human embryonic kidney cells and rat hippocampus. Interestingly, in pull-down experiments increasing concentrations of calcium drastically reduced the ability of these two proteins to interact, suggesting that NECAB2 binds to mGlu(5) receptor in a calcium-regulated manner. Immunoelectron microscopy detection of NECAB2 and mGlu(5) receptor in the rat hippocampal formation indicated that both proteins are codistributed in the same subcellular compartment of pyramidal cells. In addition, the NECAB2/mGlu(5) receptor interaction regulated mGlu(5b)-mediated activation of both inositol phosphate accumulation and the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. Overall, these findings indicate that NECAB2 by its physical interaction with mGlu(5b) receptor modulates receptor function.

  2. The ectodomain of Toll-like receptor 9 is cleaved to generate a functional receptor.

    Science.gov (United States)

    Ewald, Sarah E; Lee, Bettina L; Lau, Laura; Wickliffe, Katherine E; Shi, Guo-Ping; Chapman, Harold A; Barton, Gregory M

    2008-12-04

    Mammalian Toll-like receptors (TLRs) 3, 7, 8 and 9 initiate immune responses to infection by recognizing microbial nucleic acids; however, these responses come at the cost of potential autoimmunity owing to inappropriate recognition of self nucleic acids. The localization of TLR9 and TLR7 to intracellular compartments seems to have a role in facilitating responses to viral nucleic acids while maintaining tolerance to self nucleic acids, yet the cell biology regulating the transport and localization of these receptors remains poorly understood. Here we define the route by which TLR9 and TLR7 exit the endoplasmic reticulum and travel to endolysosomes in mouse macrophages and dendritic cells. The ectodomains of TLR9 and TLR7 are cleaved in the endolysosome, such that no full-length protein is detectable in the compartment where ligand is recognized. Notably, although both the full-length and cleaved forms of TLR9 are capable of binding ligand, only the processed form recruits MyD88 on activation, indicating that this truncated receptor, rather than the full-length form, is functional. Furthermore, conditions that prevent receptor proteolysis, including forced TLR9 surface localization, render the receptor non-functional. We propose that ectodomain cleavage represents a strategy to restrict receptor activation to endolysosomal compartments and prevent TLRs from responding to self nucleic acids.

  3. Recent Progress in Understanding Subtype Specific Regulation of NMDA Receptors by G Protein Coupled Receptors (GPCRs

    Directory of Open Access Journals (Sweden)

    Kai Yang

    2014-02-01

    Full Text Available G Protein Coupled Receptors (GPCRs are the largest family of receptors whose ligands constitute nearly a third of prescription drugs in the market. They are widely involved in diverse physiological functions including learning and memory. NMDA receptors (NMDARs, which belong to the ionotropic glutamate receptor family, are likewise ubiquitously expressed in the central nervous system (CNS and play a pivotal role in learning and memory. Despite its critical contribution to physiological and pathophysiological processes, few pharmacological interventions aimed directly at regulating NMDAR function have been developed to date. However, it is well established that NMDAR function is precisely regulated by cellular signalling cascades recruited downstream of G protein coupled receptor (GPCR stimulation. Accordingly, the downstream regulation of NMDARs likely represents an important determinant of outcome following treatment with neuropsychiatric agents that target selected GPCRs. Importantly, the functional consequence of such regulation on NMDAR function varies, based not only on the identity of the GPCR, but also on the cell type in which relevant receptors are expressed. Indeed, the mechanisms responsible for regulating NMDARs by GPCRs involve numerous intracellular signalling molecules and regulatory proteins that vary from one cell type to another. In the present article, we highlight recent findings from studies that have uncovered novel mechanisms by which selected GPCRs regulate NMDAR function and consequently NMDAR-dependent plasticity.

  4. Real-time visualization of intracellular hydrodynamics in single living cells

    NARCIS (Netherlands)

    Potma, Eric O.; Boeij, Wim P. de; Haastert, Peter J.M. van; Wiersma, Douwe A.

    2001-01-01

    Intracellular water concentrations in single living cells were visualized by nonlinear coherent anti-Stokes Raman scattering (CARS) microscopy. In combination with isotopic exchange measurements, CARS microscopy allowed the real-time observation of transient intracellular hydrodynamics at a high

  5. Cloning and expression of a widely expressed receptor tyrosine phosphatase

    DEFF Research Database (Denmark)

    Sap, J; D'Eustachio, P; Givol, D

    1990-01-01

    and Bmp-2a loci. The corresponding mRNA (3.0 kilobases) is expressed in most murine tissues and most abundantly expressed in brain and kidney. Antibodies against a synthetic peptide of R-PTP-alpha identified a 130-kDa protein in cells transfected with the R-PTP-alpha cDNA.......We describe the identification of a widely expressed receptor-type (transmembrane) protein tyrosine phosphatase (PTPase; EC 3.1.3.48). Screening of a mouse brain cDNA library under low-stringency conditions with a probe encompassing the intracellular (phosphatase) domain of the CD45 lymphocyte...... antigen yielded cDNA clones coding for a 794-amino acid transmembrane protein [hereafter referred to as receptor protein tyrosine phosphatase alpha (R-PTP-alpha)] with an intracellular domain displaying clear homology to the catalytic domains of CD45 and LAR (45% and 53%, respectively). The 142-amino acid...

  6. Intracellular interactions of umeclidinium and vilanterol in human airway smooth muscle

    Directory of Open Access Journals (Sweden)

    Shaikh N

    2017-06-01

    Full Text Available Nooreen Shaikh,1,2 Malcolm Johnson,3 David A Hall,4 Kian Fan Chung,1,2 John H Riley,3 Sally Worsley,5 Pankaj K Bhavsar1,2 1Experimental Studies, National Heart and Lung Institute, Imperial College London, 2Biomedical Research Unit, Royal Brompton and Harefield NHS Trust, London, 3Respiratory Global Franchise, GlaxoSmithKline, Uxbridge, 4Fibrosis and Lung Injury Development Planning Unit, GlaxoSmithKline, Stevenage, 5Respiratory Research & Development, GlaxoSmithKline, Uxbridge, UK Background: Intracellular mechanisms of action of umeclidinium (UMEC, a long-acting muscarinic receptor antagonist, and vilanterol (VI, a long-acting β2-adrenoceptor (β2R agonist, were investigated in target cells: human airway smooth-muscle cells (ASMCs. Materials and methods: ASMCs from tracheas of healthy lung-transplant donors were treated with VI, UMEC, UMEC and VI combined, or control compounds (salmeterol, propranolol, ICI 118.551, or methacholine [MCh]. Cyclic adenosine monophosphate (cAMP was measured using an enzyme-linked immunosorbent assay, intracellular free calcium ([Ca2+]i using a fluorescence assay, and regulator of G-protein signaling 2 (RGS2 messenger RNA using real-time quantitative polymerase chain reaction. Results: VI and salmeterol (10–12–10–6 M induced cAMP production from ASMCs in a concentration-dependent manner, which was greater for VI at all concentrations. β2R antagonism by propranolol or ICI 118.551 (10–12–10–4 M resulted in concentration-dependent inhibition of VI-induced cAMP production, and ICI 118.551 was more potent. MCh (5×10–6 M, 30 minutes attenuated VI-induced cAMP production (P<0.05, whereas pretreatment with UMEC (10–8 M, 1 hour restored the magnitude of VI-induced cAMP production. ASMC stimulation with MCh (10–11–5×10–6 M resulted in a concentration-dependent increase in [Ca2+]i, which was attenuated with UMEC pretreatment. Reduction of MCh-induced [Ca2+]i release was greater with UMEC + VI versus

  7. Two zebrafish G2A homologs activate multiple intracellular signaling pathways in acidic environment

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-01-01

    Human G2A is activated by various stimuli such as lysophosphatidylcholine (LPC), 9-hydroxyoctadecadienoic acid (9-HODE), and protons. The receptor is coupled to multiple intracellular signaling pathways, including the G{sub s}-protein/cAMP/CRE, G{sub 12/13}-protein/Rho/SRE, and G{sub q}-protein/phospholipase C/NFAT pathways. In the present study, we examined whether zebrafish G2A homologs (zG2A-a and zG2A-b) could respond to these stimuli and activate multiple intracellular signaling pathways. We also examined whether histidine residue and basic amino acid residue in the N-terminus of the homologs also play roles similar to those played by human G2A residues if the homologs sense protons. We found that the zG2A-a showed the high CRE, SRE, and NFAT activities, however, zG2A-b showed only the high SRE activity under a pH of 8.0. Extracellular acidification from pH 7.4 to 6.3 ameliorated these activities in zG2A-a-expressing cells. On the other hand, acidification ameliorated the SRE activity but not the CRE and NFAT activities in zG2A-b-expressing cells. LPC or 9-HODE did not modify any activity of either homolog. The substitution of histidine residue at the 174{sup th} position from the N-terminus of zG2A-a to asparagine residue attenuated proton-induced CRE and NFAT activities but not SRE activity. The substitution of arginine residue at the 32nd position from the N-terminus of zG2A-a to the alanine residue also attenuated its high and the proton-induced CRE and NFAT activities. On the contrary, the substitution did not attenuate SRE activity. The substitution of the arginine residue at the 10th position from the N-terminus of zG2A-b to the alanine residue also did not attenuate its high or the proton-induced SRE activity. These results indicate that zebrafish G2A homologs were activated by protons but not by LPC and 9-HODE, and the activation mechanisms of the homologs were similar to those of human G2A. - Highlights: • Zebrafish two G2A homologs are proton

  8. Intracellular diffusion restrictions in isolated cardiomyocytes from rainbow trout

    Directory of Open Access Journals (Sweden)

    Birkedal Rikke

    2009-12-01

    Full Text Available Abstract Background Restriction of intracellular diffusion of adenine nucleotides has been studied intensively on adult rat cardiomyocytes. However, their cause and role in vivo is still uncertain. Intracellular membrane structures have been suggested to play a role. We therefore chose to study cardiomyocytes from rainbow trout (Oncorhynchus mykiss, which are thinner and have fewer intracellular membrane structures than adult rat cardiomyocytes. Previous studies suggest that trout permeabilized cardiac fibers also have diffusion restrictions. However, results from fibers may be affected by incomplete separation of the cells. This is avoided when studying permeabilized, isolated cardiomyocytes. The aim of this study was to verify the existence of diffusion restrictions in trout cardiomyocytes by comparing ADP-kinetics of mitochondrial respiration in permeabilized fibers, permeabilized cardiomyocytes and isolated mitochondria from rainbow trout heart. Experiments were performed at 10, 15 and 20°C in the absence and presence of creatine. Results Trout cardiomyocytes hypercontracted in the solutions used for mammalian cardiomyocytes. We developed a new solution in which they retained their shape and showed stable steady state respiration rates throughout an experiment. The apparent ADP-affinity of permeabilized cardiomyocytes was different from that of fibers. It was higher, independent of temperature and not increased by creatine. However, it was still about ten times lower than in isolated mitochondria. Conclusions The differences between fibers and cardiomyocytes suggest that results from trout heart fibers were affected by incomplete separation of the cells. However, the lower ADP-affinity of cardiomyocytes compared to isolated mitochondria indicate that intracellular diffusion restrictions are still present in trout cardiomyocytes despite their lower density of intracellular membrane structures. The lack of a creatine effect indicates that

  9. Molecular cloning and pharmacology of functionally distinct isoforms of the human histamine H(3) receptor

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Goodman, M W; Burstein, E S

    2002-01-01

    The pharmacology of histamine H(3) receptors suggests the presence of distinct receptor isoforms or subtypes. We herein describe multiple, functionally distinct, alternatively spliced isoforms of the human H(3) receptor. Combinatorial splicing at three different sites creates at least six distinct...... receptor isoforms, of which isoforms 1, 2, and 4, encode functional proteins. Detailed pharmacology on isoforms 1 (unspliced receptor), and 2 (which has an 80 amino acid deletion within the third intracellular loop of the protein) revealed that both isoforms displayed robust responses to a series of known...... revealed a rank order of potency at both isoforms of clobenpropit>iodophenpropit>thioperamide, and these drugs are fivefold less potent at isoform 2 than isoform 1. To further explore the pharmacology of H(3) receptor function, we screened 150 clinically relevant neuropsychiatric drugs for H(3) receptor...

  10. Dimers of G-Protein Coupled Receptors as Versatile Storage and Response Units

    Directory of Open Access Journals (Sweden)

    Michael S. Parker

    2014-03-01

    Full Text Available The status and use of transmembrane, extracellular and intracellular domains in oligomerization of heptahelical G-protein coupled receptors (GPCRs are reviewed and for transmembrane assemblies also supplemented by new experimental evidence. The transmembrane-linked GPCR oligomers typically have as the minimal unit an asymmetric ~180 kDa pentamer consisting of receptor homodimer or heterodimer and a G-protein αβγ subunit heterotrimer. With neuropeptide Y (NPY receptors, this assembly is converted to ~90 kDa receptor monomer-Gα complex by receptor and Gα agonists, and dimers/heteropentamers are depleted by neutralization of Gαi subunits by pertussis toxin. Employing gradient centrifugation, quantification and other characterization of GPCR dimers at the level of physically isolated and identified heteropentamers is feasible with labeled agonists that do not dissociate upon solubilization. This is demonstrated with three neuropeptide Y (NPY receptors and could apply to many receptors that use large peptidic agonists.

  11. Estrogen-induced disruption of intracellular iron metabolism leads to oxidative stress, membrane damage, and cell cycle arrest in MCF-7 cells.

    Science.gov (United States)

    Bajbouj, Khuloud; Shafarin, Jasmin; Abdalla, Maher Y; Ahmad, Iman M; Hamad, Mawieh

    2017-10-01

    It is well established that several forms of cancer associate with significant iron overload. Recent studies have suggested that estrogen (E2) disrupts intracellular iron homeostasis by reducing hepcidin synthesis and maintaining ferroportin integrity. Here, the ability of E2 to alter intracellular iron status and cell growth potential was investigated in MCF-7 cells treated with increasing concentrations of E2. Treated cells were assessed for intracellular iron status, the expression of key proteins involved in iron metabolism, oxidative stress, cell survival, growth, and apoptosis. E2 treatment resulted in a significant reduction in hepcidin expression and a significant increase in hypoxia-inducible factor 1 alpha, ferroportin, transferrin receptor, and ferritin expression; a transient decrease in labile iron pool; and a significant increase in total intracellular iron content mainly at 20 nM/48 h E2 dose. Treated cells also showed increased total glutathione and oxidized glutathione levels, increased superoxide dismutase activity, and increased hemoxygenase 1 expression. Treatment with E2 at 20 nM for 48 h resulted in a significant reduction in cell growth (0.35/1 migration rate) and decreased cell survival (iron metabolism and precipitates adverse effects concerning cell viability, membrane integrity, and growth potential.

  12. DMPD: Intracellular DNA sensors in immunity. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18573338 Intracellular DNA sensors in immunity. Takeshita F, Ishii KJ. Curr Opin Im...munol. 2008 Aug;20(4):383-8. Epub 2008 Jun 23. (.png) (.svg) (.html) (.csml) Show Intracellular DNA sensors ...in immunity. PubmedID 18573338 Title Intracellular DNA sensors in immunity. Authors Takeshita F, Ishii KJ. P

  13. The anti-asthmatic drug pranlukast suppresses the delayed-phase vomiting and reverses intracellular indices of emesis evoked by cisplatin in the least shrew (Cryptotis parva).

    Science.gov (United States)

    Darmani, Nissar A; Chebolu, Seetha; Zhong, Weixia; Kim, William D; Narlesky, Matthew; Adams, Joia; Dong, Fanglong

    2017-08-15

    The introduction of second generation serotonin 5-HT 3 receptor (5-HT 3 ) antagonist palonosetron combined with long-acting substance P neurokinin NK 1 receptor (NK 1 ) antagonists (e.g. netupitant) has substantially improved antiemetic therapy against early- and delayed-phases of emesis caused by highly emetogenic chemotherapeutics such as cisplatin. However, the improved efficacy comes at a cost that many patients cannot afford. We introduce a new class of antiemetic, the antiasthmatic leukotriene CysLT1 receptor antagonist pranlukast for the suppression of cisplatin-evoked vomiting. Pranlukast (10mg/kg) by itself significantly reduced the mean frequency of vomits (70%) and fully protected least shrews from vomiting (46%) during the delayed-phase of cisplatin (10mg/kg)-evoked vomiting. Although, pranlukast tended to substantially reduce both the mean frequency of vomits and the number of shrews vomiting during the early-phase, these reductions failed to attain significance. When combined with a first (tropisetron)- or a second (palonosetron)-generation 5-HT 3 receptor antagonist, pranlukast potentiated their antiemetic efficacy during both phases of vomiting. In addition, pranlukast by itself prevented several intracellular signal markers of cisplatin-evoked delayed-vomiting such as phosphorylation of ERK1/2 and PKA. When pranlukast was combined with either palonosetron or tropisetron, these combinations suppressed the evoked phosphorylation of: i) ERK1/2 during both acute- and delayed-phase, ii) PKCα/β at the peak acute-phase, and iii) PKA at the peak delayed-phase. The current and our published findings suggest that overall behavioral and intracellular signaling effects of pranlukast via blockade of CysLT1 receptors generally appear to be similar to the NK 1 receptor antagonist netupitant with some differences. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Role of sigma-1 receptors in neurodegenerative diseases

    Directory of Open Access Journals (Sweden)

    Linda Nguyen

    2015-01-01

    Full Text Available Neurodegenerative diseases with distinct genetic etiologies and pathological phenotypes appear to share common mechanisms of neuronal cellular dysfunction, including excitotoxicity, calcium dysregulation, oxidative damage, ER stress and mitochondrial dysfunction. Glial cells, including microglia and astrocytes, play an increasingly recognized role in both the promotion and prevention of neurodegeneration. Sigma receptors, particularly the sigma-1 receptor subtype, which are expressed in both neurons and glia of multiple regions within the central nervous system, are a unique class of intracellular proteins that can modulate many biological mechanisms associated with neurodegeneration. These receptors therefore represent compelling putative targets for pharmacologically treating neurodegenerative disorders. In this review, we provide an overview of the biological mechanisms frequently associated with neurodegeneration, and discuss how sigma-1 receptors may alter these mechanisms to preserve or restore neuronal function. In addition, we speculate on their therapeutic potential in the treatment of various neurodegenerative disorders.

  15. KDEL Receptors Assist Dengue Virus Exit from the Endoplasmic Reticulum

    Directory of Open Access Journals (Sweden)

    Ming Yuan Li

    2015-03-01

    Full Text Available Membrane receptors at the surface of target cells are key host factors for virion entry; however, it is unknown whether trafficking and secretion of progeny virus requires host intracellular receptors. In this study, we demonstrate that dengue virus (DENV interacts with KDEL receptors (KDELR, which cycle between the ER and Golgi apparatus, for vesicular transport from ER to Golgi. Depletion of KDELR by siRNA reduced egress of both DENV progeny and recombinant subviral particles (RSPs. Coimmunoprecipitation of KDELR with dengue structural protein prM required three positively charged residues at the N terminus, whose mutation disrupted protein interaction and inhibited RSP transport from the ER to the Golgi. Finally, siRNA depletion of class II Arfs, which results in KDELR accumulation in the Golgi, phenocopied results obtained with mutagenized prME and KDELR knockdown. Our results have uncovered a function for KDELR as an internal receptor involved in DENV trafficking.

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

    Science.gov (United States)

    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

  17. Molecular mechanisms of glucocorticoid receptor signaling Mecanismos moleculares de señalización del receptor de glucocorticoides

    OpenAIRE

    Marta Labeur; Florian Holsboer

    2010-01-01

    This review highlights the most recent findings on the molecular mechanisms of the glucocorticoid receptor (GR). Most effects of glucocorticoids are mediated by the intracellular GR which is present in almost every tissue and controls transcriptional activation via direct and indirect mechanisms. Nevertheless the glucocorticoid responses are tissue -and gene- specific. GR associates selectively with corticosteroid ligands produced in the adrenal gland in response to changes of humoral homeost...

  18. Interactions between Calmodulin, Adenosine A2A, and Dopamine D2 Receptors*

    Science.gov (United States)

    Navarro, Gemma; Aymerich, Marisol S.; Marcellino, Daniel; Cortés, Antoni; Casadó, Vicent; Mallol, Josefa; Canela, Enric I.; Agnati, Luigi; Woods, Amina S.; Fuxe, Kjell; Lluís, Carmen; Lanciego, Jose Luis; Ferré, Sergi; Franco, Rafael

    2009-01-01

    The Ca2+-binding protein calmodulin (CaM) has been shown to bind directly to cytoplasmic domains of some G protein-coupled receptors, including the dopamine D2 receptor. CaM binds to the N-terminal portion of the long third intracellular loop of the D2 receptor, within an Arg-rich epitope that is also involved in the binding to Gi/o proteins and to the adenosine A2A receptor, with the formation of A2A-D2 receptor heteromers. In the present work, by using proteomics and bioluminescence resonance energy transfer (BRET) techniques, we provide evidence for the binding of CaM to the A2A receptor. By using BRET and sequential resonance energy transfer techniques, evidence was obtained for CaM-A2A-D2 receptor oligomerization. BRET competition experiments indicated that, in the A2A-D2 receptor heteromer, CaM binds preferentially to a proximal C terminus epitope of the A2A receptor. Furthermore, Ca2+ was found to induce conformational changes in the CaM-A2A-D2 receptor oligomer and to selectively modulate A2A and D2 receptor-mediated MAPK signaling in the A2A-D2 receptor heteromer. These results may have implications for basal ganglia disorders, since A2A-D2 receptor heteromers are being considered as a target for anti-parkinsonian agents. PMID:19632986

  19. Receptor activity-independent recruitment of βarrestin2 reveals specific signalling modes

    Science.gov (United States)

    Terrillon, Sonia; Bouvier, Michel

    2004-01-01

    The roles of βarrestins in regulating G protein coupling and receptor endocytosis following agonist stimulation of G protein-coupled receptors are well characterised. However, their ability to act on their own as direct modulators or activators of signalling remains poorly characterised. Here, βarrestin2 intrinsic signalling properties were assessed by forcing the recruitment of this accessory protein to vasopressin V1a or V2 receptors independently of agonist-promoted activation of the receptors. Such induction of a stable interaction with βarrestin2 initiated receptor endocytosis leading to intracellular accumulation of the βarrestin/receptor complexes. Interestingly, βarrestin2 association to a single receptor protomer was sufficient to elicit receptor dimer internalisation. In addition to recapitulating βarrestin2 classical actions on receptor trafficking, the receptor activity-independent recruitment of βarrestin2 activated the extracellular signal-regulated kinases. In the latter case, recruitment to the receptor itself was not required since kinase activation could be mediated by βarrestin2 translocation to the plasma membrane in the absence of any interacting receptor. These data demonstrate that βarrestin2 can act as a ‘bonafide' signalling molecule even in the absence of activated receptor. PMID:15385966

  20. Reduced levels of intracellular calcium releasing in spermatozoa from asthenozoospermic patients

    Science.gov (United States)

    Espino, Javier; Mediero, Matías; Lozano, Graciela M; Bejarano, Ignacio; Ortiz, Águeda; García, Juan F; Pariente, José A; Rodríguez, Ana B

    2009-01-01

    Background Asthenozoospermia is one of the most common findings present in infertile males characterized by reduced or absent sperm motility, but its aetiology remains unknown in most cases. In addition, calcium is one of the most important ions regulating sperm motility. In this study we have investigated the progesterone-evoked intracellular calcium signal in ejaculated spermatozoa from men with normospermia or asthenozoospermia. Methods Human ejaculates were obtained from healthy volunteers and asthenospermic men by masturbation after 4–5 days of abstinence. For determination of cytosolic free calcium concentration, spermatozoa were loaded with the fluorescent ratiometric calcium indicator Fura-2. Results Treatment of spermatozoa from normospermic men with 20 micromolar progesterone plus 1 micromolar thapsigargin in a calcium free medium induced a typical transient increase in cytosolic free calcium concentration due to calcium release from internal stores. Similar results were obtained when spermatozoa were stimulated with progesterone alone. Subsequent addition of calcium to the external medium evoked a sustained elevation in cytosolic free calcium concentration indicative of capacitative calcium entry. However, when progesterone plus thapsigargin were administered to spermatozoa from patients with asthenozoospermia, calcium signal and subsequent calcium entry was much smaller compared to normospermic patients. As expected, pretreatment of normospermic spermatozoa with both the anti-progesterone receptor c262 antibody and with progesterone receptor antagonist RU-38486 decreased the calcium release induced by progesterone. Treatment of spermatozoa with cytochalasin D or jasplakinolide decreased the calcium entry evoked by depletion of internal calcium stores in normospermic patients, whereas these treatments proved to be ineffective at modifying the calcium entry in patients with asthenozoospermia. Conclusion Our results suggest that spermatozoa from

  1. 11β-hydroxysteroid dehydrogenases: intracellular gate-keepers of tissue glucocorticoid action.

    Science.gov (United States)

    Chapman, Karen; Holmes, Megan; Seckl, Jonathan

    2013-07-01

    Glucocorticoid action on target tissues is determined by the density of "nuclear" receptors and intracellular metabolism by the two isozymes of 11β-hydroxysteroid dehydrogenase (11β-HSD) which catalyze interconversion of active cortisol and corticosterone with inert cortisone and 11-dehydrocorticosterone. 11β-HSD type 1, a predominant reductase in most intact cells, catalyzes the regeneration of active glucocorticoids, thus amplifying cellular action. 11β-HSD1 is widely expressed in liver, adipose tissue, muscle, pancreatic islets, adult brain, inflammatory cells, and gonads. 11β-HSD1 is selectively elevated in adipose tissue in obesity where it contributes to metabolic complications. Similarly, 11β-HSD1 is elevated in the ageing brain where it exacerbates glucocorticoid-associated cognitive decline. Deficiency or selective inhibition of 11β-HSD1 improves multiple metabolic syndrome parameters in rodent models and human clinical trials and similarly improves cognitive function with ageing. The efficacy of inhibitors in human therapy remains unclear. 11β-HSD2 is a high-affinity dehydrogenase that inactivates glucocorticoids. In the distal nephron, 11β-HSD2 ensures that only aldosterone is an agonist at mineralocorticoid receptors (MR). 11β-HSD2 inhibition or genetic deficiency causes apparent mineralocorticoid excess and hypertension due to inappropriate glucocorticoid activation of renal MR. The placenta and fetus also highly express 11β-HSD2 which, by inactivating glucocorticoids, prevents premature maturation of fetal tissues and consequent developmental "programming." The role of 11β-HSD2 as a marker of programming is being explored. The 11β-HSDs thus illuminate the emerging biology of intracrine control, afford important insights into human pathogenesis, and offer new tissue-restricted therapeutic avenues.

  2. 11β-Hydroxysteroid Dehydrogenases: Intracellular Gate-Keepers of Tissue Glucocorticoid Action

    Science.gov (United States)

    Chapman, Karen; Holmes, Megan

    2013-01-01

    Glucocorticoid action on target tissues is determined by the density of “nuclear” receptors and intracellular metabolism by the two isozymes of 11β-hydroxysteroid dehydrogenase (11β-HSD) which catalyze interconversion of active cortisol and corticosterone with inert cortisone and 11-dehydrocorticosterone. 11β-HSD type 1, a predominant reductase in most intact cells, catalyzes the regeneration of active glucocorticoids, thus amplifying cellular action. 11β-HSD1 is widely expressed in liver, adipose tissue, muscle, pancreatic islets, adult brain, inflammatory cells, and gonads. 11β-HSD1 is selectively elevated in adipose tissue in obesity where it contributes to metabolic complications. Similarly, 11β-HSD1 is elevated in the ageing brain where it exacerbates glucocorticoid-associated cognitive decline. Deficiency or selective inhibition of 11β-HSD1 improves multiple metabolic syndrome parameters in rodent models and human clinical trials and similarly improves cognitive function with ageing. The efficacy of inhibitors in human therapy remains unclear. 11β-HSD2 is a high-affinity dehydrogenase that inactivates glucocorticoids. In the distal nephron, 11β-HSD2 ensures that only aldosterone is an agonist at mineralocorticoid receptors (MR). 11β-HSD2 inhibition or genetic deficiency causes apparent mineralocorticoid excess and hypertension due to inappropriate glucocorticoid activation of renal MR. The placenta and fetus also highly express 11β-HSD2 which, by inactivating glucocorticoids, prevents premature maturation of fetal tissues and consequent developmental “programming.” The role of 11β-HSD2 as a marker of programming is being explored. The 11β-HSDs thus illuminate the emerging biology of intracrine control, afford important insights into human pathogenesis, and offer new tissue-restricted therapeutic avenues. PMID:23899562

  3. A single amino acid substitution is sufficient to modify the mitogenic properties of the epidermal growth factor receptor to resemble that of gp185erbB-2

    DEFF Research Database (Denmark)

    Di Fiore, P P; Helin, K; Kraus, M H

    1992-01-01

    The epidermal growth factor (EGF) receptor (EGFR) and the erbB-2 gene product, gp185erbB-2, exhibit distinct abilities to stimulate mitogenesis in different target cells. By using chimeric molecules between these two receptors, we have previously shown that their intracellular juxtamembrane regio...

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

    DEFF Research Database (Denmark)

    Amstrup, Jan; Novak, Ivana

    2003-01-01

    to the plasma membrane, clusters within the membrane and intracellularly. Stimulation of P2X7 receptors in HEK-293 cells led to an activation of extracellular signal-regulated kinases ERK1 and ERK2 and this activation was seen after just 1 min of stimulation with ATP. Using C- and N-terminal P2X7-receptor...

  5. AGONIST ANTAGONIST INTERACTIONS WITH CLONED HUMAN 5-HT1A RECEPTORS - VARIATIONS IN INTRINSIC ACTIVITY STUDIED IN TRANSFECTED HELA-CELLS

    NARCIS (Netherlands)

    BODDEKE, HWGM; FARGIN, A; RAYMOND, J.; SCHOEFFTER, P; HOYER, D

    The characteristics of 5-HT1A-recognition sites and receptor-mediated release of intracellular calcium were established in two transfected HeLa cell lines (HA 6 and HA 7) expressing different levels of human 5-HT1A receptors (about 3000 and 500 fmol/mg protein, Fargin et al. 1989; 1991; Raymond et

  6. THE ROLE OF INOSITOL 1,3,4,5-TETRAKISPHOSPHATE IN INTERNAL CA2+ MOBILIZATION FOLLOWING HISTAMINE H-1 RECEPTOR STIMULATION IN DDT1 MF-2 CELLS

    NARCIS (Netherlands)

    VANDERZEE, L; SIPMA, H; NELEMANS, A; DENHERTOG, A

    1995-01-01

    Receptor-activated formation of inositol phosphates results in mobilization of intracellular stored Ca2+ in a variety of cells, including vas deferens derived DDT1 MF-2 cells. Stimulation of the histamine H-1 receptor on these cells caused a pronounced formation of inositol 1,3,4,5-tetrakisphosphate

  7. Structure-function analysis of barley NLR immune receptor MLA10 reveals its cell compartment specific activity in cell death and disease resistance.

    NARCIS (Netherlands)

    Bai, S.; Liu, J.; Chang, C.; Zhang, L.; Maekawa, T.; Wang, Q.; Xiao, W.; Liu, Y.; Chai, J.; Takken, F.L.W.; Schulze-Lefert, P.; Shen, Q.H.

    2012-01-01

    Plant intracellular immune receptors comprise a large number of multi-domain proteins resembling animal NOD-like receptors (NLRs). Plant NLRs typically recognize isolate-specific pathogen-derived effectors, encoded by avirulence (AVR) genes, and trigger defense responses often associated with

  8. Agonist/antagonist interactions with cloned human 5-HT(1A) receptors: Variations in intrinsic activity studied in transfected HeLa cells

    NARCIS (Netherlands)

    Boddeke, H.W.G.M.; Fargin, A.; Raymond, J.R.; Schoeffter, P.; Hoyer, D.

    1992-01-01

    The characteristics of 5-HT(1A)-recognition sites and receptor-mediated release of intracellular calcium were established in two transfected HeLa cell lines (HA 6 and HA 7) expressing different levels of human 5-HT(1A) receptors (about 3000 and 500 fmol/mg protein, Fargin et al. 1989; 1991; Raymond

  9. 3D structure of muscle dihydropyridine receptor

    Directory of Open Access Journals (Sweden)

    Montserrat Samsó

    2015-01-01

    Full Text Available Excitation contraction coupling, the rapid and massive Ca2+ release under control of an action potential that triggers muscle contraction, takes places at specialized regions of the cell called triad junctions. There, a highly ordered supramolecular complex between the dihydropyridine receptor (DHPR and the ryanodine receptor (RyR1 mediates the quasi‐instantaneous conversion from T‐tubule depolarization into Ca2+ release from the sarcoplasmic reticulum (SR. The DHPR has several key modules required for EC coupling: the voltage sensors and II‐III loop in the alpha1s subunit, and the beta subunit. To gain insight into their molecular organization, this review examines the most updated 3D structure of the DHPR as obtained by transmission electron microscopy and image reconstruction. Although structure determination of a heteromeric membrane protein such as the DHPR is challenging, novel technical advances in protein expression and 3D labeling facilitated this task. The 3D structure of the DHPR complex consists of a main body with five irregular corners around its perimeter encompassing the transmembrane alpha 1s subunit besides the intracellular beta subunit, an extended extracellular alpha 2 subunit, and a bulky intracellular II‐III loop. The structural definition attained at 19 Å resolution enabled docking of the atomic coordinates of structural homologs of the alpha1s and beta subunits. These structural features, together with their relative location with respect to the RyR1, are discussed in the context of the functional data.

  10. Antibiotic susceptibility and intracellular localization of Diplorickettsia massiliensis.

    Science.gov (United States)

    Subramanian, Geetha; Barry, Abdoulaye O; Ghigo, Eric; Raoult, Didier; Mediannikov, Oleg

    2012-02-01

    Diplorickettsia massiliensis is an obligate intracellular bacterium from the Coxiellaceae family recently isolated from Ixodes ricinus ticks. The inhibitory effects of antimicrobial agents were assessed by two different methods, immunofluorescence and Gimenez staining assay. Different markers (EEA1, Lamp-1, Cathepsin D, and LysoTracker Red DND99) were used to reveal the nature of the vacuole containing the bacterium. Ciprofloxacin, levofloxacin, and rifampin had MIC values of 2 lg mL(-1). We found that 4 lg mL(-1) of Doxycycline inhibited the growth of D. massiliensis strain. Surprisingly, D. massiliensis was resistant to chloramphenicol up to the concentration of 64 lg mL(-1). We found that penicillin G, ammonium chloride, gentamycin, omeprazole, bafilomycin A1, and chloroquine were not active against D. massiliensis. Studies performed with markers EEA1, Lamp-1, Cathepsin D, and LysoTracker Red DND99 showed that D. massiliensis is localized within an acidic compartment that is not an early phagosome, but a late phagosome or a phagolysosome. Gimenez staining stays a good method that will work with a very low number of bacteria and can be used to determine the MICs of new therapeutic antibiotics precisely. The resistance profile of D. massiliensis was found to be quite unusual for intracellular Gram-negative bacterium with marked resistance to chloramphenicol. Despite of localization in acidic compartment, pH-neutralizing agents do not significantly inhibit intracellular growth of bacterium. The results of these studies prove that antibiotic resistance does not depend on pH of vacuole. This pH-related mechanism seems not to play a contributing role in the overall resistance of D. massiliensis.

  11. Intracellular Mono-ADP-Ribosylation in Signaling and Disease

    Science.gov (United States)

    Bütepage, Mareike; Eckei, Laura; Verheugd, Patricia; Lüscher, Bernhard

    2015-01-01

    A key process in the regulation of protein activities and thus cellular signaling pathways is the modification of proteins by post-translational mechanisms. Knowledge about the enzymes (writers and erasers) that attach and remove post-translational modifications, the targets that are modified and the functional consequences elicited by specific modifications, is crucial for understanding cell biological processes. Moreover detailed knowledge about these mechanisms and pathways helps to elucidate the molecular causes of various diseases and in defining potential targets for therapeutic approaches. Intracellular adenosine diphosphate (ADP)-ribosylation refers to the nicotinamide adenine dinucleotide (NAD+)-dependent modification of proteins with ADP-ribose and is catalyzed by enzymes of the ARTD (ADP-ribosyltransferase diphtheria toxin like, also known as PARP) family as well as some members of the Sirtuin family. Poly-ADP-ribosylation is relatively well understood with inhibitors being used as anti-cancer agents. However, the majority of ARTD enzymes and the ADP-ribosylating Sirtuins are restricted to catalyzing mono-ADP-ribosylation. Although writers, readers and erasers of intracellular mono-ADP-ribosylation have been identified only recently, it is becoming more and more evident that this reversible post-translational modification is capable of modulating key intracellular processes and signaling pathways. These include signal transduction mechanisms, stress pathways associated with the endoplasmic reticulum and stress granules, and chromatin-associated processes such as transcription and DNA repair. We hypothesize that mono-ADP-ribosylation controls, through these different pathways, the development of cancer and infectious diseases. PMID:26426055

  12. Domain-dependent effects of insulin and IGF-1 receptors on signalling and gene expression

    Science.gov (United States)

    Cai, Weikang; Sakaguchi, Masaji; Kleinridders, Andre; Gonzalez-Del Pino, Gonzalo; Dreyfuss, Jonathan M.; O'Neill, Brian T.; Ramirez, Alfred K.; Pan, Hui; Winnay, Jonathon N.; Boucher, Jeremie; Eck, Michael J.; Kahn, C. Ronald

    2017-01-01

    Despite a high degree of homology, insulin receptor (IR) and IGF-1 receptor (IGF1R) mediate distinct cellular and physiological functions. Here, we demonstrate how domain differences between IR and IGF1R contribute to the distinct functions of these receptors using chimeric and site-mutated receptors. Receptors with the intracellular domain of IGF1R show increased activation of Shc and Gab-1 and more potent regulation of genes involved in proliferation, corresponding to their higher mitogenic activity. Conversely, receptors with the intracellular domain of IR display higher IRS-1 phosphorylation, stronger regulation of genes in metabolic pathways and more dramatic glycolytic responses to hormonal stimulation. Strikingly, replacement of leucine973 in the juxtamembrane region of IR to phenylalanine, which is present in IGF1R, mimics many of these signalling and gene expression responses. Overall, we show that the distinct activities of the closely related IR and IGF1R are mediated by their intracellular juxtamembrane region and substrate binding to this region. PMID:28345670

  13. Deletion of GPR40 Impairs Glucose-Induced Insulin Secretion In Vivo in Mice Without Affecting Intracellular Fuel Metabolism in Islets

    Energy Technology Data Exchange (ETDEWEB)

    Alquier, Thierry; Peyot, Marie-Line; Latour, M. G.; Kebede, Melkam; Sorensen, Christina M.; Gesta, Stephane; Kahn, C. R.; Smith, Richard D.; Jetton, Thomas L.; Metz, Thomas O.; Prentki, Marc; Poitout, Vincent J.

    2009-11-01

    The G protein-coupled receptor GPR40 mediates fatty-acid potentiation of glucose-stimulated insulin secretion, but its contribution to insulin secretion in vivo and mechanisms of action remain uncertain. This study was aimed to ascertain whether GPR40 controls insulin secretion in vivo and modulates intracellular fuel metabolism in islets. We observed that glucose- and arginine-stimulated insulin secretion, assessed by hyperglycemic clamps, was decreased by approximately 60% in GPR40 knock-out (KO) fasted and fed mice, without changes in insulin sensitivity assessed by hyperinsulinemic-euglycemic clamps. Glucose and palmitate metabolism were not affected by GPR40 deletion. Lipid profiling revealed a similar increase in triglyceride and decrease in lysophosphatidylethanolamine species in WT and KO islets in response to palmitate. These results demonstrate that GPR40 regulates insulin secretion in vivo not only in response to fatty acids but also to glucose and arginine, without altering intracellular fuel metabolism.

  14. Subcellular site and nature of intracellular cadmium in plants

    International Nuclear Information System (INIS)

    Wagner, G.J.

    1979-01-01

    The mechanisms underlying heavy metal accumulation, toxicity, and tolerance in higher plants are poorly understood. Since subcellular processes are undoubtedly involved in all these phenomena, it is of interest to study the extent, subcellular site and nature of intracellularly accumulated cadmium in higher plants. Whole plants supplied 109 CdCl 2 or 112 CdSO 4 accumulated Cd into roots and aerial tissues. Preparation of protoplasts from aerial tissues followed by subcellular fractionation of the protoplasts to obtain intact vacuoles, chloroplasts and cytosol revealed the presence of Cd in the cytosol but not in vacuoles or chloroplasts. No evidence was obtained for the production of volatile Cd complexes in tobacco

  15. Intracellular Fluid Mechanics: Coupling Cytoplasmic Flow with Active Cytoskeletal Gel

    Science.gov (United States)

    Mogilner, Alex; Manhart, Angelika

    2018-01-01

    The cell is a mechanical machine, and continuum mechanics of the fluid cytoplasm and the viscoelastic deforming cytoskeleton play key roles in cell physiology. We review mathematical models of intracellular fluid mechanics, from cytoplasmic fluid flows, to the flow of a viscous active cytoskeletal gel, to models of two-phase poroviscous flows, to poroelastic models. We discuss application of these models to cell biological phenomena, such as organelle positioning, blebbing, and cell motility. We also discuss challenges of understanding fluid mechanics on the cellular scale.

  16. Bullous pemphigoid antigen localization suggests an intracellular association with hemidesmosomes

    DEFF Research Database (Denmark)

    Westgate, G E; Weaver, A C; Couchman, J R

    1985-01-01

    immunoelectron microscopy using both peroxidase and colloidal gold labeling techniques with patients' sera or IgG, revealed that BPA is associated with hemidesmosomes--putative adhesion structures at the BMZ, based on their similarity in ultrastructure to desmosomes. More specifically BPA was immunolocalized...... to the cytoplasmic face of hemidesmosomes and was not observed extracellularly in the basement membrane. In stratifying and nonstratifying cultures of rat keratinocytes, BPA is expressed intracellularly and not in the cell-derived matrix, unlike other known basement membrane components. These cells also synthesize...

  17. Tatp-mediated intracellular delivery of pharmaceutical nanocarriers.

    Science.gov (United States)

    Torchilin, V P

    2007-08-01

    CPPs (cell-penetrating peptides), including Tatp (transactivator of transcription peptide), have been successfully used for intracellular delivery of a wide variety of cargoes including various nanoparticulate pharmaceutical carriers such as liposomes, micelles and nanoparticles. Here, we will consider the major results obtained in this area with emphasis on Tatp-mediated delivery of liposomes and various transfection vectors. We will also address the development of 'smart' stimuli-sensitive nanocarriers, where the cell-penetrating function can only be activated when the nanocarrier is inside the biological target, thus minimizing the interaction with non-target cells.

  18. Intracellular transport driven by cytoskeletal motors: General mechanisms and defects

    Science.gov (United States)

    Appert-Rolland, C.; Ebbinghaus, M.; Santen, L.

    2015-09-01

    Cells are the elementary units of living organisms, which are able to carry out many vital functions. These functions rely on active processes on a microscopic scale. Therefore, they are strongly out-of-equilibrium systems, which are driven by continuous energy supply. The tasks that have to be performed in order to maintain the cell alive require transportation of various ingredients, some being small, others being large. Intracellular transport processes are able to induce concentration gradients and to carry objects to specific targets. These processes cannot be carried out only by diffusion, as cells may be crowded, and quite elongated on molecular scales. Therefore active transport has to be organized. The cytoskeleton, which is composed of three types of filaments (microtubules, actin and intermediate filaments), determines the shape of the cell, and plays a role in cell motion. It also serves as a road network for a special kind of vehicles, namely the cytoskeletal motors. These molecules can attach to a cytoskeletal filament, perform directed motion, possibly carrying along some cargo, and then detach. It is a central issue to understand how intracellular transport driven by molecular motors is regulated. The interest for this type of question was enhanced when it was discovered that intracellular transport breakdown is one of the signatures of some neuronal diseases like the Alzheimer. We give a survey of the current knowledge on microtubule based intracellular transport. Our review includes on the one hand an overview of biological facts, obtained from experiments, and on the other hand a presentation of some modeling attempts based on cellular automata. We present some background knowledge on the original and variants of the TASEP (Totally Asymmetric Simple Exclusion Process), before turning to more application oriented models. After addressing microtubule based transport in general, with a focus on in vitro experiments, and on cooperative effects in the

  19. Progesterone receptor and dopamine receptors are required in Δ9-tetrahydrocannabinol modulation of sexual receptivity in female rats

    Science.gov (United States)

    Mani, Shailaja K.; Mitchell, Andrea; O'Malley, Bert W.

    2001-01-01

    Ovarian steroids, estrogen and progesterone, influence the sensitivity of certain neural processes to cannabinoid treatment by modulation of brain dopaminergic activity. We examined the effects of the active ingredient of cannabis, Δ9-tetrahydrocannabinol (THC), on sexual behavior in female rats and its influence on steroid hormone receptors and neurotransmitters in the facilitation of sexual receptivity. Our results revealed that the facilitatory effect of THC was inhibited by antagonists to both progesterone and dopamine D1 receptors. To test further the idea that progesterone receptors (PR) and/or dopamine receptors (D1R) in the hypothalamus are required for THC-facilitated sexual behavior in rodents, antisense and sense oligonucleotides to PR and D1R were administered intracerebroventricularly (ICV) into the third cerebral ventricle of ovariectomized, estradiol benzoate-primed rats. Progesterone- and THC-facilitated sexual behavior was inhibited in animals treated with antisense oligonucleotides to PR or to D1R. Antagonists to cannabinoid receptor-1 subtype (CB1), but not to cannabinoid receptor-2 subtype (CB2) inhibited progesterone- and dopamine-facilitated sexual receptivity in female rats. Our studies indicate that THC acts on the CB1 cannabinoid receptor to initiate a signal transduction response that requires both membrane dopamine and intracellular progesterone receptors for effective induction of sexual behavior. PMID:11158625

  20. Methylphenidate enhances NMDA-receptor response in medial prefrontal cortex via sigma-1 receptor: a novel mechanism for methylphenidate action.

    Directory of Open Access Journals (Sweden)

    Chun-Lei Zhang

    Full Text Available Methylphenidate (MPH, commercially called Ritalin or Concerta, has been widely used as a drug for Attention Deficit Hyperactivity Disorder (ADHD. Noteworthily, growing numbers of young people using prescribed MPH improperly for pleasurable enhancement, take high risk of addiction. Thus, understanding the mechanism underlying high level of MPH action in the brain becomes an important goal nowadays. As a blocker of catecholamine transporters, its therapeutic effect is explained as being due to proper modulation of D1 and α2A receptor. Here we showed that higher dose of MPH facilitates NMDA-receptor mediated synaptic transmission via a catecholamine-independent mechanism, in layer V∼VI pyramidal cells of the rat medial prefrontal cortex (PFC. To indicate its postsynaptic action, we next found that MPH facilitates NMDA-induced current and such facilitation could be blocked by σ1 but not D1/5 and α2 receptor antagonists. And this MPH eliciting enhancement of NMDA-receptor activity involves PLC, PKC and IP3 receptor mediated intracellular Ca(2+ increase, but does not require PKA and extracellular Ca(2+ influx. Our additional pharmacological studies confirmed that higher dose of MPH increases locomotor activity via interacting with σ1 receptor. Together, the present study demonstrates for the first time that MPH facilitates NMDA-receptor mediated synaptic transmission via σ1 receptor, and such facilitation requires PLC/IP3/PKC signaling pathway. This novel mechanism possibly explains the underlying mechanism for MPH induced addictive potential and other psychiatric side effects.

  1. Canine adenovirus type 2 vector generation via I-Sce1-mediated intracellular genome release.

    Directory of Open Access Journals (Sweden)

    Sandy Ibanes

    Full Text Available When canine adenovirus type 2 (CAdV-2, or also commonly referred to as CAV-2 vectors are injected into the brain parenchyma they preferentially transduce neurons, are capable of efficient axonal transport to afferent regions, and allow transgene expression for at last >1 yr. Yet, translating these data into a user-friendly vector platform has been limited because CAV-2 vector generation is challenging. Generation of E1-deleted adenovirus vectors often requires transfection of linear DNA fragments of >30 kb containing the vector genome into an E1-transcomplementing cell line. In contrast to human adenovirus type 5 vector generation, CAV-2 vector generation is less efficient due, in part, to a reduced ability to initiate replication and poor transfectibility of canine cells with large, linear DNA fragments. To improve CAV-2 vector generation, we generated an E1-transcomplementing cell line expressing the estrogen receptor (ER fused to I-SceI, a yeast meganuclease, and plasmids containing the I-SceI recognition sites flanking the CAV-2 vector genome. Using transfection of supercoiled plasmid and intracellular genome release via 4-OH-tamoxifen-induced nuclear translocation of I-SceI, we improved CAV-2 vector titers 1,000 fold, and in turn increased the efficacy of CAV-2 vector generation.

  2. IMPDH2 Is an Intracellular Target of the Cyclophilin A and Sanglifehrin A Complex

    Directory of Open Access Journals (Sweden)

    Khian Hong Pua

    2017-01-01

    Full Text Available Natural products have demonstrated utility in the clinic and can also act as probes to understand complex cellular pathways. Sanglifehrin A (SFA is a mixed polyketide and non-ribosomal peptide synthase natural product with sub-nano-molar affinity for its receptor cyclophilin A (PPIA. It has been shown to behave in vitro as an immune suppressant. Here, we identify inosine-5′-monophosphate dehydrogenase 2 (IMPDH2 as an intracellular target of the PPIA-SFA binary complex. The formation of this ternary complex does not inhibit the enzymatic activity of IMPDH2. Rather, ternary complex formation modulates cell growth through interaction with the cystathionine-β-synthase (CBS domain of IMPDH2. We further demonstrate that the SFA complex is highly isoform selective for IMPDH2 (versus IMPDH1. This work reveals a role for the CBS domains of IMPDH2 in cellular proliferation, suggesting a more complex role than previously suspected for IMPDH2 in T cell activation and proliferation.

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

    Science.gov (United States)

    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

    2016-01-01

    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 that Irp2 mRNA 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 (CLOCKΔ19) 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 CLOCKΔ19 expression. Our findings suggest that circadian organization contributes to tumor cell proliferation by regulating iron metabolism in the tumor. PMID:26797126

  4. Intracellular fluorescent light-up bioprobes with different morphology for image-guided photothermal cancer therapy.

    Science.gov (United States)

    Li, Bangbang; Zhang, Peng; Du, Jianwei; Zhao, Xiao; Wang, Youxiang

    2017-06-01

    Multifunctional nanoprobe was drawing increased attention in tumor diagnosis and therapy. The simple and effective establishment of the theranostic nanoplatforms was still under urgent need. Meanwhile, the targeting ability and morphology of nanoprobe were essential for the effective endocytosis, which could further affect the diagnosis. In this work, two morphologies of nanoprobes were fabricated using gold nanorods (AuNRs) and gold nanospheres (AuNSs). Thiolated-hyaluronic acid labeled with nile blue (HS-HA-NB), a near-infrared (NIR) fluorescence dye, was coated on the surface of the gold nanoparticles to form stable nanoprobes (AuNR@HS-HA-NB, AuNS@HS-HA-NB). The fluorescence of NB molecules quenched outside cells due to the fluorescence resonance energy transfer (FRET), and recovered after the HA degradation inside the cells. HA also could enhance cellular uptake in CD44 receptor highly expressed human breast carcinoma cells (MCF-7). In this way, bioprobes realized the MCF-7 cell images through intracellular fluorescent light-up. Comparing with the sphere bioprobe, the rod-shaped bioprobe dramatically promoted endocytosis to achieve a better diagnosis effect in a short time. After NIR light irradiation, severe MCF-7 apoptosis was observed with AuNR@HS-HA-NB existed. Our studies suggested that the AuNR@HS-HA-NB nanoparticles were the excellent candidates of versatile bioprobes to realize rapid, precise image and photothermal therapy to MCF-7 cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Intracellular and extracellular adenosine triphosphate in regulation of insulin secretion from pancreatic β cells (β).

    Science.gov (United States)

    Wang, Chunjiong; Geng, Bin; Cui, Qinghua; Guan, Youfei; Yang, Jichun

    2014-03-01

    Adenosine triphosphate (ATP) synthesis and release in mitochondria play critical roles in regulating insulin secretion in pancreatic β cells. Mitochondrial dysfunction is mainly characterized by a decrease in ATP production, which is a central event in the progression of pancreatic β cell dysfunction and diabetes. ATP has been demonstrated to regulate insulin secretion via several pathways: (i) Intracellular ATP directly closes ATP-sensitive potassium channel to open L-type calcium channel, leading to an increase in free cytosolic calcium levels and exocytosis of insulin granules; (ii) A decrease in ATP production is always associated with an increase in production of reactive oxygen species, which exerts deleterious effects on pancreatic β cell survival and insulin secretion; and (iii) ATP can be co-secreted with insulin from pancreatic β cells, and the released ATP functions as an autocrine signal to modulate insulin secretory process via P2 receptors on the cell membrane. In this review, the recent findings regarding the role and mechanism of ATP synthesis and release in regulation of insulin secretion from pancreatic β cells will be summarized and discussed. © 2013 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd.

  6. Energetics of copper trafficking between the Atx1 metallochaperone and the intracellular copper transporter, Ccc2.

    Science.gov (United States)

    Huffman, D L; O'Halloran, T V

    2000-06-23

    The Atx1 metallochaperone protein is a cytoplasmic Cu(I) receptor that functions in intracellular copper trafficking pathways in plants, microbes, and humans. A key physiological partner of the Saccharomyces cerevisiae Atx1 is Ccc2, a cation transporting P-type ATPase located in secretory vesicles. Here, we show that Atx1 donates its metal ion cargo to the first N-terminal Atx1-like domain of Ccc2 in a direct and reversible manner. The thermodynamic gradient for metal transfer is shallow (K(exchange) = 1.4 +/- 0.2), establishing that vectorial delivery of copper by Atx1 is not based on a higher copper affinity of the target domain. Instead, Atx1 allows rapid metal transfer to its partner. This equilibrium is unaffected by a 50-fold excess of the Cu(I) competitor, glutathione, indicating that Atx1 also protects Cu(I) from nonspecific reactions. Mechanistically, we propose that a low activation barrier for transfer between partners results from complementary electrostatic forces that ultimately orient the metal-binding loops of Atx1 and Ccc2 for formation of copper-bridged intermediates. These thermodynamic and kinetic considerations suggest that copper trafficking proteins overcome the extraordinary copper chelation capacity of the eukaryotic cytoplasm by catalyzing the rate of copper transfer between physiological partners. In this sense, metallochaperones work like enzymes, carefully tailoring energetic barriers along specific reaction pathways but not others.

  7. Evolution of the duplicated intracellular lipid-binding protein genes of teleost fishes.

    Science.gov (United States)

    Venkatachalam, Ananda B; Parmar, Manoj B; Wright, Jonathan M

    2017-08-01

    Increasing organismal complexity during the evolution of life has been attributed to the duplication of genes and entire genomes. More recently, theoretical models have been proposed that postulate the fate of duplicated genes, among them the duplication-degeneration-complementation (DDC) model. In the DDC model, the common fate of a duplicated gene is lost from the genome owing to nonfunctionalization. Duplicated genes are retained in the genome either by subfunctionalization, where the functions of the ancestral gene are sub-divided between the sister duplicate genes, or by neofunctionalization, where one of the duplicate genes acquires a new function. Both processes occur either by loss or gain of regulatory elements in the promoters of duplicated genes. Here, we review the genomic organization, evolution, and transcriptional regulation of the multigene family of intracellular lipid-binding protein (iLBP) genes from teleost fishes. Teleost fishes possess many copies of iLBP genes owing to a whole genome duplication (WGD) early in the teleost fish radiation. Moreover, the retention of duplicated iLBP genes is substantially higher than the retention of all other genes duplicated in the teleost genome. The fatty acid-binding protein genes, a subfamily of the iLBP multigene family in zebrafish, are differentially regulated by peroxisome proliferator-activated receptor (PPAR) isoforms, which may account for the retention of iLBP genes in the zebrafish genome by the process of subfunctionalization of cis-acting regulatory elements in iLBP gene promoters.

  8. Plasminogen activator inhibitor 1 is an intracellular inhibitor of furin proprotein convertase.

    Science.gov (United States)

    Bernot, Denis; Stalin, Jimmy; Stocker, Pierre; Bonardo, Bernadette; Scroyen, Ilse; Alessi, Marie-Christine; Peiretti, Franck

    2011-04-15

    Proprotein convertases (PCs) are a family of serine proteases that are involved in the post-translational processing and activation of a wide range of regulatory proteins. The upstream role of PCs in the control of many physiological and pathological processes generates a growing interest in understanding their regulation. Here, we demonstrate that the serine protease inhibitor plasminogen activator inhibitor 1 (PAI-1) forms an SDS-stable complex with the PC furin, which leads to the inhibition of the intra-Golgi activity of furin. It is known that elevated PAI-1 plasma levels are correlated with the occurrence of the metabolic syndrome and type 2 diabetes, and we show that PAI-1 reduces the furin-dependent maturation and activity of the insulin receptor and ADAM17: two proteins involved in the onset of these metabolic disorders. In addition to demonstrating that PAI-1 is an intracellular inhibitor of furin, this study also provides arguments in favor of an active role for PAI-1 in the development of metabolic disorders.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-09-17

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

  10. Targeting the Receptor for Advanced Glycation Endproducts (RAGE): A Medicinal Chemistry Perspective

    Science.gov (United States)

    2017-01-01

    The receptor for advanced glycation endproducts (RAGE) is an ubiquitous, transmembrane, immunoglobulin-like receptor that exists in multiple isoforms and binds to a diverse range of endogenous extracellular ligands and intracellular effectors. Ligand binding at the extracellular domain of RAGE initiates a complex intracellular signaling cascade, resulting in the production of reactive oxygen species (ROS), immunoinflammatory effects, cellular proliferation, or apoptosis with concomitant upregulation of RAGE itself. To date, research has mainly focused on the correlation between RAGE activity and pathological conditions, such as cancer, diabetes, cardiovascular diseases, and neurodegeneration. Because RAGE plays a role in many pathological disorders, it has become an attractive target for the development of inhibitors at the extracellular and intracellular domains. This review describes the role of endogenous RAGE ligands/effectors in normo- and pathophysiological processes, summarizes the current status of exogenous small-molecule inhibitors of RAGE and concludes by identifying key strategies for future therapeutic intervention. PMID:28482155

  11. The Janus face of Bartonella quintana recognition by Toll-like receptors (TLRs): a review.

    NARCIS (Netherlands)

    Matera, G.; Liberto, M.C.; Joosten, L.A.B.; Vinci, M.; Quirino, A.; Pulicari, M.C.; Kullberg, B.J.; Meer, J.W.M. van der; Netea, M.G.; Foca, A.

    2008-01-01

    Bartonella quintana (B. quintana) is a facultative, intracellular bacterium, which causes trench fever, chronic bacteraemia and bacillary angiomatosis. Little is known about the recognition of B. quintana by the innate immune system. In this review, we address the impact of Toll-like receptors

  12. Novel mutation in the interferon-gamma-receptor gene and susceptibility to mycobacterial infections

    DEFF Research Database (Denmark)

    Storgaard, M; Varming, K; Herlin, Troels

    2006-01-01

    In 1981 we presented a patient with Mycobacterium intracellulare osteomyelitis and depressed monocyte cytotoxicity. It is now demonstrated that the molecular defect was a never-before-described nucleotide deletion at position 794 (794delT) in the interferon-gamma-receptor alpha-1 gene. The genetic...

  13. Calcium regulation of androgen receptor expression in the human prostate cancer cell line LNCaP

    NARCIS (Netherlands)

    L.J. Blok (Leen); J.E. Perry; J.K. Lindzey; D.J. Tindall; Y. Gong (Yuewen)

    1995-01-01

    textabstractElevation of intracellular calcium levels in the presence of normal androgen levels has been implicated in apoptotic prostate cell death. Since the androgen receptor (AR) plays a critical role in the regulation of growth and differentiation of the prostate, it was of

  14. Functional and molecular evidence for heteromeric association of P2Y1 receptor with P2Y2 and P2Y4 receptors in mouse granulocytes.

    Science.gov (United States)

    Ribeiro-Filho, Antonio Carlos; Buri, Marcus Vinicius; Barros, Carlos Castilho; Dreyfuss, Juliana Luporini; Nader, Helena Bonciani; Justo, Giselle Zenker; Craveiro, Rogério Bastos; Pesquero, João Bosco; Miranda, Antonio; Ferreira, Alice Teixeira; Paredes-Gamero, Edgar Julian

    2016-07-07

    All hematopoietic cells express P2 receptors, however pharmacological characteristics such as expression and affinity in granulocytes are unknown. Pharmacological characteristics of P2 receptors were evaluated by Ca(2+) measurements using Fura-2 fluorophore. P2 receptors expression were analyzed by flow cytometry and RT-PCR. P2 interaction were shown by coimmunoprecipitation, western blotting and FRET. Granulocytes were responsive to P2Y agonists, whereas P2X agonists were ineffective. Ca(2+) increase, elicited by ADP and UTP was dependent on intracellular stocks and sensitive to G-coupled receptor inhibition. Moreover, MRS2179, a specific antagonist of the P2Y1 receptor, abolished ADP response. Interestingly, ADP and UTP exhibited full heterologous desensitization, suggesting that these agonists interact with the same receptor. The heteromeric association between P2Y1 receptor and the P2Y2 and P2Y4 receptors was shown by immunoprecipitation and FRET analysis. Clear evidence of heteromeric association of P2Y receptors was found during the evaluation of P2 receptors present in mice granulocytes, which could impact in the classical pharmacology of P2Y receptors in granulocytes.

  15. Intracellular transport of low density lipoprotein-derived cholesterol is defective in Niemann-Pick type C fibroblasts

    International Nuclear Information System (INIS)

    Liscum, L.; Ruggiero, R.M.; Faust, J.R.

    1989-01-01

    Niemann-Pick disease type C (NPC) is characterized by substantial intracellular accumulation of unesterified cholesterol. The accumulation of unesterified cholesterol in NPC fibroblasts cultured with low density lipoprotein (LDL) appears to result from the inability of LDL to stimulate cholesterol esterification in addition to impaired LDL-mediated downregulation of LDL receptor activity and cellular cholesterol synthesis. Although a defect in cholesterol transport in NPC cells has been inferred from previous studies, no experiments have been reported that measure the intracellular movement of LDL-cholesterol specifically. We have used four approaches to assess intracellular cholesterol transport in normal and NPC cells and have determined the following: (a) mevinolin-inhibited NPC cells are defective in using LDL-cholesterol for growth. However, exogenously added mevalonate restores cell growth equally in normal and NPC cells; (b) the transport of LDL-derived [3H]cholesterol to the plasma membrane is slower in NPC cells, while the rate of appearance of [3H]acetate-derived, endogenously synthesized [3H]cholesterol at the plasma membrane is the same for normal and NPC cells; (c) in NPC cells, LDL-derived [3H]cholesterol accumulates in lysosomes to higher levels than normal, resulting in defective movement to other cell membranes; and (d) incubation of cells with LDL causes an increase in cholesterol content of NPC lysosomes that is threefold greater than that observed in normal lysosomes. Our results indicate that a cholesterol transport defect exists in NPC that is specific for LDL-derived cholesterol

  16. To Eat and to Be Eaten: Mutual Metabolic Adaptations of Immune Cells and Intracellular Bacterial Pathogens upon Infection

    Science.gov (United States)

    Eisenreich, Wolfgang; Rudel, Thomas; Heesemann, Jürgen; Goebel, Werner

    2017-01-01

    Intracellular bacterial pathogens (IBPs) invade and replicate in different cell types including immune cells, in particular of the innate immune system (IIS) during infection in the acute phase. However, immune cells primarily function as essential players in the highly effective and integrated host defense systems comprising the IIS and the adaptive immune system (AIS), which cooperatively protect the host against invading microbes including IBPs. As countermeasures, the bacterial pathogens (and in particular the IBPs) have developed strategies to evade or reprogram the IIS at various steps. The intracellular replication capacity and the anti-immune defense responses of the IBP's as well as the specific antimicrobial responses of the immune cells of the innate and the AIS depend on specific metabolic programs of the IBPs and their host cells. The metabolic programs of the immune cells supporting or counteracting replication of the IBPs appear to be mutually exclusive. Indeed, recent studies show that upon interaction of naïve, metabolically quiescent immune cells with IBPs, different metabolic activation processes occur which may result in the provision of a survival and replication niche for the pathogen or its eradication. It is therefore likely that within a possible host cell population subsets exist that are metabolically programmed for pro- or anti-microbial conditions. These metabolic programs may be triggered by the interactions between different bacterial agonistic components and host cell receptors. In this review, we summarize the current status in the field and discuss metabolic adaptation processes within immune cells of the IIS and the IBPs that support or restrict the intracellular replication of the pathogens. PMID:28752080

  17. Horizontal Transmission of Intracellular Insect Symbionts via Plants

    Directory of Open Access Journals (Sweden)

    Ewa Chrostek

    2017-11-01

    Full Text Available Experimental evidence is accumulating that endosymbionts of phytophagous insects may transmit horizontally via plants. Intracellular symbionts known for manipulating insect reproduction and altering fitness (Rickettsia, Cardinium, Wolbachia, and bacterial parasite of the leafhopper Euscelidius variegatus have been found to travel from infected insects into plants. Other insects, either of the same or different species can acquire the symbiont from the plant through feeding, and in some cases transfer it to their progeny. These reports prompt many questions regarding how intracellular insect symbionts are delivered to plants and how they affect them. Are symbionts passively transported along the insect-plant-insect path, or do they actively participate in the process? How widespread are these interactions? How does symbiont presence influence the plant? And what conditions are required for the new infection to establish in an insect? From an ecological, evolutionary, and applied perspective, this mode of horizontal transmission could have profound implications if occurring frequently enough or if new stable symbiont infections are established. Transmission of symbionts through plants likely represents an underappreciated means of infection, both in terms of symbiont epidemiology and the movement of symbionts to new host species.

  18. Raman spectroscopy for intracellular monitoring of carotenoid in Blakeslea trispora.

    Science.gov (United States)

    Papaioannou, Emmanouil H; Liakopoulou-Kyriakides, Maria; Christofilos, Dimitrios; Arvanitidis, Ioannis; Kourouklis, Gerasimos

    2009-11-01

    In the present study, we explore the feasibility of Raman spectroscopy for intracellular monitoring of carotenoid in filamentous fungi Blakeslea trispora. Although carotenoid production from this fungus has been extensively studied through various chromatographic methods and ultraviolet-visible spectroscopy, no intracellular monitoring has been demonstrated until now. The intensity of the Raman spectrum, and more conveniently that of the strongest nu(1) carotenoid band at approximately 1,519 cm(-1), exhibits a good linear correlation with the carotenoid content of the sample as determined by high-performance liquid chromatography (HPLC) and ultraviolet-visible (UV-Vis) spectroscopy. Our results suggest that Raman spectroscopy can serve as an alternative method for the study and quantification of carotenoid in batch-mated submerged cultivations of B. trispora and similar organisms. Although not as accurate as HPLC, it allows a rapid sampling and analysis, avoiding the prolonged and tedious classical isolation procedures required for carotenoid determination by HPLC and UV-Vis spectroscopy.

  19. Cell fate reprogramming by control of intracellular network dynamics

    Science.gov (United States)

    Zanudo, Jorge G. T.; Albert, Reka

    Identifying control strategies for biological networks is paramount for practical applications that involve reprogramming a cell's fate, such as disease therapeutics and stem cell reprogramming. Although the topic of controlling the dynamics of a system has a long history in control theory, most of this work is not directly applicable to intracellular networks. Here we present a network control method that integrates the structural and functional information available for intracellular networks to predict control targets. Formulated in a logical dynamic scheme, our control method takes advantage of certain function-dependent network components and their relation to steady states in order to identify control targets, which are guaranteed to drive any initial state to the target state with 100% effectiveness and need to be applied only transiently for the system to reach and stay in the desired state. We illustrate our method's potential to find intervention targets for cancer treatment and cell differentiation by applying it to a leukemia signaling network and to the network controlling the differentiation of T cells. We find that the predicted control targets are effective in a broad dynamic framework. Moreover, several of the predicted interventions are supported by experiments. This work was supported by NSF Grant PHY 1205840.

  20. Influenza vaccine induces intracellular immune memory of human NK cells.

    Science.gov (United States)

    Dou, Yaling; Fu, Binqing; Sun, Rui; Li, Wenting; Hu, Wanfu; Tian, Zhigang; Wei, Haiming

    2015-01-01

    Influenza vaccines elicit antigen-specific antibodies and immune memory to protect humans from infection with drift variants. However, what supports or limits vaccine efficacy and duration is unclear. Here, we vaccinated healthy volunteers with annual vaccine formulations and investigated the dynamics of T cell, natural killer (NK) cell and antibody responses upon restimulation with heterologous or homologous influenza virus strains. Influenza vaccines induced potential memory NK cells with increased antigen-specific recall IFN-γ responses during the first 6 months. In the absence of significant changes in other NK cell markers (CD45RO, NKp44, CXCR6, CD57, NKG2C, CCR7, CD62L and CD27), influenza vaccines induced memory NK cells with the distinct feature of intracellular NKp46 expression. Indeed, surface NKp46 was internalized, and the dynamic increase in NKp46(intracellular)+CD56dim NK cells positively correlated with increased IFN-γ production to influenza virus restimulation after vaccination. In addition, anti-NKp46 antibodies blocked IFN-γ responses. These findings provide insights into a novel mechanism underlying vaccine-induced immunity and NK-related diseases, which may help to design persisting and universal vaccines in the future.

  1. Horizontal Transmission of Intracellular Insect Symbionts via Plants.

    Science.gov (United States)

    Chrostek, Ewa; Pelz-Stelinski, Kirsten; Hurst, Gregory D D; Hughes, Grant L

    2017-01-01

    Experimental evidence is accumulating that endosymbionts of phytophagous insects may transmit horizontally via plants. Intracellular symbionts known for manipulating insect reproduction and altering fitness ( Rickettsia, Cardinium, Wolbachia , and bacterial parasite of the leafhopper Euscelidius variegatus ) have been found to travel from infected insects into plants. Other insects, either of the same or different species can acquire the symbiont from the plant through feeding, and in some cases transfer it to their progeny. These reports prompt many questions regarding how intracellular insect symbionts are delivered to plants and how they affect them. Are symbionts passively transported along the insect-plant-insect path, or do they actively participate in the process? How widespread are these interactions? How does symbiont presence influence the plant? And what conditions are required for the new infection to establish in an insect? From an ecological, evolutionary, and applied perspective, this mode of horizontal transmission could have profound implications if occurring frequently enough or if new stable symbiont infections are established. Transmission of symbionts through plants likely represents an underappreciated means of infection, both in terms of symbiont epidemiology and the movement of symbionts to new host species.

  2. Hybrid micro-/nanogels for optical sensing and intracellular imaging

    Directory of Open Access Journals (Sweden)

    Shuiqin Zhou

    2010-12-01

    Full Text Available Hybrid micro-/nanogels are playing an increasing important part in a diverse range of applications, due to their tunable dimensions, large surface area, stable interior network structure, and a very short response time. We review recent advances and challenges in the developments of hybrid micro-/nanogels toward applications for optical sensing of pH, temperature, glucose, ions, and other species as well as for intracellular imaging. Due to their unique advantages, hybrid micro-/nanogels as optical probes are attracting substantial interests for continuous monitoring of chemical parameters in complex samples such as blood and bioreactor fluids, in chemical research and industry, and in food quality control. In particular, their intracellular probing ability enables the monitoring of the biochemistry and biophysics of live cells over time and space, thus contributing to the explanation of intricate biological processes and the development of novel diagnoses. Unlike most other probes, hybrid micro-/nanogels could also combine other multiple functions into a single probe. The rational design of hybrid micro-/nanogels will not only improve the probing applications as desirable, but also implement their applications in new arenas. With ongoing rapid advances in bionanotechnology, the well-designed hybrid micro-/nanogel probes will be able to provide simultaneous sensing, imaging diagnosis, and therapy toward clinical applications.

  3. Crystallographic study of FABP5 as an intracellular endocannabinoid transporter

    Energy Technology Data Exchange (ETDEWEB)

    Sanson, Benoît; Wang, Tao [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Sun, Jing; Wang, Liqun; Kaczocha, Martin [Stony Brook University, Stony Brook, NY 11794-5213 (United States); Ojima, Iwao [Stony Brook University, Stony Brook, NY 1794-3400 (United States); Stony Brook University, Stony Brook, NY 11794-3400 (United States); Deutsch, Dale, E-mail: dale.deutsch@stonybrook.edu [Stony Brook University, Stony Brook, NY 11794-5213 (United States); Stony Brook University, Stony Brook, NY 11794-3400 (United States); Li, Huilin, E-mail: dale.deutsch@stonybrook.edu [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Stony Brook University, Stony Brook, NY 11794-5213 (United States); Stony Brook University, Stony Brook, NY 11794-3400 (United States)

    2014-02-01

    FABP5 was recently found to intracellularly transport endocannabinoid signaling lipids. The structures of FABP5 complexed with two endocannabinoids and an inhibitor were solved. Human FABP5 was found to dimerize via a domain-swapping mechanism. This work will help in the development of inhibitors to raise endocannabinoid levels. In addition to binding intracellular fatty acids, fatty-acid-binding proteins (FABPs) have recently been reported to also transport the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG), arachidonic acid derivatives that function as neurotransmitters and mediate a diverse set of physiological and psychological processes. To understand how the endocannabinoids bind to FABPs, the crystal structures of FABP5 in complex with AEA, 2-AG and the inhibitor BMS-309403 were determined. These ligands are shown to interact primarily with the substrate-binding pocket via hydrophobic interactions as well as a common hydrogen bond to the Tyr131 residue. This work advances our understanding of FABP5–endocannabinoid interactions and may be useful for future efforts in the development of small-molecule inhibitors to raise endocannabinoid levels.

  4. Imaging the intracellular degradation of biodegradable polymer nanoparticles

    Directory of Open Access Journals (Sweden)

    Anne-Kathrin Barthel

    2014-10-01

    Full Text Available In recent years, the development of smart drug delivery systems based on biodegradable polymeric nanoparticles has become of great interest. Drug-loaded nanoparticles can be introduced into the cell interior via endocytotic processes followed by the slow release of the drug due to degradation of the nanoparticle. In this work, poly(L-lactic acid (PLLA was chosen as the biodegradable polymer. Although common degradation of PLLA has been studied in various biological environments, intracellular degradation processes have been examined only to a very limited extent. PLLA nanoparticles with an average diameter of approximately 120 nm were decorated with magnetite nanocrystals and introduced into mesenchymal stem cells (MSCs. The release of the magnetite particles from the surface of the PLLA nanoparticles during the intracellular residence was monitored by transmission electron microscopy (TEM over a period of 14 days. It was demonstrated by the release of the magnetite nanocrystals from the PLLA surface that the PLLA nanoparticles do in fact undergo degradation within the cell. Furthermore, even after 14 days of residence, the PLLA nanoparticles were found in the MSCs. Additionally, the ultrastructural TEM examinations yield insight into the long term intercellular fate of these nanoparticles. From the statistical analysis of ultrastructural details (e.g., number of detached magnetite crystals, and the number of nanoparticles in one endosome, we demonstrate the importance of TEM studies for such applications in addition to fluorescence studies (flow cytometry and confocal laser scanning microscopy.

  5. Modeling nanoparticle uptake and intracellular distribution using stochastic process algebras

    Energy Technology Data Exchange (ETDEWEB)

    Dobay, M. P. D., E-mail: maria.pamela.david@physik.uni-muenchen.de; Alberola, A. Piera; Mendoza, E. R.; Raedler, J. O., E-mail: joachim.raedler@physik.uni-muenchen.de [Ludwig-Maximilians University, Faculty of Physics, Center for NanoScience (Germany)

    2012-03-15

    Computational modeling is increasingly important to help understand the interaction and movement of nanoparticles (NPs) within living cells, and to come to terms with the wealth of data that microscopy imaging yields. A quantitative description of the spatio-temporal distribution of NPs inside cells; however, it is challenging due to the complexity of multiple compartments such as endosomes and nuclei, which themselves are dynamic and can undergo fusion and fission and exchange their content. Here, we show that stochastic pi calculus, a widely-used process algebra, is well suited for mapping surface and intracellular NP interactions and distributions. In stochastic pi calculus, each NP is represented as a process, which can adopt various states such as bound or aggregated, as well as be passed between processes representing location, as a function of predefined stochastic channels. We created a pi calculus model of gold NP uptake and intracellular movement and compared the evolution of surface-bound, cytosolic, endosomal, and nuclear NP densities with electron microscopy data. We demonstrate that the computational approach can be extended to include specific molecular binding and potential interaction with signaling cascades as characteristic for NP-cell interactions in a wide range of applications such as nanotoxicity, viral infection, and drug delivery.

  6. Modeling nanoparticle uptake and intracellular distribution using stochastic process algebras

    International Nuclear Information System (INIS)

    Dobay, M. P. D.; Alberola, A. Piera; Mendoza, E. R.; Rädler, J. O.

    2012-01-01

    Computational modeling is increasingly important to help understand the interaction and movement of nanoparticles (NPs) within living cells, and to come to terms with the wealth of data that microscopy imaging yields. A quantitative description of the spatio-temporal distribution of NPs inside cells; however, it is challenging due to the complexity of multiple compartments such as endosomes and nuclei, which themselves are dynamic and can undergo fusion and fission and exchange their content. Here, we show that stochastic pi calculus, a widely-used process algebra, is well suited for mapping surface and intracellular NP interactions and distributions. In stochastic pi calculus, each NP is represented as a process, which can adopt various states such as bound or aggregated, as well as be passed between processes representing location, as a function of predefined stochastic channels. We created a pi calculus model of gold NP uptake and intracellular movement and compared the evolution of surface-bound, cytosolic, endosomal, and nuclear NP densities with electron microscopy data. We demonstrate that the computational approach can be extended to include specific molecular binding and potential interaction with signaling cascades as characteristic for NP-cell interactions in a wide range of applications such as nanotoxicity, viral infection, and drug delivery.

  7. Modeling nanoparticle uptake and intracellular distribution using stochastic process algebras

    Science.gov (United States)

    Dobay, M. P. D.; Alberola, A. Piera; Mendoza, E. R.; Rädler, J. O.

    2012-03-01

    Computational modeling is increasingly important to help understand the interaction and movement of nanoparticles (NPs) within living cells, and to come to terms with the wealth of data that microscopy imaging yields. A quantitative description of the spatio-temporal distribution of NPs inside cells; however, it is challenging due to the complexity of multiple compartments such as endosomes and nuclei, which themselves are dynamic and can undergo fusion and fission and exchange their content. Here, we show that stochastic pi calculus, a widely-used process algebra, is well suited for mapping surface and intracellular NP interactions and distributions. In stochastic pi calculus, each NP is represented as a process, which can adopt various states such as bound or aggregated, as well as be passed between processes representing location, as a function of predefined stochastic channels. We created a pi calculus model of gold NP uptake and intracellular movement and compared the evolution of surface-bound, cytosolic, endosomal, and nuclear NP densities with electron microscopy data. We demonstrate that the computational approach can be extended to include specific molecular binding and potential interaction with signaling cascades as characteristic for NP-cell interactions in a wide range of applications such as nanotoxicity, viral infection, and drug delivery.

  8. CIRRHOSIS INDUCES APOPTOSIS IN RENAL TISSUE THROUGH INTRACELLULAR OXIDATIVE STRESS

    Directory of Open Access Journals (Sweden)

    Keli Cristina Simões da SILVEIRA

    2015-03-01

    Full Text Available Background Renal failure is a frequent and serious complication in patients with decompensated cirrhosis. Objectives We aimed to evaluate the renal oxidative stress, cell damage and impaired cell function in animal model of cirrhosis. Methods Secondary biliary cirrhosis was induced in rats by ligation of the common bile duct. We measured TBARS, ROS and mitochondrial membrane potential in kidney as markers of oxidative stress, and activities of the antioxidant enzymes. Relative cell viability was determined by trypan blue dye-exclusion assay. Annexin V-PE was used with a vital dye, 7-AAD, to distinguish apoptotic from necrotic cells and comet assay was used for determined DNA integrity in single cells. Results In bile duct ligation animals there was significant increase in the kidney lipoperoxidation and an increase of the level of intracellular ROS. There was too an increase in the activity of all antioxidant enzymes evaluated in the kidney. The percentage viability was above 90% in the control group and in bile duct ligation was 64.66% and the dominant cell death type was apoptosis. DNA damage was observed in the bile duct ligation. There was a decreased in the mitochondrial membrane potential from 71.40% ± 6.35% to 34.48% ± 11.40% in bile duct ligation. Conclusions These results indicate that intracellular increase of ROS cause damage in the DNA and apoptosis getting worse the renal function in cirrhosis.

  9. Dynamic intracellular localization of Dazl protein during Xenopus germline development.

    Science.gov (United States)

    Tada, Haru; Orii, Hidefumi

    2015-08-01

    Xenopus dazl encoding an RNA-binding protein has been identified as a component of the germ plasm and is involved in the migration and differentiation of the primordial germ cells (PGCs). Here, we investigated the intracellular localization of Dazl in germline cells throughout the lifetime of Xenopus. In early embryogenesis, Dazl was detected initially in the germ plasm and then translocated to a perinuclear region. Then, it was detected within the nucleus in PGCs. Dazl was observed only in the cytoplasm in PGCs when sex differentiation began in the gonads. Dazl was distributed in both the nucleus and cytoplasm of the primary oogonium and spermatogonium, but only in the cytoplasm of the secondary oogonium and spermatogonium. In spermatocytes, Dazl was distributed throughout cytoplasm and localized at the spindles and cytoplasm during meiosis. Then, it was detected as speckles in the nucleus in the round spermatid. The dynamic intracellular localization suggests that Dazl is a multifunctional protein regulating RNA metabolism required for Xenopus germline development.

  10. Roles of rho GTPases in intracellular transport and cellular transformation.

    Science.gov (United States)

    Chi, Xiaojuan; Wang, Song; Huang, Yifan; Stamnes, Mark; Chen, Ji-Long

    2013-03-28

    Rho family GTPases belong to the Ras GTPase superfamily and transduce intracellular signals known to regulate a variety of cellular processes, including cell polarity, morphogenesis, migration, apoptosis, vesicle trafficking, viral transport and cellular transformation. The three best-characterized Rho family members are Cdc42, RhoA and Rac1. Cdc42 regulates endocytosis, the transport between the endoplasmic reticulum and Golgi apparatus, post-Golgi transport and exocytosis. Cdc42 influences trafficking through interaction with Wiskott-Aldrich syndrome protein (N-WASP) and the Arp2/3 complex, leading to changes in actin dynamics. Rac1 mediates endocytic and exocytic vesicle trafficking by interaction with its effectors, PI3kinase, synaptojanin 2, IQGAP1 and phospholipase D1. RhoA participates in the regulation of endocytosis through controlling its downstream target, Rho kinase. Interestingly, these GTPases play important roles at different stages of viral protein and genome transport in infected host cells. Importantly, dysregulation of Cdc42, Rac1 and RhoA leads to numerous disorders, including malignant transformation. In some cases, hyperactivation of Rho GTPases is required for cellular transformation. In this article, we review a number of findings related to Rho GTPase function in intracellular transport and cellular transformation.

  11. Detection of ubiquitinated huntingtin species in intracellular aggregates

    Directory of Open Access Journals (Sweden)

    Katrin eJuenemann

    2015-01-01

    Full Text Available Protein conformation diseases, including polyglutamine diseases, result from the accumulation and aggregation of misfolded proteins. Huntington’s disease is one of nine diseases caused by an expanded polyglutamine repeat within the affected protein and is hallmarked by intracellular inclusion bodies composed of aggregated N-terminal huntingtin fragments and other sequestered proteins. Fluorescence microscopy and filter trap assay are conventional methods to study protein aggregates, but cannot be used to analyze the presence and levels of post-translational modifications of aggregated huntingtin such as ubiquitination. Ubiquitination of proteins can be a signal for degradation and intracellular localization, but also affects protein activity and protein-protein interactions. The function of ubiquitination relies on its mono- and polymeric isoforms attached to protein substrates. Studying the ubiquitination pattern of aggregated huntingtin fragments offers an important possibility to understand huntingtin degradation and aggregation processes within the cell. For the identification of aggregated huntingtin and its ubiquitinated species, solubilization of the cellular aggregates is mandatory. Here we describe methods to identify post-translational modifications such as ubiquitination of aggregated mutant huntingtin. This approach is specifically described for use with mammalian cell culture and is suitable to study other disease-related proteins prone to aggregate.

  12. Transient light-induced intracellular oxidation revealed by redox biosensor

    International Nuclear Information System (INIS)

    Kolossov, Vladimir L.; Beaudoin, Jessica N.; Hanafin, William P.; DiLiberto, Stephen J.; Kenis, Paul J.A.; Rex Gaskins, H.

    2013-01-01

    Highlights: •Time-resolved live cell imaging revealed light-induced oxidation. •Only the roGFP probe fused with glutaredoxin reveals photooxidation. •The transient oxidation is rapidly reduced by the cytosolic antioxidant system. •Intracellular photooxidation is media-dependent. •Oxidation is triggered exclusively by exposure to short wavelength excitation. -- Abstract: We have implemented a ratiometric, genetically encoded redox-sensitive green fluorescent protein fused to human glutaredoxin (Grx1-roGFP2) to monitor real time intracellular glutathione redox potentials of mammalian cells. This probe enabled detection of media-dependent oxidation of the cytosol triggered by short wavelength excitation. The transient nature of light-induced oxidation was revealed by time-lapse live cell imaging when time intervals of less than 30 s were implemented. In contrast, transient ROS generation was not observed with the parental roGFP2 probe without Grx1, which exhibits slower thiol-disulfide exchange. These data demonstrate that the enhanced sensitivity of the Grx1-roGFP2 fusion protein enables the detection of short-lived ROS in living cells. The superior sensitivity of Grx1-roGFP2, however, also enhances responsiveness to environmental cues introducing a greater likelihood of false positive results during image acquisition

  13. Transient light-induced intracellular oxidation revealed by redox biosensor

    Energy Technology Data Exchange (ETDEWEB)

    Kolossov, Vladimir L., E-mail: viadimer@illinois.edu [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); Beaudoin, Jessica N. [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); Department of Animal Sciences, University of Illinois at Urbana-Champaign, 1207 W. Gregory Drive, Urbana, IL 61801 (United States); Hanafin, William P. [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); DiLiberto, Stephen J. [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); Department of Animal Sciences, University of Illinois at Urbana-Champaign, 1207 W. Gregory Drive, Urbana, IL 61801 (United States); Kenis, Paul J.A. [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 S. Mathews Avenue, Urbana, IL 61801 (United States); Rex Gaskins, H. [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); Department of Animal Sciences, University of Illinois at Urbana-Champaign, 1207 W. Gregory Drive, Urbana, IL 61801 (United States); Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 S. Lincoln Avenue, Urbana, IL 61801 (United States); Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, 905 S. Goodwin Avenue, Urbana, IL 61801 (United States)

    2013-10-04

    Highlights: •Time-resolved live cell imaging revealed light-induced oxidation. •Only the roGFP probe fused with glutaredoxin reveals photooxidation. •The transient oxidation is rapidly reduced by the cytosolic antioxidant system. •Intracellular photooxidation is media-dependent. •Oxidation is triggered exclusively by exposure to short wavelength excitation. -- Abstract: We have implemented a ratiometric, genetically encoded redox-sensitive green fluorescent protein fused to human glutaredoxin (Grx1-roGFP2) to monitor real time intracellular glutathione redox potentials of mammalian cells. This probe enabled detection of media-dependent oxidation of the cytosol triggered by short wavelength excitation. The transient nature of light-induced oxidation was revealed by time-lapse live cell imaging when time intervals of less than 30 s were implemented. In contrast, transient ROS generation was not observed with the parental roGFP2 probe without Grx1, which exhibits slower thiol-disulfide exchange. These data demonstrate that the enhanced sensitivity of the Grx1-roGFP2 fusion protein enables the detection of short-lived ROS in living cells. The superior sensitivity of Grx1-roGFP2, however, also enhances responsiveness to environmental cues introducing a greater likelihood of false positive results during image acquisition.

  14. Charcot–Marie–Tooth disease and intracellular traffic

    Science.gov (United States)

    Bucci, Cecilia; Bakke, Oddmund; Progida, Cinzia

    2012-01-01

    Mutations of genes whose primary function is the regulation of membrane traffic are increasingly being identified as the underlying causes of various important human disorders. Intriguingly, mutations in ubiquitously expressed membrane traffic genes often lead to cell type- or organ-specific disorders. This is particularly true for neuronal diseases, identifying the nervous system as the most sensitive tissue to alterations of membrane traffic. Charcot–Marie–Tooth (CMT) disease is one of the most common inherited peripheral neuropathies. It is also known as hereditary motor and sensory neuropathy (HMSN), which comprises a group of disorders specifically affecting peripheral nerves. This peripheral neuropathy, highly heterogeneous both clinically and genetically, is characterized by a slowly progressive degeneration of the muscle of the foot, lower leg, hand and forearm, accompanied by sensory loss in the toes, fingers and limbs. More than 30 genes have been identified as targets of mutations that cause CMT neuropathy. A number of these genes encode proteins directly or indirectly involved in the regulation of intracellular traffic. Indeed, the list of genes linked to CMT disease includes genes important for vesicle formation, phosphoinositide metabolism, lysosomal degradation, mitochondrial fission and fusion, and also genes encoding endosomal and cytoskeletal proteins. This review focuses on the link between intracellular transport and CMT disease, highlighting the molecular mechanisms that underlie the different forms of this peripheral neuropathy and discussing the pathophysiological impact of membrane transport genetic defects as well as possible future ways to counteract these defects. PMID:22465036

  15. Prolactin receptor and signal transduction to milk protein genes

    Energy Technology Data Exchange (ETDEWEB)

    Djiane, J.; Daniel, N.; Bignon, C. [Unite d`Endocrinologie Moleculaire, Jouy en Josas (France)] [and others

    1994-06-01

    After cloning of the mammary gland prolactin (PRL) receptor cDNA, a functional assay was established using co-transfection of PRL receptor cDNA together with a milk protein promoter/chloramphenicol acetyl transferase (CAT) construct in Chinese hamster ovary (CHO) cells. Different mutants of the PRL receptor were tested in this CAT assay to delimit the domains in the receptor necessary for signal transduction to milk protein genes. In CHO cells stably transfected with PRL receptor cDNA, high numbers of PRL receptor are expressed. By metabolic labeling and immunoprecipitation, expressed PRL receptor was identified as a single species of 100 kDa. Using these cells, we analyzed the effects of PRL on intracellular free Ca{sup ++} concentration. PRL stimulates Ca{sup ++} entry and induces secondary Ca{sup ++} mobilization. The entry of Ca{sup ++} is a result of an increase in K{sup +} conductance that hyperpolarizes the membranes. We have also analyzed tyrosine phosphorylation induced by PRL. In CHO cells stably transfected with PRL receptor cDNA, PRL induced a very rapid and transient tyrosine phosphorylation of a 100-kDa protein which is most probably the PRL receptor. The same finding was obtained in mammary membranes after PRL injection to lactating rabbits. Whereas tyrosine kinase inhibitors genistein and lavendustin were without effect, PRL stimulation of milk protein gene promoters was partially inhibited by 2 {mu}M herbimycin in CHO cells co-transfected with PRL receptor cDNA and the {Beta} lactoglobulin CAT construct. Taken together these observations indicate that the cytoplasmic domain of the PRL receptor interacts with one or several tyrosine kinases, which may represent early postreceptor events necessary for PRL signal transduction to milk protein genes. 14 refs., 4 figs.

  16. Purinergic receptors in the endocrine and exocrine pancreas.

    Science.gov (United States)

    Novak, I

    2008-09-01

    The pancreas is a complex gland performing both endocrine and exocrine functions. In recent years there has been increasing evidence that both endocrine and exocrine cells possess purinergic receptors, which influence processes such as insulin secretion and epithelial ion transport. Most commonly, these processes have been viewed separately. In beta cells, stimulation of P2Y(1) receptors amplifies secretion of insulin in the presence of glucose. Nucleotides released from secretory granules could also contribute to autocrine/paracrine regulation in pancreatic islets. In addition to P2Y(1) receptors, there is also evidence for other P2 and adenosine receptors in beta cells (P2Y(2), P2Y(4), P2Y(6), P2X subtypes and A(1) receptors) and in glucagon-secreting alpha cells (P2X(7), A(2) receptors). In the exocrine pancreas, acini release ATP and ATP-hydrolysing and ATP-generating enzymes. P2 receptors are prominent in pancreatic ducts, and several studies indicate that P2Y(2), P2Y(4), P2Y(11), P2X(4) and P2X(7) receptors could regulate secretion, primarily by affecting Cl(-) and K(+) channels and intracellular Ca(2+) signalling. In order to understand the physiology of the whole organ, it is necessary to consider the full complement of purinergic receptors on different cells as well as the structural and functional relation between various cells within the whole organ. In addition to the possible physiological function of purinergic receptors, this review analyses whether the receptors could be potential therapeutic targets for drug design aimed at treatment of pancreatic diseases.

  17. Control of endothelial cell tube formation by Notch ligand intracellular domain interactions with activator protein 1 (AP-1)

    DEFF Research Database (Denmark)

    Forghany, Zary; Robertson, Francesca; Lundby, Alicia

    2018-01-01

    work further demonstrated that JUN strongly stimulates endothelial cell tube formation and that DLL4 constrains this process. These results raise the possibility that Notch/DLL4 signaling is bi-directional and suggest that the DLL4 ICD could represent a point of cross-talk between Notch and receptor...... characterized, accumulating evidence points to the existence of multiple, less well-defined, layers of regulation. In this study, we investigated the function of the intracellular domain (ICD) of the Notch ligand Delta-like 4 (DLL4). We provide evidence that the DLL4 ICD is required for normal DLL4 subcellular......Notch signaling is a ubiquitous signal transduction pathway found in most if not all metazoan cell types characterized to date. It is indispensable for cell differentiation as well as tissue growth, tissue remodelling and apoptosis. Although the canonical Notch signaling pathway is well...

  18. Characterization of Intracellular and Extracellular Saxitoxin Levels in Both Field and Cultured Alexandrium spp. Samples from Sequim Bay, Washington

    Directory of Open Access Journals (Sweden)

    Vera L. Trainer

    2008-05-01

    Full Text Available Traditionally, harmful algal bloom studies have primarily focused on quantifying toxin levels contained within the phytoplankton cells of interest. In the case of paralytic shellfish poisoning toxins (PSTs, intracellular toxin levels and the effects of dietary consumption of toxic cells by planktivores have been well documented. However, little information is available regarding the levels of extracellular PSTs that may leak or be released into seawater from toxic cells during blooms. In order to fully evaluate the risks of harmful algal bloom toxins in the marine food web, it is necessary to understand all potential routes of exposure. In the present study, extracellular and intracellular PST levels were measured in field seawater samples (collected weekly from June to October 2004- 2007 and in Alexandrium spp. culture samples isolated from Sequim Bay, Washington. Measurable levels of intra- and extra-cellular toxins were detected in both field and culture samples via receptor binding assay (RBA and an enzyme-linked immunosorbent assay (ELISA. Characterization of the PST toxin profile in the Sequim Bay isolates by preMar. column oxidation and HPLC-fluorescence detection revealed that gonyautoxin 1 and 4 made up 65 ± 9.7 % of the total PSTs present. Collectively, these data confirm that extracellular PSTs are present during blooms of Alexandrium spp. in the Sequim Bay region.

  19. Tyrosine-phosphorylation of AAV2 vectors and its consequences on viral intracellular trafficking and transgene expression

    International Nuclear Information System (INIS)

    Zhong Li; Li Baozheng; Jayandharan, Giridhararao; Mah, Cathryn S.; Govindasamy, Lakshmanan; Agbandje-McKenna, Mavis; Herzog, Roland W.

    2008-01-01

    We have documented that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects intracellular trafficking and transduction efficiency of recombinant adeno-associated virus 2 (AAV2) vectors. Specifically, inhibition of EGFR-PTK signaling leads to decreased ubiquitination of AAV2 capsid proteins, which in turn, facilitates viral nuclear transport by limiting proteasome-mediated degradation of AAV2 vectors. In the present studies, we observed that AAV capsids can indeed be phosphorylated at tyrosine residues by EGFR-PTK in in vitro phosphorylation assays and that phosphorylated AAV capsids retain their structural integrity. However, although phosphorylated AAV vectors enter cells as efficiently as their unphosphorylated counterparts, their transduction efficiency is significantly reduced. This reduction is not due to impaired viral second-strand DNA synthesis since transduction efficiency of both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors is decreased by ∼ 68% and ∼ 74%, respectively. We also observed that intracellular trafficking of tyrosine-phosphorylated AAV vectors from cytoplasm to nucleus is significantly decreased, which results from ubiquitination of AAV capsids followed by proteasome-mediated degradation, although downstream consequences of capsid ubiquitination may also be affected by tyrosine-phosphorylation. These studies provide new insights into the role of tyrosine-phosphorylation of AAV capsids in various steps in the virus life cycle, which has implications in the optimal use of recombinant AAV vectors in human gene therapy

  20. Heat shock protein 70 protects PC12 cells against ischemia-hypoxia/reoxygenation by maintaining intracellular Ca2+ homeostasis

    Directory of Open Access Journals (Sweden)

    Yuan Liu

    2016-01-01

    Full Text Available Heat shock protein 70 (HSP70 maintains Ca2+ homeostasis in PC12 cells, which may protect against apoptosis; however, the mechanisms of neuroprotection are unclear. Therefore, in this study, we examined Ca2+ levels in PC12 cells transfected with an exogenous lentiviral HSP70 gene expression construct, and we subsequently subjected the cells to ischemia-hypoxia/reoxygenation injury. HSP70 overexpression increased neuronal viability and ATPase activity, and it decreased cellular reactive oxygen species levels and intracellular Ca2+ concentration after hypoxia/reoxygenation. HSP70 overexpression enhanced the protein and mRNA expression levels of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA, but it decreased the protein and mRNA levels of inositol 1,4,5-trisphosphate receptor (IP3R, thereby leading to decreased intracellular Ca2+ concentration after ischemia-hypoxia/reoxygenation. These results suggest that exogenous HSP70 protects against ischemia-hypoxia/reoxygenation injury, at least in part, by maintaining cellular Ca2+ homeostasis, by upregulating SERCA expression and by downregulating IP3R expression.

  1. Missense mutations in the growth hormone receptor dimerization region in Laron syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Berg, M.A.; Francke, U. [Howard Hughes Medical Institute, Stanford, CA (United States)]|[Univ. of Stanford, CA (United States); Geffner, M.E.; Bersch, N. [Univ. of California, Los Angeles, CA (United States)] [and others

    1994-09-01

    Laron syndrome (LS) is an autosomal recessively inherited condition characterized by insensitivity to endogenous and exogenous GH. Affected individuals have severe episodes and other characteristic features. GH receptor gene mutations are present in all affected individuals in whom molecular studies have been reported. The GH receptor is a plasma membrane-spanning protein in which the extracellular domain binds circulating GH and the intracellular domain interacts with the JAK-2 kinase and possibly other intracellular signaling molecules. GH receptor dimerization occurs on GH binding and is thought to be required for normal signal transduction. We have studied the GH receptor genes of four unrelated individuals affected with LS from the United States, Italy, Saudi Arabia, and India. We have identified four different missense mutations that alter consecutive amino acids 152 to 155 in or near the dimerization domain of the GH receptor. One of these mutations, D152H, has been reported previously in Asian LS patients and, in in vitro studies, the mutant receptor was unable to dimerize. This report increases to over 20 the number of different GH receptor gene mutations that have been reported in LS patients and defines the first apparent mutational {open_quotes}hotspot{close_quotes} region in this gene. This cluster of mutations in patients with classic LS phenotype provides additional in vivo evidence that receptor dimerization plays an important role in signaling GH`s growth promoting and metabolic effects. Further in vitro studies of the mutations in this region are in progress.

  2. Internalization of the human CRF receptor 1 is independent of classical phosphorylation sites and of beta-arrestin 1 recruitment

    DEFF Research Database (Denmark)

    Rasmussen, Trine N; Novak, Ivana; Nielsen, Søren M

    2004-01-01

    AMP-dependent protein kinase and protein kinase C are not prerequisites for CRFR1 internalization. Surprisingly, deletion of all putative phosphorylation sites in the C-terminal tail, as well as a cluster of putative phosphorylation sites in the third intracellular loop, did not affect receptor internalization. However......, these mutations almost abolished the recruitment of beta-arrestin 1 following receptor activation. In conclusion, we demonstrate that CRFR1 internalization is independent of phosphorylation sites in the C-terminal tail and third intracellular loop, and the degree of beta-arrestin 1 recruitment....

  3. Subtype-specific mechanisms for functional interaction between α6β4* nicotinic acetylcholine receptors and P2X receptors.

    Science.gov (United States)

    Limapichat, Walrati; Dougherty, Dennis A; Lester, Henry A

    2014-09-01

    P2X receptors and nicotinic acetylcholine receptors (nAChRs) display functional and physical interactions in many cell types and heterologous expression systems, but interactions between α6β4-containing (α6β4*) nAChRs and P2X2 receptors and/or P2X3 receptors have not been fully characterized. We measured several types of crosstalk in oocytes coexpressing α6β4 nAChRs and P2X2, P2X3, or P2X2/3 receptors. A novel form of crosstalk occurs between α6β4 nAChRs and P2X2 receptors. P2X2 receptors were forced into a prolonged desensitized state upon activation by ATP through a mechanism that does not depend on the intracellular C terminus of the P2X2 receptors. Coexpression of α6β4 nAChRs with P2X3 receptors shifts the ATP dose-response relation to the right, even in the absence of acetylcholine (ACh). Moreover, currents become nonadditive when ACh and ATP are coapplied, as previously reported for other Cys-loop receptors interacting with P2X receptors, and this crosstalk is dependent on the presence of the P2X3 C-terminal domain. P2X2 receptors also functionally interact with α6β4β3 but through a different mechanism from α6β4. The interaction with P2X3 receptors is less pronounced for the α6β4β3 nAChR than the α6β4 nAChR. We also measured a functional interaction between the α6β4 nAChRs and the heteromeric P2X2/3 receptor. Experiments with the nAChR channel blocker mecamylamine on P2X2-α6β4 oocytes point to the loss of P2X2 channel activity during the crosstalk, whereas the ion channel pores of the P2X receptors were fully functional and unaltered by the receptor interaction for P2X2-α6β4β3, P2X2/3-α6β4, and P2X2/3-α6β4β3. These results may be relevant to dorsal root ganglion cells and to other neurons that coexpress these receptor subunits. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

  4. Distribution of voltage-dependent and intracellular Ca2+ channels in submucosal neurons from rat distal colon.

    Science.gov (United States)

    Rehn, Matthias; Bader, Sandra; Bell, Anna; Diener, Martin

    2013-09-01

    We recently observed a bradykinin-induced increase in the cytosolic Ca2+ concentration in submucosal neurons of rat colon, an increase inhibited by blockers of voltage-dependent Ca2+ (Ca(v)) channels. As the types of Ca(v) channels used by this part of the enteric nervous system are unknown, the expression of various Ca(v) subunits has been investigated in whole-mount submucosal preparations by immunohistochemistry. Submucosal neurons, identified by a neuronal marker (microtubule-associated protein 2), are immunoreactive for Ca(v)1.2, Ca(v)1.3 and Ca(v)2.2, expression being confirmed by reverse transcription plus the polymerase chain reaction. These data agree with previous observations that the inhibition of L- and N-type Ca2+ currents strongly inhibits the response to bradykinin. However, whole-cell patch-clamp experiments have revealed that bradykinin does not enhance Ca2+ inward currents under voltage-clamp conditions. Consequently, bradykinin does not directly interact with Ca(v) channels. Instead, the kinin-induced Ca2+ influx is caused indirectly by the membrane depolarization evoked by this peptide. As intracellular Ca2+ channels on Ca(2+)-storing organelles can also contribute to Ca2+ signaling, their expression has been investigated by imaging experiments and immunohistochemistry. Inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) have been functionally demonstrated in submucosal neurons loaded with the Ca(2+)-sensitive fluorescent dye, fura-2. Histamine, a typical agonist coupled to the phospholipase C pathway, induces an increase in the fura-2 signal ratio, which is suppressed by 2-aminophenylborate, a blocker of IP3 receptors. The expression of IP3R1 has been confirmed by immunohistochemistry. In contrast, ryanodine, tested over a wide concentration range, evokes no increase in the cytosolic Ca2+ concentration nor is there immunohistochemical evidence for the expression of ryanodine receptors in these neurons. Thus, rat submucosal neurons are equipped

  5. Tumour Cell Labelling by Magnetic Nanoparticles with Determination of Intracellular Iron Content and Spatial Distribution of the Intracellular Iron

    Directory of Open Access Journals (Sweden)

    Alfred Cuschieri

    2013-04-01

    Full Text Available Magnetically labelled cells are used for in vivo cell tracking by MRI, used for the clinical translation of cell-base therapies. Studies involving magnetic labelled cells may include separation of labelled cells, targeted delivery and controlled release of drugs, contrast enhanced MRI and magnetic hyperthermia for the in situ ablation of tumours. Dextran-coated super-paramagnetic iron oxide (SPIO ferumoxides are used clinically as an MR contrast agents primarily for hepatic imaging. The material is also widely used for in vitro cell labelling, as are other SPIO-based particles. Our results on the uptake by human cancer cell lines of ferumoxides indicate that electroporation in the presence of protamine sulphate (PS results in rapid high uptake of SPIO nanoparticles (SPIONs by parenchymal tumour cells without significant impairment of cell viability. Quantitative determination of cellular iron uptake performed by colorimetric assay is in agreement with data from the literature. These results on intracellular iron content together with the intracellular distribution of SPIONs by magnetic force microscopy (MFM following in vitro uptake by parenchymal tumour cells confirm the potential of this technique for clinical tumour cell detection and destruction.

  6. Intracellular Trafficking Modulation by Ginsenoside Rg3 Inhibits Brucella abortus Uptake and Intracellular Survival within RAW 264.7 Cells.

    Science.gov (United States)

    Huy, Tran Xuan Ngoc; Reyes, Alisha Wehdnesday Bernardo; Hop, Huynh Tan; Arayan, Lauren Togonon; Min, WonGi; Lee, Hu Jang; Rhee, Man Hee; Chang, Hong Hee; Kim, Suk

    2017-03-28

    Ginsenoside Rg3, a saponin extracted from ginseng, has various pharmacological and biological activities; however, its effects against Brucella infection are still unclear. Herein, the inhibitory effects of ginsenoside Rg3 against intracellular parasitic Brucella infection were evaluated through bacterial infection, adherence assays, and LAMP-1 colocalization, as well as immunoblotting and FACS for detecting MAPK signaling proteins and F-actin polymerization, respectively. The internalization, intracellular growth, and adherence of Brucella abortus in Rg3-treated RAW 264.7 cells were significantly decreased compared with the Rg3-untreated control. Furthermore, an apparent reduction of F-actin content and intensity of F-actin fluorescence in Rg3-treated cells was observed compared with B. abortus -infected cells without treatment by flow cytometry analysis and confocal microscopy, respectively. In addition, treating cells with Rg3 decreased the phosphorylation of MAPK signaling proteins such as ERK 1/2 and p38 compared with untreated cells. Moreover, the colocalization of B. abortus -containing phagosomes with LAMP-1 was markedly increased in Rg3-treated cells. These findings suggest that ginsenoside Rg3 inhibits B. abortus infection in mammalian cells and can be used as an alternative approach in the treatment of brucellosis.