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Sample records for cftr chloride channel

  1. CFTR chloride channel as a molecular target of anthraquinone compounds in herbal laxatives

    Yang, Hong; Xu, Li-na; He, Cheng-yan; Liu, Xin; Fang, Rou-yu; Ma, Tong-hui

    2011-01-01

    Aim: To clarify whether CFTR is a molecular target of intestinal fluid secretion caused by the anthraquinone compounds from laxative herbal plants. Methods: A cell-based fluorescent assay to measure I− influx through CFTR chloride channel. A short-circuit current assay to measure transcellular Cl− current across single layer FRT cells and freshly isolated colon mucosa. A closed loop experiment to measure colon fluid secretion in vivo. Results: Anthraquinone compounds rhein, aloe-emodin and 1,8-dihydroxyanthraquinone (DHAN) stimulated I− influx through CFTR chloride channel in a dose-dependent manner in the presence of physiological concentration of cAMP. In the short-circuit current assay, the three compound enhanced Cl− currents in epithelia formed by CFTR-expressing FRT cells with EC50 values of 73±1.4, 56±1.7, and 50±0.5μmol/L, respectively, and Rhein also enhanced Cl− current in freshly isolated rat colonic mucosa with a similar potency. These effects were completely reversed by the CFTR selective blocker CFTRinh-172. In in vivo closed loop experiments, rhein 2 mmol/L stimulated colonic fluid accumulation that was largely blocked by CFTRinh-172. The anthraquinone compounds did not elevate cAMP level in cultured FRT cells and rat colonic mucosa, suggesting a direct effect on CFTR activity. Conclusion: Natural anthraquinone compounds in vegetable laxative drugs are CFTR potentiators that stimulated colonic chloride and fluid secretion. Thus CFTR chloride channel is a molecular target of vegetable laxative drugs. PMID:21602836

  2. The hypertonic environment differentially regulates wild-type CFTR and TNR-CFTR chloride channels.

    Lassance-Soares, Roberta M; Cheng, Jie; Krasnov, Kristina; Cebotaru, Liudmila; Cutting, Garry R; Souza-Menezes, Jackson; Morales, Marcelo M; Guggino, William B

    2010-01-01

    This study tested the hypotheses that the hypertonic environment of the renal medulla regulates the expression of cystic fibrosis transmembrane conductance regulator protein (CFTR) and its natural splice variant, TNR-CFTR. To accomplish this, Madin-Darby canine kidney (MDCK) stable cell lines expressing TNR-CFTR or CFTR were used. The cells were treated with hypertonic medium made with either NaCl or urea or sucrose (480 mOsm/kg or 560 mOsm/kg) to mimic the tonicity of the renal medulla environment. Western blot data showed that CFTR and TNR-CFTR total cell protein is increased by hypertonic medium, but using the surface biotinylation technique, only CFTR was found to be increased in cell plasma membrane. Confocal microscopy showed TNR-CFTR localization primarily at the endoplasmic reticulum and plasma membrane. In conclusion, CFTR and TNR-CFTR have different patterns of distribution in MDCK cells and they are modulated by a hypertonic environment, suggesting their physiological importance in renal medulla. Copyright © 2010 S. Karger AG, Basel.

  3. Mercury toxicity in the shark (Squalus acanthias) rectal gland: apical CFTR chloride channels are inhibited by mercuric chloride.

    Ratner, Martha A; Decker, Sarah E; Aller, Stephen G; Weber, Gerhard; Forrest, John N

    2006-03-01

    In the shark rectal gland, basolateral membrane proteins have been suggested as targets for mercury. To examine the membrane polarity of mercury toxicity, we performed experiments in three preparations: isolated perfused rectal glands, primary monolayer cultures of rectal gland epithelial cells, and Xenopus oocytes expressing the shark cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. In perfused rectal glands we observed: (1) a dose-dependent inhibition by mercury of forskolin/3-isobutyl-1-methylxanthine (IBMX)-stimulated chloride secretion; (2) inhibition was maximal when mercury was added before stimulation with forskolin/IBMX; (3) dithiothrietol (DTT) and glutathione (GSH) completely prevented inhibition of chloride secretion. Short-circuit current (Isc) measurements in monolayers of rectal gland epithelial cells were performed to examine the membrane polarity of this effect. Mercuric chloride inhibited Isc more potently when applied to the solution bathing the apical vs. the basolateral membrane (23 +/- 5% and 68 +/- 5% inhibition at 1 and 10 microM HgCl2 in the apical solution vs. 2 +/- 0.9% and 14 +/- 5% in the basolateral solution). This inhibition was prevented by pre-treatment with apical DTT or GSH; however, only the permeant reducing agent DTT reversed mercury inhibition when added after exposure. When the shark rectal gland CFTR channel was expressed in Xenopus oocytes and chloride conductance was measured by two-electrode voltage clamping, we found that 1 microM HgCl2 inhibited forskolin/IBMX conductance by 69.2 +/- 2.0%. We conclude that in the shark rectal gland, mercury inhibits chloride secretion by interacting with the apical membrane and that CFTR is the likely site of this action. Copyright 2006 Wiley-Liss, Inc.

  4. Stimulation of wild-type, F508del- and G551D-CFTR chloride channels by non toxic modified pyrrolo[2,3-b]pyrazine derivatives

    Luc eDannhoffer

    2011-08-01

    Full Text Available Cystic Fibrosis is a major inherited disorder involving abnormalities of fluid and electrolyte transport in a number of different organs due to abnormal function of Cystic Fibrosis Transmembrane conductance Regulator (CFTR protein. We recently identified a family of CFTR activators, which contains the hit: RP107 [7-n-butyl-6-(4-hydroxyphenyl[5H]-pyrrolo[2,3-b]pyrazine]. Here, we further evaluated the effect of the chemical modifications of the RP107-OH radical on CFTR activation. The replacement of the OH radical by a fluorine atom at position 2 (RP193 or 4 (RP185 significantly decreased the toxicity of the compounds without altering the ability to activate CFTR, especially for RP193. The non-toxic compound RP193 has no effect on cAMP production but stimulates the channel activity of wild-type CFTR in stably transfected CHO cells, in human bronchial epithelial NuLi-1 cells and in primary culture of human bronchial epithelial cells. Whole cell and single patch clamp recordings showed that RP193 induced a linear, time and voltage-independent current, which was fully inhibited by two different and selective CFTR inhibitors (CFTRinh-172 and GPinh-5a. Moreover, RP193 stimulates CFTR in temperature-rescued CuFi-1 (F508del/F508del human bronchial epithelial cells and in CHO cells stably expressing G551D-CFTR. This study shows that it is feasible to reduce cytotoxicity of chemical compounds without affecting their potency to activate CFTR and to rescue the class 2 F508del-CFTR and class 3 G551D-CFTR CF mutant activities.

  5. Transmembrane helical interactions in the CFTR channel pore.

    Jhuma Das

    2017-06-01

    Full Text Available Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR gene affect CFTR protein biogenesis or its function as a chloride channel, resulting in dysregulation of epithelial fluid transport in the lung, pancreas and other organs in cystic fibrosis (CF. Development of pharmaceutical strategies to treat CF requires understanding of the mechanisms underlying channel function. However, incomplete 3D structural information on the unique ABC ion channel, CFTR, hinders elucidation of its functional mechanism and correction of cystic fibrosis causing mutants. Several CFTR homology models have been developed using bacterial ABC transporters as templates but these have low sequence similarity to CFTR and are not ion channels. Here, we refine an earlier model in an outward (OWF and develop an inward (IWF facing model employing an integrated experimental-molecular dynamics simulation (200 ns approach. Our IWF structure agrees well with a recently solved cryo-EM structure of a CFTR IWF state. We utilize cysteine cross-linking to verify positions and orientations of residues within trans-membrane helices (TMHs of the OWF conformation and to reconstruct a physiologically relevant pore structure. Comparison of pore profiles of the two conformations reveal a radius sufficient to permit passage of hydrated Cl- ions in the OWF but not the IWF model. To identify structural determinants that distinguish the two conformations and possible rearrangements of TMHs within them responsible for channel gating, we perform cross-linking by bifunctional reagents of multiple predicted pairs of cysteines in TMH 6 and 12 and 6 and 9. To determine whether the effects of cross-linking on gating observed are the result of switching of the channel from open to close state, we also treat the same residue pairs with monofunctional reagents in separate experiments. Both types of reagents prevent ion currents indicating that pore blockage is primarily responsible.

  6. Steviol reduces MDCK Cyst formation and growth by inhibiting CFTR channel activity and promoting proteasome-mediated CFTR degradation.

    Chaowalit Yuajit

    Full Text Available Cyst enlargement in polycystic kidney disease (PKD involves cAMP-activated proliferation of cyst-lining epithelial cells and transepithelial fluid secretion into the cyst lumen via cystic fibrosis transmembrane conductance regulator (CFTR chloride channel. This study aimed to investigate an inhibitory effect and detailed mechanisms of steviol and its derivatives on cyst growth using a cyst model in Madin-Darby canine kidney (MDCK cells. Among 4 steviol-related compounds tested, steviol was found to be the most potent at inhibiting MDCK cyst growth. Steviol inhibition of cyst growth was dose-dependent; steviol (100 microM reversibly inhibited cyst formation and cyst growth by 72.53.6% and 38.2±8.5%, respectively. Steviol at doses up to 200 microM had no effect on MDCK cell viability, proliferation and apoptosis. However, steviol acutely inhibited forskolin-stimulated apical chloride current in MDCK epithelia, measured with the Ussing chamber technique, in a dose-dependent manner. Prolonged treatment (24 h with steviol (100 microM also strongly inhibited forskolin-stimulated apical chloride current, in part by reducing CFTR protein expression in MDCK cells. Interestingly, proteasome inhibitor, MG-132, abolished the effect of steviol on CFTR protein expression. Immunofluorescence studies demonstrated that prolonged treatment (24 h with steviol (100 microM markedly reduced CFTR expression at the plasma membrane. Taken together, the data suggest that steviol retards MDCK cyst progression in two ways: first by directly inhibiting CFTR chloride channel activity and second by reducing CFTR expression, in part, by promoting proteasomal degradation of CFTR. Steviol and related compounds therefore represent drug candidates for treatment of polycystic kidney disease.

  7. Rab4GTPase modulates CFTR function by impairing channel expression at plasma membrane

    Saxena, Sunil K.; Kaur, Simarna; George, Constantine

    2006-01-01

    Cystic fibrosis (CF), an autosomal recessive disorder, is caused by the disruption of biosynthesis or the function of a membrane cAMP-activated chloride channel, CFTR. CFTR regulatory mechanisms include recruitment of channel proteins to the cell surface from intracellular pools and by protein-protein interactions. Rab proteins are small GTPases involved in regulated trafficking controlling vesicle docking and fusion. Rab4 controls recycling events from endosome to the plasma membrane, fusion, and degradation. The colorectal cell line HT-29 natively expresses CFTR and responds to cAMP stimulation with an increase in CFTR-mediated currents. Rab4 over-expression in HT-29 cells inhibits both basal and cAMP-stimulated CFTR-mediated currents. GTPase-deficient Rab4Q67L and GDP locked Rab4S22N both inhibit channel activity, which appears characteristically different. Active status of Rab4 was confirmed by GTP overlay assay, while its expression was verified by Western blotting. The pull-down and immunoprecipitation experiments suggest that Rab4 physically interacts with CFTR through protein-protein interaction. Biotinylation with cell impermeant NHS-Sulfo-SS-Biotin implies that Rab4 impairs CFTR expression at cell surface. The enhanced cytosolic CFTR indicates that Rab4 expression restrains CFTR appearance at the cell membrane. The study suggests that Rab4 regulates the channel through multiple mechanisms that include protein-protein interaction, GTP/GDP exchange, and channel protein trafficking. We propose that Rab4 is a dynamic molecule with a significant role in CFTR function

  8. Sweat chloride as a biomarker of CFTR activity: proof of concept and ivacaftor clinical trial data.

    Accurso, Frank J; Van Goor, Fredrick; Zha, Jiuhong; Stone, Anne J; Dong, Qunming; Ordonez, Claudia L; Rowe, Steven M; Clancy, John Paul; Konstan, Michael W; Hoch, Heather E; Heltshe, Sonya L; Ramsey, Bonnie W; Campbell, Preston W; Ashlock, Melissa A

    2014-03-01

    We examined data from a Phase 2 trial {NCT00457821} of ivacaftor, a CFTR potentiator, in cystic fibrosis (CF) patients with aG551D mutation to evaluate standardized approaches to sweat chloride measurement and to explore the use of sweat chloride and nasal potential difference (NPD) to estimate CFTR activity. Sweat chloride and NPD were secondary endpoints in this placebo-controlled, multicenter trial. Standardization of sweat collection, processing,and analysis was employed for the first time. Sweat chloride and chloride ion transport (NPD) were integrated into a model of CFTR activity. Within-patient sweat chloride determinations showed sufficient precision to detect differences between dose-groups and assess ivacaftor treatment effects. Analysis of changes in sweat chloride and NPD demonstrated that patients treated with ivacaftor achieved CFTR activity equivalent to approximately 35%–40% of normal. Sweat chloride is useful in multicenter trials as a biomarker of CFTR activity and to test the effect of CFTR potentiators.

  9. Antibodies to the CFTR modulate the turgor pressure of guard cell protoplasts via slow anion channels.

    Leonhardt, N; Bazin, I; Richaud, P; Marin, E; Vavasseur, A; Forestier, C

    2001-04-06

    The plasma membrane guard cell slow anion channel is a key element at the basis of water loss control in plants allowing prolonged osmolite efflux necessary for stomatal closure. This channel has been extensively studied by electrophysiological approaches but its molecular identification is still lacking. Recently, we described that this channel was sharing some similarities with the mammalian ATP-binding cassette protein, cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel [Leonhardt, N. et al. (1999) Plant Cell 11, 1141-1151]. Here, using the patch-clamp technique and a bioassay, consisting in the observation of the change in guard cell protoplasts volume, we demonstrated that a functional antibody raised against the mammalian CFTR prevented ABA-induced guard cell protoplasts shrinking and partially inhibited the slow anion current. Moreover, this antibody immunoprecipitated a polypeptide from guard cell protein extracts and immunolabeled stomata in Vicia faba leaf sections. These results indicate that the guard cell slow anion channel is, or is closely controlled by a polypeptide, exhibiting one epitope shared with the mammalian CFTR.

  10. Gap Junctions Are Involved in the Rescue of CFTR-Dependent Chloride Efflux by Amniotic Mesenchymal Stem Cells in Coculture with Cystic Fibrosis CFBE41o- Cells

    Annalucia Carbone

    2018-01-01

    Full Text Available We previously found that human amniotic mesenchymal stem cells (hAMSCs in coculture with CF immortalised airway epithelial cells (CFBE41o- line, CFBE on Transwell® filters acquired an epithelial phenotype and led to the expression of a mature and functional CFTR protein. In order to explore the role of gap junction- (GJ- mediated intercellular communication (GJIC in this rescue, cocultures (hAMSC : CFBE, 1 : 5 ratio were studied for the formation of GJIC, before and after silencing connexin 43 (Cx43, a major component of GJs. Functional GJs in cocultures were inhibited when the expression of the Cx43 protein was downregulated. Transfection of cocultures with siRNA against Cx43 resulted in the absence of specific CFTR signal on the apical membrane and reduction in the mature form of CFTR (band C, and in parallel, the CFTR-dependent chloride channel activity was significantly decreased. Cx43 downregulation determined also a decrease in transepithelial resistance and an increase in paracellular permeability as compared with control cocultures, implying that GJIC may regulate CFTR expression and function that in turn modulate airway epithelium tightness. These results indicate that GJIC is involved in the correction of CFTR chloride channel activity upon the acquisition of an epithelial phenotype by hAMSCs in coculture with CF cells.

  11. Gap Junctions Are Involved in the Rescue of CFTR-Dependent Chloride Efflux by Amniotic Mesenchymal Stem Cells in Coculture with Cystic Fibrosis CFBE41o- Cells.

    Carbone, Annalucia; Zefferino, Roberto; Beccia, Elisa; Casavola, Valeria; Castellani, Stefano; Di Gioia, Sante; Giannone, Valentina; Seia, Manuela; Angiolillo, Antonella; Colombo, Carla; Favia, Maria; Conese, Massimo

    2018-01-01

    We previously found that human amniotic mesenchymal stem cells (hAMSCs) in coculture with CF immortalised airway epithelial cells (CFBE41o- line, CFBE) on Transwell® filters acquired an epithelial phenotype and led to the expression of a mature and functional CFTR protein. In order to explore the role of gap junction- (GJ-) mediated intercellular communication (GJIC) in this rescue, cocultures (hAMSC : CFBE, 1 : 5 ratio) were studied for the formation of GJIC, before and after silencing connexin 43 (Cx43), a major component of GJs. Functional GJs in cocultures were inhibited when the expression of the Cx43 protein was downregulated. Transfection of cocultures with siRNA against Cx43 resulted in the absence of specific CFTR signal on the apical membrane and reduction in the mature form of CFTR (band C), and in parallel, the CFTR-dependent chloride channel activity was significantly decreased. Cx43 downregulation determined also a decrease in transepithelial resistance and an increase in paracellular permeability as compared with control cocultures, implying that GJIC may regulate CFTR expression and function that in turn modulate airway epithelium tightness. These results indicate that GJIC is involved in the correction of CFTR chloride channel activity upon the acquisition of an epithelial phenotype by hAMSCs in coculture with CF cells.

  12. Cellular chloride and bicarbonate retention alters intracellular pH regulation in Cftr KO crypt epithelium

    Walker, Nancy M.; Liu, Jinghua; Stein, Sydney R.; Stefanski, Casey D.; Strubberg, Ashlee M.

    2015-01-01

    Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), an anion channel providing a major pathway for Cl− and HCO3− efflux across the apical membrane of the epithelium. In the intestine, CF manifests as obstructive syndromes, dysbiosis, inflammation, and an increased risk for gastrointestinal cancer. Cftr knockout (KO) mice recapitulate CF intestinal disease, including intestinal hyperproliferation. Previous studies using Cftr KO intestinal organoids (enteroids) indicate that crypt epithelium maintains an alkaline intracellular pH (pHi). We hypothesized that Cftr has a cell-autonomous role in downregulating pHi that is incompletely compensated by acid-base regulation in its absence. Here, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein microfluorimetry of enteroids showed that Cftr KO crypt epithelium sustains an alkaline pHi and resistance to cell acidification relative to wild-type. Quantitative real-time PCR revealed that Cftr KO enteroids exhibit downregulated transcription of base (HCO3−)-loading proteins and upregulation of the basolateral membrane HCO3−-unloader anion exchanger 2 (Ae2). Although Cftr KO crypt epithelium had increased Ae2 expression and Ae2-mediated Cl−/HCO3− exchange with maximized gradients, it also had increased intracellular Cl− concentration relative to wild-type. Pharmacological reduction of intracellular Cl− concentration in Cftr KO crypt epithelium normalized pHi, which was largely Ae2-dependent. We conclude that Cftr KO crypt epithelium maintains an alkaline pHi as a consequence of losing both Cl− and HCO3− efflux, which impairs pHi regulation by Ae2. Retention of Cl− and an alkaline pHi in crypt epithelium may alter several cellular processes in the proliferative compartment of Cftr KO intestine. PMID:26542396

  13. Cellular chloride and bicarbonate retention alters intracellular pH regulation in Cftr KO crypt epithelium.

    Walker, Nancy M; Liu, Jinghua; Stein, Sydney R; Stefanski, Casey D; Strubberg, Ashlee M; Clarke, Lane L

    2016-01-15

    Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), an anion channel providing a major pathway for Cl(-) and HCO3 (-) efflux across the apical membrane of the epithelium. In the intestine, CF manifests as obstructive syndromes, dysbiosis, inflammation, and an increased risk for gastrointestinal cancer. Cftr knockout (KO) mice recapitulate CF intestinal disease, including intestinal hyperproliferation. Previous studies using Cftr KO intestinal organoids (enteroids) indicate that crypt epithelium maintains an alkaline intracellular pH (pHi). We hypothesized that Cftr has a cell-autonomous role in downregulating pHi that is incompletely compensated by acid-base regulation in its absence. Here, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein microfluorimetry of enteroids showed that Cftr KO crypt epithelium sustains an alkaline pHi and resistance to cell acidification relative to wild-type. Quantitative real-time PCR revealed that Cftr KO enteroids exhibit downregulated transcription of base (HCO3 (-))-loading proteins and upregulation of the basolateral membrane HCO3 (-)-unloader anion exchanger 2 (Ae2). Although Cftr KO crypt epithelium had increased Ae2 expression and Ae2-mediated Cl(-)/HCO3 (-) exchange with maximized gradients, it also had increased intracellular Cl(-) concentration relative to wild-type. Pharmacological reduction of intracellular Cl(-) concentration in Cftr KO crypt epithelium normalized pHi, which was largely Ae2-dependent. We conclude that Cftr KO crypt epithelium maintains an alkaline pHi as a consequence of losing both Cl(-) and HCO3 (-) efflux, which impairs pHi regulation by Ae2. Retention of Cl(-) and an alkaline pHi in crypt epithelium may alter several cellular processes in the proliferative compartment of Cftr KO intestine. Copyright © 2016 the American Physiological Society.

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

    Yuning Hou; Xiaoqing Guan; Zhe Yang; Chunying Li

    2016-01-01

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

  15. CFTR-dependent chloride efflux in cystic fibrosis mononuclear cells is increased by ivacaftor therapy.

    Guerra, Lorenzo; D'Oria, Susanna; Favia, Maria; Castellani, Stefano; Santostasi, Teresa; Polizzi, Angela M; Mariggiò, Maria A; Gallo, Crescenzio; Casavola, Valeria; Montemurro, Pasqualina; Leonetti, Giuseppina; Manca, Antonio; Conese, Massimo

    2017-07-01

    The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) potentiator ivacaftor (Kalydeco®) improves clinical outcome in G551D cystic fibrosis (CF) patients. Here, we have investigated whether ivacaftor has a clinical impact on non-G551D gating mutations and function of circulating leukocytes as well. Seven patients were treated with ivacaftor and evaluated at baseline, and at 1-3 and 6 months. Besides clinical and systemic inflammatory parameters, circulating mononuclear cells (MNC) were evaluated for CFTR-dependent chloride efflux by spectrofluorimetry, neutrophils for oxidative burst by cytofluorimetry and HVCN1 mRNA expression by real time PCR. Ivacaftor determined a significant decrease in sweat chloride concentrations at all time points during treatment. Body mass index (BMI), FEV 1 , and FVC showed an increasing trend. While C-reactive protein decreased significantly at 2 months, the opposite behavior was noticed for circulating monocytes. CFTR activity in MNC was found to increase significantly at 3 and 6 months. Neutrophil oxidative burst peaked at 2 months and then decreased to baseline. HVCN1 mRNA expression was significantly higher than baseline at 1-3 months and decreased after 6 months of treatment. The chloride efflux in MNC correlated positively with both FEV 1 and FVC. On the other hand, sweat chloride correlated positively with CRP and WBC, and negatively with both respiratory function tests. A cluster analysis confirmed that sweat chloride, FEV 1 , FVC, BMI, and MNC chloride efflux behaved as a single entity over time. In patients with non-G551D mutations, ivacaftor improved both chloride transport in sweat ducts and chloride efflux in MNC, that is, functions directly imputed to CFTR. © 2017 Wiley Periodicals, Inc.

  16. Microparticle-mediated transfer of the viral receptors CAR and CD46, and the CFTR channel in a CHO cell model confers new functions to target cells.

    Gaëlle Gonzalez

    Full Text Available Cell microparticles (MPs released in the extracellular milieu can embark plasma membrane and intracellular components which are specific of their cellular origin, and transfer them to target cells. The MP-mediated, cell-to-cell transfer of three human membrane glycoproteins of different degrees of complexity was investigated in the present study, using a CHO cell model system. We first tested the delivery of CAR and CD46, two monospanins which act as adenovirus receptors, to target CHO cells. CHO cells lack CAR and CD46, high affinity receptors for human adenovirus serotype 5 (HAdV5, and serotype 35 (HAdV35, respectively. We found that MPs derived from CHO cells (MP-donor cells constitutively expressing CAR (MP-CAR or CD46 (MP-CD46 were able to transfer CAR and CD46 to target CHO cells, and conferred selective permissiveness to HAdV5 and HAdV35. In addition, target CHO cells incubated with MP-CD46 acquired the CD46-associated function in complement regulation. We also explored the MP-mediated delivery of a dodecaspanin membrane glycoprotein, the CFTR to target CHO cells. CFTR functions as a chloride channel in human cells and is implicated in the genetic disease cystic fibrosis. Target CHO cells incubated with MPs produced by CHO cells constitutively expressing GFP-tagged CFTR (MP-GFP-CFTR were found to gain a new cellular function, the chloride channel activity associated to CFTR. Time-course analysis of the appearance of GFP-CFTR in target cells suggested that MPs could achieve the delivery of CFTR to target cells via two mechanisms: the transfer of mature, membrane-inserted CFTR glycoprotein, and the transfer of CFTR-encoding mRNA. These results confirmed that cell-derived MPs represent a new class of promising therapeutic vehicles for the delivery of bioactive macromolecules, proteins or mRNAs, the latter exerting the desired therapeutic effect in target cells via de novo synthesis of their encoded proteins.

  17. Two Salt Bridges Differentially Contribute to the Maintenance of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel Function*

    Cui, Guiying; Freeman, Cody S.; Knotts, Taylor; Prince, Chengyu Z.; Kuang, Christopher; McCarty, Nael A.

    2013-01-01

    Previous studies have identified two salt bridges in human CFTR chloride ion channels, Arg352-Asp993 and Arg347-Asp924, that are required for normal channel function. In the present study, we determined how the two salt bridges cooperate to maintain the open pore architecture of CFTR. Our data suggest that Arg347 not only interacts with Asp924 but also interacts with Asp993. The tripartite interaction Arg347-Asp924-Asp993 mainly contributes to maintaining a stable s2 open subconductance state. The Arg352-Asp993 salt bridge, in contrast, is involved in stabilizing both the s2 and full (f) open conductance states, with the main contribution being to the f state. The s1 subconductance state does not require either salt bridge. In confirmation of the role of Arg352 and Asp993, channels bearing cysteines at these sites could be latched into a full open state using the bifunctional cross-linker 1,2-ethanediyl bismethanethiosulfonate, but only when applied in the open state. Channels remained latched open even after washout of ATP. The results suggest that these interacting residues contribute differently to stabilizing the open pore in different phases of the gating cycle. PMID:23709221

  18. Mechanisms of CFTR Functional Variants That Impair Regulated Bicarbonate Permeation and Increase Risk for Pancreatitis but Not for Cystic Fibrosis

    LaRusch, Jessica; Jung, Jinsei; General, Ignacio J.; Lewis, Michele D.; Park, Hyun Woo; Brand, Randall E.; Gelrud, Andres; Anderson, Michelle A.; Banks, Peter A.; Conwell, Darwin; Lawrence, Christopher; Romagnuolo, Joseph; Baillie, John; Alkaade, Samer; Cote, Gregory

    2014-01-01

    CFTR is a dynamically regulated anion channel. Intracellular WNK1-SPAK activation causes CFTR to change permeability and conductance characteristics from a chloride-preferring to bicarbonate-preferring channel through unknown mechanisms. Two severe CFTR mutations (CFTRsev ) cause complete loss of CFTR function and result in cystic fibrosis (CF), a severe genetic disorder affecting sweat glands, nasal sinuses, lungs, pancreas, liver, intestines, and male reproductive system. We hypothesize tha...

  19. Sweat chloride and immunoreactive trypsinogen in infants carrying two CFTR mutations and not affected by cystic fibrosis.

    Castellani, Carlo; Tridello, Gloria; Tamanini, Anna; Assael, Baroukh M

    2017-07-01

    Newborns with raised immunotrypsinogen levels who have non-pathological sweat chloride values and carry two cystic fibrosis transmembrane regulator ( CFTR ) mutations of which at least one is not acknowledged to be cystic fibrosis (CF)-causing are at risk of developing clinical manifestations consistent with CFTR-related disorders or even CF. It is not known whether newborns with similar genotypes and normal immunoreactive trypsinogen (IRT) may share the same risk. This study found that newborns with these characteristics and normal IRT have lower sweat chloride values than those with raised IRT (p=0.007). Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  20. Functional modifications of acid-sensing ion channels by ligand-gated chloride channels.

    Xuanmao Chen

    Full Text Available Together, acid-sensing ion channels (ASICs and epithelial sodium channels (ENaC constitute the majority of voltage-independent sodium channels in mammals. ENaC is regulated by a chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR. Here we show that ASICs were reversibly inhibited by activation of GABA(A receptors in murine hippocampal neurons. This inhibition of ASICs required opening of the chloride channels but occurred with both outward and inward GABA(A receptor-mediated currents. Moreover, activation of the GABA(A receptors modified the pharmacological features and kinetic properties of the ASIC currents, including the time course of activation, desensitization and deactivation. Modification of ASICs by open GABA(A receptors was also observed in both nucleated patches and outside-out patches excised from hippocampal neurons. Interestingly, ASICs and GABA(A receptors interacted to regulate synaptic plasticity in CA1 hippocampal slices. The activation of glycine receptors, which are similar to GABA(A receptors, also modified ASICs in spinal neurons. We conclude that GABA(A receptors and glycine receptors modify ASICs in neurons through mechanisms that require the opening of chloride channels.

  1. Intra-individual biological variation in sweat chloride concentrations in CF, CFTR dysfunction, and healthy pediatric subjects.

    Cirilli, Natalia; Raia, Valeria; Rocco, Ilaria; De Gregorio, Fabiola; Tosco, Antonella; Salvadori, Laura; Sepe, Angela Ornella; Buzzetti, Roberto; Minicuci, Nadia; Castaldo, Giuseppe

    2018-04-02

    The sweat test is one of the main diagnostic tools used in newborn screening programs and as a confirmatory test, in case of suspect of Cystic Fibrosis (CF). Since sweat chloride (Cl) concentration is also considered an appropriate parameter to explore the efficacy of CFTR modulators in clinical trials, it is crucial to evaluate the biological variability of this test in healthy and pathological conditions. The aim of this pilot study was to determine the intra-individual biological variability of sweat Cl, both in healthy individuals and CF patients and to assess its correlation with diet, season, and menstrual cycle. Thirty-five out of 36 selected subjects (6-18 years) were enrolled by 2 CF care centers and assigned to 3 cohorts: CF, CFTR-related disorder (CFTR-RD) and healthy volunteers. Each participant was subjected to eight sweat tests in different conditions and time of the year. Data were analyzed using linear mixed effects models for repeated measures, taking also into account intra-individual correlations. We observed a high intra-individual variability of sweat Cl, with the lowest mean CV% values among CF patients (20.21 in CF, 29.74 in CFTR-RD, and 31.15 in healthy subjects). Gender and diet had no influence on sweat Cl variability, nor had pubertal age and menstrual phase. Results of this pilot study confirmed that sweat Cl variability is high in CF patients, although non-CF individuals displayed even higher mean CV% values. Season significantly influenced sweat test values only in CF patients, likely due to changes in their hydration status. © 2018 Wiley Periodicals, Inc.

  2. Current insights into the role of PKA phosphorylation in CFTR channel activity and the pharmacological rescue of cystic fibrosis disease-causing mutants.

    Chin, Stephanie; Hung, Maurita; Bear, Christine E

    2017-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) channel gating is predominantly regulated by protein kinase A (PKA)-dependent phosphorylation. In addition to regulating CFTR channel activity, PKA phosphorylation is also involved in enhancing CFTR trafficking and mediating conformational changes at the interdomain interfaces of the protein. The major cystic fibrosis (CF)-causing mutation is the deletion of phenylalanine at position 508 (F508del); it causes many defects that affect CFTR trafficking, stability, and gating at the cell surface. Due to the multiple roles of PKA phosphorylation, there is growing interest in targeting PKA-dependent signaling for rescuing the trafficking and functional defects of F508del-CFTR. This review will discuss the effects of PKA phosphorylation on wild-type CFTR, the consequences of CF mutations on PKA phosphorylation, and the development of therapies that target PKA-mediated signaling.

  3. The ichthyotoxic alga Chattonella marina induces Na+, K+-ATPase, and CFTR proteins expression in fish gill chloride cells in vivo

    Tang, Janet Y.M.; Wong, Chris K.C.; Au, Doris W.T.

    2007-01-01

    Our previous studies demonstrated that the ichthyotoxic Chattonella marina stimulated proliferation of branchial chloride cell (CC) and induced osmotic distress akin to hyperactive elimination of ions in fish (Rhabdosargus sarba). To ascertain the in vivo effects of C. marina on key CC ion transporters, the localization and expression of Na + , K + -ATPase (NKA) and cystic fibrosis transmembrane conductance regulator (CFTR) proteins in response to C. marina exposure were investigated, using a quantitative immunocytochemical approach. The polarized distributions of NKA (α subunit) and CFTR proteins in branchial CCs of R. sarba remained unchanged under C. marina exposure. However, significant inductions of these two ion-transporters were detected in CCs of fish after 6 h exposure. By real-time PCR, no significant changes in gill NKA and CFTR mRNA expressions were detected, suggesting a post-transcriptional pathway is likely involved in regulating the ion transporters abundance. This study is the first to demonstrate the in vivo effects of harmful algal toxin on NKA and CFTR protein expressions in gill transepithelial cells. Taken together, an augmentation of branchial CCs together with hyper-stimulation of NKA and CFTR in CCs attribute to the rapid development of osmotic distress in C. marina susceptible fish

  4. Regulatory crosstalk by protein kinases on CFTR trafficking and activity

    Farinha, Carlos Miguel; Swiatecka-Urban, Agnieszka; Brautigan, David; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e. channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease.

  5. Involvement of the Cdc42 pathway in CFTR post-translational turnover and in its plasma membrane stability in airway epithelial cells.

    Romain Ferru-Clément

    Full Text Available Cystic fibrosis transmembrane conductance regulator (CFTR is a chloride channel that is expressed on the apical plasma membrane (PM of epithelial cells. The most common deleterious allele encodes a trafficking-defective mutant protein undergoing endoplasmic reticulum-associated degradation (ERAD and presenting lower PM stability. In this study, we investigated the involvement of the Cdc42 pathway in CFTR turnover and trafficking in a human bronchiolar epithelial cell line (CFBE41o- expressing wild-type CFTR. Cdc42 is a small GTPase of the Rho family that fulfils numerous cell functions, one of which is endocytosis and recycling process via actin cytoskeleton remodelling. When we treated cells with chemical inhibitors such as ML141 against Cdc42 and wiskostatin against the downstream effector N-WASP, we observed that CFTR channel activity was inhibited, in correlation with a decrease in CFTR amount at the cell surface and an increase in dynamin-dependent CFTR endocytosis. Anchoring of CFTR to the cortical cytoskeleton was then presumably impaired by actin disorganization. When we performed siRNA-mediated depletion of Cdc42, actin polymerization was not impacted, but we observed actin-independent consequences upon CFTR. Total and PM CFTR amounts were increased, resulting in greater activation of CFTR. Pulse-chase experiments showed that while CFTR degradation was slowed, CFTR maturation through the Golgi apparatus remained unaffected. In addition, we observed increased stability of CFTR in PM and reduction of its endocytosis. This study highlights the involvement of the Cdc42 pathway at several levels of CFTR biogenesis and trafficking: (i Cdc42 is implicated in the first steps of CFTR biosynthesis and processing; (ii it contributes to the stability of CFTR in PM via its anchoring to cortical actin; (iii it promotes CFTR endocytosis and presumably its sorting toward lysosomal degradation.

  6. The effect of NO-donors on chloride efflux, intracellular Ca(2+) concentration and mRNA expression of CFTR and ENaC in cystic fibrosis airway epithelial cells.

    Oliynyk, Igor; Hussain, Rashida; Amin, Ahmad; Johannesson, Marie; Roomans, Godfried M

    2013-06-01

    Since previous studies showed that the endogenous bronchodilator, S-nitrosglutathione (GSNO), caused a marked increase in CFTR-mediated chloride (Cl(-)) efflux and improved the trafficking of CFTR to the plasma membrane, and that also the nitric oxide (NO)-donor GEA3162 had a similar, but smaller, effect on Cl(-) efflux, it was investigated whether the NO-donor properties of GSNO were relevant for its effect on Cl(-) efflux from airway epithelial cells. Hence, the effect of a number of other NO-donors, sodium nitroprusside (SNP), S-nitroso-N-acetyl-DL-penicillamine (SNAP), diethylenetriamine/nitric oxide adduct (DETA-NO), and diethylenetriamine/nitric oxide adduct (DEA-NONOate) on Cl(-) efflux from CFBE (∆F508/∆F508-CFTR) airway epithelial cells was tested. Cl(-) efflux was determined using the fluorescent N-(ethoxycarbonylmethyl)-6-methoxyquinoliniu bromide (MQAE)-technique. Possible changes in the intracellular Ca(2+) concentration were tested by the fluorescent fluo-4 method in a confocal microscope system. Like previously with GSNO, after 4 h incubation with the NO-donor, an increased Cl(-) efflux was found (in the order SNAP>DETA-NO>SNP). The effect of DEA-NONOate on Cl(-) efflux was not significant, and the compound may have (unspecific) deleterious effects on the cells. Again, as with GSNO, after a short (5 min) incubation, SNP had no significant effect on Cl(-) efflux. None of the NO-donors that had a significant effect on Cl(-) efflux caused significant changes in the intracellular Ca(2+) concentration. After 4 h preincubation, SNP caused a significant increase in the mRNA expression of CFTR. SNAP and DEA-NONOate decreased the mRNA expression of all ENaC subunits significantly. DETA-NO caused a significant decrease only in α-ENaC expression. After a short preincubation, none of the NO-donors had a significant effect, neither on the expression of CFTR, nor on that of the ENaC subunits in the presence and absence of L-cysteine. It can be concluded that

  7. The CFTR-Associated Ligand Arrests the Trafficking of the Mutant ΔF508 CFTR Channel in the ER Contributing to Cystic Fibrosis

    Emily Bergbower

    2018-01-01

    Full Text Available Background/Aims: The CFTR-Associated Ligand (CAL, a PDZ domain containing protein with two coiled-coil domains, reduces cell surface WT CFTR through degradation in the lysosome by a well-characterized mechanism. However, CAL’s regulatory effect on ΔF508 CFTR has remained almost entirely uninvestigated. Methods: In this study, we describe a previously unknown pathway for CAL by which it regulates the membrane expression of ΔF508 CFTR through arrest of ΔF508 CFTR trafficking in the endoplasmic reticulum (ER using a combination of cell biology, biochemistry and electrophysiology. Results: We demonstrate that CAL is an ER localized protein that binds to ΔF508 CFTR and is degraded in the 26S proteasome. When CAL is inhibited, ΔF508 CFTR retention in the ER decreases and cell surface expression of mature functional ΔF508 CFTR is observed alongside of enhanced expression of plasma membrane scaffolding protein NHERF1. Chaperone proteins regulate this novel process, and ΔF508 CFTR binding to HSP40, HSP90, HSP70, VCP, and Aha1 changes to improve ΔF508 CFTR cell surface trafficking. Conclusion: Our results reveal a pathway in which CAL regulates the cell surface availability and intracellular retention of ΔF508 CFTR.

  8. Purinergic regulation of CFTR and Ca2+ -activated Cl- channels and K+ channels in human pancreatic duct epithelium

    Wang, Jing; Haanes, Kristian A; Novak, Ivana

    2013-01-01

    mutated CFTR, basolateral ATP and UTP had negligible effects. In addition to Cl(-) transport in Capan-1 cells, the effects of 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (DC-EBIO) and clotrimazole indicated functional expression of the intermediate conductance K(+) channels (IK, KCa3...

  9. General anesthetic octanol and related compounds activate wild-type and delF508 cystic fibrosis chloride channels

    Marcet, Brice; Becq, Frédéric; Norez, Caroline; Delmas, Patrick; Verrier, Bernard

    2004-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is defective during cystic fibrosis (CF). Activators of the CFTR Cl− channel may be useful for therapy of CF. Here, we demonstrate that a range of general anesthetics like normal-alkanols (n-alkanols) and related compounds can stimulate the Cl− channel activity of wild-type CFTR and delF508-CFTR mutant.The effects of n-alkanols like octanol on CFTR activity were measured by iodide (125I) efflux and patch-clamp techniques o...

  10. Clusters of Cl- channels in CFTR-expressing em>Sf>9 cells switch spontaneously between slow and fast gating modes

    Larsen, Erik Hviid; Price, E. M.; Gabriel, S. E.

    1996-01-01

    channel. Excised outside-out patches of CFTR-infected and forskolin-stimulated cells exhibited wave-like gating kinetics of well-resolved current transitions. All-point Gaussian distributions revealed contributions from several (five to nine) identical channels. Such channels, in excised outside...

  11. From the endoplasmic reticulum to the plasma membrane: mechanisms of CFTR folding and trafficking.

    Farinha, Carlos M; Canato, Sara

    2017-01-01

    CFTR biogenesis starts with its co-translational insertion into the membrane of endoplasmic reticulum and folding of the cytosolic domains, towards the acquisition of a fully folded compact native structure. Efficiency of this process is assessed by the ER quality control system that allows the exit of folded proteins but targets unfolded/misfolded CFTR to degradation. If allowed to leave the ER, CFTR is modified at the Golgi and reaches the post-Golgi compartments to be delivered to the plasma membrane where it functions as a cAMP- and phosphorylation-regulated chloride/bicarbonate channel. CFTR residence at the membrane is a balance of membrane delivery, endocytosis, and recycling. Several adaptors, motor, and scaffold proteins contribute to the regulation of CFTR stability and are involved in continuously assessing its structure through peripheral quality control systems. Regulation of CFTR biogenesis and traffic (and its dysregulation by mutations, such as the most common F508del) determine its overall activity and thus contribute to the fine modulation of chloride secretion and hydration of epithelial surfaces. This review covers old and recent knowledge on CFTR folding and trafficking from its synthesis to the regulation of its stability at the plasma membrane and highlights how several of these steps can be modulated to promote the rescue of mutant CFTR.

  12. Chloride channels as tools for developing selective insecticides.

    Bloomquist, Jeffrey R

    2003-12-01

    Ligand-gated chloride channels underlie inhibition in excitable membranes and are proven target sites for insecticides. The gamma-aminobutyric acid (GABA(1)) receptor/chloride ionophore complex is the primary site of action for a number of currently used insecticides, such as lindane, endosulfan, and fipronil. These compounds act as antagonists by stabilizing nonconducting conformations of the chloride channel. Blockage of the GABA-gated chloride channel reduces neuronal inhibition, which leads to hyperexcitation of the central nervous system, convulsions, and death. We recently investigated the mode of action of the silphinenes, plant-derived natural compounds that structurally resemble picrotoxinin. These materials antagonize the action of GABA on insect neurons and block GABA-mediated chloride uptake into mouse brain synaptoneurosomes in a noncompetitive manner. In mammals, avermectins have a blocking action on the GABA-gated chloride channel consistent with a coarse tremor, whereas at longer times and higher concentrations, activation of the channel suppresses neuronal activity. Invertebrates display ataxia, paralysis, and death as the predominant signs of poisoning, with a glutamate-gated chloride channel playing a major role. Additional target sites for the avermectins or other chloride channel-directed compounds might include receptors gated by histamine, serotonin, or acetylcholine.The voltage-sensitive chloride channels form another large gene family of chloride channels. Voltage-dependent chloride channels are involved in a number of physiological processes including: maintenance of electrical excitability, chloride ion secretion and resorption, intravesicular acidification, and cell volume regulation. A subset of these channels is affected by convulsants and insecticides in mammals, although the role they play in acute lethality in insects is unclear. Given the wide range of functions that they mediate, these channels are also potential targets for

  13. Determination of CFTR densities in erythrocyte plasma membranes using recognition imaging

    Ebner, Andreas; Hinterdorfer, Peter [Institute for Biophysics, University of Linz, A-4040 Linz (Austria); Nikova, Dessy; Lange, Tobias; Bruns, Reimer; Oberleithner, Hans; Schillers, Hermann [Institute of Physiology II, University of Muenster, D-48149 Muenster (Germany); Haeberle, Johannes; Falk, Sabine; Duebbers, Angelika [Department of Pediatrics, University Hospitals of Muenster, D-48149 Muenster (Germany)], E-mail: schille@uni-muenster.de

    2008-09-24

    CFTR (cystic fibrosis transmembrane conductance regulator) is a cAMP-regulated chloride (Cl{sup -}) channel that plays an important role in salt and fluid movement across epithelia. Cystic fibrosis (CF), the most common genetic disease among Caucasians, is caused by mutations in the gene encoding CFTR. The most predominant mutation, F508del, disturbs CFTR protein trafficking, resulting in a reduced number of CFTR in the plasma membrane. Recent studies indicate that CFTR is not only found in epithelia but also in human erythrocytes. Although considerable attempts have been made to quantify CFTR in cells, conclusions on numbers of CFTR molecules localized in the plasma membrane have been drawn indirectly. AFM has the power to provide the needed information, since both sub-molecular spatial resolution and direct protein recognition via antibody-antigen interaction can be observed. We performed a quantification study of the CFTR copies in erythrocyte membranes at the single molecule level, and compared the difference between healthy donors and CF patients. We detected that the number of CFTR molecules is reduced by 70% in erythrocytes of cystic fibrosis patients.

  14. Determination of CFTR densities in erythrocyte plasma membranes using recognition imaging

    Ebner, Andreas; Hinterdorfer, Peter; Nikova, Dessy; Lange, Tobias; Bruns, Reimer; Oberleithner, Hans; Schillers, Hermann; Haeberle, Johannes; Falk, Sabine; Duebbers, Angelika

    2008-01-01

    CFTR (cystic fibrosis transmembrane conductance regulator) is a cAMP-regulated chloride (Cl - ) channel that plays an important role in salt and fluid movement across epithelia. Cystic fibrosis (CF), the most common genetic disease among Caucasians, is caused by mutations in the gene encoding CFTR. The most predominant mutation, F508del, disturbs CFTR protein trafficking, resulting in a reduced number of CFTR in the plasma membrane. Recent studies indicate that CFTR is not only found in epithelia but also in human erythrocytes. Although considerable attempts have been made to quantify CFTR in cells, conclusions on numbers of CFTR molecules localized in the plasma membrane have been drawn indirectly. AFM has the power to provide the needed information, since both sub-molecular spatial resolution and direct protein recognition via antibody-antigen interaction can be observed. We performed a quantification study of the CFTR copies in erythrocyte membranes at the single molecule level, and compared the difference between healthy donors and CF patients. We detected that the number of CFTR molecules is reduced by 70% in erythrocytes of cystic fibrosis patients

  15. Conserved allosteric hot spots in the transmembrane domains of cystic fibrosis transmembrane conductance regulator (CFTR) channels and multidrug resistance protein (MRP) pumps.

    Wei, Shipeng; Roessler, Bryan C; Chauvet, Sylvain; Guo, Jingyu; Hartman, John L; Kirk, Kevin L

    2014-07-18

    ATP-binding cassette (ABC) transporters are an ancient family of transmembrane proteins that utilize ATPase activity to move substrates across cell membranes. The ABCC subfamily of the ABC transporters includes active drug exporters (the multidrug resistance proteins (MRPs)) and a unique ATP-gated ion channel (cystic fibrosis transmembrane conductance regulator (CFTR)). The CFTR channel shares gating principles with conventional ligand-gated ion channels, but the allosteric network that couples ATP binding at its nucleotide binding domains (NBDs) with conformational changes in its transmembrane helices (TMs) is poorly defined. It is also unclear whether the mechanisms that govern CFTR gating are conserved with the thermodynamically distinct MRPs. Here we report a new class of gain of function (GOF) mutation of a conserved proline at the base of the pore-lining TM6. Multiple substitutions of this proline promoted ATP-free CFTR activity and activation by the weak agonist, 5'-adenylyl-β,γ-imidodiphosphate (AMP-PNP). TM6 proline mutations exhibited additive GOF effects when combined with a previously reported GOF mutation located in an outer collar of TMs that surrounds the pore-lining TMs. Each TM substitution allosterically rescued the ATP sensitivity of CFTR gating when introduced into an NBD mutant with defective ATP binding. Both classes of GOF mutations also rescued defective drug export by a yeast MRP (Yor1p) with ATP binding defects in its NBDs. We conclude that the conserved TM6 proline helps set the energy barrier to both CFTR channel opening and MRP-mediated drug efflux and that CFTR channels and MRP pumps utilize similar allosteric mechanisms for coupling conformational changes in their translocation pathways to ATP binding at their NBDs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. The human CFTR protein expressed in CHO cells activates aquaporin-3 in a cAMP-dependent pathway: study by digital holographic microscopy

    Jourdain, P.

    2013-12-11

    The transmembrane water movements during cellular processes and their relationship to ionic channel activity remain largely unknown. As an example, in epithelial cells it was proposed that the movement of water could be directly linked to cystic fibrosis transmembrane conductance regulator (CFTR) protein activity through a cAMP-stimulated aqueous pore, or be dependent on aquaporin. Here, we used digital holographic microscopy (DHM) an interferometric technique to quantify in situ the transmembrane water fluxes during the activity of the epithelial chloride channel, CFTR, measured by patch-clamp and iodide efflux techniques. We showed that the water transport measured by DHM is fully inhibited by the selective CFTR blocker CFTRinh172 and is absent in cells lacking CFTR. Of note, in cells expressing the mutated version of CFTR (F508del-CFTR), which mimics the most common genetic alteration encountered in cystic fibrosis, we also show that the water movement is profoundly altered but restored by pharmacological manipulation of F508del-CFTR-defective trafficking. Importantly, whereas activation of this endogenous water channel required a cAMP-dependent stimulation of CFTR, activation of CFTR or F508del-CFTR by two cAMP-independent CFTR activators, genistein and MPB91, failed to trigger water movements. Finally, using a specific small-interfering RNA against the endogenous aquaporin AQP3, the water transport accompanying CFTR activity decreased. We conclude that water fluxes accompanying CFTR activity are linked to AQP3 but not to a cAMP-stimulated aqueous pore in the CFTR protein.

  17. General anesthetic octanol and related compounds activate wild-type and delF508 cystic fibrosis chloride channels.

    Marcet, Brice; Becq, Frédéric; Norez, Caroline; Delmas, Patrick; Verrier, Bernard

    2004-03-01

    1. Cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is defective during cystic fibrosis (CF). Activators of the CFTR Cl(-) channel may be useful for therapy of CF. Here, we demonstrate that a range of general anesthetics like normal-alkanols (n-alkanols) and related compounds can stimulate the Cl(-) channel activity of wild-type CFTR and delF508-CFTR mutant. 2. The effects of n-alkanols like octanol on CFTR activity were measured by iodide ((125)I) efflux and patch-clamp techniques on three distinct cellular models: (1). CFTR-expressing Chinese hamster ovary cells, (2). human airway Calu-3 epithelial cells and (3). human airway JME/CF15 epithelial cells which express the delF508-CFTR mutant. 3. Our data show for the first time that n-alkanols activate both wild-type CFTR and delF508-CFTR mutant. Octanol stimulated (125)I efflux in a dose-dependent manner in CFTR-expressing cells (wild-type and delF508) but not in cell lines lacking CFTR. (125)I efflux and Cl(-) currents induced by octanol were blocked by glibenclamide but insensitive to 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, as expected for a CFTR Cl(-) current. 4. CFTR activation by octanol was neither due to cell-to-cell uncoupling properties of octanol nor to an intracellular cAMP increase. CFTR activation by octanol requires phosphorylation by protein kinase-A (PKA) since it was prevented by H-89, a PKA inhibitor. 5. n-Alkanols chain length was an important determinant for channel activation, with rank order of potencies: 1-heptanoloctanoloctanol<1-decanol. Our findings may be of valuable interest for developing novel therapeutic strategies for CF.

  18. Biophysical characterisation of calumenin as a charged F508del-CFTR folding modulator.

    Rashmi Tripathi

    Full Text Available The cystic fibrosis transmembrane regulator (CFTR is a cyclic-AMP dependent chloride channel expressed at the apical surface of epithelial cells lining various organs such as the respiratory tract. Defective processing and functioning of this protein caused by mutations in the CFTR gene results in loss of ionic balance, defective mucus clearance, increased proliferation of biofilms and inflammation of human airways observed in cystic fibrosis (CF patients. The process by which CFTR folds and matures under the influence of various chaperones in the secretory pathway remains incompletely understood. Recently, calumenin, a secretory protein, belonging to the CREC family of low affinity calcium binding proteins has been identified as a putative CFTR chaperone whose biophysical properties and functions remain uncharacterized. We compared hydropathy, instability, charge, unfoldability, disorder and aggregation propensity of calumenin and other CREC family members with CFTR associated chaperones and calcium binding proteins, wild-type and mutant CFTR proteins and intrinsically disordered proteins (IDPs. We observed that calumenin, along with other CREC proteins, was significantly more charged and less folded compared to CFTR associated chaperones. Moreover like IDPs, calumenin and other CREC proteins were found to be less hydrophobic and aggregation prone. Phylogenetic analysis revealed a close link between calumenin and other CREC proteins indicating how evolution might have shaped their similar biophysical properties. Experimentally, calumenin was observed to significantly reduce F508del-CFTR aggregation in a manner similar to AavLEA1, a well-characterized IDP. Fluorescence microscopy based imaging analysis also revealed altered trafficking of calumenin in bronchial cells expressing F508del-CFTR, indicating its direct role in the pathophysiology of CF. In conclusion, calumenin is characterized as a charged protein exhibiting close similarity with

  19. Optimizing nasal potential difference analysis for CFTR modulator development: assessment of ivacaftor in CF subjects with the G551D-CFTR mutation.

    Steven M Rowe

    Full Text Available Nasal potential difference (NPD is used as a biomarker of the cystic fibrosis transmembrane conductance regulator (CFTR and epithelial sodium channel (ENaC activity. We evaluated methods to detect changes in chloride and sodium transport by NPD based on a secondary analysis of a Phase II CFTR-modulator study. Thirty-nine subjects with CF who also had the G551D-CFTR mutation were randomized to receive ivacaftor (Kalydeco™; also known as VX-770 in four doses or placebo twice daily for at least 14 days. All data were analyzed by a single investigator who was blinded to treatment assignment. We compared three analysis methods to determine the best approach to quantify changes in chloride and sodium transport: (1 the average of both nostrils; (2 the most-polarized nostril at each visit; and (3 the most-polarized nostril at screening carried forward. Parameters of ion transport included the PD change with zero chloride plus isoproterenol (CFTR activity, the basal PD, Ringer's PD, and change in PD with amiloride (measurements of ENaC activity, and the delta NPD (measuring CFTR and ENaC activity. The average and most-polarized nostril at each visit were most sensitive to changes in chloride and sodium transport, whereas the most-polarized nostril at screening carried forward was less discriminatory. Based on our findings, NPD studies should assess both nostrils rather than a single nostril. We also found that changes in CFTR activity were more readily detected than changes in ENaC activity, and that rigorous standardization was associated with relatively good within-subject reproducibility in placebo-treated subjects (± 2.8 mV. Therefore, we have confirmed an assay of reasonable reproducibility for detecting chloride-transport improvements in response to CFTR modulation.

  20. Mechanisms of CFTR functional variants that impair regulated bicarbonate permeation and increase risk for pancreatitis but not for cystic fibrosis.

    LaRusch, Jessica; Jung, Jinsei; General, Ignacio J; Lewis, Michele D; Park, Hyun Woo; Brand, Randall E; Gelrud, Andres; Anderson, Michelle A; Banks, Peter A; Conwell, Darwin; Lawrence, Christopher; Romagnuolo, Joseph; Baillie, John; Alkaade, Samer; Cote, Gregory; Gardner, Timothy B; Amann, Stephen T; Slivka, Adam; Sandhu, Bimaljit; Aloe, Amy; Kienholz, Michelle L; Yadav, Dhiraj; Barmada, M Michael; Bahar, Ivet; Lee, Min Goo; Whitcomb, David C

    2014-07-01

    CFTR is a dynamically regulated anion channel. Intracellular WNK1-SPAK activation causes CFTR to change permeability and conductance characteristics from a chloride-preferring to bicarbonate-preferring channel through unknown mechanisms. Two severe CFTR mutations (CFTRsev) cause complete loss of CFTR function and result in cystic fibrosis (CF), a severe genetic disorder affecting sweat glands, nasal sinuses, lungs, pancreas, liver, intestines, and male reproductive system. We hypothesize that those CFTR mutations that disrupt the WNK1-SPAK activation mechanisms cause a selective, bicarbonate defect in channel function (CFTRBD) affecting organs that utilize CFTR for bicarbonate secretion (e.g. the pancreas, nasal sinus, vas deferens) but do not cause typical CF. To understand the structural and functional requirements of the CFTR bicarbonate-preferring channel, we (a) screened 984 well-phenotyped pancreatitis cases for candidate CFTRBD mutations from among 81 previously described CFTR variants; (b) conducted electrophysiology studies on clones of variants found in pancreatitis but not CF; (c) computationally constructed a new, complete structural model of CFTR for molecular dynamics simulation of wild-type and mutant variants; and (d) tested the newly defined CFTRBD variants for disease in non-pancreas organs utilizing CFTR for bicarbonate secretion. Nine variants (CFTR R74Q, R75Q, R117H, R170H, L967S, L997F, D1152H, S1235R, and D1270N) not associated with typical CF were associated with pancreatitis (OR 1.5, p = 0.002). Clones expressed in HEK 293T cells had normal chloride but not bicarbonate permeability and conductance with WNK1-SPAK activation. Molecular dynamics simulations suggest physical restriction of the CFTR channel and altered dynamic channel regulation. Comparing pancreatitis patients and controls, CFTRBD increased risk for rhinosinusitis (OR 2.3, p<0.005) and male infertility (OR 395, p<0.0001). WNK1-SPAK pathway-activated increases in CFTR

  1. Mechanisms of CFTR functional variants that impair regulated bicarbonate permeation and increase risk for pancreatitis but not for cystic fibrosis.

    Jessica LaRusch

    2014-07-01

    Full Text Available CFTR is a dynamically regulated anion channel. Intracellular WNK1-SPAK activation causes CFTR to change permeability and conductance characteristics from a chloride-preferring to bicarbonate-preferring channel through unknown mechanisms. Two severe CFTR mutations (CFTRsev cause complete loss of CFTR function and result in cystic fibrosis (CF, a severe genetic disorder affecting sweat glands, nasal sinuses, lungs, pancreas, liver, intestines, and male reproductive system. We hypothesize that those CFTR mutations that disrupt the WNK1-SPAK activation mechanisms cause a selective, bicarbonate defect in channel function (CFTRBD affecting organs that utilize CFTR for bicarbonate secretion (e.g. the pancreas, nasal sinus, vas deferens but do not cause typical CF. To understand the structural and functional requirements of the CFTR bicarbonate-preferring channel, we (a screened 984 well-phenotyped pancreatitis cases for candidate CFTRBD mutations from among 81 previously described CFTR variants; (b conducted electrophysiology studies on clones of variants found in pancreatitis but not CF; (c computationally constructed a new, complete structural model of CFTR for molecular dynamics simulation of wild-type and mutant variants; and (d tested the newly defined CFTRBD variants for disease in non-pancreas organs utilizing CFTR for bicarbonate secretion. Nine variants (CFTR R74Q, R75Q, R117H, R170H, L967S, L997F, D1152H, S1235R, and D1270N not associated with typical CF were associated with pancreatitis (OR 1.5, p = 0.002. Clones expressed in HEK 293T cells had normal chloride but not bicarbonate permeability and conductance with WNK1-SPAK activation. Molecular dynamics simulations suggest physical restriction of the CFTR channel and altered dynamic channel regulation. Comparing pancreatitis patients and controls, CFTRBD increased risk for rhinosinusitis (OR 2.3, p<0.005 and male infertility (OR 395, p<<0.0001. WNK1-SPAK pathway-activated increases in

  2. Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia.

    Dong, Qian; Ernst, Sarah E; Ostedgaard, Lynda S; Shah, Viral S; Ver Heul, Amanda R; Welsh, Michael J; Randak, Christoph O

    2015-05-29

    The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P(1),P(5)-di(adenosine-5') pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5'-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5'-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl(-) channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Manipulating proteostasis to repair the F508del-CFTR defect in cystic fibrosis.

    Esposito, Speranza; Tosco, Antonella; Villella, Valeria R; Raia, Valeria; Kroemer, Guido; Maiuri, Luigi

    2016-12-01

    Cystic fibrosis (CF) is a lethal monogenic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that entails the (diagnostic) increase in sweat electrolyte concentrations, progressive lung disease with chronic inflammation and recurrent bacterial infections, pancreatic insufficiency, and male infertility. Therapies aimed at restoring the CFTR defect have emerged. Thus, a small molecule which facilitates chloride channel opening, the potentiator Ivacaftor, has been approved for the treatment of CF patients bearing a particular class of rare CFTR mutations. However, small molecules that directly target the most common misfolded CFTR mutant, F508del, and improve its intracellular trafficking in vitro, have been less effective than expected when tested in CF patients, even in combination with Ivacaftor. Thus, new strategies are required to circumvent the F508del-CFTR defect. Airway and intestinal epithelial cells from CF patients bearing the F508del-CFTR mutation exhibit an impressive derangement of cellular proteostasis, with oxidative stress, overactivation of the tissue transglutaminase (TG2), and disabled autophagy. Proteostasis regulators such as cysteamine can rescue and stabilize a functional F508del-CFTR protein through suppressing TG2 activation and restoring autophagy in vivo in F508del-CFTR homozygous mice, in vitro in CF patient-derived cell lines, ex vivo in freshly collected primary patient's nasal cells, as well as in a pilot clinical trial involving homozygous F508del-CFTR patients. Here, we discuss how the therapeutic normalization of defective proteostasis can be harnessed for the treatment of CF patients with the F508del-CFTR mutation.

  4. Subunit Stoichiometry of Human Muscle Chloride Channels

    Fahlke, Christoph; Knittle, Timothy; Gurnett, Christina A.; Campbell, Kevin P.; George, Alfred L.

    1997-01-01

    Voltage-gated Cl? channels belonging to the ClC family appear to function as homomultimers, but the number of subunits needed to form a functional channel is controversial. To determine subunit stoichiometry, we constructed dimeric human skeletal muscle Cl? channels in which one subunit was tagged by a mutation (D136G) that causes profound changes in voltage-dependent gating. Sucrose-density gradient centrifugation experiments indicate that both monomeric and dimeric hClC-1 channels in their ...

  5. Computational design of a PDZ domain peptide inhibitor that rescues CFTR activity.

    Kyle E Roberts

    Full Text Available The cystic fibrosis transmembrane conductance regulator (CFTR is an epithelial chloride channel mutated in patients with cystic fibrosis (CF. The most prevalent CFTR mutation, ΔF508, blocks folding in the endoplasmic reticulum. Recent work has shown that some ΔF508-CFTR channel activity can be recovered by pharmaceutical modulators ("potentiators" and "correctors", but ΔF508-CFTR can still be rapidly degraded via a lysosomal pathway involving the CFTR-associated ligand (CAL, which binds CFTR via a PDZ interaction domain. We present a study that goes from theory, to new structure-based computational design algorithms, to computational predictions, to biochemical testing and ultimately to epithelial-cell validation of novel, effective CAL PDZ inhibitors (called "stabilizers" that rescue ΔF508-CFTR activity. To design the "stabilizers", we extended our structural ensemble-based computational protein redesign algorithm K* to encompass protein-protein and protein-peptide interactions. The computational predictions achieved high accuracy: all of the top-predicted peptide inhibitors bound well to CAL. Furthermore, when compared to state-of-the-art CAL inhibitors, our design methodology achieved higher affinity and increased binding efficiency. The designed inhibitor with the highest affinity for CAL (kCAL01 binds six-fold more tightly than the previous best hexamer (iCAL35, and 170-fold more tightly than the CFTR C-terminus. We show that kCAL01 has physiological activity and can rescue chloride efflux in CF patient-derived airway epithelial cells. Since stabilizers address a different cellular CF defect from potentiators and correctors, our inhibitors provide an additional therapeutic pathway that can be used in conjunction with current methods.

  6. Chloride channels in myotonia congenita assessed by velocity recovery cycles.

    Tan, S Veronica; Z'Graggen, Werner J; Boërio, Delphine; Rayan, Dipa Raja; Norwood, Fiona; Ruddy, Deborah; Howard, R; Hanna, Michael G; Bostock, Hugh

    2014-06-01

    Myotonia congenita (MC) is caused by congenital defects in the muscle chloride channel CLC-1. This study used muscle velocity recovery cycles (MVRCs) to investigate how membrane function is affected. MVRCs and responses to repetitive stimulation were compared between 18 patients with genetically confirmed MC (13 recessive, 7 dominant) and 30 age-matched, normal controls. MC patients exhibited increased early supernormality, but this was prevented by treatment with sodium channel blockers. After multiple conditioning stimuli, late supernormality was enhanced in all MC patients, indicating delayed repolarization. These abnormalities were similar between the MC subtypes, but recessive patients showed a greater drop in amplitude during repetitive stimulation. MVRCs indicate that chloride conductance only becomes important when muscle fibers are depolarized. The differential responses to repetitive stimulation suggest that, in dominant MC, the affected chloride channels are activated by strong depolarization, consistent with a positive shift of the CLC-1 activation curve. Copyright © 2013 Wiley Periodicals, Inc.

  7. Crystallographic and single-particle analyses of native- and nucleotide-bound forms of the cystic fibrosis transmembrane conductance regulator (CFTR) protein.

    Awayn, N H; Rosenberg, M F; Kamis, A B; Aleksandrov, L A; Riordan, J R; Ford, R C

    2005-11-01

    Cystic fibrosis, one of the major human inherited diseases, is caused by defects in the CFTR (cystic fibrosis transmembrane conductance regulator), a cell-membrane protein. CFTR acts as a chloride channel which can be opened by ATP. Low-resolution structural studies of purified recombinant human CFTR are described in the present paper. Localization of the C-terminal decahistidine tag in CFTR was achieved by Ni2+-nitriloacetate nanogold labelling, followed by electron microscopy and single-particle analysis. The presence of the gold label appears to improve the single-particle-alignment procedure. Projection structures of CFTR from two-dimensional crystals analysed by electron crystallography displayed two alternative conformational states in the presence of nucleotide and nanogold, but only one form of the protein was observed in the quiescent (nucleotide-free) state.

  8. Evidence that CFTR is expressed in rat tracheal smooth muscle cells and contributes to bronchodilation

    Mettey Yvette

    2006-08-01

    Full Text Available Abstract Background The airway functions are profoundly affected in many diseases including asthma, chronic obstructive pulmonary disease (COPD and cystic fibrosis (CF. CF the most common lethal autosomal recessive genetic disease is caused by mutations of the CFTR gene, which normally encodes a multifunctional and integral membrane protein, the CF transmembrane conductance regulator (CFTR expressed in airway epithelial cells. Methods To demonstrate that CFTR is also expressed in tracheal smooth muscle cells (TSMC, we used iodide efflux assay to analyse the chloride transports in organ culture of rat TSMC, immunofluorescence study to localize CFTR proteins and isometric contraction measurement on isolated tracheal rings to observe the implication of CFTR in the bronchodilation. Results We characterized three different pathways stimulated by the cAMP agonist forskolin and the isoflavone agent genistein, by the calcium ionophore A23187 and by hypo-osmotic challenge. The pharmacology of the cAMP-dependent iodide efflux was investigated in detail. We demonstrated in rat TSMC that it is remarkably similar to that of the epithelial CFTR, both for activation (using three benzo [c]quinolizinium derivatives and for inhibition (glibenclamide, DPC and CFTRinh-172. Using rat tracheal rings, we observed that the activation of CFTR by benzoquinolizinium derivatives in TSMC leads to CFTRinh-172-sensitive bronchodilation after constriction with carbachol. An immunolocalisation study confirmed expression of CFTR in tracheal myocytes. Conclusion Altogether, these observations revealed that CFTR in the airways of rat is expressed not only in the epithelial cells but also in tracheal smooth muscle cells leading to the hypothesis that this ionic channel could contribute to bronchodilation.

  9. Chloride channel inhibition by a red wine extract and a synthetic small molecule prevents rotaviral secretory diarrhoea in neonatal mice

    Ko, Eun-A; Jin, Byung-Ju; Namkung, Wan; Ma, Tonghui; Thiagarajah, Jay R.; Verkman, A. S.

    2014-01-01

    Background Rotavirus is the most common cause of severe secretory diarrhoea in infants and young children globally. The rotaviral enterotoxin, NSP4, has been proposed to stimulate calcium-activated chloride channels (CaCC) on the apical plasma membrane of intestinal epithelial cells. We previously identified red wine and small molecule CaCC inhibitors. Objective To investigate the efficacy of a red wine extract and a synthetic small molecule, CaCCinh-A01, in inhibiting intestinal CaCCs and rotaviral diarrhoea. Design Inhibition of CaCC-dependent current was measured in T84 cells and mouse ileum. The effectiveness of an orally administered wine extract and CaCCinh-A01 in inhibiting diarrhoea in vivo was determined in a neonatal mouse model of rotaviral infection. Results Screening of ~150 red wines revealed a Cabernet Sauvignon that inhibited CaCC current in T84 cells with IC50 at a ~1:200 dilution, and higher concentrations producing 100% inhibition. A >1 kdalton wine extract prepared by dialysis, which retained full inhibition activity, blocked CaCC current in T84 cells and mouse intestine. In rotavirus-inoculated mice, oral administration of the wine extract prevented diarrhoea by inhibition of intestinal fluid secretion without affecting rotaviral infection. The wine extract did not inhibit the cystic fibrosis chloride channel (CFTR) in cell cultures, nor did it prevent watery stools in neonatal mice administered cholera toxin, which activates CFTR-dependent fluid secretion. CaCCinh-A01 also inhibited rotaviral diarrhoea. Conclusions Our results support a pathogenic role for enterocyte CaCCs in rotaviral diarrhoea and demonstrate the antidiarrhoeal action of CaCC inhibition by an alcohol-free, red wine extract and by a synthetic small molecule. PMID:24052273

  10. Swell activated chloride channel function in human neutrophils

    Salmon, Michael D. [Leukocyte and Ion Channel Research Laboratory, School of Health and Biosciences, University of East London, Stratford Campus, London E15 4LZ (United Kingdom); Ahluwalia, Jatinder, E-mail: j.ahluwalia@uel.ac.uk [Leukocyte and Ion Channel Research Laboratory, School of Health and Biosciences, University of East London, Stratford Campus, London E15 4LZ (United Kingdom)

    2009-04-17

    Non-excitable cells such as neutrophil granulocytes are the archetypal inflammatory immune cell involved in critical functions of the innate immune system. The electron current generated (I{sub e}) by the neutrophil NADPH oxidase is electrogenic and rapidly depolarises the membrane potential. For continuous function of the NADPH oxidase, I{sub e} has to be balanced to preserve electroneutrality, if not; sufficient depolarisation would prevent electrons from leaving the cell and neutrophil function would be abrogated. Subsequently, the depolarisation generated by the neutrophil NADPH oxidase I{sub e} must be counteracted by ion transport. The finding that depolarisation required counter-ions to compensate electron transport was followed by the observation that chloride channels activated by swell can counteract the NADPH oxidase membrane depolarisation. In this mini review, we discuss the research findings that revealed the essential role of swell activated chloride channels in human neutrophil function.

  11. Chloride channels regulate chondrogenesis in chicken mandibular mesenchymal cells.

    Tian, Meiyu; Duan, Yinzhong; Duan, Xiaohong

    2010-12-01

    Voltage gated chloride channels (ClCs) play an important role in the regulation of intracellular pH and cell volume homeostasis. Mutations of these genes result in genetic diseases with abnormal bone deformation and body size, indicating that ClCs may have a role in chondrogenesis. In the present study, we isolated chicken mandibular mesenchymal cells (CMMC) from Hamburg-Hamilton (HH) stage 26 chick embryos and induced chondrocyte maturation by using ascorbic acid and β-glycerophosphate (AA-BGP). We also determined the effect of the chloride channel inhibitor NPPB [5-nitro-2-(3-phenylpropylamino) benzoic acid] on regulation of growth, differentiation, and gene expression in these cells using MTT and real-time PCR assays. We found that CLCN1 and CLCN3-7 mRNA were expressed in CMMC and NPPB reduced expression of CLCN3, CLCN5, and CLCN7 mRNA in these cells. At the same time, NPPB inhibited the growth of the CMMC, but had no effect on the mRNA level of cyclin D1 and cyclin E (P>0.05) with/without AA-BGP treatment. AA-BGP increased markers for early chondrocyte differentiation including type II collagen, aggrecan (Ptype X collagen. NPPB antagonized AA-BGP-induced expression of type II collagen and aggrecan (Ptype X collagen (PType X collagen might function as a target of chloride channel inhibitors during the differentiation process. Copyright © 2010 Elsevier Ltd. All rights reserved.

  12. Functional interaction between CFTR and the sodium-phosphate co-transport type 2a in Xenopus laevis oocytes.

    Naziha Bakouh

    Full Text Available A growing number of proteins, including ion transporters, have been shown to interact with Cystic Fibrosis Transmembrane conductance Regulator (CFTR. CFTR is an epithelial chloride channel that is involved in Cystic Fibrosis (CF when mutated; thus a better knowledge of its functional interactome may help to understand the pathophysiology of this complex disease. In the present study, we investigated if CFTR and the sodium-phosphate co-transporter type 2a (NPT2a functionally interact after heterologous expression of both proteins in Xenopus laevis oocytes.NPT2a was expressed alone or in combination with CFTR in X. laevis oocytes. Using the two-electrode voltage-clamp technique, the inorganic phosphate-induced current (IPi was measured and taken as an index of NPT2a activity. The maximal IPi for NPT2a substrates was reduced when CFTR was co-expressed with NPT2a, suggesting a decrease in its expression at the oolemna. This was consistent with Western blot analysis showing reduced NPT2a plasma membrane expression in oocytes co-expressing both proteins, whereas NPT2a protein level in total cell lysate was the same in NPT2a- and NPT2a+CFTR-oocytes. In NPT2a+CFTR- but not in NPT2a-oocytes, IPi and NPT2a surface expression were increased upon PKA stimulation, whereas stimulation of Exchange Protein directly Activated by cAMP (EPAC had no effect. When NPT2a-oocytes were injected with NEG2, a short amino-acid sequence from the CFTR regulatory domain that regulates PKA-dependent CFTR trafficking to the plasma membrane, IPi values and NPT2a membrane expression were diminished, and could be enhanced by PKA stimulation, thereby mimicking the effects of CFTR co-expression.We conclude that when both CFTR and NPT2a are expressed in X. laevis oocytes, CFTR confers to NPT2a a cAMPi-dependent trafficking to the membrane. This functional interaction raises the hypothesis that CFTR may play a role in phosphate homeostasis.

  13. Prevention of secretory diarrhea by ethanol extract of Bistortae rhizoma through inhibition of chloride channel

    Bo Yu

    2015-08-01

    Full Text Available Inhibition of cystic fibrosis transmembrane conductance regulator (CFTR and Ca2+-activated Cl- channel (CaCC represents an attractive approach for the treatment of secretory diarrhea. The aim of the study is to investigate the molecular basis of the anti-diarrheal effect of traditional Chinese herbal anti-diarrheal medicine Bistortae rhizoma. Fluorescence quenching assay indicated that the 40% methanol /water fraction (D5 dose-dependently inhibited both CFTR and CaCC function in transfected Fischer rat thyroid (FRT cells. Ex vivo studies indicated that D5 inhibited both forskolin (FSK-activated CFTR current and CCh-induced CaCC current in rat colonic mucosa. In the mouse closed-loop model, intraluminal application of D5 (200 µg/mL significantly reduced cholera toxin-stimulated fluid secretion. In the intestinal motility model, D5 significantly delayed intestinal peristalsis in mice. Our research suggests that CFTR and CaCC-mediated intestinal epithelial Cl- secretion inhibiting and gastrointestinal motility delaying may account for the anti-diarrheal activity of B. rhizoma.

  14. Characterization of nasal potential difference in cftr knockout and F508del-CFTR mice.

    Emilie Lyne Saussereau

    Full Text Available BACKGROUND: Treatments designed to correct cystic fibrosis transmembrane conductance regulator (CFTR defects must first be evaluated in preclinical experiments in the mouse model of cystic fibrosis (CF. Mice nasal mucosa mimics the bioelectric defect seen in humans. The use of nasal potential difference (V(TE to assess ionic transport is a powerful test evaluating the restoration of CFTR function. Nasal V(TE in CF mice must be well characterized for correct interpretation. METHODS: We performed V(TE measurements in large-scale studies of two mouse models of CF--B6;129 cftr knockout and FVB F508del-CFTR--and their respective wild-type (WT littermates. We assessed the repeatability of the test for cftr knockout mice and defined cutoff points distinguishing between WT and F508del-CFTR mice. RESULTS: We determined the typical V(TE values for CF and WT mice and demonstrated the existence of residual CFTR activity in F508del-CFTR mice. We characterized intra-animal variability in B6;129 mice and defined the cutoff points for F508del-CFTR chloride secretion rescue. Hyperpolarization of more than -2.15 mV after perfusion with a low-concentration Cl(- solution was considered to indicate a normal response. CONCLUSIONS: These data will make it possible to interpret changes in nasal V(TE in mouse models of CF, in future preclinical studies.

  15. CFTR is a tumor suppressor gene in murine and human intestinal cancer

    Than, B. L. N.; Linnekamp, J. F.; Starr, T. K.; Largaespada, D. A.; Rod, A.; Zhang, Y.; Bruner, V.; Abrahante, J.; Schumann, A.; Luczak, T.; Niemczyk, A.; O'Sullivan, M. G.; Medema, J. P.; Fijneman, R. J. A.; Meijer, G. A.; van den Broek, E.; Hodges, C. A.; Scott, P. M.; Vermeulen, L.; Cormier, R. T.

    2016-01-01

    CFTR, the cystic fibrosis (CF) gene, encodes for the CFTR protein that plays an essential role in anion regulation and tissue homeostasis of various epithelia. In the gastrointestinal (GI) tract CFTR promotes chloride and bicarbonate secretion, playing an essential role in ion and acid-base

  16. Molecular determinants of ivermectin sensitivity at the glycine receptor chloride channel

    Lynagh, Timothy; Webb, Timothy I.; Dixon, Christine L.

    2011-01-01

    Ivermectin is an anthelmintic drug that works by activating glutamate-gated chloride channel receptors (GluClRs) in nematode parasites. GluClRs belong to the Cys-loop receptor family that also includes glycine receptor (GlyR) chloride channels. GluClRs and A288G mutant GlyRs are both activated...

  17. The gastric H,K-ATPase blocker lansoprazole is an inhibitor of chloride channels

    Schmarda, Andreas; Dinkhauser, Patrick; Gschwentner, Martin; Ritter, Markus; Fürst, Johannes; Scandella, Elke; Wöll, Ewald; Laich, Andreas; Rossmann, Heidi; Seidler, Ursula; Lang, Florian; Paulmichl, Markus

    2000-01-01

    It was postulated that swelling dependent chloride channels are involved in the proton secretion of parietal cells. Since omeprazole, lansoprazole and its acid activated sulphenamide form AG2000 are structurally related to phenol derivatives known to block swelling dependent chloride channels, we set out to test, whether these substances – which are known to block the H,K-ATPase – could also lead to an inhibition of swelling-dependent chloride channels. Swelling-dependent chloride channels – characterized in many different cell types – show highly conserved biophysical and pharmacological features, therefore we investigated the effect of omeprazole, lansoprazole and its acid activated sulphenamide form AG2000 on swelling-dependent chloride channels elicited in fibroblasts, after the reduction of the extracellular osmolarity. Omeprazole, lansoprazole and its acid activated sulphenamide form AG2000 are able to block swelling-dependent chloride channels (IClswell). Lansoprazole and its protonated metabolite AG2000 act on at least two different sites of the IClswell protein: on an extracellular site which seems to be in a functional proximity to the nucleotide binding site, and on an intracellular site which allows the formation of disulfide-bridges. The inhibition of the proton pump and the simultaneous blocking of chloride channels by omeprazole, lansoprazole and its acid activated sulphenamide form AG2000, as described here could be an effective mode to restrict proton secretion in parietal cells. PMID:10711360

  18. Anoctamin Calcium-Activated Chloride Channels May Modulate Inhibitory Transmission in the Cerebellar Cortex.

    Weiping Zhang

    Full Text Available Calcium-activated chloride channels of the anoctamin (alias TMEM16 protein family fulfill critical functions in epithelial fluid transport, smooth muscle contraction and sensory signal processing. Little is known, however, about their contribution to information processing in the central nervous system. Here we examined the recent finding that a calcium-dependent chloride conductance impacts on GABAergic synaptic inhibition in Purkinje cells of the cerebellum. We asked whether anoctamin channels may underlie this chloride conductance. We identified two anoctamin channel proteins, ANO1 and ANO2, in the cerebellar cortex. ANO1 was expressed in inhibitory interneurons of the molecular layer and the granule cell layer. Both channels were expressed in Purkinje cells but, while ANO1 appeared to be retained in the cell body, ANO2 was targeted to the dendritic tree. Functional studies confirmed that ANO2 was involved in a calcium-dependent mode of ionic plasticity that reduces the efficacy of GABAergic synapses. ANO2 channels attenuated GABAergic transmission by increasing the postsynaptic chloride concentration, hence reducing the driving force for chloride influx. Our data suggest that ANO2 channels are involved in a Ca2+-dependent regulation of synaptic weight in GABAergic inhibition. Thus, in balance with the chloride extrusion mechanism via the co-transporter KCC2, ANO2 appears to regulate ionic plasticity in the cerebellum.

  19. Subconductance states of mitochondrial chloride channels: implication for functionally-coupled tetramers.

    Tomasek, Milan; Misak, Anton; Grman, Marian; Tomaskova, Zuzana

    2017-08-01

    Recently, it has been discovered that isoforms of intracellular chloride channels (CLIC) are present in cardiac mitochondria. By reconstituting rat cardiac mitochondrial chloride channels into bilayer lipid membranes, we detected three equally separated subconductance states with conductance increment of 45 pS and < 2% occupancy. The observed rare events of channel decomposition into substates, accompanied by disrupted gating, provide an insight into channel quaternary structure. Our findings suggest that the observed channels work as four functionally coupled subunits with synchronized gating. We discuss the putative connection of channel activity from native mitochondria with the recombinant CLIC channels. However, conclusive evidence is needed to prove this connection. © 2017 Federation of European Biochemical Societies.

  20. Mechanism of HERG potassium channel inhibition by tetra-n-octylammonium bromide and benzethonium chloride

    Long, Yan; Lin, Zuoxian; Xia, Menghang; Zheng, Wei; Li, Zhiyuan

    2013-01-01

    Tetra-n-octylammonium bromide and benzethonium chloride are synthetic quaternary ammonium salts that are widely used in hospitals and industries for the disinfection and surface treatment and as the preservative agent. Recently, the activities of HERG channel inhibition by these compounds have been found to have potential risks to induce the long QT syndrome and cardiac arrhythmia, although the mechanism of action is still elusive. This study was conducted to investigate the mechanism of HERG channel inhibition by these compounds by using whole-cell patch clamp experiments in a CHO cell line stably expressing HERG channels. Tetra-n-octylammonium bromide and benzethonium chloride exhibited concentration-dependent inhibitions of HERG channel currents with IC 50 values of 4 nM and 17 nM, respectively, which were also voltage-dependent and use-dependent. Both compounds shifted the channel activation I–V curves in a hyperpolarized direction for 10–15 mV and accelerated channel activation and inactivation processes by 2-fold. In addition, tetra-n-octylammonium bromide shifted the inactivation I–V curve in a hyperpolarized direction for 24.4 mV and slowed the rate of channel deactivation by 2-fold, whereas benzethonium chloride did not. The results indicate that tetra-n-octylammonium bromide and benzethonium chloride are open-channel blockers that inhibit HERG channels in the voltage-dependent, use-dependent and state-dependent manners. - Highlights: ► Tetra-n-octylammonium and benzethonium are potent HERG channel inhibitors. ► Channel activation and inactivation processes are accelerated by the two compounds. ► Both compounds are the open-channel blockers to HERG channels. ► HERG channel inhibition by both compounds is use-, voltage- and state dependent. ► The in vivo risk of QT prolongation needs to be studied for the two compounds

  1. Mechanism of HERG potassium channel inhibition by tetra-n-octylammonium bromide and benzethonium chloride

    Long, Yan; Lin, Zuoxian [Key Laboratory of Regenerative Biology, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530 (China); Xia, Menghang; Zheng, Wei [National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD 20892 (United States); Li, Zhiyuan, E-mail: li_zhiyuan@gibh.ac.cn [Key Laboratory of Regenerative Biology, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530 (China)

    2013-03-01

    Tetra-n-octylammonium bromide and benzethonium chloride are synthetic quaternary ammonium salts that are widely used in hospitals and industries for the disinfection and surface treatment and as the preservative agent. Recently, the activities of HERG channel inhibition by these compounds have been found to have potential risks to induce the long QT syndrome and cardiac arrhythmia, although the mechanism of action is still elusive. This study was conducted to investigate the mechanism of HERG channel inhibition by these compounds by using whole-cell patch clamp experiments in a CHO cell line stably expressing HERG channels. Tetra-n-octylammonium bromide and benzethonium chloride exhibited concentration-dependent inhibitions of HERG channel currents with IC{sub 50} values of 4 nM and 17 nM, respectively, which were also voltage-dependent and use-dependent. Both compounds shifted the channel activation I–V curves in a hyperpolarized direction for 10–15 mV and accelerated channel activation and inactivation processes by 2-fold. In addition, tetra-n-octylammonium bromide shifted the inactivation I–V curve in a hyperpolarized direction for 24.4 mV and slowed the rate of channel deactivation by 2-fold, whereas benzethonium chloride did not. The results indicate that tetra-n-octylammonium bromide and benzethonium chloride are open-channel blockers that inhibit HERG channels in the voltage-dependent, use-dependent and state-dependent manners. - Highlights: ► Tetra-n-octylammonium and benzethonium are potent HERG channel inhibitors. ► Channel activation and inactivation processes are accelerated by the two compounds. ► Both compounds are the open-channel blockers to HERG channels. ► HERG channel inhibition by both compounds is use-, voltage- and state dependent. ► The in vivo risk of QT prolongation needs to be studied for the two compounds.

  2. Spatial distribution of calcium-gated chloride channels in olfactory cilia.

    French, Donald A; Badamdorj, Dorjsuren; Kleene, Steven J

    2010-12-30

    In vertebrate olfactory receptor neurons, sensory cilia transduce odor stimuli into changes in neuronal membrane potential. The voltage changes are primarily caused by the sequential openings of two types of channel: a cyclic-nucleotide-gated (CNG) cationic channel and a calcium-gated chloride channel. In frog, the cilia are 25 to 200 µm in length, so the spatial distributions of the channels may be an important determinant of odor sensitivity. To determine the spatial distribution of the chloride channels, we recorded from single cilia as calcium was allowed to diffuse down the length of the cilium and activate the channels. A computational model of this experiment allowed an estimate of the spatial distribution of the chloride channels. On average, the channels were concentrated in a narrow band centered at a distance of 29% of the ciliary length, measured from the base of the cilium. This matches the location of the CNG channels determined previously. This non-uniform distribution of transduction proteins is consistent with similar findings in other cilia. On average, the two types of olfactory transduction channel are concentrated in the same region of the cilium. This may contribute to the efficient detection of weak stimuli.

  3. Antisense oligonucleotides suppress cell-volume-induced activation of chloride channels.

    Gschwentner, M; Nagl, U O; Wöll, E; Schmarda, A; Ritter, M; Paulmichl, M

    1995-08-01

    Cell volume regulation is an essential feature of most cells. After swelling in hypotonic media, the simultaneous activation of potassium and chloride channels is believed to be the initial, time-determining step in cell volume regulation. The activation of both pathways is functionally linked and enables the cells to lose ions and water, subsequently leading to cell shrinkage and readjustment of the initial volume. NIH 3T3 fibroblasts efficiently regulate their volume after swelling and bear chloride channels that are activated by decreasing extracellular osmolarity. The chloride current elicited in these cells after swelling is reminiscent of the current found in oocytes expressing an outwardly rectifying chloride current termed ICln. Introduction of antisense oligodeoxynucleotides complementary to the first 30 nucleotides of the coding region of the ICln channel into NIH 3T3 fibroblasts suppresses the activation of the swelling-induced chloride current. The experiments directly demonstrate an unambiguous link between a volume-activated chloride current and a cloned protein involved in chloride transport.

  4. Structure of the CLC-1 chloride channel from Homo sapiens.

    Park, Eunyong; MacKinnon, Roderick

    2018-05-29

    CLC channels mediate passive Cl - conduction, while CLC transporters mediate active Cl - transport coupled to H + transport in the opposite direction. The distinction between CLC-0/1/2 channels and CLC transporters seems undetectable by amino acid sequence. To understand why they are different functionally we determined the structure of the human CLC-1 channel. Its 'glutamate gate' residue, known to mediate proton transfer in CLC transporters, adopts a location in the structure that appears to preclude it from its transport function. Furthermore, smaller side chains produce a wider pore near the intracellular surface, potentially reducing a kinetic barrier for Cl - conduction. When the corresponding residues are mutated in a transporter, it is converted to a channel. Finally, Cl - at key sites in the pore appear to interact with reduced affinity compared to transporters. Thus, subtle differences in glutamate gate conformation, internal pore diameter and Cl - affinity distinguish CLC channels and transporters. © 2018, Park & MacKinnon.

  5. Evidence for a role of GABA- and glutamate-gated chloride channels in olfactory memory.

    Boumghar, Katia; Couret-Fauvel, Thomas; Garcia, Mikael; Armengaud, Catherine

    2012-11-01

    In the honeybee, we investigated the role of transmissions mediated by GABA-gated chloride channels and glutamate-gated chloride channels (GluCls) of the mushroom bodies (MBs) on olfactory learning using a single-trial olfactory conditioning paradigm. The GABAergic antagonist picrotoxin (PTX) or the GluCl antagonist L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC) was injected alone or in combination into the α-lobes of MBs. PTX impaired early long-term olfactory memory when injected before conditioning or before testing. L-trans-PDC alone induced no significant effect on learning and memory but induced a less specific response to the conditioned odor. When injected before PTX, L-trans-PDC was able to modulate PTX effects. These results emphasize the role of MB GABA-gated chloride channels in consolidation processes and strongly support that GluCls are involved in the perception of the conditioned stimulus.

  6. Gating of human ClC-2 chloride channels and regulation by carboxy-terminal domains.

    Garcia-Olivares, Jennie; Alekov, Alexi; Boroumand, Mohammad Reza; Begemann, Birgit; Hidalgo, Patricia; Fahlke, Christoph

    2008-11-15

    Eukaryotic ClC channels are dimeric proteins with each subunit forming an individual protopore. Single protopores are gated by a fast gate, whereas the slow gate is assumed to control both protopores through a cooperative movement of the two carboxy-terminal domains. We here study the role of the carboxy-terminal domain in modulating fast and slow gating of human ClC-2 channels, a ubiquitously expressed ClC-type chloride channel involved in transepithelial solute transport and in neuronal chloride homeostasis. Partial truncation of the carboxy-terminus abolishes function of ClC-2 by locking the channel in a closed position. However, unlike other isoforms, its complete removal preserves function of ClC-2. ClC-2 channels without the carboxy-terminus exhibit fast and slow gates that activate and deactivate significantly faster than in WT channels. In contrast to the prevalent view, a single carboxy-terminus suffices for normal slow gating, whereas both domains regulate fast gating of individual protopores. Our findings demonstrate that the carboxy-terminus is not strictly required for slow gating and that the cooperative gating resides in other regions of the channel protein. ClC-2 is expressed in neurons and believed to open at negative potentials and increased internal chloride concentrations after intense synaptic activity. We propose that the function of the ClC-2 carboxy-terminus is to slow down the time course of channel activation in order to stabilize neuronal excitability.

  7. The antiparasitic isoxazoline A1443 is a potent blocker of insect ligand-gated chloride channels.

    Ozoe, Yoshihisa; Asahi, Miho; Ozoe, Fumiyo; Nakahira, Kunimitsu; Mita, Takeshi

    2010-01-01

    A structurally unique isoxazoline class compound, A1443, exhibits antiparasitic activity against cat fleas and dog ticks comparable to that of the commercial ectoparasiticide fipronil. This isoxazoline compound inhibits specific binding of the gamma-aminobutyric acid (GABA) receptor channel blocker [(3)H]4'-ethynyl-4-n-propylbicycloorthobenzoate (EBOB) to housefly-head membranes, with an IC(50) value of 455pM. In contrast, the IC(50) value in rat-brain membranes is>10muM. To study the mode of action of this isoxazoline, we utilized MdGBCl and MdGluCl cDNAs, which encode the subunits of housefly GABA- and glutamate-gated chloride channels, respectively. Two-electrode voltage clamp electrophysiology was used to confirm that A1443 blocks GABA- and glutamate-induced chloride currents in Xenopus oocytes expressing MdGBCl or MdGluCl channels, with IC(50) values of 5.32 and 79.9 nM, respectively. Blockade by A1443 was observed in A2'S-MdGBCl and S2'A-MdGluCl mutant channels at levels similar to those of the respective wild-types, and houseflies expressing A2'S-MdGBCl channels were as susceptible to A1443 as standard houseflies. These findings indicate that A1443 is a novel and specific blocker of insect ligand-gated chloride channels. Copyright 2009 Elsevier Inc. All rights reserved.

  8. Chloride Channel 3 Channels in the Activation and Migration of Human Blood Eosinophils in Allergic Asthma.

    Gaurav, Rohit; Bewtra, Againdra K; Agrawal, Devendra K

    2015-08-01

    Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is responsible for respiratory burst in immune cells. Chloride channel 3 (CLC3) has been linked to the respiratory burst in eosinophils and neutrophils. The effect of cytokines and the involvement of CLC3 in the regulation of NADPH-dependent oxidative stress and on cytokine-mediated migration of eosinophils are not known. Human peripheral blood eosinophils were isolated from healthy individuals and from individuals with asthma by negative selection. Real-time PCR was used to detect the expression of NADPH oxidases in eosinophils. Intracellular reactive oxygen species (ROS) measurement was done with flow cytometry. Superoxide generation was measured with transforming growth factor (TGF)-β, eotaxin, and CLC3 blockers. CLC3 dependence of eosinophils in TGF-β- and eotaxin-induced migration was also examined. The messenger RNA (mRNA) transcripts of NADPH oxidase (NOX) 2, dual oxidase (DUOX) 1, and DUOX2 were detected in blood eosinophils, with very low expression of NOX1, NOX3, and NOX5 and no NOX4 mRNA. The level of NOX2 mRNA transcripts increased with disease severity in the eosinophils of subjects with asthma compared with healthy nonatopic volunteers. Change in granularity and size in eosinophils, but no change in intracellular ROS, was observed with phorbol myristate acetate (PMA). PMA, TGF-β, and eotaxin used the CLC3-dependent pathway to increase superoxide radicals. TGF-β and eotaxin induced CLC3-dependent chemotaxis of eosinophils. These findings support the requirement of CLC3 in the activation and migration of human blood eosinophils and may provide a potential novel therapeutic target to regulate eosinophil hyperactivity in allergic airway inflammation in asthma.

  9. Comparative pharmacology of flatworm and roundworm glutamate-gated chloride channels

    Lynagh, Timothy; Cromer, Brett A.; Dufour, Vanessa

    2014-01-01

    Pharmacological targeting of glutamate-gated chloride channels (GluCls) is a potent anthelmintic strategy, evidenced by macrocyclic lactones that eliminate numerous roundworm infections by activating roundworm GluCls. Given the recent identification of flatworm GluCls and the urgent need for drugs...

  10. Activation of AMPK inhibits cholera toxin stimulated chloride secretion in human and murine intestine.

    Ailín C Rogers

    Full Text Available Increased intestinal chloride secretion through chloride channels, such as the cystic fibrosis transmembrane conductance regulator (CFTR, is one of the major molecular mechanisms underlying enterotoxigenic diarrhea. It has been demonstrated in the past that the intracellular energy sensing kinase, the AMP-activated protein kinase (AMPK, can inhibit CFTR opening. We hypothesized that pharmacological activation of AMPK can abrogate the increased chloride flux through CFTR occurring during cholera toxin (CTX mediated diarrhea. Chloride efflux was measured in isolated rat colonic crypts using real-time fluorescence imaging. AICAR and metformin were used to activate AMPK in the presence of the secretagogues CTX or forskolin (FSK. In order to substantiate our findings on the whole tissue level, short-circuit current (SCC was monitored in human and murine colonic mucosa using Ussing chambers. Furthermore, fluid accumulation was measured in excised intestinal loops. CTX and forskolin (FSK significantly increased chloride efflux in isolated colonic crypts. The increase in chloride efflux could be offset by using the AMPK activators AICAR and metformin. In human and mouse mucosal sheets, CTX and FSK increased SCC. AICAR and metformin inhibited the secretagogue induced rise in SCC, thereby confirming the findings made in isolated crypts. Moreover, AICAR decreased CTX stimulated fluid accumulation in excised intestinal segments. The present study suggests that pharmacological activation of AMPK effectively reduces CTX mediated increases in intestinal chloride secretion, which is a key factor for intestinal water accumulation. AMPK activators may therefore represent a supplemental treatment strategy for acute diarrheal illness.

  11. CLC Chloride Channels and Transporters: Structure, Function, Physiology, and Disease.

    Jentsch, Thomas J; Pusch, Michael

    2018-07-01

    CLC anion transporters are found in all phyla and form a gene family of eight members in mammals. Two CLC proteins, each of which completely contains an ion translocation parthway, assemble to homo- or heteromeric dimers that sometimes require accessory β-subunits for function. CLC proteins come in two flavors: anion channels and anion/proton exchangers. Structures of these two CLC protein classes are surprisingly similar. Extensive structure-function analysis identified residues involved in ion permeation, anion-proton coupling and gating and led to attractive biophysical models. In mammals, ClC-1, -2, -Ka/-Kb are plasma membrane Cl - channels, whereas ClC-3 through ClC-7 are 2Cl - /H + -exchangers in endolysosomal membranes. Biological roles of CLCs were mostly studied in mammals, but also in plants and model organisms like yeast and Caenorhabditis elegans. CLC Cl - channels have roles in the control of electrical excitability, extra- and intracellular ion homeostasis, and transepithelial transport, whereas anion/proton exchangers influence vesicular ion composition and impinge on endocytosis and lysosomal function. The surprisingly diverse roles of CLCs are highlighted by human and mouse disorders elicited by mutations in their genes. These pathologies include neurodegeneration, leukodystrophy, mental retardation, deafness, blindness, myotonia, hyperaldosteronism, renal salt loss, proteinuria, kidney stones, male infertility, and osteopetrosis. In this review, emphasis is laid on biophysical structure-function analysis and on the cell biological and organismal roles of mammalian CLCs and their role in disease.

  12. Inhibition of herpes simplex virus type 1 entry by chloride channel inhibitors tamoxifen and NPPB

    Zheng, Kai; Chen, Maoyun; Xiang, Yangfei; Ma, Kaiqi; Jin, Fujun; Wang, Xiao; Wang, Xiaoyan; Wang, Shaoxiang; Wang, Yifei

    2014-01-01

    Highlights: • We analyze the anti-HSV potential of chloride channel inhibitors. • Tamoxifen and NPPB show anti-HSV-1 and anti-ACV-resistant HSV-1 activities. • HSV-1 infection induces intracellular chloride concentration increasing. • Tamoxifen and NPPB inhibit HSV-1 early infection. • Tamoxifen and NPPB prevent the fusion process of HSV-1. - Abstract: Herpes simplex virus type 1 (HSV-1) infection is very common worldwide and can cause significant health problems from periodic skin and corneal lesions to encephalitis. Appearance of drug-resistant viruses in clinical therapy has made exploring novel antiviral agents emergent. Here we show that chloride channel inhibitors, including tamoxifen and 5-nitro-2-(3-phenyl-propylamino) benzoic acid (NPPB), exhibited extensive antiviral activities toward HSV-1 and ACV-resistant HSV viruses. HSV-1 infection induced chloride ion influx while treatment with inhibitors reduced the increase of intracellular chloride ion concentration. Pretreatment or treatment of inhibitors at different time points during HSV-1 infection all suppressed viral RNA synthesis, protein expression and virus production. More detailed studies demonstrated that tamoxifen and NPPB acted as potent inhibitors of HSV-1 early entry step by preventing viral binding, penetration and nuclear translocation. Specifically the compounds appeared to affect viral fusion process by inhibiting virus binding to lipid rafts and interrupting calcium homeostasis. Taken together, the observation that tamoxifen and NPPB can block viral entry suggests a stronger potential for these compounds as well as other ion channel inhibitors in antiviral therapy against HSV-1, especially the compound tamoxifen is an immediately actionable drug that can be reused for treatment of HSV-1 infections

  13. Inhibition of herpes simplex virus type 1 entry by chloride channel inhibitors tamoxifen and NPPB

    Zheng, Kai [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China); College of Life Science and Technology, Jinan University, Guangzhou (China); Chen, Maoyun [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China); College of pharmacy, Jinan University, Guangzhou (China); Xiang, Yangfei; Ma, Kaiqi [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China); Jin, Fujun [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China); College of pharmacy, Jinan University, Guangzhou (China); Wang, Xiao [School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006 (China); Wang, Xiaoyan; Wang, Shaoxiang [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China); Wang, Yifei, E-mail: twang-yf@163.com [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China)

    2014-04-18

    Highlights: • We analyze the anti-HSV potential of chloride channel inhibitors. • Tamoxifen and NPPB show anti-HSV-1 and anti-ACV-resistant HSV-1 activities. • HSV-1 infection induces intracellular chloride concentration increasing. • Tamoxifen and NPPB inhibit HSV-1 early infection. • Tamoxifen and NPPB prevent the fusion process of HSV-1. - Abstract: Herpes simplex virus type 1 (HSV-1) infection is very common worldwide and can cause significant health problems from periodic skin and corneal lesions to encephalitis. Appearance of drug-resistant viruses in clinical therapy has made exploring novel antiviral agents emergent. Here we show that chloride channel inhibitors, including tamoxifen and 5-nitro-2-(3-phenyl-propylamino) benzoic acid (NPPB), exhibited extensive antiviral activities toward HSV-1 and ACV-resistant HSV viruses. HSV-1 infection induced chloride ion influx while treatment with inhibitors reduced the increase of intracellular chloride ion concentration. Pretreatment or treatment of inhibitors at different time points during HSV-1 infection all suppressed viral RNA synthesis, protein expression and virus production. More detailed studies demonstrated that tamoxifen and NPPB acted as potent inhibitors of HSV-1 early entry step by preventing viral binding, penetration and nuclear translocation. Specifically the compounds appeared to affect viral fusion process by inhibiting virus binding to lipid rafts and interrupting calcium homeostasis. Taken together, the observation that tamoxifen and NPPB can block viral entry suggests a stronger potential for these compounds as well as other ion channel inhibitors in antiviral therapy against HSV-1, especially the compound tamoxifen is an immediately actionable drug that can be reused for treatment of HSV-1 infections.

  14. Insulin-like growth factor 1 (IGF-1 enhances the protein expression of CFTR.

    Ha Won Lee

    Full Text Available Low levels of insulin-like growth factor 1 (IGF-1 have been observed in the serum of cystic fibrosis (CF patients. However, the effects of low serum IGF-1 on the cystic fibrosis transmembrane conductance regulator (CFTR, whose defective function is the primary cause of cystic fibrosis, have not been studied. Here, we show in human cells that IGF-1 increases the steady-state levels of mature wildtype CFTR in a CFTR-associated ligand (CAL- and TC10-dependent manner; moreover, IGF-1 increases CFTR-mediated chloride transport. Using an acceptor photobleaching fluorescence resonance energy transfer (FRET assay, we have confirmed the binding of CAL and CFTR in the Golgi. We also show that CAL overexpression inhibits forskolin-induced increases in the cell-surface expression of CFTR. We found that IGF-1 activates TC10, and active TC10 alters the functional association between CAL and CFTR. Furthermore, IGF-1 and active TC10 can reverse the CAL-mediated reduction in the cell-surface expression of CFTR. IGF-1 does not increase the expression of ΔF508 CFTR, whose processing is arrested in the ER. This finding is consistent with our observation that IGF-1 alters the functional interaction of CAL and CFTR in the Golgi. However, when ΔF508 CFTR is rescued with low temperature or the corrector VRT-325 and proceeds to the Golgi, IGF-1 can increase the expression of the rescued ΔF508 CFTR. Our data support a model indicating that CAL-CFTR binding in the Golgi inhibits CFTR trafficking to the cell surface, leading CFTR to the degradation pathway instead. IGF-1-activated TC10 changes the interaction of CFTR and CAL, allowing CFTR to progress to the plasma membrane. These findings offer a potential strategy using a combinational treatment of IGF-1 and correctors to increase the post-Golgi expression of CFTR in cystic fibrosis patients bearing the ΔF508 mutation.

  15. ClC-K chloride channels: emerging pathophysiology of Bartter syndrome type 3.

    Andrini, Olga; Keck, Mathilde; Briones, Rodolfo; Lourdel, Stéphane; Vargas-Poussou, Rosa; Teulon, Jacques

    2015-06-15

    The mutations in the CLCNKB gene encoding the ClC-Kb chloride channel are responsible for Bartter syndrome type 3, one of the four variants of Bartter syndrome in the genetically based nomenclature. All forms of Bartter syndrome are characterized by hypokalemia, metabolic alkalosis, and secondary hyperaldosteronism, but Bartter syndrome type 3 has the most heterogeneous presentation, extending from severe to very mild. A relatively large number of CLCNKB mutations have been reported, including gene deletions and nonsense or missense mutations. However, only 20 CLCNKB mutations have been functionally analyzed, due to technical difficulties regarding ClC-Kb functional expression in heterologous systems. This review provides an overview of recent progress in the functional consequences of CLCNKB mutations on ClC-Kb chloride channel activity. It has been observed that 1) all ClC-Kb mutants have an impaired expression at the membrane; and 2) a minority of the mutants combines reduced membrane expression with altered pH-dependent channel gating. Although further investigation is needed to fully characterize disease pathogenesis, Bartter syndrome type 3 probably belongs to the large family of conformational diseases, in which the mutations destabilize channel structure, inducing ClC-Kb retention in the endoplasmic reticulum and accelerated channel degradation. Copyright © 2015 the American Physiological Society.

  16. Cysteamine re-establishes the clearance of Pseudomonas aeruginosa by macrophages bearing the cystic fibrosis-relevant F508del-CFTR mutation.

    Ferrari, Eleonora; Monzani, Romina; Villella, Valeria R; Esposito, Speranza; Saluzzo, Francesca; Rossin, Federica; D'Eletto, Manuela; Tosco, Antonella; De Gregorio, Fabiola; Izzo, Valentina; Maiuri, Maria C; Kroemer, Guido; Raia, Valeria; Maiuri, Luigi

    2017-01-12

    Cystic fibrosis (CF), the most common lethal monogenic disease in Caucasians, is characterized by recurrent bacterial infections and colonization, mainly by Pseudomonas aeruginosa, resulting in unresolved airway inflammation. CF is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which functions as a chloride channel in epithelial cells, macrophages, and other cell types. Impaired bacterial handling by macrophages is a feature of CF airways, although it is still debated how defective CFTR impairs bacterial killing. Recent evidence indicates that a defective autophagy in CF macrophages leads to alterations of bacterial clearance upon infection. Here we use bone marrow-derived macrophages from transgenic mice to provide the genetic proof that defective CFTR compromises both uptake and clearance of internalized Pseudomonas aeruginosa. We demonstrate that the proteostasis regulator cysteamine, which rescues the function of the most common F508del-CFTR mutant and hence reduces lung inflammation in CF patients, can also repair the defects of CF macrophages, thus restoring both bacterial internalization and clearance through a process that involves upregulation of the pro-autophagic protein Beclin 1 and re-establishment of the autophagic pathway. Altogether these results indicate that cysteamine restores the function of several distinct cell types, including that of macrophages, which might contribute to its beneficial effects on CF.

  17. The cystic fibrosis transmembrane recruiter the alter ego of CFTR as a multi-kinase anchor.

    Mehta, Anil

    2007-11-01

    This review focuses on a newly discovered interaction between protein kinases involved in cellular energetics, a process that may be disturbed in cystic fibrosis for unknown reasons. I propose a new model where kinase-mediated cellular transmission of energy provides mechanistic insight to a latent role of the cystic fibrosis transmembrane conductance regulator (CFTR). I suggest that CFTR acts as a multi-kinase recruiter to the apical epithelial membrane. My group finds that, in the cytosol, two protein kinases involved in cell energy homeostasis, nucleoside diphosphate kinase (NDPK) and AMP-activated kinase (AMPK), bind one another. Preliminary data suggest that both can also bind CFTR (function unclear). The disrupted role of this CFTR-kinase complex as 'membrane transmitter to the cell' is proposed as an alternative paradigm to the conventional ion transport mediated and CFTR/chloride-centric view of cystic fibrosis pathogenesis. Chloride remains important, but instead, chloride-induced control of the phosphohistidine content of one kinase component (NDPK, via a multi-kinase complex that also includes a third kinase, CK2; formerly casein kinase 2). I suggest that this complex provides the necessary near-equilibrium conditions needed for efficient transmission of phosphate energy to proteins controlling cellular energetics. Crucially, a new role for CFTR as a kinase controller is proposed with ionic concentration acting as a signal. The model posits a regulatory control relay for energy sensing involving a cascade of protein kinases bound to CFTR.

  18. Dynamic [Cl-]i measurement with chloride sensing quantum dots nanosensor in epithelial cells

    Wang Yuchi; Mao Hua; Wong, Lid B

    2010-01-01

    We have synthesized a chloride sensing quantum dots (QD) nanosensor, Cl-QD, for the dynamic measurements of chloride ion concentration in the millimolar range, a sensitivity that is applicable to most physiological intracellular chloride ion concentration ([Cl - ] i ) measurements in epithelial cells. The Cl-QD is synthesized by conjugating an anion receptor, 1-(2-mercapto-ethyl)-3-phenyl-thiourea (MEPTU) to a water soluble CdSe/ZnS QD at an emission wavelength of 620 nm. Upon binding of chloride ions to the Cl-QD, a photo-induced electron transfer mechanism caused the fluorescence of the QD to quench. This resulted in an inversely proportional relationship between the chloride ion concentration and the fluorescence intensity of the Cl-QD. We have utilized this Cl-QD to measure [Cl - ] i in T84 and CF-PAC cultured cells, with either the C1C-2 or CFTR chloride channels being manipulated by pharmacological chloride channel activators and inhibitors. Activations of C1C-2 and CFTR chloride channels in T84 by the respective lubiprostone and genistein caused predictive increases in the fluorescence of the Cl-QD, i.e., a decrease of [Cl - ] i . Conversely, glibenclamide, a chloride channel inhibitor, applied to the CF-PAC cells caused a predictable decrease in the fluorescence of Cl-QD due to the increase of [Cl - ] i . These are the first data in using QD-based chloride ion sensors for dynamic measurements of intracellular chloride ion concentrations in epithelial cells.

  19. pH-modulation of chloride channels from the sarcoplasmic reticulum of skeletal muscle.

    Kourie, J I

    1999-01-01

    The understanding of the role of cytoplasmic pH in modulating sarcoplasmic reticulum (SR) ion channels involved in Ca2+ regulation is important for the understanding of the function of normal and adversely affected muscles. The dependency of the SR small chloride (SCl) channel from rabbit skeletal muscle on cytoplasmic pH (pHcis) and luminal pH (pHtrans) was investigated using the lipid bilayer-vesicle fusion technique. Low pHcis 6.75-4.28 modifies the operational mode of this multiconductance channel (conductance levels between 5 and 75 pS). At pHcis 7.26-7.37 the channel mode is dominated by the conductance and kinetics of the main conductance state (65-75 pS) whereas at low pHcis 6.75-4.28 the channel mode is dominated by the conductance and kinetics of subconductance states (5-40 pS). Similarly, low pHtrans 4.07, but not pHtrans 6.28, modified the activity of SCl channels. The effects of low pHcis are pronounced at 10(-3) and 10(-4) M [Ca2+]cis but are not apparent at 10(-5) M [Ca2+]cis, where the subconductances of the channel are already prominent. Low pHcis-induced mode shift in the SCl channel activity is due to modification of the channel proteins that cause the uncoupling of the subconductance states. The results in this study suggest that low pHcis can modify the functional properties of the skeletal SR ion channels and hence contribute, at least partly, to the malfunction in the contraction-relaxation mechanism in skeletal muscle under low cytoplasmic pH levels.

  20. Investigating CFTR and KCa3.1 Protein/Protein Interactions.

    Hélène Klein

    Full Text Available In epithelia, Cl- channels play a prominent role in fluid and electrolyte transport. Of particular importance is the cAMP-dependent cystic fibrosis transmembrane conductance regulator Cl- channel (CFTR with mutations of the CFTR encoding gene causing cystic fibrosis. The bulk transepithelial transport of Cl- ions and electrolytes needs however to be coupled to an increase in K+ conductance in order to recycle K+ and maintain an electrical driving force for anion exit across the apical membrane. In several epithelia, this K+ efflux is ensured by K+ channels, including KCa3.1, which is expressed at both the apical and basolateral membranes. We show here for the first time that CFTR and KCa3.1 can physically interact. We first performed a two-hybrid screen to identify which KCa3.1 cytosolic domains might mediate an interaction with CFTR. Our results showed that both the N-terminal fragment M1-M40 of KCa3.1 and part of the KCa3.1 calmodulin binding domain (residues L345-A400 interact with the NBD2 segment (G1237-Y1420 and C- region of CFTR (residues T1387-L1480, respectively. An association of CFTR and F508del-CFTR with KCa3.1 was further confirmed in co-immunoprecipitation experiments demonstrating the formation of immunoprecipitable CFTR/KCa3.1 complexes in CFBE cells. Co-expression of KCa3.1 and CFTR in HEK cells did not impact CFTR expression at the cell surface, and KCa3.1 trafficking appeared independent of CFTR stimulation. Finally, evidence is presented through cross-correlation spectroscopy measurements that KCa3.1 and CFTR colocalize at the plasma membrane and that KCa3.1 channels tend to aggregate consequent to an enhanced interaction with CFTR channels at the plasma membrane following an increase in intracellular Ca2+ concentration. Altogether, these results suggest 1 that the physical interaction KCa3.1/CFTR can occur early during the biogenesis of both proteins and 2 that KCa3.1 and CFTR form a dynamic complex, the formation of which

  1. The Arabidopsis thylakoid chloride channel AtCLCe functions in chloride homeostasis and regulation of photosynthetic electron transport

    Andrei eHerdean

    2016-02-01

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

  2. Novel short chain fatty acids restore chloride secretion in cystic fibrosis

    Nguyen, Toan D.; Kim, Ug-Sung; Perrine, Susan P.

    2006-01-01

    Phenylalanine deletion at position 508 of the cystic fibrosis transmembrane conductance regulator (ΔF508-CFTR), the most common mutation in cystic fibrosis (CF), causes a misfolded protein exhibiting partial chloride conductance and impaired trafficking to the plasma membrane. 4-Phenylbutyrate corrects defective ΔF508-CFTR trafficking in vitro, but is not clinically efficacious. From a panel of short chain fatty acid derivatives, we showed that 2,2-dimethyl-butyrate (ST20) and α-methylhydrocinnamic acid (ST7), exhibiting high oral bioavailability and sustained plasma levels, correct the ΔF508-CFTR defect. Pre-incubation (≥6 h) of CF IB3-1 airway cells with ≥1 mM ST7 or ST20 restored the ability of 100 μM forskolin to stimulate an 125 I - efflux. This efflux was fully inhibited by NPPB, DPC, or glibenclamide, suggesting mediation through CFTR. Partial inhibition by DIDS suggests possible contribution from an additional Cl - channel regulated by CFTR. Thus, ST7 and ST20 offer treatment potential for CF caused by the ΔF508 mutation

  3. The chloride channel inhibitor NS3736 [corrected] prevents bone resorption in ovariectomized rats without changing bone formation

    Schaller, Sophie; Henriksen, Kim; Sveigaard, Christina

    2004-01-01

    , appearing mainly in osteoclasts, ovaries, appendix, and Purkinje cells. This highly selective distribution predicts that inhibition of ClC-7 should specifically target osteoclasts in vivo. We suggest that NS3736 is inhibiting ClC-7, leading to a bone-specific effect in vivo. RESULTS AND CONCLUSION......Chloride channel activity is essential for osteoclast function. Consequently, inhibition of the osteoclastic chloride channel should prevent bone resorption. Accordingly, we tested a chloride channel inhibitor on bone turnover and found that it inhibits bone resorption without affecting bone...... for osteoporosis, daily treatment with 30 mg/kg orally protected bone strength and BMD by approximately 50% 6 weeks after surgery. Most interestingly, bone formation assessed by osteocalcin, mineral apposition rate, and mineralized surface index was not inhibited. MATERIALS AND METHODS: Analysis of chloride...

  4. Chloride ions in the pore of glycine and GABA channels shape the time course and voltage dependence of agonist currents

    Moroni, Mirko; Biro, Istvan; Giugliano, Michele; Vijayan, Ranjit; Biggin, Philip C.; Beato, Marco; Sivilotti, Lucia G.

    2011-01-01

    In the vertebrate CNS, fast synaptic inhibition is mediated by GABA and glycine receptors. We recently reported that the time course of these synaptic currents is slower when intracellular chloride is high. Here we extend these findings to measure the effects of both extracellular and intracellular chloride on the deactivation of glycine and GABA currents at both negative and positive holding potentials. Currents were elicited by fast agonist application to outside-out patches from HEK293 cells expressing rat glycine or GABA receptors. The slowing effect of high extracellular chloride on current decay was detectable only in low intracellular chloride (4 mM). Our main finding is that glycine and GABA receptors “sense” chloride concentrations because of interactions between the M2 pore-lining domain and the permeating ions. This hypothesis is supported by the observation that the sensitivity of channel gating to intracellular chloride is abolished if the channel is engineered to become cation-selective, or if positive charges in the external pore vestibule are eliminated by mutagenesis. The appropriate interaction between permeating ions and channel pore is also necessary to maintain the channel voltage sensitivity of gating, which prolongs current decay at depolarized potentials. Voltage-dependence is abolished by the same mutations that suppress the effect of intracellular chloride and also by replacing chloride with another permeant ion, thiocyanate. These observations suggest that permeant chloride affects gating by a foot-in-the-door effect, binding to a channel site with asymmetrical access from the intracellular and extracellular sides of the membrane. PMID:21976494

  5. A proton-activated, outwardly rectifying chloride channel in human umbilical vein endothelial cells

    Ma Zhiyong; Zhang Wei; Chen Liang; Wang Rong; Kan Xiaohong; Sun Guizhen; Liu Chunxi; Li Li; Zhang Yun

    2008-01-01

    Extracellular acidic pH-activated chloride channel I Cl,acid , has been characterized in HEK 293 cells and mammalian cardiac myocytes. This study was designed to characterize I Cl,acid in human umbilical vein endothelial cells(HUVECs). The activation and deactivation of the current rapidly and repeatedly follows the change of the extracellular solution at pH 4.3, with the threshold pH 5.3. In addition, at very positive potentials, the current displays a time-dependent facilitation. pH-response relationship for I Cl,acid revealed that EC 50 is pH 4.764 with a threshold pH value of pH 5.3 and nH of 14.545. The current can be blocked by the Cl - channel inhibitor DIDS (100 μM). In summary, for the first time we report the presence of proton-activated, outwardly rectifying chloride channel in HUVECs. Because an acidic environment can develop in local myocardium under pathological conditions such as myocardial ischemia, I Cl,acid would play a role in regulation of EC function under these pathological conditions

  6. Molecular, biophysical, and pharmacological properties of calcium-activated chloride channels.

    Kamaleddin, Mohammad Amin

    2018-02-01

    Calcium-activated chloride channels (CaCCs) are a family of anionic transmembrane ion channels. They are mainly responsible for the movement of Cl - and other anions across the biological membranes, and they are widely expressed in different tissues. Since the Cl - flow into or out of the cell plays a crucial role in hyperpolarizing or depolarizing the cells, respectively, the impact of intracellular Ca 2+ concentration on these channels is attracting a lot of attentions. After summarizing the molecular, biophysical, and pharmacological properties of CaCCs, the role of CaCCs in normal cellular functions will be discussed, and I will emphasize how dysregulation of CaCCs in pathological conditions can account for different diseases. A better understanding of CaCCs and a pivotal regulatory role of Ca 2+ can shed more light on the therapeutic strategies for different neurological disorders that arise from chloride dysregulation, such as asthma, cystic fibrosis, and neuropathic pain. © 2017 Wiley Periodicals, Inc.

  7. EG-VEGF, BV8, and their receptor expression in human bronchi and their modification in cystic fibrosis: Impact of CFTR mutation (delF508).

    Chauvet, Sylvain; Traboulsi, Wael; Thevenon, Laura; Kouadri, Amal; Feige, Jean-Jacques; Camara, Boubou; Alfaidy, Nadia; Benharouga, Mohamed

    2015-08-01

    Enhanced lung angiogenesis has been reported in cystic fibrosis (CF). Recently, two highly homologous ligands, endocrine gland vascular endothelial growth factor (EG-VEGF) and mammalian Bv8, have been described as new angiogenic factors. Both ligands bind and activate two closely related G protein-coupled receptors, the prokineticin receptor (PROKR) 1 and 2. Yet, the expression, regulation, and potential role of EG-VEGF, BV8, and their receptors in normal and CF lung are still unknown. The expression of the receptors and their ligands was examined using molecular, biochemical, and immunocytochemistry analyses in lungs obtained from CF patients vs. control and in normal and CF bronchial epithelial cells. Cystic fibrosis transmembrane conductance regulator (CFTR) activity was evaluated in relation to both ligands, and concentrations of EG-VEGF were measured by ELISA. At the mRNA level, EG-VEGF, BV8, and PROKR2 gene expression was, respectively, approximately five, four, and two times higher in CF lungs compared with the controls. At the cellular level, both the ligands and their receptors showed elevated expressions in the CF condition. Similar results were observed at the protein level. The EG-VEGF secretion was apical and was approximately two times higher in CF compared with the normal epithelial cells. This secretion was increased following the inhibition of CFTR chloride channel activity. More importantly, EG-VEGF and BV8 increased the intracellular concentration of Ca(2+) and cAMP and stimulated CFTR-chloride channel activity. Altogether, these data suggest local roles for epithelial BV8 and EG-VEGF in the CF airway peribronchial vascular remodeling and highlighted the role of CFTR activity in both ligand biosynthesis and secretion. Copyright © 2015 the American Physiological Society.

  8. Contribution of chloride channel permease to fluoride resistance in Streptococcus mutans.

    Murata, Takatoshi; Hanada, Nobuhiro

    2016-06-01

    Genes encoding fluoride transporters have been identified in bacterial and archaeal species. The genome sequence of the cariogenic Streptococcus mutans bacteria suggests the presence of a putative fluoride transporter, which is referred to as a chloride channel permease. Two homologues of this gene (GenBank locus tags SMU_1290c and SMU_1289c) reside in tandem in the genome of S. mutans The aim of this study was to determine whether the chloride channel permeases contribute to fluoride resistance. We constructed SMU_1290c- and SMU_1289c-knockout S. mutans UA159 strains. We also constructed a double-knockout strain lacking both genes. SMU_1290c or SMU_1289c was transformed into a fluoride transporter- disrupted Escherichia coli strain. All bacterial strains were cultured under appropriate conditions with or without sodium fluoride, and fluoride resistance was evaluated. All three gene-knockout S. mutans strains showed lower resistance to sodium fluoride than did the wild-type strain. No significant changes in resistance to other sodium halides were recognized between the wild-type and double-knockout strains. Both SMU_1290c and SMU_1289c transformation rescued fluoride transporter-disrupted E. coli cell from fluoride toxicity. We conclude that the chloride channel permeases contribute to fluoride resistance in S. mutans. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  9. Targeting the intracellular environment in cystic fibrosis: restoring autophagy as a novel strategy to circumvent the CFTR defect

    Valeria Rachela Villella

    2013-01-01

    Full Text Available Cystic fibrosis (CF patients harboring the most common deletion mutation of the cystic fibrosis transmembrane conductance regulator (CFTR, F508del, are poor responders to potentiators of CFTR channel activity which can be used to treat a small subset of CF patients who genetically carry plasma membrane-resident CFTR mutants. The misfolded F508del-CFTR protein is unstable in the plasma membrane even if rescued by pharmacological agents that prevent its intracellular retention and degradation. CF is a conformational disease in which defective CFTR induces an impressive derangement of general proteostasis resulting from disabled autophagy. In this review, we discuss how rescuing Beclin 1 (BECN1, a major player of autophagosome formation, either by means of direct gene transfer or indirectly by administration of proteostasis regulators, could stabilize F508del-CFTR at the plasma membrane. We focus on the relationship between the improvement of peripheral proteostasis and CFTR plasma membrane stability in F508del-CFTR homozygous bronchial epithelia or mouse lungs. Moreover, this article reviews recent preclinical evidence indicating that targeting the intracellular environment surrounding the misfolded mutant CFTR instead of protein itself could constitute an attractive therapeutic option to sensitize patients carrying the F508del-CFTR mutation to the beneficial action of CFTR potentiators on lung inflammation.

  10. Targeting the Intracellular Environment in Cystic Fibrosis: Restoring Autophagy as a Novel Strategy to Circumvent the CFTR Defect

    Villella, Valeria Rachela; Esposito, Speranza; Bruscia, Emanuela M.; Maiuri, Maria Chiara; Raia, Valeria; Kroemer, Guido; Maiuri, Luigi

    2013-01-01

    Cystic fibrosis (CF) patients harboring the most common deletion mutation of the CF transmembrane conductance regulator (CFTR), F508del, are poor responders to potentiators of CFTR channel activity which can be used to treat a small subset of CF patients who genetically carry plasma membrane (PM)-resident CFTR mutants. The misfolded F508del-CFTR protein is unstable in the PM even if rescued by pharmacological agents that prevent its intracellular retention and degradation. CF is a conformational disease in which defective CFTR induces an impressive derangement of general proteostasis resulting from disabled autophagy. In this review, we discuss how rescuing Beclin 1 (BECN1), a major player of autophagosome formation, either by means of direct gene transfer or indirectly by administration of proteostasis regulators, could stabilize F508del-CFTR at the PM. We focus on the relationship between the improvement of peripheral proteostasis and CFTR PM stability in F508del-CFTR homozygous bronchial epithelia or mouse lungs. Moreover, this article reviews recent pre-clinical evidence indicating that targeting the intracellular environment surrounding the misfolded mutant CFTR instead of protein itself could constitute an attractive therapeutic option to sensitize patients carrying the F508del-CFTR mutation to the beneficial action of CFTR potentiators on lung inflammation. PMID:23346057

  11. Restoration of CFTR function in patients with cystic fibrosis carrying the F508del-CFTR mutation.

    De Stefano, Daniela; Villella, Valeria R; Esposito, Speranza; Tosco, Antonella; Sepe, Angela; De Gregorio, Fabiola; Salvadori, Laura; Grassia, Rosa; Leone, Carlo A; De Rosa, Giuseppe; Maiuri, Maria C; Pettoello-Mantovani, Massimo; Guido, Stefano; Bossi, Anna; Zolin, Anna; Venerando, Andrea; Pinna, Lorenzo A; Mehta, Anil; Bona, Gianni; Kroemer, Guido; Maiuri, Luigi; Raia, Valeria

    2014-01-01

    Restoration of BECN1/Beclin 1-dependent autophagy and depletion of SQSTM1/p62 by genetic manipulation or autophagy-stimulatory proteostasis regulators, such as cystamine, have positive effects on mouse models of human cystic fibrosis (CF). These measures rescue the functional expression of the most frequent pathogenic CFTR mutant, F508del, at the respiratory epithelial surface and reduce lung inflammation in Cftr(F508del) homozygous mice. Cysteamine, the reduced form of cystamine, is an FDA-approved drug. Here, we report that oral treatment with cysteamine greatly reduces the mortality rate and improves the phenotype of newborn mice bearing the F508del-CFTR mutation. Cysteamine was also able to increase the plasma membrane expression of the F508del-CFTR protein in nasal epithelial cells from F508del homozygous CF patients, and these effects persisted for 24 h after cysteamine withdrawal. Importantly, this cysteamine effect after washout was further sustained by the sequential administration of epigallocatechin gallate (EGCG), a green tea flavonoid, both in vivo, in mice, and in vitro, in primary epithelial cells from CF patients. In a pilot clinical trial involving 10 F508del-CFTR homozygous CF patients, the combination of cysteamine and EGCG restored BECN1, reduced SQSTM1 levels and improved CFTR function from nasal epithelial cells in vivo, correlating with a decrease of chloride concentrations in sweat, as well as with a reduction of the abundance of TNF/TNF-alpha (tumor necrosis factor) and CXCL8 (chemokine [C-X-C motif] ligand 8) transcripts in nasal brushing and TNF and CXCL8 protein levels in the sputum. Altogether, these results suggest that optimal schedules of cysteamine plus EGCG might be used for the treatment of CF caused by the F508del-CFTR mutation.

  12. Differential distribution of glutamate- and GABA-gated chloride channels in the housefly Musca domestica.

    Kita, Tomo; Ozoe, Fumiyo; Azuma, Masaaki; Ozoe, Yoshihisa

    2013-09-01

    l-Glutamic acid (glutamate) mediates fast inhibitory neurotransmission by affecting glutamate-gated chloride channels (GluCls) in invertebrates. The molecular function and pharmacological properties of GluCls have been well studied, but not much is known about their physiological role and localization in the insect body. The distribution of GluCls in the housefly (Musca domestica L.) was thus compared with the distribution of γ-aminobutyric acid (GABA)-gated chloride channels (GABACls). Quantitative PCR and ligand-binding experiments indicate that the GluCl and GABACl transcripts and proteins are predominantly expressed in the adult head. Intense GluCl immunostaining was detected in the lamina, leg motor neurons, and legs of adult houseflies. The GABACl (Rdl) immunostaining was more widely distributed, and was found in the medulla, lobula, lobula plate, mushroom body, antennal lobe, and ellipsoid body. The present findings suggest that GluCls have physiological roles in different tissues than GABACls. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Dietary Factors Modulate Colonic Tumorigenesis Through the Interaction of Gut Microbiota and Host Chloride Channels.

    Zhang, Yong; Kang, Chao; Wang, Xiao-Lan; Zhou, Min; Chen, Meng-Ting; Zhu, Xiao-Hui; Liu, Kai; Wang, Bin; Zhang, Qian-Yong; Zhu, Jun-Dong; Mi, Man-Tian

    2018-03-01

    In recent decades, the association among diet, gut microbiota, and the risk of colorectal cancer (CRC) has been established. Gut microbiota and associated metabolites, such as bile acids and butyrate, are now known to play a key role in CRC development. The aim of this study is to identify that the progression to CRC is influenced by cholic acid, sodium butyrate, a high-fat diet, or different dose of dihydromyricetin (DMY) interacted with gut microbiota. An AOM/DSS (azoxymethan/dextran sodium sulfate) model is established to study the gut microbiota compsition before and after tumor formation during colitis-induced tumorigenesis. All above dietary factors profoundly influence the composition of gut microbiota and host colonic tumorigenesis. In addition, mice with DMY-modified initial microbiota display different degrees of chemically induced tumorigenesis. Mechanism analysis reveals that gut microbiota-associated chloride channels participated in colon tumorigenesis. Gut microbiota changes occur in the hyperproliferative stage before tumor formation. Gut microbiota and host chloride channels, both of which are regulated by dietary factors, are associated with CRC development. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Activation of CFTR by ASBT-mediated bile salt absorption

    Bijvelds, MJC; Jorna, H; Verkade, HJ; Bot, AGM; Hofmann, F; Agellon, LB; Sinaasappel, M; de Jonge, HR

    2005-01-01

    In cholangiocytes, bile salt (BS) uptake via the apical sodium-dependent bile acid transporter (ASBT) may evoke ductular flow by enhancing cAMP-mediated signaling to the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. We considered that ASBT-mediated BS uptake in the distal

  15. Cloning and functional expression of a plant voltage-dependent chloride channel.

    Lurin, C; Geelen, D; Barbier-Brygoo, H; Guern, J; Maurel, C

    1996-01-01

    Plant cell membrane anion channels participate in basic physiological functions, such as cell volume regulation and signal transduction. However, nothing is known about their molecular structure. Using a polymerase chain reaction strategy, we have cloned a tobacco cDNA (CIC-Nt1) encoding a 780-amino acid protein with several putative transmembrane domains. CIC-Nt1 displays 24 to 32% amino acid identity with members of the animal voltage-dependent chloride channel (CIC) family, whose archetype is CIC-0 from the Torpedo marmorata electric organ. Injection of CIC-Nt1 complementary RNA into Xenopus oocytes elicited slowly activating inward currents upon membrane hyperpolarization more negative than -120 mV. These currents were carried mainly by anions, modulated by extracellular anions, and totally blocked by 10 mM extracellular calcium. The identification of CIC-Nt1 extends the CIC family to higher plants and provides a molecular probe for the study of voltage-dependent anion channels in plants. PMID:8624442

  16. Participation of GABAA Chloride Channels in the Anxiolytic-Like Effects of a Fatty Acid Mixture

    Juan Francisco Rodríguez-Landa

    2013-01-01

    Full Text Available Human amniotic fluid and a mixture of eight fatty acids (FAT-M identified in this maternal fluid (C12:0, lauric acid, 0.9 μg%; C14:0, myristic acid, 6.9 μg%; C16:0, palmitic acid, 35.3 μg%; C16:1, palmitoleic acid, 16.4 μg%; C18:0, stearic acid, 8.5 μg%; C18:1cis, oleic acid, 18.4 μg%; C18:1trans, elaidic acid, 3.5 μg%; C18:2, linoleic acid, 10.1 μg% produce anxiolytic-like effects that are comparable to diazepam in Wistar rats, suggesting the involvement of γ-aminobutyric acid-A (GABAA receptors, a possibility not yet explored. Wistar rats were subjected to the defensive burying test, elevated plus maze, and open field test. In different groups, three GABAA receptor antagonists were administered 30 min before FAT-M administration, including the competitive GABA binding antagonist bicuculline (1 mg/kg, GABAA benzodiazepine antagonist flumazenil (5 mg/kg, and noncompetitive GABAA chloride channel antagonist picrotoxin (1 mg/kg. The FAT-M exerted anxiolytic-like effects in the defensive burying test and elevated plus maze, without affecting locomotor activity in the open field test. The GABAA antagonists alone did not produce significant changes in the behavioral tests. Picrotoxin but not bicuculline or flumazenil blocked the anxiolytic-like effect of the FAT-M. Based on the specific blocking action of picrotoxin on the effects of the FAT-M, we conclude that the FAT-M exerted its anxiolytic-like effects through GABAA receptor chloride channels.

  17. Cholesterol modulates CFTR confinement in the plasma membrane of primary epithelial cells.

    Abu-Arish, Asmahan; Pandzic, Elvis; Goepp, Julie; Matthes, Elizabeth; Hanrahan, John W; Wiseman, Paul W

    2015-07-07

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma-membrane anion channel that, when mutated, causes the disease cystic fibrosis. Although CFTR has been detected in a detergent-resistant membrane fraction prepared from airway epithelial cells, suggesting that it may partition into cholesterol-rich membrane microdomains (lipid rafts), its compartmentalization has not been demonstrated in intact cells and the influence of microdomains on CFTR lateral mobility is unknown. We used live-cell imaging, spatial image correlation spectroscopy, and k-space image correlation spectroscopy to examine the aggregation state of CFTR and its dynamics both within and outside microdomains in the plasma membrane of primary human bronchial epithelial cells. These studies were also performed during treatments that augment or deplete membrane cholesterol. We found two populations of CFTR molecules that were distinguishable based on their dynamics at the cell surface. One population showed confinement and had slow dynamics that were highly cholesterol dependent. The other, more abundant population was less confined and diffused more rapidly. Treatments that deplete the membrane of cholesterol caused the confined fraction and average number of CFTR molecules per cluster to decrease. Elevating cholesterol had the opposite effect, increasing channel aggregation and the fraction of channels displaying confinement, consistent with CFTR recruitment into cholesterol-rich microdomains with dimensions below the optical resolution limit. Viral infection caused the nanoscale microdomains to fuse into large platforms and reduced CFTR mobility. To our knowledge, these results provide the first biophysical evidence for multiple CFTR populations and have implications for regulation of their surface expression and channel function. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  18. Cryo-EM structures of the TMEM16A calcium-activated chloride channel.

    Dang, Shangyu; Feng, Shengjie; Tien, Jason; Peters, Christian J; Bulkley, David; Lolicato, Marco; Zhao, Jianhua; Zuberbühler, Kathrin; Ye, Wenlei; Qi, Lijun; Chen, Tingxu; Craik, Charles S; Jan, Yuh Nung; Minor, Daniel L; Cheng, Yifan; Jan, Lily Yeh

    2017-12-21

    Calcium-activated chloride channels (CaCCs) encoded by TMEM16A control neuronal signalling, smooth muscle contraction, airway and exocrine gland secretion, and rhythmic movements of the gastrointestinal system. To understand how CaCCs mediate and control anion permeation to fulfil these physiological functions, knowledge of the mammalian TMEM16A structure and identification of its pore-lining residues are essential. TMEM16A forms a dimer with two pores. Previous CaCC structural analyses have relied on homology modelling of a homologue (nhTMEM16) from the fungus Nectria haematococca that functions primarily as a lipid scramblase, as well as subnanometre-resolution electron cryo-microscopy. Here we present de novo atomic structures of the transmembrane domains of mouse TMEM16A in nanodiscs and in lauryl maltose neopentyl glycol as determined by single-particle electron cryo-microscopy. These structures reveal the ion permeation pore and represent different functional states. The structure in lauryl maltose neopentyl glycol has one Ca 2+ ion resolved within each monomer with a constricted pore; this is likely to correspond to a closed state, because a CaCC with a single Ca 2+ occupancy requires membrane depolarization in order to open (C.J.P. et al., manuscript submitted). The structure in nanodiscs has two Ca 2+ ions per monomer and its pore is in a closed conformation; this probably reflects channel rundown, which is the gradual loss of channel activity that follows prolonged CaCC activation in 1 mM Ca 2+ . Our mutagenesis and electrophysiological studies, prompted by analyses of the structures, identified ten residues distributed along the pore that interact with permeant anions and affect anion selectivity, as well as seven pore-lining residues that cluster near pore constrictions and regulate channel gating. Together, these results clarify the basis of CaCC anion conduction.

  19. Purification and crystallization of the cystic fibrosis transmembrane conductance regulator (CFTR).

    Rosenberg, Mark F; Kamis, Alhaji Bukar; Aleksandrov, Luba A; Ford, Robert C; Riordan, John R

    2004-09-10

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane protein that is mutated in patients suffering from cystic fibrosis. Here we report the purification and first crystallization of wild-type human CFTR. Functional characterization of the material showed it to be highly active. Electron crystallography of negatively stained two-dimensional crystals of CFTR has revealed the overall architecture of this channel for two different conformational states. These show a strong structural homology to two conformational states of another eukaryotic ATP-binding cassette transporter, P-glycoprotein. In contrast to P-glycoprotein, however, both conformational states can be observed in the presence of a nucleotide, which may be related to the role of CFTR as an ion channel rather than a transporter. The hypothesis that the two conformations could represent the "open" and "closed" states of the channel is considered.

  20. CFTR, Mucins, and Mucus Obstruction in Cystic Fibrosis

    Kreda, Silvia M.; Davis, C. William; Rose, Mary Callaghan

    2012-01-01

    Mucus pathology in cystic fibrosis (CF) has been known for as long as the disease has been recognized and is sometimes called mucoviscidosis. The disease is marked by mucus hyperproduction and plugging in many organs, which are usually most fatal in the airways of CF patients, once the problem of meconium ileus at birth is resolved. After the CF gene, CFTR, was cloned and its protein product identified as a cAMP-regulated Cl− channel, causal mechanisms underlying the strong mucus phenotype of the disease became obscure. Here we focus on mucin genes and polymeric mucin glycoproteins, examining their regulation and potential relationships to a dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR). Detailed examination of CFTR expression in organs and different cell types indicates that changes in CFTR expression do not always correlate with the severity of CF disease or mucus accumulation. Thus, the mucus hyperproduction that typifies CF does not appear to be a direct cause of a defective CFTR but, rather, to be a downstream consequence. In organs like the lung, up-regulation of mucin gene expression by inflammation results from chronic infection; however, in other instances and organs, the inflammation may have a non-infectious origin. The mucus plugging phenotype of the β-subunit of the epithelial Na+ channel (βENaC)-overexpressing mouse is proving to be an archetypal example of this kind of inflammation, with a dehydrated airway surface/concentrated mucus gel apparently providing the inflammatory stimulus. Data indicate that the luminal HCO3 − deficiency recently described for CF epithelia may also provide such a stimulus, perhaps by causing a mal-maturation of mucins as they are released onto luminal surfaces. In any event, the path between CFTR dysfunction and mucus hyperproduction has proven tortuous, and its unraveling continues to offer its own twists and turns, along with fascinating glimpses into biology. PMID:22951447

  1. Oxidation promotes insertion of the CLIC1 chloride intracellular channel into the membrane.

    Goodchild, Sophia C; Howell, Michael W; Cordina, Nicole M; Littler, Dene R; Breit, Samuel N; Curmi, Paul M G; Brown, Louise Jennifer

    2009-12-01

    Members of the chloride intracellular channel (CLIC) family exist primarily as soluble proteins but can also auto-insert into cellular membranes to form ion channels. While little is known about the process of CLIC membrane insertion, a unique feature of mammalian CLIC1 is its ability to undergo a dramatic structural metamorphosis between a monomeric glutathione-S-transferase homolog and an all-helical dimer upon oxidation in solution. Whether this oxidation-induced metamorphosis facilitates CLIC1 membrane insertion is unclear. In this work, we have sought to characterise the role of oxidation in the process of CLIC1 membrane insertion. We examined how redox conditions modify the ability of CLIC1 to associate with and insert into the membrane using fluorescence quenching studies and a sucrose-loaded vesicle sedimentation assay to measure membrane binding. Our results suggest that oxidation of monomeric CLIC1, in the presence of membranes, promotes insertion into the bilayer more effectively than the oxidised CLIC1 dimer.

  2. Suppression of adenosine-activated chloride transport by ethanol in airway epithelia.

    Sammeta V Raju

    Full Text Available Alcohol abuse is associated with increased lung infections. Molecular understanding of the underlying mechanisms is not complete. Airway epithelial ion transport regulates the homeostasis of airway surface liquid, essential for airway mucosal immunity and lung host defense. Here, air-liquid interface cultures of Calu-3 epithelial cells were basolaterally exposed to physiologically relevant concentrations of ethanol (0, 25, 50 and 100 mM for 24 hours and adenosine-stimulated ion transport was measured by Ussing chamber. The ethanol exposure reduced the epithelial short-circuit currents (I(SC in a dose-dependent manner. The ion currents activated by adenosine were chloride conductance mediated by cystic fibrosis transmembrane conductance regulator (CFTR, a cAMP-activated chloride channel. Alloxazine, a specific inhibitor for A(2B adenosine receptor (A(2BAR, largely abolished the adenosine-stimulated chloride transport, suggesting that A(2BAR is a major receptor responsible for regulating the chloride transport of the cells. Ethanol significantly reduced intracellular cAMP production upon adenosine stimulation. Moreover, ethanol-suppression of the chloride secretion was able to be restored by cAMP analogs or by inhibitors to block cAMP degradation. These results imply that ethanol exposure dysregulates CFTR-mediated chloride transport in airways by suppression of adenosine-A(2BAR-cAMP signaling pathway, which might contribute to alcohol-associated lung infections.

  3. Advancing clinical development pathways for new CFTR modulators in cystic fibrosis.

    Mayer-Hamblett, Nicole; Boyle, Michael; VanDevanter, Donald

    2016-05-01

    Cystic fibrosis (CF) is a life-shortening genetic disease affecting approximately 70,000 individuals worldwide. Until recently, drug development efforts have emphasised therapies treating downstream signs and symptoms resulting from the underlying CF biological defect: reduced function of the CF transmembrane conductance regulator (CFTR) protein. The current CF drug development landscape has expanded to include therapies that enhance CFTR function by either restoring wild-type CFTR protein expression or increasing (modulating) the function of mutant CFTR proteins in cells. To date, two systemic small-molecule CFTR modulators have been evaluated in pivotal clinical trials in individuals with CF and specific mutant CFTR genotypes that have led to regulatory review and/or approval. Advances in the discovery of CFTR modulators as a promising new class of therapies have been impressive, yet work remains to develop highly effective, disease-modifying modulators for individuals of all CF genotypes. The objectives of this review are to outline the challenges and opportunities in drug development created by systemic genotype-specific CFTR modulators, highlight the advantages of sweat chloride as an established biomarker of CFTR activity to streamline early-phase development and summarise options for later phase clinical trial designs that respond to the adoption of approved genotype-specific modulators into standard of care. An optimal development framework will be needed to move the most promising therapies efficiently through the drug development pipeline and ultimately deliver efficacious and safe therapies to all individuals with CF. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  4. Cellular distribution and function of ion channels involved in transport processes in rat tracheal epithelium.

    Hahn, Anne; Faulhaber, Johannes; Srisawang, Lalita; Stortz, Andreas; Salomon, Johanna J; Mall, Marcus A; Frings, Stephan; Möhrlen, Frank

    2017-06-01

    Transport of water and electrolytes in airway epithelia involves chloride-selective ion channels, which are controlled either by cytosolic Ca 2+ or by cAMP The contributions of the two pathways to chloride transport differ among vertebrate species. Because rats are becoming more important as animal model for cystic fibrosis, we have examined how Ca 2+ - dependent and cAMP- dependent Cl - secretion is organized in the rat tracheal epithelium. We examined the expression of the Ca 2+ -gated Cl - channel anoctamin 1 (ANO1), the cystic fibrosis transmembrane conductance regulator (CFTR) Cl - channel, the epithelial Na + channel ENaC, and the water channel aquaporin 5 (AQP5) in rat tracheal epithelium. The contribution of ANO1 channels to nucleotide-stimulated Cl - secretion was determined using the channel blocker Ani9 in short-circuit current recordings obtained from primary cultures of rat tracheal epithelial cells in Ussing chambers. We found that ANO1, CFTR and AQP5 proteins were expressed in nonciliated cells of the tracheal epithelium, whereas ENaC was expressed in ciliated cells. Among nonciliated cells, ANO1 occurred together with CFTR and Muc5b and, in addition, in a different cell type without CFTR and Muc5b. Bioelectrical studies with the ANO1-blocker Ani9 indicated that ANO1 mediated the secretory response to the nucleotide uridine-5'-triphosphate. Our data demonstrate that, in rat tracheal epithelium, Cl - secretion and Na + absorption are routed through different cell types, and that ANO1 channels form the molecular basis of Ca 2+ -dependent Cl - secretion in this tissue. These characteristic features of Cl - -dependent secretion reveal similarities and distinct differences to secretory processes in human airways. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

  5. The ClC-K2 Chloride Channel Is Critical for Salt Handling in the Distal Nephron.

    Hennings, J Christopher; Andrini, Olga; Picard, Nicolas; Paulais, Marc; Huebner, Antje K; Cayuqueo, Irma Karen Lopez; Bignon, Yohan; Keck, Mathilde; Cornière, Nicolas; Böhm, David; Jentsch, Thomas J; Chambrey, Régine; Teulon, Jacques; Hübner, Christian A; Eladari, Dominique

    2017-01-01

    Chloride transport by the renal tubule is critical for blood pressure (BP), acid-base, and potassium homeostasis. Chloride uptake from the urinary fluid is mediated by various apical transporters, whereas basolateral chloride exit is thought to be mediated by ClC-Ka/K1 and ClC-Kb/K2, two chloride channels from the ClC family, or by KCl cotransporters from the SLC12 gene family. Nevertheless, the localization and role of ClC-K channels is not fully resolved. Because inactivating mutations in ClC-Kb/K2 cause Bartter syndrome, a disease that mimics the effects of the loop diuretic furosemide, ClC-Kb/K2 is assumed to have a critical role in salt handling by the thick ascending limb. To dissect the role of this channel in detail, we generated a mouse model with a targeted disruption of the murine ortholog ClC-K2. Mutant mice developed a Bartter syndrome phenotype, characterized by renal salt loss, marked hypokalemia, and metabolic alkalosis. Patch-clamp analysis of tubules isolated from knockout (KO) mice suggested that ClC-K2 is the main basolateral chloride channel in the thick ascending limb and in the aldosterone-sensitive distal nephron. Accordingly, ClC-K2 KO mice did not exhibit the natriuretic response to furosemide and exhibited a severely blunted response to thiazide. We conclude that ClC-Kb/K2 is critical for salt absorption not only by the thick ascending limb, but also by the distal convoluted tubule. Copyright © 2016 by the American Society of Nephrology.

  6. Inhibition of the voltage-dependent chloride channel of Torpedo electric organ by diisopropylfluorophosphate and its reversal by oximes

    Abalis, I.M.; Chiang, P.K.; Wirtz, R.A.; Andre, R.G.

    1986-01-01

    Diisopropylfluorophosphate (DFP), a potent organophosphate inhibitor of cholinesterases, was found to inhibit the specific binding of [ 35 S]t-butylbicyclophosphorothionate (TBPS), specific chloride channels ligand, to the electric organ membranes of Torpedo, with a Ki of 21 +/- 3 μM. The binding sites of [ 35 S]TBPS in the Torpedo membranes were found not to be GABA receptors or nicotinic acetylcholine receptors as previously described. Interestingly, a stimulation of the binding of [ 35 S]TBPS was observed in the presence of atropine and three oximes, monopyridinium oxime 2-PAM, bispyridinium bis-oxime TMB-4 and H-oxime HI-6. The maximal stimulation was 300-500% of control, after which, the stimulation was reversed at higher concentrations. The three oximes protected by more than 95% the inhibition by 1 mM DFP of the binding of [ 35 S]TBPS to the voltage-dependent chloride channel. However, atropine protected only 20% of the inhibited channel. These results, thus, suggest that the protection against the toxic effects of DFP or other anticholinesterase agents by the tested oximes may not be solely a result of the reactivation of cholinesterases but also the protection of the voltage-dependent chloride channel

  7. Myotonia congenita-associated mutations in chloride channel-1 affect zebrafish body wave swimming kinematics.

    Cheng, Wei; Tian, Jing; Burgunder, Jean-Marc; Hunziker, Walter; Eng, How-Lung

    2014-01-01

    Myotonia congenita is a human muscle disorder caused by mutations in CLCN1, which encodes human chloride channel 1 (CLCN1). Zebrafish is becoming an increasingly useful model for human diseases, including muscle disorders. In this study, we generated transgenic zebrafish expressing, under the control of a muscle specific promoter, human CLCN1 carrying mutations that have been identified in human patients suffering from myotonia congenita. We developed video analytic tools that are able to provide precise quantitative measurements of movement abnormalities in order to analyse the effect of these CLCN1 mutations on adult transgenic zebrafish swimming. Two new parameters for body-wave kinematics of swimming reveal changes in body curvature and tail offset in transgenic zebrafish expressing the disease-associated CLCN1 mutants, presumably due to their effect on muscle function. The capability of the developed video analytic tool to distinguish wild-type from transgenic zebrafish could provide a useful asset to screen for compounds that reverse the disease phenotype, and may be applicable to other movement disorders besides myotonia congenita.

  8. Myotonia congenita-associated mutations in chloride channel-1 affect zebrafish body wave swimming kinematics.

    Wei Cheng

    Full Text Available Myotonia congenita is a human muscle disorder caused by mutations in CLCN1, which encodes human chloride channel 1 (CLCN1. Zebrafish is becoming an increasingly useful model for human diseases, including muscle disorders. In this study, we generated transgenic zebrafish expressing, under the control of a muscle specific promoter, human CLCN1 carrying mutations that have been identified in human patients suffering from myotonia congenita. We developed video analytic tools that are able to provide precise quantitative measurements of movement abnormalities in order to analyse the effect of these CLCN1 mutations on adult transgenic zebrafish swimming. Two new parameters for body-wave kinematics of swimming reveal changes in body curvature and tail offset in transgenic zebrafish expressing the disease-associated CLCN1 mutants, presumably due to their effect on muscle function. The capability of the developed video analytic tool to distinguish wild-type from transgenic zebrafish could provide a useful asset to screen for compounds that reverse the disease phenotype, and may be applicable to other movement disorders besides myotonia congenita.

  9. The HDAC inhibitor SAHA does not rescue CFTR membrane expression in Cystic Fibrosis.

    Bergougnoux, Anne; Petit, Aurélie; Knabe, Lucie; Bribes, Estelle; Chiron, Raphaël; De Sario, Albertina; Claustres, Mireille; Molinari, Nicolas; Vachier, Isabelle; Taulan-Cadars, Magali; Bourdin, Arnaud

    2017-07-01

    The development of suitable Cystic Fibrosis (CF) models for preclinical bench tests of therapeutic candidates is challenging. Indeed, the validation of molecules to rescue the p.Phe508del-CFTR channel (encoded by the Cystic Fibrosis Transmembrane conductance Regulator gene carrying the p.Phe508del mutation) requires taking into account their overall effects on the epithelium. Suberoylanilide Hydroxamic Acid (SAHA), a histone deacetylase inhibitor (HDACi), was previously shown to be a CFTR corrector via proteostasis modulation in CFTR-deficient immortalized cells. Here, we tested SAHA effects on goblet cell metaplasia using an ex vivo model based on the air-liquid interface (ALI) culture of differentiated airway epithelial cells obtained by nasal scraping from CF patients and healthy controls. Ex vivo epithelium grew successfully in ALI cultures with significant rise in the expression of CFTR and of markers of airway epithelial differentiation compared to monolayer cell culture. SAHA decreased CFTR transcript and protein levels in CF and non-CF epithelia. Whereas SAHA induced lysine hyperacetylation, it did not change histone modifications at the CFTR promoter. SAHA reduced MUC5AC and MUC5B expression and inhibited goblet epithelial cell differentiation. Similar effects were obtained in CF and non-CF epithelia. All the effects were fully reversible within five days from SAHA withdrawal. We conclude that, ex vivo, SAHA modulate the structure of airway epithelia without specific effect on CFTR gene and protein suggesting that HDACi cannot be useful for CF treatment. Copyright © 2017. Published by Elsevier Ltd.

  10. cGMP inhibition of type 3 phosphodiesterase is the major mechanism by which C-type natriuretic peptide activates CFTR in the shark rectal gland

    De Jonge, Hugo R.; Tilly, Ben C.; Hogema, Boris M.; Pfau, Daniel J.; Kelley, Catherine A.; Kelley, Megan H.; Melita, August M.; Morris, Montana T.; Viola, Ryan M.

    2013-01-01

    The in vitro perfused rectal gland of the dogfish shark (Squalus acanthias) and filter-grown monolayers of primary cultures of shark rectal gland (SRG) epithelial cells were used to analyze the signal transduction pathway by which C-type natriuretic peptide (CNP) stimulates chloride secretion. CNP binds to natriuretic receptors in the basolateral membrane, elevates cellular cGMP, and opens cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels in the apical membrane. CNP-provoked chloride secretion was completely inhibitable by the nonspecific protein kinase inhibitor staurosporine and the PKA inhibitor H89 but insensitive to H8, an inhibitor of type I and II isoforms of cGMP-dependent protein kinase (cGKI and cGKII). CNP-induced secretion could not be mimicked by nonhydrolyzable cGMP analogs added alone or in combination with the protein kinase C activator phorbolester, arguing against a role for cGK or for cGMP-induced PKC signaling. We failed to detect a dogfish ortholog of cGKII by molecular cloning and affinity chromatography. However, inhibitors of the cGMP-inhibitable isoform of phosphodiesterase (PDE3) including milrinone, amrinone, and cilostamide but not inhibitors of other PDE isoenzymes mimicked the effect of CNP on chloride secretion in perfused glands and monolayers. CNP raised cGMP and cAMP levels in the SRG epithelial cells. This rise in cAMP as well as the CNP and amrinone-provoked chloride secretion, but not the rise in cGMP, was almost completely blocked by the Gαi-coupled adenylyl cyclase inhibitor somatostatin, arguing against a role for cGMP cross-activation of PKA in CNP action. These data provide molecular, functional, and pharmacological evidence for a CNP/cGMP/PDE3/cAMP/PKA signaling cascade coupled to CFTR in the SRG. PMID:24259420

  11. Thermodynamic study of the native and phosphorylated regulatory domain of the CFTR

    Marasini, Carlotta, E-mail: marasini@ge.ibf.cnr.it [Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Via De Marini 6, 16149 Genova (Italy); Galeno, Lauretta; Moran, Oscar [Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Via De Marini 6, 16149 Genova (Italy)

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer CFTR mutations produce cystic fibrosis. Black-Right-Pointing-Pointer Chloride transport depends on the regulatory domain phosphorylation. Black-Right-Pointing-Pointer Regulatory domain is intrinsically disordered. Black-Right-Pointing-Pointer Secondary structure and protein stability change upon phosphorylation. -- Abstract: The regulatory domain (RD) of the cystic fibrosis transmembrane conductance regulator (CFTR), the defective protein in cystic fibrosis, is the region of the channel that regulates the CFTR activity with multiple phosphorylation sites. This domain is an intrinsically disordered protein, characterized by lack of stable or unique tertiary structure. The disordered character of a protein is directly correlated with its function. The flexibility of RD may be important for its regulatory role: the continuous conformational change may be necessary for the progressive phosphorylation, and thus activation, of the channel. However, the lack of a defined and stable structure results in a considerable limitation when trying to in build a unique molecular model for the RD. Moreover, several evidences indicate significant structural differences between the native, non-phosphorylated state, and the multiple phosphorylated state of the protein. The aim of our work is to provide data to describe the conformations and the thermodynamic properties in these two functional states of RD. We have done the circular dichroism (CD) spectra in samples with a different degree of phosphorylation, from the non-phosphorylated state to a bona fide completely phosphorylated state. Analysis of CD spectra showed that the random coil and {beta}-sheets secondary structure decreased with the polypeptide phosphorylation, at expenses of an increase of {alpha}-helix. This observation lead to interpret phosphorylation as a mechanism favoring a more structured state. We also studied the thermal denaturation curves of the protein in the two

  12. Thermodynamic study of the native and phosphorylated regulatory domain of the CFTR

    Marasini, Carlotta; Galeno, Lauretta; Moran, Oscar

    2012-01-01

    Highlights: ► CFTR mutations produce cystic fibrosis. ► Chloride transport depends on the regulatory domain phosphorylation. ► Regulatory domain is intrinsically disordered. ► Secondary structure and protein stability change upon phosphorylation. -- Abstract: The regulatory domain (RD) of the cystic fibrosis transmembrane conductance regulator (CFTR), the defective protein in cystic fibrosis, is the region of the channel that regulates the CFTR activity with multiple phosphorylation sites. This domain is an intrinsically disordered protein, characterized by lack of stable or unique tertiary structure. The disordered character of a protein is directly correlated with its function. The flexibility of RD may be important for its regulatory role: the continuous conformational change may be necessary for the progressive phosphorylation, and thus activation, of the channel. However, the lack of a defined and stable structure results in a considerable limitation when trying to in build a unique molecular model for the RD. Moreover, several evidences indicate significant structural differences between the native, non-phosphorylated state, and the multiple phosphorylated state of the protein. The aim of our work is to provide data to describe the conformations and the thermodynamic properties in these two functional states of RD. We have done the circular dichroism (CD) spectra in samples with a different degree of phosphorylation, from the non-phosphorylated state to a bona fide completely phosphorylated state. Analysis of CD spectra showed that the random coil and β-sheets secondary structure decreased with the polypeptide phosphorylation, at expenses of an increase of α-helix. This observation lead to interpret phosphorylation as a mechanism favoring a more structured state. We also studied the thermal denaturation curves of the protein in the two conditions, monitoring the changes of the mean residue ellipticity measured at 222 nm as a function of temperature

  13. Exterior Site Occupancy Infers Chloride-Induced Proton Gating in a Prokaryotic Homolog of the ClC Chloride Channel

    Bostick, David L.; Berkowitz, Max L.

    2004-01-01

    The ClC family of anion channels mediates the efficient, selective permeation of Cl− across the biological membranes of living cells under the driving force of an electrochemical gradient. In some eukaryotes, these channels are known to exhibit a unique gating mechanism, which appears to be triggered by the permeant Cl− anion. We infer details of this gating mechanism by studying the free energetics of Cl− occupancy in the pore of a prokaryotic ClC homolog. These free energetics were gleaned from 30 ns of molecular dynamics simulation on an ∼133,000-atom system consisting of a hydrated membrane embedded StClC transporter. The binding sites for Cl− in the transporter were determined for the cases where the putative gating residue, Glu148, was protonated and unprotonated. When the glutamate gate is protonated, Cl− favorably occupies an exterior site, Sext, to form a queue of anions in the pore. However, when the glutamate gate is unprotonated, Cl− cannot occupy this site nor, consequently, pass through the pore. An additional, previously undetected, site was found in the pore near the outer membrane that exists regardless of the protonation state of Glu148. Although this suggests that, for the prokaryotic homolog, protonation of Glu148 may be the first step in transporting Cl− at the expense of H+ transport in the opposite direction, an evolutionary argument might suggest that Cl− opens the ClC gate in eukaryotic channels by inducing the conserved glutamate's protonation. During an additional 20 ns free dynamics simulation, the newly discovered outermost site, Sout, and the innermost site, Sint, were seen to allow spontaneous exchange of Cl− ions with the bulk electrolyte while under depolarization conditions. PMID:15345547

  14. The novel isoxazoline ectoparasiticide lotilaner (Credelio™): a non-competitive antagonist specific to invertebrates γ-aminobutyric acid-gated chloride channels (GABACls)

    Rufener, Lucien; Danelli, Vanessa; Bertrand, Daniel; Sager, Heinz

    2017-01-01

    Background The isoxazolines are a novel class of parasiticides that are potent inhibitors of γ-aminobutyric acid (GABA)-gated chloride channels (GABACls) and, to a lesser extent, of inhibitory glutamate-gated chloride channels (GluCls). Lotilaner (Credelio™), a novel representative of this chemical class, is currently evaluated for its excellent ectoparasiticide properties. Methods In this study, we investigated the molecular mode of action and pharmacology of lotilaner. We report the success...

  15. CFTR mediates noradrenaline-induced ATP efflux from DRG neurons.

    Kanno, Takeshi; Nishizaki, Tomoyuki

    2011-09-24

    In our earlier study, noradrenaline (NA) stimulated ATP release from dorsal root ganglion (DRG) neurons as mediated via β(3) adrenoceptors linked to G(s) protein involving protein kinase A (PKA) activation, to cause allodynia. The present study was conducted to understand how ATP is released from DRG neurons. In an outside-out patch-clamp configuration from acutely dissociated rat DRG neurons, single-channel currents, sensitive to the P2X receptor inhibitor PPADS, were evoked by approaching the patch-electrode tip close to a neuron, indicating that ATP is released from DRG neurons, to activate P2X receptor. NA increased the frequency of the single-channel events, but such NA effect was not found for DRG neurons transfected with the siRNA to silence the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In the immunocytochemical study using acutely dissociated rat DRG cells, CFTR was expressed in neurons alone, but not satellite cells, fibroblasts, or Schwann cells. It is concluded from these results that CFTR mediates NA-induced ATP efflux from DRG neurons as an ATP channel.

  16. [Application of Brownian dynamics to the description of transmembrane ion flow as exemplified by the chloride channel of glycine receptor].

    Boronovskiĭ, S E; Nartsissov, Ia R

    2009-01-01

    Using the Brownian dynamics of the movement of hydrated ion in a viscous water solution, a mathematical model has been built, which describes the transport of charged particles through a single protein pore in a lipid membrane. The dependences of transmembrane ion currents on ion concentrations in solution have been obtained. It was shown that, if the geometry of a membrane pore is identical to that of the inner part of the glycine receptor channel and there is no ion selectivity, then the values of both chloride and sodium currents are not greater than 0.5 pA at the physiological concentrations of these ions. If local charge heterogeneity caused by charged amino acid residues of transmembrane protein segments is included into the model calculations, the chloride current increases to about 3.7 pA, which exceeds more than seven times the value for sodium ions under the conditions of the complex channel geometry in the range of physiological concentrations of ions in the solution. The model takes changes in the density of charge distribution both inside the channel and near the protein surface into account. The alteration of pore geometry can be also considered as a parameter at the researcher's option. Thus, the model appears as an effective tool for the description of transmembrane currents for other types of membrane channels.

  17. NM23 proteins: innocent bystanders or local energy boosters for CFTR?

    Muimo, Richmond; Alothaid, Hani Mm; Mehta, Anil

    2018-03-01

    NM23 proteins NDPK-A and -B bind to the cystic fibrosis (CF) protein CFTR in different ways from kinases such as PKA, CK2 and AMPK or linkers to cell calcium such as calmodulin and annexins. NDPK-A (not -B) interacts with CFTR through reciprocal AMPK binding/control, whereas NDPK-B (not -A) binds directly to CFTR. NDPK-B can activate G proteins without ligand-receptor coupling, so perhaps NDPK-B's binding influences energy supply local to a nucleotide-binding site (NBD1) needed for CFTR to function. Curiously, CFTR (ABC-C7) is a member of the ATP-binding cassette (ABC) protein family that does not obey 'clan rules'; CFTR channels anions and is not a pump, regulates disparate processes, is itself regulated by multiple means and is so pleiotropic that it acts as a hub that orchestrates calcium signaling through its consorts such as calmodulin/annexins. Furthermore, its multiple partners make CFTR dance to different tunes in different cellular and subcellular locations as it recycles from the plasma membrane to endosomes. CFTR function in airway apical membranes is inhibited by smoking which has been dubbed 'acquired CF'. CFTR alone among family members possesses a trap for other proteins that it unfurls as a 'fish-net' and which bears consensus phosphorylation sites for many protein kinases, with PKA being the most canonical. Recently, the site of CFTR's commonest mutation has been proposed as a knock-in mutant that alters allosteric control of kinase CK2 by log orders of activity towards calmodulin and other substrates after CFTR fragmentation. This link from CK2 to calmodulin that binds the R region invokes molecular paths that control lumen formation, which is incomplete in the tracheas of some CF-affected babies. Thus, we are poised to understand the many roles of NDPK-A and -B in CFTR function and, especially lumen formation, which is defective in the gut and lungs of many CF babies.

  18. CLC-Nt1, a putative chloride channel protein of tobacco, co-localizes with mitochondrial membrane markers.

    Lurin, C; Güclü, J; Cheniclet, C; Carde, J P; Barbier-Brygoo, H; Maurel, C

    2000-06-01

    The voltage-dependent chloride channel (CLC) family of membrane proteins has cognates in animals, yeast, bacteria and plants, and chloride-channel activity has been assigned to most of the animal homologues. Lack of evidence of CLC functions in plants prompted us to characterize the cellular localization of the tobacco CLC-Nt1 protein. Specific polyclonal antibodies were raised against an N-terminal polypeptide of CLC-Nt1. These antibodies were used to probe membrane proteins prepared by various cell-fractionation methods. These included aqueous two-phase partitioning (for plasma membranes), free-flow electrophoresis (for vacuolar and plasma membranes), intact vacuole isolation, Percoll-gradient centrifugation (for plastids and mitochondria) and stepped, linear, sucrose-density-gradient centrifugation (for mitochondria). Each purified membrane fraction was characterized with specific marker enzyme activities or antibodies. Our studies ruled out the possibility that the major cell localization of CLC-Nt1 was the vacuolar or plasma membranes, the endoplasmic reticulum, the Golgi apparatus or the plastids. In contrast, we showed that the tobacco CLC-Nt1 specifically co-localized with the markers of the mitochondrial inner membrane, cytochrome c oxidase and NAD9 protein. CLC-Nt1 may correspond to the inner membrane anion channel ('IMAC') described previously in animal and plant mitochondria.

  19. Use of the chloride channel activator lubiprostone for constipation in adults with cystic fibrosis: a case series.

    O'Brien, Catherine E; Anderson, Paula J; Stowe, Cindy D

    2010-03-01

    To describe the use of lubiprostone for constipation in 3 adults with cystic fibrosis (CF). This case series describes the use of lubiprostone for the treatment of constipation in 3 adults with CF (mean +/- SD length of therapy 17.3 +/- 1.5 mo). All 3 patients were prescribed lubiprostone 24 microg twice daily after hospitalization for treatment of intestinal obstruction. Patient 1 continues on chronic polyethylene glycol (PEG) 3350 and lubiprostone and has not had a recurrence of obstruction. Patient 2 requires aggressive chronic therapy with PEG 3350, lubiprostone, and methylnaltrexone. She has had 1 recurrence of obstruction. Patient 3 continues with lubiprostone taken several times per week with good control of constipation and no recurrence of obstruction to date. The adverse effect profile has been tolerable in all 3 patients. CF is caused by a genetic mutation resulting in a dysfunctional or absent CF transmembrane conductance regulator that normally functions as a chloride channel. This results in viscous secretions in multiple organ systems including the lungs and intestinal tract. Accumulation of viscous intestinal contents contributes to constipation, which is common among adults with CF and can sometimes lead to intestinal obstruction. Lubiprostone is indicated for chronic constipation and works by activating type 2 chloride channels (ClC-2) in the intestinal tract. Because it utilizes an alternate chloride channel, lubiprostone may be especially effective for constipation in patients with CF. Lubiprostone provides an additional option for the treatment of constipation in adults with CF. Its use in the CF population deserves further study.

  20. Chloride channels in the plasma membrane of a foetal Drosophila cell line, S2

    Asmild, Margit; Willumsen, Niels J.

    2000-01-01

    S2 cells, Cl- Channels, Expression system, Drosophila, Inward rectifier, Outward rectifier, Patch clamp......S2 cells, Cl- Channels, Expression system, Drosophila, Inward rectifier, Outward rectifier, Patch clamp...

  1. A host defense mechanism involving CFTR-mediated bicarbonate secretion in bacterial prostatitis.

    Chen Xie

    Full Text Available BACKGROUND: Prostatitis is associated with a characteristic increase in prostatic fluid pH; however, the underlying mechanism and its physiological significance have not been elucidated. METHODOLOGY/PRINCIPAL FINDINGS: In this study a primary culture of rat prostatic epithelial cells and a rat prostatitis model were used. Here we reported the involvement of CFTR, a cAMP-activated anion channel conducting both Cl(- and HCO(3(-, in mediating prostate HCO(3(- secretion and its possible role in bacterial killing. Upon Escherichia coli (E. coli-LPS challenge, the expression of CFTR and carbonic anhydrase II (CA II, along with several pro-inflammatory cytokines was up-regulated in the primary culture of rat prostate epithelial cells. Inhibiting CFTR function in vitro or in vivo resulted in reduced bacterial killing by prostate epithelial cells or the prostate. High HCO(3(- content (>50 mM, rather than alkaline pH, was found to be responsible for bacterial killing. The direct action of HCO(3(- on bacterial killing was confirmed by its ability to increase cAMP production and suppress bacterial initiation factors in E. coli. The relevance of the CFTR-mediated HCO(3(- secretion in humans was demonstrated by the upregulated expression of CFTR and CAII in human prostatitis tissues. CONCLUSIONS/SIGNIFICANCE: The CFTR and its mediated HCO(3(- secretion may be up-regulated in prostatitis as a host defense mechanism.

  2. Unique contributions of an arginine side chain to ligand recognition in a glutamate-gated chloride channel

    Lynagh, Timothy; Komnatnyy, Vitaly V; Pless, Stephan A

    2017-01-01

    Glutamate recognition by neurotransmitter receptors often relies on arginine (Arg) residues in the binding site, leading to the assumption that charge-charge interactions underlie ligand recognition. However, assessing the precise chemical contribution of Arg side chains to protein function......-gated chloride channel from the nematode Haemonchus contortus. Our data unveil a surprisingly small contribution of charge at a conserved arginine side chain previously suggested to form a salt bridge with the ligand, glutamate. Instead, our data show that Arg contributes crucially to ligand sensitivity via...

  3. A comprehensive search for calcium binding sites critical for TMEM16A calcium-activated chloride channel activity

    Tien, Jason; Peters, Christian J; Wong, Xiu Ming; Cheng, Tong; Jan, Yuh Nung; Jan, Lily Yeh; Yang, Huanghe

    2014-01-01

    TMEM16A forms calcium-activated chloride channels (CaCCs) that regulate physiological processes such as the secretions of airway epithelia and exocrine glands, the contraction of smooth muscles, and the excitability of neurons. Notwithstanding intense interest in the mechanism behind TMEM16A-CaCC calcium-dependent gating, comprehensive surveys to identify and characterize potential calcium sensors of this channel are still lacking. By aligning distantly related calcium-activated ion channels in the TMEM16 family and conducting systematic mutagenesis of all conserved acidic residues thought to be exposed to the cytoplasm, we identify four acidic amino acids as putative calcium-binding residues. Alterations of the charge, polarity, and size of amino acid side chains at these sites alter the ability of different divalent cations to activate the channel. Furthermore, TMEM16A mutant channels containing double cysteine substitutions at these residues are sensitive to the redox potential of the internal solution, providing evidence for their physical proximity and solvent accessibility. DOI: http://dx.doi.org/10.7554/eLife.02772.001 PMID:24980701

  4. Cell swelling activates separate taurine and chloride channels in Ehrlich mouse ascites tumor cells

    Lambert, Ian Henry; Hoffmann, Else Kay

    1994-01-01

    The taurine efflux from Ehrlich ascites tumor cells is stimulated by hypotonic cell swelling. The swelling-activated taurine efflux is unaffected by substitution of gluconate for extracellular Cl– but inhibited by addition of MK196 (anion channel blocker) and 4,4 -diisothiocyanostilbene-2......,2 -disulfonic acid (DIDS; anion channel and anion exchange blocker) and by depolarization of the cell membrane. This is taken to indicate that taurine does not leave the osmotically swollen Ehrlich cells in exchange for extracellular Cl–, i.e., via the anion exchanger but via a MK196- and DIDS-sensitive channel...... that is potential dependent. An additional stimulation of the swelling-activated taurine efflux is seen after addition of arachidonic acid and oleic acid. Cell swelling also activates a Mini Cl– channel. The Cl– efflux via this Cl– channel, in contrast to the swelling-activated taurine efflux, is unaffected by DIDS...

  5. Changes in cationic selectivity of the nicotinic channel at the rat ganglionic synapse: a role for chloride ions?

    Sacchi, Oscar; Rossi, Maria Lisa; Canella, Rita; Fesce, Riccardo

    2011-02-25

    The permeability of the nicotinic channel (nAChR) at the ganglionic synapse has been examined, in the intact rat superior cervical ganglion in vitro, by fitting the Goldman current equation to the synaptic current (EPSC) I-V relationship. Subsynaptic nAChRs, activated by neurally-released acetylcholine (ACh), were thus analyzed in an intact environment as natively expressed by the mature sympathetic neuron. Postsynaptic neuron hyperpolarization (from -40 to -90 mV) resulted in a change of the synaptic potassium/sodium permeability ratio (P(K)/P(Na)) from 1.40 to 0.92, corresponding to a reversible shift of the apparent acetylcholine equilibrium potential, E(ACh), by about +10 mV. The effect was accompanied by a decrease of the peak synaptic conductance (g(syn)) and of the EPSC decay time constant. Reduction of [Cl(-)](o) to 18 mM resulted in a change of P(K)/P(Na) from 1.57 (control) to 2.26, associated with a reversible shift of E(ACh) by about -10 mV. Application of 200 nM αBgTx evoked P(K)/P(Na) and g(syn) modifications similar to those observed in reduced [Cl(-)](o). The two treatments were overlapping and complementary, as if the same site/mechanism were involved. The difference current before and after chloride reduction or toxin application exhibited a strongly positive equilibrium potential, which could not be explained by the block of a calcium component of the EPSC. Observations under current-clamp conditions suggest that the driving force modification of the EPSC due to P(K)/P(Na) changes represent an additional powerful integrative mechanism of neuron behavior. A possible role for chloride ions is suggested: the nAChR selectivity was actually reduced by increased chloride gradient (membrane hyperpolarization), while it was increased, moving towards a channel preferentially permeable for potassium, when the chloride gradient was reduced.

  6. Changes in cationic selectivity of the nicotinic channel at the rat ganglionic synapse: a role for chloride ions?

    Oscar Sacchi

    Full Text Available The permeability of the nicotinic channel (nAChR at the ganglionic synapse has been examined, in the intact rat superior cervical ganglion in vitro, by fitting the Goldman current equation to the synaptic current (EPSC I-V relationship. Subsynaptic nAChRs, activated by neurally-released acetylcholine (ACh, were thus analyzed in an intact environment as natively expressed by the mature sympathetic neuron. Postsynaptic neuron hyperpolarization (from -40 to -90 mV resulted in a change of the synaptic potassium/sodium permeability ratio (P(K/P(Na from 1.40 to 0.92, corresponding to a reversible shift of the apparent acetylcholine equilibrium potential, E(ACh, by about +10 mV. The effect was accompanied by a decrease of the peak synaptic conductance (g(syn and of the EPSC decay time constant. Reduction of [Cl(-](o to 18 mM resulted in a change of P(K/P(Na from 1.57 (control to 2.26, associated with a reversible shift of E(ACh by about -10 mV. Application of 200 nM αBgTx evoked P(K/P(Na and g(syn modifications similar to those observed in reduced [Cl(-](o. The two treatments were overlapping and complementary, as if the same site/mechanism were involved. The difference current before and after chloride reduction or toxin application exhibited a strongly positive equilibrium potential, which could not be explained by the block of a calcium component of the EPSC. Observations under current-clamp conditions suggest that the driving force modification of the EPSC due to P(K/P(Na changes represent an additional powerful integrative mechanism of neuron behavior. A possible role for chloride ions is suggested: the nAChR selectivity was actually reduced by increased chloride gradient (membrane hyperpolarization, while it was increased, moving towards a channel preferentially permeable for potassium, when the chloride gradient was reduced.

  7. Blockade of chloride channels by DIDS stimulates renin release and inhibits contraction of afferent arterioles

    Jensen, B L; Skøtt, O

    1996-01-01

    or without ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] and DIDS were not additive. In the absence of chloride, basal renin release was suppressed and the stimulatory effect of DIDS was abolished. The DIDS-induced enhancement of renin release was not dependent on bicarbonate....... Norepinephrine (5 x 10(-7)-1 x 10(-6) M) and angiotensin II (1 x 10(-8)-10(-6) M) evoked reversible and dose-dependent contractions of microperfused rabbit afferent arterioles. DIDS (0.5 mM) did not affect the basal diameter of the arterioles but strongly inhibited the response to angiotensin II and attenuated...

  8. Activation of a cGMP-sensitive calcium-dependent chloride channel may cause transition from calcium waves to whole-cell oscillations in smooth muscle cells

    Jacobsen, Jens Christian; Aalkjær, Christian; Nilsson, Holger

    2007-01-01

    approximately doubles. In this transition, the simulated results point to a key role for a recently discovered cGMP-sensitive calcium-dependent chloride channel. This channel depolarizes the membrane in response to calcium released from the SR. In turn, depolarization causes uniform opening of L-type calcium...... channels on the cell surface stimulating synchronized release of SR-calcium and inducing the shift from waves to whole-cell oscillations. The effect of the channel is therefore to couple the processes of the SR with those of the membrane. We hypothesize that the shift in oscillatory mode and the associated...

  9. Ribosomal Stalk Protein Silencing Partially Corrects the ΔF508-CFTR Functional Expression Defect.

    Guido Veit

    2016-05-01

    Full Text Available The most common cystic fibrosis (CF causing mutation, deletion of phenylalanine 508 (ΔF508 or Phe508del, results in functional expression defect of the CF transmembrane conductance regulator (CFTR at the apical plasma membrane (PM of secretory epithelia, which is attributed to the degradation of the misfolded channel at the endoplasmic reticulum (ER. Deletion of phenylalanine 670 (ΔF670 in the yeast oligomycin resistance 1 gene (YOR1, an ABC transporter of Saccharomyces cerevisiae phenocopies the ΔF508-CFTR folding and trafficking defects. Genome-wide phenotypic (phenomic analysis of the Yor1-ΔF670 biogenesis identified several modifier genes of mRNA processing and translation, which conferred oligomycin resistance to yeast. Silencing of orthologues of these candidate genes enhanced the ΔF508-CFTR functional expression at the apical PM in human CF bronchial epithelia. Although knockdown of RPL12, a component of the ribosomal stalk, attenuated the translational elongation rate, it increased the folding efficiency as well as the conformational stability of the ΔF508-CFTR, manifesting in 3-fold augmented PM density and function of the mutant. Combination of RPL12 knockdown with the corrector drug, VX-809 (lumacaftor restored the mutant function to ~50% of the wild-type channel in primary CFTRΔF508/ΔF508 human bronchial epithelia. These results and the observation that silencing of other ribosomal stalk proteins partially rescue the loss-of-function phenotype of ΔF508-CFTR suggest that the ribosomal stalk modulates the folding efficiency of the mutant and is a potential therapeutic target for correction of the ΔF508-CFTR folding defect.

  10. Differential involvement of glutamate-gated chloride channel splice variants in the olfactory memory processes of the honeybee Apis mellifera.

    Démares, Fabien; Drouard, Florian; Massou, Isabelle; Crattelet, Cindy; Lœuillet, Aurore; Bettiol, Célia; Raymond, Valérie; Armengaud, Catherine

    2014-09-01

    Glutamate-gated chloride channels (GluCl) belong to the cys-loop ligand-gated ion channel superfamily and their expression had been described in several invertebrate nervous systems. In the honeybee, a unique gene amel_glucl encodes two alternatively spliced subunits, Amel_GluCl A and Amel_GluCl B. The expression and differential localization of those variants in the honeybee brain had been previously reported. Here we characterized the involvement of each variant in olfactory learning and memory processes, using specific small-interfering RNA (siRNA) targeting each variant. Firstly, the efficacy of the two siRNAs to decrease their targets' expression was tested, both at mRNA and protein levels. The two proteins showed a decrease of their respective expression 24h after injection. Secondly, each siRNA was injected into the brain to test whether or not it affected olfactory memory by using a classical paradigm of conditioning the proboscis extension reflex (PER). Amel_GluCl A was found to be involved only in retrieval of 1-nonanol, whereas Amel_GluCl B was involved in the PER response to 2-hexanol used as a conditioned stimulus or as new odorant. Here for the first time, a differential behavioral involvement of two highly similar GluCl subunits has been characterized in an invertebrate species. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Functional classification of mitochondrion-rich cells in euryhaline Mozambique tilapia (Oreochromis mossambicus) embryos, by means of triple immunofluorescence staining for Na+/K+-ATPase, Na +/K+/2Cl- cotransporter and CFTR anion channel

    Hiroi, J.; McCormick, S.D.; Ohtani-Kaneko, R.; Kaneko, T.

    2005-01-01

    Mozambique tilapia Oreochromis mossambicus embryos were transferred from freshwater to seawater and vice versa, and short-term changes in the localization of three major ion transport proteins, Na+/K +-ATPase, Na+/K+/2Cl- cotransporter (NKCC) and cystic fibrosis transmembrane conductance regulator (CFTR) were examined within mitochondrion-rich cells (MRCs) in the embryonic yolk-sac membrane. Triple-color immunofluorescence staining allowed us to classify MRCs into four types: type I, showing only basolateral Na+/K +-ATPase staining; type II, basolateral Na+/K +-ATPase and apical NKCC; type III, basolateral Na+/K +-ATPase and basolateral NKCC; type IV, basolateral Na +/K+-ATPase, basolateral NKCC and apical CFTR. In freshwater, type-I, type-II and type-III cells were observed. Following transfer from freshwater to seawater, type-IV cells appeared at 12 h and showed a remarkable increase in number between 24 h and 48 h, whereas type-III cells disappeared. When transferred from seawater back to freshwater, type-IV cells decreased and disappeared at 48 h, type-III cells increased, and type-II cells, which were not found in seawater, appeared at 12 h and increased in number thereafter. Type-I cells existed consistently irrespective of salinity changes. These results suggest that type I is an immature MRC, type II is a freshwater-type ion absorptive cell, type III is a dormant type-IV cell and/or an ion absorptive cell (with a different mechanism from type II), and type IV is a seawater-type ion secretory cell. The intracellular localization of the three ion transport proteins in type-IV cells is completely consistent with a widely accepted model for ion secretion by MRCs. A new model for ion absorption is proposed based on type-II cells possessing apical NKCC.

  12. Cl- channels in apoptosis

    Wanitchakool, Podchanart; Ousingsawat, Jiraporn; Sirianant, Lalida

    2016-01-01

    A remarkable feature of apoptosis is the initial massive cell shrinkage, which requires opening of ion channels to allow release of K(+), Cl(-), and organic osmolytes to drive osmotic water movement and cell shrinkage. This article focuses on the role of the Cl(-) channels LRRC8, TMEM16/anoctamin......, and cystic fibrosis transmembrane conductance regulator (CFTR) in cellular apoptosis. LRRC8A-E has been identified as a volume-regulated anion channel expressed in many cell types. It was shown to be required for regulatory and apoptotic volume decrease (RVD, AVD) in cultured cell lines. Its presence also......(-) channels or as regulators of other apoptotic Cl(-) channels, such as LRRC8. CFTR has been known for its proapoptotic effects for some time, and this effect may be based on glutathione release from the cell and increase in cytosolic reactive oxygen species (ROS). Although we find that CFTR is activated...

  13. Activation of a cGMP-sensitive calcium-dependent chloride channel may cause transition from calcium waves to whole-cell oscillations in smooth muscle cells

    Jacobsen, Jens Christian; Aalkjær, Christian; Nilsson, Holger

    2007-01-01

    waves sweeping through the cytoplasm when the SR is stimulated to release calcium. A rise in cyclic guanosine monophosphate (cGMP) leads to the experimentally observed transition from waves to whole-cell calcium oscillations. At the same time membrane potential starts to oscillate and the frequency...... approximately doubles. In this transition, the simulated results point to a key role for a recently discovered cGMP-sensitive calcium-dependent chloride channel. This channel depolarizes the membrane in response to calcium released from the SR. In turn, depolarization causes uniform opening of L-type calcium...... onset of oscillations in membrane potential within the individual cell may underlie sudden intercellular synchronization and the appearance of vasomotion. Key words: Vasomotion, Chloride channel, cGMP, Mathematical model, Calcium waves....

  14. The transition from proliferation to differentiation in colorectal cancer is regulated by the calcium activated chloride channel A1.

    Bo Yang

    Full Text Available Breaking the balance between proliferation and differentiation in animal cells can lead to cancer, but the mechanisms maintaining this balance remain largely undefined. The calcium activated chloride channel A1 (CLCA1 is a member of the calcium sensitive chloride conductance family of proteins and is expressed mainly in the colon, small intestine and appendix. We show that CLCA1 plays a functional role in differentiation and proliferation of Caco-2 cells and of intestinal tissue. Caco-2 cells spontaneously differentiate either in confluent culture or when treated with butyrate, a molecule present naturally in the diet. Here, we compared CLCA1 expressional levels between patients with and without colorectal cancer (CRC and determined the functional role of CLCA1 in differentiation and proliferation of Caco-2 cells. We showed that: 1 CLCA1 and CLCA4 expression were down-regulated significantly in CRC patients; 2 CLCA1 expression was up-regulated in Caco-2 cells induced to differentiate by confluent culture or by treatment with sodium butyrate (NaBT; 3 Knockdown of CLCA1 with siRNA significantly inhibited cell differentiation and promoted cell proliferation in Caco-2 confluent cultures, and 4 In Caco-2 3D culture, suppression of CLCA1 significantly increased cell proliferation and compromised NaBT-induced inhibition of proliferation. In conclusion, CLCA1 may contribute to promoting spontaneous differentiation and reducing proliferation of Caco-2 cells and may be a target of NaBT-induced inhibition of proliferation and therefore a potential diagnostic marker for CRC prognosis.

  15. A little CFTR goes a long way: CFTR-dependent sweat secretion from G551D and R117H-5T cystic fibrosis subjects taking ivacaftor.

    Jessica E Char

    Full Text Available To determine if oral dosing with the CFTR-potentiator ivacaftor (VX-770, Kalydeco improves CFTR-dependent sweating in CF subjects carrying G551D or R117H-5T mutations, we optically measured sweat secretion from 32-143 individually identified glands in each of 8 CF subjects; 6 F508del/G551D, one G551D/R117H-5T, and one I507del/R117H-5T. Two subjects were tested only (- ivacaftor, 3 only (+ ivacaftor and 3 (+/- ivacaftor (1-5 tests per condition. The total number of gland measurements was 852 (- ivacaftor and 906 (+ ivacaftor. A healthy control was tested 4 times (51 glands. For each gland we measured both CFTR-independent (M-sweat and CFTR-dependent (C-sweat; C-sweat was stimulated with a β-adrenergic cocktail that elevated [cAMP]i while blocking muscarinic receptors. Absent ivacaftor, almost all CF glands produced M-sweat on all tests, but only 1/593 glands produced C-sweat (10 tests, 5 subjects. By contrast, 6/6 subjects (113/342 glands produced C-sweat in the (+ ivacaftor condition, but with large inter-subject differences; 3-74% of glands responded with C/M sweat ratios 0.04%-2.57% of the average WT ratio of 0.265. Sweat volume losses cause proportionally larger underestimates of CFTR function at lower sweat rates. The losses were reduced by measuring C/M ratios in 12 glands from each subject that had the highest M-sweat rates. Remaining losses were estimated from single channel data and used to correct the C/M ratios, giving estimates of CFTR function (+ ivacaftor  = 1.6%-7.7% of the WT average. These estimates are in accord with single channel data and transcript analysis, and suggest that significant clinical benefit can be produced by low levels of CFTR function.

  16. Meroterpenoid Chrodrimanins Are Selective and Potent Blockers of Insect GABA-Gated Chloride Channels.

    Yan Xu

    Full Text Available Meroterpenoid chrodrimanins, produced from Talaromyces sp. YO-2, are known to paralyze silkworm (Bombyx mori larvae, but their target is unknown. We have investigated the actions of chrodrimanin B on ligand-gated ion channels of silkworm larval neurons using patch-clamp electrophysiology. Chrodrimanin B had no effect on membrane currents when tested alone at 1 μM. However, it completely blocked the γ-aminobutyric acid (GABA-induced current and showed less pronounced actions on acetylcholine- and L-glutamate-induced currents, when delivered at 1 μM for 1 min prior to co-application with transmitter GABA. Thus, chrodrimanins were also tested on a wild-type isoform of the B. mori GABA receptor (GABAR RDL using two-electrode voltage-clamp electrophysiology. Chrodrimanin B attenuated the peak current amplitude of the GABA response of RDL with an IC50 of 1.66 nM. The order of the GABAR-blocking potency of chrodrimanins B > D > A was in accordance with their reported insecticidal potency. Chrodrimanin B had no open channel blocking action when tested at 3 nM on the GABA response of RDL. Co-application with 3 nM chrodrimanin B shifted the GABA concentration response curve to a higher concentration and further increase of chrodrimanin B concentration to 10 nM; it reduced maximum current amplitude of the GABA response, pointing to a high-affinity competitive action and a lower affinity non-competitive action. The A282S;T286V double mutation of RDL, which impairs the actions of fipronil, hardly affected the blocking action of chrodrimanin B, indicating a binding site of chrodrimanin B distinct from that of fipronil. Chrodrimanin B showed approximately 1,000-fold lower blocking action on human α1β2γ2 GABAR compared to RDL and thus is a selective blocker of insect GABARs.

  17. Hyperthyroidism enhances 5-HT-induced contraction of the rat pulmonary artery: role of calcium-activated chloride channel activation.

    Oriowo, Mabayoje A; Oommen, Elsie; Khan, Islam

    2011-11-01

    Experimentally-induced hyperthyroidism in rodents is associated with signs and symptoms of pulmonary hypertension. The main objective of the present study was to investigate the effect of thyroxine-induced pulmonary hypertension on the contractile response of the pulmonary artery to 5-HT and the possible underlying signaling pathway. 5-HT concentration-dependently contracted artery segments from control and thyroxine-treated rats with pD(2) values of 5.04 ± 0.19 and 5.34 ± 0.14, respectively. The maximum response was significantly greater in artery segments from thyroxine-treated rats. Neither BW 723C86 (5-HT(2B)-receptor agonist) nor CP 93129 (5-HT(1B)-receptor agonist) contracted ring segments of the pulmonary artery from control and thyroxine-treated rats at concentrations up to 10(-4)M. There was no significant difference in the level of expression of 5-HT(2A)-receptor protein between the two groups. Ketanserin (3 × 10(-8)M) produced a rightward shift of the concentration-response curve to 5-HT in both groups with equal potency (-logK(B) values were 8.1 ± 0.2 and 7.9 ± 0.1 in control and thyroxine-treated rats, respectively). Nifedipine (10(-6)M) inhibited 5-HT-induced contractions in artery segments from control and thyroxine-treated rats and was more effective against 5-HT-induced contraction in artery segments for thyroxine-treated rats. The calcium-activated chloride channel blocker, niflumic acid (10(-4)M) also inhibited 5-HT-induced contractions in artery segments from control and thyroxine-treated rats and was more effective against 5-HT-induced contraction in artery segments for thyroxine-treated rats. It was concluded that hyperthyroidism enhanced 5-HT-induced contractions of the rat pulmonary artery by a mechanism involving increased activity of calcium-activated chloride channels. Copyright © 2011 Elsevier B.V. All rights reserved.

  18. Combined Bicarbonate Conductance-Impairing Variants in CFTR and SPINK1 Are Associated with Chronic Pancreatitis in Patients without Cystic Fibrosis

    Schneider, Alexander; LaRusch, Jessica; Sun, Xiumei; Aloe, Amy; Lamb, Janette; Hawes, Robert; Cotton, Peter; Brand, Randall E.; Anderson, Michelle A.; Money, Mary E.; Banks, Peter A.; Lewis, Michele D.; Baillie, John; Sherman, Stuart; DiSario, James; Burton, Frank R.; Gardner, Timothy B.; Amann, Stephen T.; Gelrud, Andres; George, Ryan; Kassabian, Sirvart; Martinson, Jeremy; Slivka, Adam; Yadav, Dhiraj; Oruc, Nevin; Barmada, M. Michael; Frizzell, Raymond; Whitcomb, David C.

    2010-01-01

    Background & Aims Idiopathic chronic pancreatitis (ICP) is a complex inflammatory disorder associated with multiple genetic and environmental factors. In individuals without cystic fibrosis (CF), variants of CFTR that inhibit bicarbonate conductance but maintain chloride conductance might selectively impair secretion of pancreatic juice, leading to trypsin activation and pancreatitis. We investigated whether sequence variants in the gene encoding the pancreatic secretory trypsin inhibitor, SPINK1, further increase the risk of pancreatitis in these patients. Methods We screened patients with ICP (sporadic or familial) and controls for variants in SPINK1 associated with chronic pancreatitis (CP) risk (in exon 3) and in all 27 exons of CFTR. The final study group included 53 patients with sporadic ICP, 27 probands with familial ICP, and 150 unrelated controls, plus 503 controls for limited genotyping. CFTR wild-type (wt) and p.R75Q were cloned and expressed in HEK293 cells and relative conductances of HCO3− and Cl− were measured. Results SPINK1 variants were identified in 36% of subjects and 3% controls (odds ratio [OR]=16.5). One variant of CFTR that has not been associated with CF, p.R75Q, was found in 16% of subjects and 5.4% controls (OR=3.4). Co-inheritance of CFTR p.R75Q and SPINK1 variants occurred in 8.75% of patients and 0.15% controls (OR=62.5). Patch-clamp recordings of cells that expressed CFTR p.R75Q demonstrated normal chloride currents but significantly reduced bicarbonate currents (P=0.0001). Conclusions The CFTR variant p.R75Q causes a selective defect in bicarbonate conductance and increases risk for pancreatitis. Co-inheritance of CF-associated, and some not associated, CFTR variants with SPINK1 variants significantly increase risk of ICP. PMID:20977904

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

    El Hiani, Yassine; Linsdell, Paul

    2015-06-19

    As an ion channel, the cystic fibrosis transmembrane conductance regulator must form a continuous pathway for the movement of Cl(-) and other anions between the cytoplasm and the extracellular solution. Both the structure and the function of the membrane-spanning part of this pathway are well defined. In contrast, the structure of the pathway that connects the cytoplasm to the membrane-spanning regions is unknown, and functional roles for different parts of the protein forming this pathway have not been described. We used patch clamp recording and substituted cysteine accessibility mutagenesis to identify positively charged amino acid side chains that attract cytoplasmic Cl(-) ions to the inner mouth of the pore. Our results indicate that the side chains of Lys-190, Arg-248, Arg-303, Lys-370, Lys-1041, and Arg-1048, located in different intracellular loops of the protein, play important roles in the electrostatic attraction of Cl(-) ions. Mutation and covalent modification of these residues have charge-dependent effects on the rate of Cl(-) permeation, demonstrating their functional role in maximization of Cl(-) flux. Other nearby positively charged side chains were not involved in electrostatic interactions with Cl(-). The location of these Cl(-)-attractive residues suggests that cytoplasmic Cl(-) ions enter the pore via a lateral portal located between the cytoplasmic extensions to the fourth and sixth transmembrane helices; a secondary, functionally less relevant portal might exist between the extensions to the 10th and 12th transmembrane helices. These results define the cytoplasmic mouth of the pore and show how it attracts Cl(-) ions from the cytoplasm. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Identification of Chloride Channels CLCN3 and CLCN5 Mediating the Excitatory Cl− Currents Activated by Sphingosine-1-Phosphate in Sensory Neurons

    Qi, Yanmei; Mair, Norbert; Kummer, Kai K.; Leitner, Michael G.; Camprubí-Robles, María; Langeslag, Michiel; Kress, Michaela

    2018-01-01

    Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid involved in numerous physiological and pathophysiological processes. We have previously reported a S1P-induced nocifensive response in mice by excitation of sensory neurons via activation of an excitatory chloride current. The underlying molecular mechanism for the S1P-induced chloride conductance remains elusive. In the present study, we identified two CLCN voltage-gated chloride channels, CLCN3 and CLCN5, which mediated a S1P-induced excitatory Cl− current in sensory neurons by combining RNA-seq, adenovirus-based gene silencing and whole-cell electrophysiological voltage-clamp recordings. Downregulation of CLCN3 and CLCN5 channels by adenovirus-mediated delivery of shRNA dramatically reduced S1P-induced Cl− current and membrane depolarization in sensory neurons. The mechanism of S1P-induced activation of the chloride current involved Rho GTPase but not Rho-associated protein kinase. Although S1P-induced potentiation of TRPV1-mediated ionic currents also involved Rho-dependent process, the lack of correlation of the S1P-activated Cl− current and the potentiation of TRPV1 by S1P suggests that CLCN3 and CLCN5 are necessary components for S1P-induced excitatory Cl− currents but not for the amplification of TRPV1-mediated currents in sensory neurons. This study provides a novel mechanistic insight into the importance of bioactive sphingolipids in nociception. PMID:29479306

  1. Identification of Chloride Channels CLCN3 and CLCN5 Mediating the Excitatory Cl− Currents Activated by Sphingosine-1-Phosphate in Sensory Neurons

    Yanmei Qi

    2018-02-01

    Full Text Available Sphingosine-1-phosphate (S1P is a bioactive sphingolipid involved in numerous physiological and pathophysiological processes. We have previously reported a S1P-induced nocifensive response in mice by excitation of sensory neurons via activation of an excitatory chloride current. The underlying molecular mechanism for the S1P-induced chloride conductance remains elusive. In the present study, we identified two CLCN voltage-gated chloride channels, CLCN3 and CLCN5, which mediated a S1P-induced excitatory Cl− current in sensory neurons by combining RNA-seq, adenovirus-based gene silencing and whole-cell electrophysiological voltage-clamp recordings. Downregulation of CLCN3 and CLCN5 channels by adenovirus-mediated delivery of shRNA dramatically reduced S1P-induced Cl− current and membrane depolarization in sensory neurons. The mechanism of S1P-induced activation of the chloride current involved Rho GTPase but not Rho-associated protein kinase. Although S1P-induced potentiation of TRPV1-mediated ionic currents also involved Rho-dependent process, the lack of correlation of the S1P-activated Cl− current and the potentiation of TRPV1 by S1P suggests that CLCN3 and CLCN5 are necessary components for S1P-induced excitatory Cl− currents but not for the amplification of TRPV1-mediated currents in sensory neurons. This study provides a novel mechanistic insight into the importance of bioactive sphingolipids in nociception.

  2. An L319F mutation in transmembrane region 3 (TM3) selectively reduces sensitivity to okaramine B of the Bombyx mori l-glutamate-gated chloride channel.

    Furutani, Shogo; Okuhara, Daiki; Hashimoto, Anju; Ihara, Makoto; Kai, Kenji; Hayashi, Hideo; Sattelle, David B; Matsuda, Kazuhiko

    2017-10-01

    Okaramines produced by Penicillium simplicissimum AK-40 activate l-glutamate-gated chloride channels (GluCls) and thus paralyze insects. However, the okaramine binding site on insect GluCls is poorly understood. Sequence alignment shows that the equivalent of residue Leucine319 of the okaramine B sensitive Bombyx mori (B. mori) GluCl is a phenylalanine in the okaramine B insensitive B. mori γ-aminobutyric acid-gated chloride channel of the same species. This residue is located in the third transmembrane (TM3) region, a location which in a nematode GluCl is close to the ivermectin binding site. The B. mori GluCl containing the L319F mutation retained its sensitivity to l-glutamate, but responses to ivermectin were reduced and those to okaramine B were completely blocked.

  3. Sources of Variation in Sweat Chloride Measurements in Cystic Fibrosis

    Blackman, Scott M.; Raraigh, Karen S.; Corvol, Harriet; Rommens, Johanna M.; Pace, Rhonda G.; Boelle, Pierre-Yves; McGready, John; Sosnay, Patrick R.; Strug, Lisa J.; Knowles, Michael R.; Cutting, Garry R.

    2016-01-01

    Rationale: Expanding the use of cystic fibrosis transmembrane conductance regulator (CFTR) potentiators and correctors for the treatment of cystic fibrosis (CF) requires precise and accurate biomarkers. Sweat chloride concentration provides an in vivo assessment of CFTR function, but it is unknown the degree to which CFTR mutations account for sweat chloride variation. Objectives: To estimate potential sources of variation for sweat chloride measurements, including demographic factors, testing variability, recording biases, and CFTR genotype itself. Methods: A total of 2,639 sweat chloride measurements were obtained in 1,761 twins/siblings from the CF Twin-Sibling Study, French CF Modifier Gene Study, and Canadian Consortium for Genetic Studies. Variance component estimation was performed by nested mixed modeling. Measurements and Main Results: Across the tested CF population as a whole, CFTR gene mutations were found to be the primary determinant of sweat chloride variability (56.1% of variation) with contributions from variation over time (e.g., factors related to testing on different days; 13.8%), environmental factors (e.g., climate, family diet; 13.5%), other residual factors (e.g., test variability; 9.9%), and unique individual factors (e.g., modifier genes, unique exposures; 6.8%) (likelihood ratio test, P < 0.001). Twin analysis suggested that modifier genes did not play a significant role because the heritability estimate was negligible (H2 = 0; 95% confidence interval, 0.0–0.35). For an individual with CF, variation in sweat chloride was primarily caused by variation over time (58.1%) with the remainder attributable to residual/random factors (41.9%). Conclusions: Variation in the CFTR gene is the predominant cause of sweat chloride variation; most of the non-CFTR variation is caused by testing variability and unique environmental factors. If test precision and accuracy can be improved, sweat chloride measurement could be a valuable biomarker

  4. Adrenomedullin increases the short-circuit current in the rat prostate: Receptors, chloride channels, the effects of cAMP and calcium ions and implications on fluid secretion.

    Liao, S B; Cheung, K H; Cheung, M P L; Wong, P F; O, W S; Tang, F

    2014-05-01

    In this study, we have investigated the effects of adrenomedullin on chloride and fluid secretion in the rat prostate. The presence of adrenomedullin (ADM) in rat prostate was confirmed using immunostaining, and the molecular species was determined using gel filtration chromatography coupled with an enzyme-linked assay for ADM. The effects of ADM on fluid secretion were studied by short-circuit current technique in a whole mount preparation of the prostate in an Ussing chamber. The results indicated that the ADM level was higher in the ventral than the dorso-lateral prostate and the major molecular species was the active peptide. ADM increased the short-circuit current through both the cAMP- and calcium-activated chloride channels in the ventral lobe, but only through the calcium-activated channels in the dorso-lateral lobe. These stimulatory effects were blocked by the calcitonin gene-related peptide (CGRP) receptor antagonist, hCGRP8-37. We conclude that ADM may regulate prostatic fluid secretion through the chloride channels, which may affect the composition of the seminal plasma bathing the spermatozoa and hence fertility. © 2014 American Society of Andrology and European Academy of Andrology.

  5. Role of individual histidines in the pH-dependent global stability of human chloride intracellular channel 1.

    Achilonu, Ikechukwu; Fanucchi, Sylvia; Cross, Megan; Fernandes, Manuel; Dirr, Heini W

    2012-02-07

    Chloride intracellular channel proteins exist in both a soluble cytosolic form and a membrane-bound form. The mechanism of conversion between the two forms is not properly understood, although one of the contributing factors is believed to be the variation in pH between the cytosol (~7.4) and the membrane (~5.5). We systematically mutated each of the three histidine residues in CLIC1 to an alanine at position 74 and a phenylalanine at positions 185 and 207. We examined the effect of the histidine-mediated pH dependence on the structure and global stability of CLIC1. None of the mutations were found to alter the global structure of the protein. However, the stability of H74A-CLIC1 and H185F-CLIC1, as calculated from the equilibrium unfolding data, is no longer dependent on pH because similar trends are observed at pH 7.0 and 5.5. The crystal structures show that the mutations result in changes in the local hydrogen bond coordination. Because the mutant total free energy change upon unfolding is not different from that of the wild type at pH 7.0, despite the presence of intermediates that are not seen in the wild type, we propose that it may be the stability of the intermediate state rather than the native state that is dependent on pH. On the basis of the lower stability of the intermediate in the H74A and H185F mutants compared to that of the wild type, we conclude that both His74 and His185 are involved in triggering the pH changes to the conformational stability of wild-type CLIC1 via their protonation, which stabilizes the intermediate state.

  6. Association of chloride intracellular channel 4 and Indian hedgehog proteins with survival of patients with pancreatic ductal adenocarcinoma.

    Zou, Qiong; Yang, Zhulin; Li, Daiqiang; Liu, Ziru; Yuan, Yuan

    2016-12-01

    Pancreatic cancer is the fourth most common cause of cancer-related mortality. Novel molecular biomarkers need to be identified for personalized medicine and to improve survival. The aim of this study was to examine chloride intracellular channel 4 (CLIC4) and Indian Hedgehog (Ihh) expression in benign and malignant lesions of the pancreas and to examine the eventual association between CLIC4 and Ihh expression, with clinicopathological features and prognosis of pancreatic cancer. A retrospective study of specimens collected from January 2000 to December 2011 at the Department of Pathology of the Second and Third Xiangya Hospitals, Central South University was undertaken to explore this question. Immunohistochemistry of CLIC4 and Ihh was performed with EnVision ™ in 106 pancreatic ductal adenocarcinoma specimens, 35 paracancer samples (2 cm away from the tumour, when possible or available), 55 benign lesions and 13 normal tissue samples. CLIC4 and Ihh expression in pancreatic ductal adenocarcinoma were significantly higher than in paracancer tissue and benign lesions (CLIC4: P = 0.009 and Ihh: P Ihh: P = 0.0001 respectively). CLIC4 and Ihh expression was negative in normal pancreatic tissues. The expression of CLIC4 and Ihh was associated significantly with tumour grade, lymph node metastasis, tumour invasion and poor overall survival. Thus CLIC4 and Ihh could serve as biological markers for the progression, metastasis and/or invasiveness of pancreatic ductal adenocarcinoma. © 2017 The Authors. International Journal of Experimental Pathology © 2017 International Journal of Experimental Pathology.

  7. Distribution of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR Mutations in a Cohort of Patients Residing in Palestine.

    Issa Siryani

    Full Text Available Cystic fibrosis (CF is an autosomal recessive inherited life-threatening disorder that causes severe damage to the lungs and the digestive system. In Palestine, mutations in the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR that contributes to the clinical presentation of CF are ill defined. A cohort of thirty three clinically diagnosed CF patients from twenty one different Palestinian families residing in the central and southern part of Palestine were incorporated in this study. Sweat chloride testing was performed using the Sweat Chek Conductivity Analyzer (ELITECH Group, France to confirm the clinical diagnosis of CF. In addition, nucleic acid from the patients' blood samples was extracted and the CFTR mutation profiles were assessed by direct sequencing of the CFTR 27 exons and the intron-exon boundaries. For patient's DNA samples where no homozygous or two heterozygous CFTR mutations were identified by exon sequencing, DNA samples were tested for deletions or duplications using SALSA MLPA probemix P091-D1 CFTR assay. Sweat chloride testing confirmed the clinical diagnosis of CF in those patients. All patients had NaCl conductivity >60 mmol/l. In addition, nine different CFTR mutations were identified in all 21 different families evaluated. These mutations were c.1393-1G>A, F508del, W1282X, G85E, c.313delA, N1303K, deletion exons 17a-17b-18, deletion exons 17a-17b and Q1100P. c.1393-1G>A was shown to be the most frequent occurring mutation among tested families. We have profiled the underling mutations in the CFTR gene of a cohort of 21 different families affected by CF. Unlike other studies from the Arab countries where F508del was reported to be the most common mutation, in southern/central Palestine, the c.1393-1G>A appeared to be the most common. Further studies are needed per sample size and geographic distribution to account for other possible CFTR genetic alterations and their frequencies. Genotype

  8. The novel isoxazoline ectoparasiticide lotilaner (Credelio™: a non-competitive antagonist specific to invertebrates γ-aminobutyric acid-gated chloride channels (GABACls

    Lucien Rufener

    2017-11-01

    Full Text Available Abstract Background The isoxazolines are a novel class of parasiticides that are potent inhibitors of γ-aminobutyric acid (GABA-gated chloride channels (GABACls and, to a lesser extent, of inhibitory glutamate-gated chloride channels (GluCls. Lotilaner (Credelio™, a novel representative of this chemical class, is currently evaluated for its excellent ectoparasiticide properties. Methods In this study, we investigated the molecular mode of action and pharmacology of lotilaner. We report the successful gene identification, cDNA cloning and functional expression in Xenopus oocytes of Drosohpila melanogaster (wild type and dieldrin/fipronil-resistant forms, Lepeophtheirus salmonis (an ectoparasite copepod crustacean of salmon, Rhipicephalus microplus and Canis lupus familiaris GABACls. Automated Xenopus oocyte two-electrode voltage clamp electrophysiology was used to assess GABACls functionality and to compare ion channel inhibition by lotilaner with that of established insecticides addressing GABACls as targets. Results In these assays, we demonstrated that lotilaner is a potent non-competitive antagonist of insects (fly GABACls. No cross-resistance with dieldrin or fipronil resistance mutations was detected, suggesting that lotilaner might bind to a site at least partly different from the one bound by known GABACl blockers. Using co-application experiments, we observed that lotilaner antagonism differs significantly from the classical open channel blocker fipronil. We finally confirmed for the first time that isoxazoline compounds are not only powerful antagonists of GABACls of acari (ticks but also of crustaceans (sea lice, while no activity on a dog GABAA receptor was observed up to a concentration of 10 μM. Conclusions Together, these results demonstrate that lotilaner is a non-competitive antagonist specific to invertebrate’s γ-aminobutyric acid-gated chloride channels (GABACls. They contribute to our understanding of the mode of

  9. The novel isoxazoline ectoparasiticide lotilaner (Credelio™): a non-competitive antagonist specific to invertebrates γ-aminobutyric acid-gated chloride channels (GABACls).

    Rufener, Lucien; Danelli, Vanessa; Bertrand, Daniel; Sager, Heinz

    2017-11-01

    The isoxazolines are a novel class of parasiticides that are potent inhibitors of γ-aminobutyric acid (GABA)-gated chloride channels (GABACls) and, to a lesser extent, of inhibitory glutamate-gated chloride channels (GluCls). Lotilaner (Credelio™), a novel representative of this chemical class, is currently evaluated for its excellent ectoparasiticide properties. In this study, we investigated the molecular mode of action and pharmacology of lotilaner. We report the successful gene identification, cDNA cloning and functional expression in Xenopus oocytes of Drosohpila melanogaster (wild type and dieldrin/fipronil-resistant forms), Lepeophtheirus salmonis (an ectoparasite copepod crustacean of salmon), Rhipicephalus microplus and Canis lupus familiaris GABACls. Automated Xenopus oocyte two-electrode voltage clamp electrophysiology was used to assess GABACls functionality and to compare ion channel inhibition by lotilaner with that of established insecticides addressing GABACls as targets. In these assays, we demonstrated that lotilaner is a potent non-competitive antagonist of insects (fly) GABACls. No cross-resistance with dieldrin or fipronil resistance mutations was detected, suggesting that lotilaner might bind to a site at least partly different from the one bound by known GABACl blockers. Using co-application experiments, we observed that lotilaner antagonism differs significantly from the classical open channel blocker fipronil. We finally confirmed for the first time that isoxazoline compounds are not only powerful antagonists of GABACls of acari (ticks) but also of crustaceans (sea lice), while no activity on a dog GABA A receptor was observed up to a concentration of 10 μM. Together, these results demonstrate that lotilaner is a non-competitive antagonist specific to invertebrate's γ-aminobutyric acid-gated chloride channels (GABACls). They contribute to our understanding of the mode of action of this new ectoparasiticide compound.

  10. Restoration of CFTR Activity in Ducts Rescues Acinar Cell Function and Reduces Inflammation in Pancreatic and Salivary Glands of Mice.

    Zeng, Mei; Szymczak, Mitchell; Ahuja, Malini; Zheng, Changyu; Yin, Hongen; Swaim, William; Chiorini, John A; Bridges, Robert J; Muallem, Shmuel

    2017-10-01

    Sjögren's syndrome and autoimmune pancreatitis are disorders with decreased function of salivary, lacrimal glands, and the exocrine pancreas. Nonobese diabetic/ShiLTJ mice and mice transduced with the cytokine BMP6 develop Sjögren's syndrome and chronic pancreatitis and MRL/Mp mice are models of autoimmune pancreatitis. Cystic fibrosis transmembrane conductance regulator (CFTR) is a ductal Cl -  channel essential for ductal fluid and HCO 3 - secretion. We used these models to ask the following questions: is CFTR expression altered in these diseases, does correction of CFTR correct gland function, and most notably, does correcting ductal function correct acinar function? We treated the mice models with the CFTR corrector C18 and the potentiator VX770. Glandular, ductal, and acinar cells damage, infiltration, immune cells and function were measured in vivo and in isolated duct/acini. In the disease models, CFTR expression is markedly reduced. The salivary glands and pancreas are inflamed with increased fibrosis and tissue damage. Treatment with VX770 and, in particular, C18 restored salivation, rescued CFTR expression and localization, and nearly eliminated the inflammation and tissue damage. Transgenic overexpression of CFTR exclusively in the duct had similar effects. Most notably, the markedly reduced acinar cell Ca 2+ signaling, Orai1, inositol triphosphate receptors, Aquaporin 5 expression, and fluid secretion were restored by rescuing ductal CFTR. Our findings reveal that correcting ductal function is sufficient to rescue acinar cell function and suggests that CFTR correctors are strong candidates for the treatment of Sjögren's syndrome and pancreatitis. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

  11. Molecular cloning, functional expression and subcellular localization of two putative vacuolar voltage-gated chloride channels in rice (Oryza sativa L.).

    Nakamura, Atsuko; Fukuda, Atsunori; Sakai, Shingo; Tanaka, Yoshiyuki

    2006-01-01

    We isolated two cDNA clones (OsCLC-1 and OsCLC-2) homologous to tobacco CLC-Nt1, which encodes a voltage-gated chloride channel, from rice (Oryza sativa L. ssp. japonica, cv. Nipponbare). The deduced amino acid sequences were highly conserved (87.9% identity with each other). Southern blot analysis of the rice genomic DNA revealed that OsCLC-1 and OsCLC-2 were single-copy genes on chromosomes 4 and 2, respectively. OsCLC-1 was expressed in most tissues, whereas OsCLC-2 was expressed only in the roots, nodes, internodes and leaf sheaths. The level of expression of OsCLC-1, but not of OsCLC-2, was increased by treatment with NaCl. Both genes could partly substitute for GEF1, which encodes the sole chloride channel in yeast, by restoring growth under ionic stress. These results indicate that both genes are chloride channel genes. The proteins from both genes were immunochemically detected in the tonoplast fraction. Tagged synthetic green fluorescent protein which was fused to OsCLC-1 or OsCLC-2 localized in the vacuolar membranes. These results indicate that the proteins may play a role in the transport of chloride ions across the vacuolar membrane. We isolated loss-of-function mutants of both genes from a panel of rice mutants produced by the insertion of a retrotransposon, Tos17, in the exon region, and found inhibition of growth at all life stages.

  12. S737F is a new CFTR mutation typical of patients originally from the Tuscany region in Italy.

    Terlizzi, Vito; Di Lullo, Antonella Miriam; Comegna, Marika; Centrone, Claudia; Pelo, Elisabetta; Castaldo, Giuseppe; Raia, Valeria; Braggion, Cesare

    2018-01-03

    An increasing number of patients have been described as having a number of Cystic Fibrosis Transmembrane conductance Regulator (CFTR) variants for which it lacks a clear genotype-phenotype correlation. We assesses the clinical features of patients bearing the S737F (p.Ser737Phe) CFTR missense variant and evaluated the residual function of CFTR protein on nasal epithelial cells (NEC). A retrospective database was performed from individuals homozygous or compound heterozygous for the S737F variant followed in the Cystic Fibrosis (CF) Centre of Florence. We performed a nasal brushing in cooperating patients and compared the results with those of patients followed in the pediatric CF Centre of Naples. 9/295 (3%) subjects carrying at least S737F CFTR variant on one allele were identified. Patients were diagnosed in 7/9 cases by newborn screening and in two cases for dehydration with hypochloremic metabolic alkalosis; at diagnosis sweat chloride levels (SCL) were in the pathological range in only one case. After a mean follow up of 8,6 years (range 0,5-15,8), SCL were in the pathological range in 8/9 cases (mean age at CF diagnosis: 1,5 years), all patients were pancreatic sufficiency and respiratory function was normal. The gating activity on NEC was 15.6% and 12.7% in two patients compound heterozygous for W1282X and DelE22_24, while it was ranged between 6,2% and 9,8% in CF patients. S737F is a CFTR mutation associated to hypochloremic alkalosis in childhood, mild CF phenotype in teenage years and a residual function of CFTR protein.

  13. CFTR-France, a national relational patient database for sharing genetic and phenotypic data associated with rare CFTR variants.

    Claustres, Mireille; Thèze, Corinne; des Georges, Marie; Baux, David; Girodon, Emmanuelle; Bienvenu, Thierry; Audrezet, Marie-Pierre; Dugueperoux, Ingrid; Férec, Claude; Lalau, Guy; Pagin, Adrien; Kitzis, Alain; Thoreau, Vincent; Gaston, Véronique; Bieth, Eric; Malinge, Marie-Claire; Reboul, Marie-Pierre; Fergelot, Patricia; Lemonnier, Lydie; Mekki, Chadia; Fanen, Pascale; Bergougnoux, Anne; Sasorith, Souphatta; Raynal, Caroline; Bareil, Corinne

    2017-10-01

    Most of the 2,000 variants identified in the CFTR (cystic fibrosis transmembrane regulator) gene are rare or private. Their interpretation is hampered by the lack of available data and resources, making patient care and genetic counseling challenging. We developed a patient-based database dedicated to the annotations of rare CFTR variants in the context of their cis- and trans-allelic combinations. Based on almost 30 years of experience of CFTR testing, CFTR-France (https://cftr.iurc.montp.inserm.fr/cftr) currently compiles 16,819 variant records from 4,615 individuals with cystic fibrosis (CF) or CFTR-RD (related disorders), fetuses with ultrasound bowel anomalies, newborns awaiting clinical diagnosis, and asymptomatic compound heterozygotes. For each of the 736 different variants reported in the database, patient characteristics and genetic information (other variations in cis or in trans) have been thoroughly checked by a dedicated curator. Combining updated clinical, epidemiological, in silico, or in vitro functional data helps to the interpretation of unclassified and the reassessment of misclassified variants. This comprehensive CFTR database is now an invaluable tool for diagnostic laboratories gathering information on rare variants, especially in the context of genetic counseling, prenatal and preimplantation genetic diagnosis. CFTR-France is thus highly complementary to the international database CFTR2 focused so far on the most common CF-causing alleles. © 2017 Wiley Periodicals, Inc.

  14. A Lys-Trp cation-π interaction mediates the dimerization and function of the chloride intracellular channel protein 1 transmembrane domain.

    Peter, Bradley; Polyansky, Anton A; Fanucchi, Sylvia; Dirr, Heini W

    2014-01-14

    Chloride intracellular channel protein 1 (CLIC1) is a dual-state protein that can exist either as a soluble monomer or in an integral membrane form. The oligomerization of the transmembrane domain (TMD) remains speculative despite it being implicated in pore formation. The extent to which electrostatic and van der Waals interactions drive folding and association of the dimorphic TMD is unknown and is complicated by the requirement of interactions favorable in both aqueous and membrane environments. Here we report a putative Lys37-Trp35 cation-π interaction and show that it stabilizes the dimeric form of the CLIC1 TMD in membranes. A synthetic 30-mer peptide comprising a K37M TMD mutant was examined in 2,2,2-trifluoroethanol, sodium dodecyl sulfate micelles, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes using far-ultraviolet (UV) circular dichroism, fluorescence, and UV absorbance spectroscopy. Our data suggest that Lys37 is not implicated in the folding, stability, or membrane insertion of the TMD peptide. However, removal of this residue impairs the formation of dimers and higher-order oligomers. This is accompanied by a 30-fold loss of chloride influx activity, suggesting that dimerization modulates the rate of chloride conductance. We propose that, within membranes, individual TMD helices associate via a Lys37-mediated cation-π interaction to form active dimers. The latter findings are also supported by results of modeling a putative TMD dimer conformation in which Lys37 and Trp35 form cation-π pairs at the dimer interface. Dimeric helix bundles may then associate to form fully active ion channels. Thus, within a membrane-like environment, aromatic interactions involving a polar lysine side chain provide a thermodynamic driving force for helix-helix association.

  15. Cystic fibrosis transmembrane conductance regulator (CFTR allelic variants relate to shifts in faecal microbiota of cystic fibrosis patients.

    Serena Schippa

    Full Text Available INTRODUCTION: In this study we investigated the effects of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR gene variants on the composition of faecal microbiota, in patients affected by Cystic Fibrosis (CF. CFTR mutations (F508del is the most common lead to a decreased secretion of chloride/water, and to mucus sticky secretions, in pancreas, respiratory and gastrointestinal tracts. Intestinal manifestations are underestimated in CF, leading to ileum meconium at birth, or small bowel bacterial overgrowth in adult age. METHODS: Thirty-six CF patients, fasting and under no-antibiotic treatment, were CFTR genotyped on both alleles. Faecal samples were subjected to molecular microbial profiling through Temporal Temperature Gradient Electrophoresis and species-specific PCR. Ecological parameters and multivariate algorithms were employed to find out if CFTR variants could be related to the microbiota structure. RESULTS: Patients were classified by two different criteria: 1 presence/absence of F508del mutation; 2 disease severity in heterozygous and homozygous F508del patients. We found that homozygous-F508del and severe CF patients exhibited an enhanced dysbiotic faecal microbiota composition, even within the CF cohort itself, with higher biodiversity and evenness. We also found, by species-specific PCR, that potentially harmful species (Escherichia coli and Eubacterium biforme were abundant in homozygous-F508del and severe CF patients, while beneficial species (Faecalibacterium prausnitzii, Bifidobacterium spp., and Eubacterium limosum were reduced. CONCLUSIONS: This is the first report that establishes a link among CFTR variants and shifts in faecal microbiota, opening the way to studies that perceive CF as a 'systemic disease', linking the lung and the gut in a joined axis.

  16. Cystic fibrosis transmembrane conductance regulator (CFTR) allelic variants relate to shifts in faecal microbiota of cystic fibrosis patients.

    Schippa, Serena; Iebba, Valerio; Santangelo, Floriana; Gagliardi, Antonella; De Biase, Riccardo Valerio; Stamato, Antonella; Bertasi, Serenella; Lucarelli, Marco; Conte, Maria Pia; Quattrucci, Serena

    2013-01-01

    In this study we investigated the effects of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene variants on the composition of faecal microbiota, in patients affected by Cystic Fibrosis (CF). CFTR mutations (F508del is the most common) lead to a decreased secretion of chloride/water, and to mucus sticky secretions, in pancreas, respiratory and gastrointestinal tracts. Intestinal manifestations are underestimated in CF, leading to ileum meconium at birth, or small bowel bacterial overgrowth in adult age. Thirty-six CF patients, fasting and under no-antibiotic treatment, were CFTR genotyped on both alleles. Faecal samples were subjected to molecular microbial profiling through Temporal Temperature Gradient Electrophoresis and species-specific PCR. Ecological parameters and multivariate algorithms were employed to find out if CFTR variants could be related to the microbiota structure. Patients were classified by two different criteria: 1) presence/absence of F508del mutation; 2) disease severity in heterozygous and homozygous F508del patients. We found that homozygous-F508del and severe CF patients exhibited an enhanced dysbiotic faecal microbiota composition, even within the CF cohort itself, with higher biodiversity and evenness. We also found, by species-specific PCR, that potentially harmful species (Escherichia coli and Eubacterium biforme) were abundant in homozygous-F508del and severe CF patients, while beneficial species (Faecalibacterium prausnitzii, Bifidobacterium spp., and Eubacterium limosum) were reduced. This is the first report that establishes a link among CFTR variants and shifts in faecal microbiota, opening the way to studies that perceive CF as a 'systemic disease', linking the lung and the gut in a joined axis.

  17. Differential expression of gill Na+,K+-ATPase alpha- and beta-subunits, Na+,K+,2Cl- cotransporter and CFTR anion channel in juvenile anadromous and landlocked Atlantic salmon Salmo salar

    Nilsen, Tom O.; Ebbesson, Lars O. E.; Madsen, Steffen S.

    2007-01-01

    This study examines changes in gill Na(+),K(+)-ATPase (NKA) alpha- and beta-subunit isoforms, Na(+),K(+),2Cl(-) cotransporter (NKCC) and cystic fibrosis transmembrane conductance regulator (CFTR I and II) in anadromous and landlocked strains of Atlantic salmon during parr-smolt transformation, an...

  18. Predominant constitutive CFTR conductance in small airways

    Lytle Christian

    2005-01-01

    Full Text Available Abstract Background The pathological hallmarks of chronic obstructive pulmonary disease (COPD are inflammation of the small airways (bronchiolitis and destruction of lung parenchyma (emphysema. These forms of disease arise from chronic prolonged infections, which are usually never present in the normal lung. Despite the fact that primary hygiene and defense of the airways presumably requires a well controlled fluid environment on the surface of the bronchiolar airway, very little is known of the fluid and electrolyte transport properties of airways of less than a few mm diameter. Methods We introduce a novel approach to examine some of these properties in a preparation of minimally traumatized porcine bronchioles of about 1 mm diameter by microperfusing the intact bronchiole. Results In bilateral isotonic NaCl Ringer solutions, the spontaneous transepithelial potential (TEP; lumen to bath of the bronchiole was small (mean ± sem: -3 ± 1 mV; n = 25, but when gluconate replaced luminal Cl-, the bionic Cl- diffusion potentials (-58 ± 3 mV; n = 25 were as large as -90 mV. TEP diffusion potentials from 2:1 NaCl dilution showed that epithelial Cl- permeability was at least 5 times greater than Na+ permeability. The anion selectivity sequence was similar to that of CFTR. The bionic TEP became more electronegative with stimulation by luminal forskolin (5 μM+IBMX (100 μM, ATP (100 μM, or adenosine (100 μM, but not by ionomycin. The TEP was partially inhibited by NPPB (100 μM, GlyH-101* (5–50 μM, and CFTRInh-172* (5 μM. RT-PCR gave identifying products for CFTR, α-, β-, and γ-ENaC and NKCC1. Antibodies to CFTR localized specifically to the epithelial cells lining the lumen of the small airways. Conclusion These results indicate that the small airway of the pig is characterized by a constitutively active Cl- conductance that is most likely due to CFTR.

  19. Gq activity- and β-arrestin-1 scaffolding-mediated ADGRG2/CFTR coupling are required for male fertility

    Lin, Hui; Li, Rui-Rui; Liang, Zong-Lai; Gao, Yuan; Yang, Zhao; He, Dong-Fang; Lin, Amy; Mo, Hui; Lu, Yu-Jing; Li, Meng-Jing; Kong, Wei; Chung, Ka Young; Yi, Fan; Li, Jian-Yuan; Qin, Ying-Ying; Li, Jingxin; Thomsen, Alex R B; Kahsai, Alem W; Chen, Zi-Jiang; Xu, Zhi-Gang; Liu, Mingyao

    2018-01-01

    Luminal fluid reabsorption plays a fundamental role in male fertility. We demonstrated that the ubiquitous GPCR signaling proteins Gq and β-arrestin-1 are essential for fluid reabsorption because they mediate coupling between an orphan receptor ADGRG2 (GPR64) and the ion channel CFTR. A reduction in protein level or deficiency of ADGRG2, Gq or β-arrestin-1 in a mouse model led to an imbalance in pH homeostasis in the efferent ductules due to decreased constitutive CFTR currents. Efferent ductule dysfunction was rescued by the specific activation of another GPCR, AGTR2. Further mechanistic analysis revealed that β-arrestin-1 acts as a scaffold for ADGRG2/CFTR complex formation in apical membranes, whereas specific residues of ADGRG2 confer coupling specificity for different G protein subtypes, this specificity is critical for male fertility. Therefore, manipulation of the signaling components of the ADGRG2-Gq/β-arrestin-1/CFTR complex by small molecules may be an effective therapeutic strategy for male infertility. PMID:29393851

  20. 5,5'-Dithio-bis(2-nitrobenzoic acid) modification of cysteine improves the crystal quality of human chloride intracellular channel protein 2

    Mi Wei; Li Lanfen; Su Xiaodong

    2008-01-01

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

  1. Mutations on M3 helix of Plutella xylostella glutamate-gated chloride channel confer unequal resistance to abamectin by two different mechanisms.

    Wang, Xingliang; Puinean, Alin M; O Reilly, Andrias O; Williamson, Martin S; Smelt, Charles L C; Millar, Neil S; Wu, Yidong

    2017-07-01

    Abamectin is one of the most widely used avermectins for agricultural pests control, but the emergence of resistance around the world is proving a major threat to its sustained application. Abamectin acts by directly activating glutamate-gated chloride channels (GluCls) and modulating other Cys-loop ion channels. To date, three mutations occurring in the transmembrane domain of arthropod GluCls are associated with target-site resistance to abamectin: A309V in Plutella xylostella GluCl (PxGluCl), G323D in Tetranychus urticae GluCl1 (TuGluCl1) and G326E in TuGluCl3. To compare the effects of these mutations in a single system, A309V/I/G and G315E (corresponding to G323 in TuGluCl1 and G326 in TuGluCl3) substitutions were introduced individually into the PxGluCl channel. Functional analysis using Xenopus oocytes showed that the A309V and G315E mutations reduced the sensitivity to abamectin by 4.8- and 493-fold, respectively. In contrast, the substitutions A309I/G show no significant effects on the response to abamectin. Interestingly, the A309I substitution increased the channel sensitivity to glutamate by one order of magnitude (∼12-fold). Analysis of PxGluCl homology models indicates that the G315E mutation interferes with abamectin binding through a steric hindrance mechanism. In contrast, the structural consequences of the A309 mutations are not so clear and an allosteric modification of the binding site is the most likely mechanism. Overall the results show that both A309V and G315E mutations may contribute to target-site resistance to abamectin and may be important for the future prediction and monitoring of abamectin resistance in P. xylostella and other arthropod pests. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Inhibition of Calcium-Activated Chloride Channel ANO1/TMEM16A Suppresses Tumor Growth and Invasion in Human Lung Cancer.

    Linghan Jia

    Full Text Available Lung cancer or pulmonary carcinoma is primarily derived from epithelial cells that are thin and line on the alveolar surfaces of the lung for gas exchange. ANO1/TMEM16A, initially identified from airway epithelial cells, is a member of Ca2+-activated Cl- channels (CaCCs that function to regulate epithelial secretion and cell volume for maintenance of ion and tissue homeostasis. ANO1/TMEM16A has recently been shown to be highly expressed in several epithelium originated carcinomas. However, the role of ANO1 in lung cancer remains unknown. In this study, we show that inhibition of calcium-activated chloride channel ANO1/TMEM16A suppresses tumor growth and invasion in human lung cancer. ANO1 is upregulated in different human lung cancer cell lines. Knocking-down ANO1 by small hairpin RNAs inhibited proliferation, migration and invasion of GLC82 and NCI-H520 cancel cells evaluated by CCK-8, would-healing, transwell and 3D soft agar assays. ANO1 protein is overexpressed in 77.3% cases of human lung adenocarcinoma tissues detected by immunohistochemistry. Furthermore, the tumor growth in nude mice implanted with GLC82 cells was significantly suppressed by ANO1 silencing. Taken together, our findings provide evidence that ANO1 overexpression contributes to tumor growth and invasion of lung cancer; and suppressing ANO1 overexpression may have therapeutic potential in lung cancer therapy.

  3. Identification and functional expression of a glutamate- and avermectin-gated chloride channel from Caligus rogercresseyi, a southern Hemisphere sea louse affecting farmed fish.

    Isabel Cornejo

    2014-09-01

    Full Text Available Parasitic sea lice represent a major sanitary threat to marine salmonid aquaculture, an industry accounting for 7% of world fish production. Caligus rogercresseyi is the principal sea louse species infesting farmed salmon and trout in the southern hemisphere. Most effective control of Caligus has been obtained with macrocyclic lactones (MLs ivermectin and emamectin. These drugs target glutamate-gated chloride channels (GluCl and act as irreversible non-competitive agonists causing neuronal inhibition, paralysis and death of the parasite. Here we report the cloning of a full-length CrGluClα receptor from Caligus rogercresseyi. Expression in Xenopus oocytes and electrophysiological assays show that CrGluClα is activated by glutamate and mediates chloride currents blocked by the ligand-gated anion channel inhibitor picrotoxin. Both ivermectin and emamectin activate CrGluClα in the absence of glutamate. The effects are irreversible and occur with an EC(50 value of around 200 nM, being cooperative (n(H = 2 for ivermectin but not for emamectin. Using the three-dimensional structure of a GluClα from Caenorabditis elegans, the only available for any eukaryotic ligand-gated anion channel, we have constructed a homology model for CrGluClα. Docking and molecular dynamics calculations reveal the way in which ivermectin and emamectin interact with CrGluClα. Both drugs intercalate between transmembrane domains M1 and M3 of neighbouring subunits of a pentameric structure. The structure displays three H-bonds involved in this interaction, but despite similarity in structure only of two these are conserved from the C. elegans crystal binding site. Our data strongly suggest that CrGluClα is an important target for avermectins used in the treatment of sea louse infestation in farmed salmonids and open the way for ascertaining a possible mechanism of increasing resistance to MLs in aquaculture industry. Molecular modeling could help in the design of new

  4. Salmon lice (Lepeophtheirus salmonis) showing varying emamectin benzoate susceptibilities differ in neuronal acetylcholine receptor and GABA-gated chloride channel mRNA expression.

    Carmichael, Stephen N; Bron, James E; Taggart, John B; Ireland, Jacqueline H; Bekaert, Michaël; Burgess, Stewart Tg; Skuce, Philip J; Nisbet, Alasdair J; Gharbi, Karim; Sturm, Armin

    2013-06-18

    Caligid copepods, also called sea lice, are fish ectoparasites, some species of which cause significant problems in the mariculture of salmon, where the annual cost of infection is in excess of €300 million globally. At present, caligid control on farms is mainly achieved using medicinal treatments. However, the continued use of a restricted number of medicine actives potentially favours the development of drug resistance. Here, we report transcriptional changes in a laboratory strain of the caligid Lepeophtheirus salmonis (Krøyer, 1837) that is moderately (~7-fold) resistant to the avermectin compound emamectin benzoate (EMB), a component of the anti-salmon louse agent SLICE® (Merck Animal Health). Suppression subtractive hybridisation (SSH) was used to enrich transcripts differentially expressed between EMB-resistant (PT) and drug-susceptible (S) laboratory strains of L. salmonis. SSH libraries were subjected to 454 sequencing. Further L. salmonis transcript sequences were available as expressed sequence tags (EST) from GenBank. Contiguous sequences were generated from both SSH and EST sequences and annotated. Transcriptional responses in PT and S salmon lice were investigated using custom 15 K oligonucleotide microarrays designed using the above sequence resources. In the absence of EMB exposure, 359 targets differed in transcript abundance between the two strains, these genes being enriched for functions such as calcium ion binding, chitin metabolism and muscle structure. γ-aminobutyric acid (GABA)-gated chloride channel (GABA-Cl) and neuronal acetylcholine receptor (nAChR) subunits showed significantly lower transcript levels in PT lice compared to S lice. Using RT-qPCR, the decrease in mRNA levels was estimated at ~1.4-fold for GABA-Cl and ~2.8-fold for nAChR. Salmon lice from the PT strain showed few transcriptional responses following acute exposure (1 or 3 h) to 200 μg L-1 of EMB, a drug concentration tolerated by PT lice, but toxic for S lice

  5. EBIO, an agent causing maintained epithelial chloride secretion by co-ordinate actions at both apical and basolateral membranes.

    MacVinish, L J; Keogh, J; Cuthbert, A W

    2001-01-01

    The effect of 1-ethyl-2-benzimidazolone (EBIO) on electrogenic chloride secretion in murine colonic and nasal epithelium was investigated by the short-circuit technique. In the colon, EBIO produces a sustained current increase in the presence of amiloride, which is sensitive to furosemide. In nasal epithelium EBIO causes only a small, transient current increase. Sustained increases in current were obtained in response to forskolin in both epithelia. To examine the mechanisms by which EBIO increases chloride secretion, the effects on intracellular mediators were measured in colonic crypts. There was no effect on [Ca(2+)]i but cAMP content was increased, more so in the presence of IBMX, indicating a direct effect on adenylate cyclase. In colonic epithelia in which the apical surface was permeabilized by nystatin, and the tissue subjected to an apical to basolateral K(+) gradient, EBIO caused a current increase that was entirely sensitive to charybdotoxin (ChTX). In similarly permeabilized colons Br-cAMP caused a current increase that was entirely sensitive to 293B. Thus EBIO increases chloride secretion in the colon by coordinated actions at both the apical and basolateral faces of the cells. These include direct and indirect actions on Ca(2+)-sensitive and cAMP-sensitive K(+) channels respectively, and indirect actions on the basolateral cotransporter and apical CFTR chloride channels via cAMP. In CF colonic epithelia EBIO did not evoke chloride secretion. It is not clear why the nasal epithelium responds poorly to EBIO whereas it gives a sustained response to the related compound chlorzoxazone.

  6. Similar expression patterns of bestrophin-4 and cGMP dependent Ca2+-activated chloride channel activity in the vasculature

    Bouzinova, Elena V.; Larsen, Per; Matchkov, Vladimir

    2008-01-01

    (abstract by Matchkov et. al) that siRNA mediated downregulation of bestrophin-4 is associated with the disappearance of a recently demonstrated2 cGMP-dependent Ca2+-activated Cl- current in vascular smooth muscle cells (SMCs). Here we study the distribution of bestrophin-4-and cGMP dependent Cl- channel...... expressed epitope) Western blot detected a ~65 kDa band in cell lysates from rat mesenteric small arteries and aorta, which was not seen in pulmonary arteries and when preincubated with the immunizing peptide. The distribution of bestrophin-4 mRNA and protein has a pattern similar to the cGMP-dependent Cl......- current in SMCs of different origins. Immunohistochemistry identified bestrophin-4 both in endothelial and SMCs of the vascular tree in the brain, heart, kidney and mesentery, but not in the lungs. We suggest that bestrophin-4 is important for the cGMP dependent, Ca2+ activated Cl- conductance in many...

  7. Inhibition of transmembrane member 16A calcium-activated chloride channels by natural flavonoids contributes to flavonoid anticancer effects.

    Zhang, Xuan; Li, Honglin; Zhang, Huiran; Liu, Yani; Huo, Lifang; Jia, Zhanfeng; Xue, Yucong; Sun, Xiaorun; Zhang, Wei

    2017-07-01

    Natural flavonoids are ubiquitous in dietary plants and vegetables and have been proposed to have antiviral, antioxidant, cardiovascular protective and anticancer effects. Transmembrane member 16A (TMEM16A)-encoded Ca 2+ -activated Cl - channels play a variety of physiological roles in many organs and tissues. Overexpression of TMEM16A is also believed to be associated with cancer progression. Therefore, inhibition of TMEM16A current may be a potential target for cancer therapy. In this study, we screened a broad spectrum of flavonoids for their inhibitory activities on TMEM16A currents. A whole-cell patch technique was used to record the currents. The BrdU assay and transwell technique were used to investigate cell proliferation and migration. At a concentration of 100 μM, 10 of 20 compounds caused significant (>50%) inhibition of TMEM16A currents. The four most potent compounds - luteolin, galangin, quercetin and fisetin - had IC 50 values ranging from 4.5 to 15 μM). To examine the physiological relevance of these findings, we also studied the effects of these flavonoids on endogenous TMEM16A currents in addition to cell proliferation and migration in LA795 cancer cells. Among the flavonoids tested, we detected a highly significant correlation between TMEM16A current inhibition and cell proliferation or reduction of migration. This study demonstrates that flavonoids inhibit TMEM16A currents and suggests that flavonoids could have anticancer effects via this mechanism. © 2017 The British Pharmacological Society.

  8. Is sweat chloride predictive of severity of cystic fibrosis lung disease assessed by chest computed tomography?

    Caudri, Daan; Zitter, David; Bronsveld, Inez; Tiddens, Harm

    BACKGROUND: Cystic Fibrosis (CF) lung disease is characterized by a marked heterogeneity. Sweat chloride-level is a functional marker of the CF Transmembrane Regulator (CFTR) protein and could be an important predictor of later disease severity. METHODS: In this retrospective analysis children from

  9. Identification of Potent Chloride Intracellular Channel Protein 1 Inhibitors from Traditional Chinese Medicine through Structure-Based Virtual Screening and Molecular Dynamics Analysis

    Wei Wang

    2017-01-01

    Full Text Available Chloride intracellular channel 1 (CLIC1 is involved in the development of most aggressive human tumors, including gastric, colon, lung, liver, and glioblastoma cancers. It has become an attractive new therapeutic target for several types of cancer. In this work, we aim to identify natural products as potent CLIC1 inhibitors from Traditional Chinese Medicine (TCM database using structure-based virtual screening and molecular dynamics (MD simulation. First, structure-based docking was employed to screen the refined TCM database and the top 500 TCM compounds were obtained and reranked by X-Score. Then, 30 potent hits were achieved from the top 500 TCM compounds using cluster and ligand-protein interaction analysis. Finally, MD simulation was employed to validate the stability of interactions between each hit and CLIC1 protein from docking simulation, and Molecular Mechanics/Generalized Born Surface Area (MM-GBSA analysis was used to refine the virtual hits. Six TCM compounds with top MM-GBSA scores and ideal-binding models were confirmed as the final hits. Our study provides information about the interaction between TCM compounds and CLIC1 protein, which may be helpful for further experimental investigations. In addition, the top 6 natural products structural scaffolds could serve as building blocks in designing drug-like molecules for CLIC1 inhibition.

  10. Substituted 2-Acylaminocycloalkylthiophene-3-carboxylic Acid Arylamides as Inhibitors of the Calcium-Activated Chloride Channel Transmembrane Protein 16A (TMEM16A).

    Truong, Eric C; Phuan, Puay W; Reggi, Amanda L; Ferrera, Loretta; Galietta, Luis J V; Levy, Sarah E; Moises, Alannah C; Cil, Onur; Diez-Cecilia, Elena; Lee, Sujin; Verkman, Alan S; Anderson, Marc O

    2017-06-08

    Transmembrane protein 16A (TMEM16A), also called anoctamin 1 (ANO1), is a calcium-activated chloride channel expressed widely mammalian cells, including epithelia, vascular smooth muscle tissue, electrically excitable cells, and some tumors. TMEM16A inhibitors have been proposed for treatment of disorders of epithelial fluid and mucus secretion, hypertension, asthma, and possibly cancer. Herein we report, by screening, the discovery of 2-acylaminocycloalkylthiophene-3-carboxylic acid arylamides (AACTs) as inhibitors of TMEM16A and analysis of 48 synthesized analogs (10ab-10bw) of the original AACT compound (10aa). Structure-activity studies indicated the importance of benzene substituted as 2- or 4-methyl, or 4-fluoro, and defined the significance of thiophene substituents and size of the cycloalkylthiophene core. The most potent compound (10bm), which contains an unusual bromodifluoroacetamide at the thiophene 2-position, had IC 50 of ∼30 nM, ∼3.6-fold more potent than the most potent previously reported TMEM16A inhibitor 4 (Ani9), and >10-fold improved metabolic stability. Direct and reversible inhibition of TMEM16A by 10bm was demonstrated by patch-clamp analysis. AACTs may be useful as pharmacological tools to study TMEM16A function and as potential drug development candidates.

  11. Chloride Test

    ... metabolic acidosis ) or when a person hyperventilates (causing respiratory alkalosis ). A decreased level of blood chloride (called hypochloremia) ... disease , emphysema or other chronic lung diseases (causing respiratory ... metabolic alkalosis). An increased level of urine chloride can indicate ...

  12. 21 CFR 866.5900 - Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation detection system.

    2010-04-01

    ... regulator (CFTR) gene mutation detection system. 866.5900 Section 866.5900 Food and Drugs FOOD AND DRUG...) gene mutation detection system. (a) Identification. The CFTR gene mutation detection system is a device... Guidance Document: CFTR Gene Mutation Detection System.” See § 866.1(e) for the availability of this...

  13. CFTR Modulators: Shedding Light on Precision Medicine for Cystic Fibrosis

    Lopes-Pacheco, Miquéias

    2016-01-01

    Cystic fibrosis (CF) is the most common life-threatening monogenic disease afflicting Caucasian people. It affects the respiratory, gastrointestinal, glandular and reproductive systems. The major cause of morbidity and mortality in CF is the respiratory disorder caused by a vicious cycle of obstruction of the airways, inflammation and infection that leads to epithelial damage, tissue remodeling and end-stage lung disease. Over the past decades, life expectancy of CF patients has increased due to early diagnosis and improved treatments; however, these patients still present limited quality of life. Many attempts have been made to rescue CF transmembrane conductance regulator (CFTR) expression, function and stability, thereby overcoming the molecular basis of CF. Gene and protein variances caused by CFTR mutants lead to different CF phenotypes, which then require different treatments to quell the patients’ debilitating symptoms. In order to seek better approaches to treat CF patients and maximize therapeutic effects, CFTR mutants have been stratified into six groups (although several of these mutations present pleiotropic defects). The research with CFTR modulators (read-through agents, correctors, potentiators, stabilizers and amplifiers) has achieved remarkable progress, and these drugs are translating into pharmaceuticals and personalized treatments for CF patients. This review summarizes the main molecular and clinical features of CF, emphasizes the latest clinical trials using CFTR modulators, sheds light on the molecular mechanisms underlying these new and emerging treatments, and discusses the major breakthroughs and challenges to treating all CF patients. PMID:27656143

  14. Functional modulation of cerebral gamma-aminobutyric acidA receptor/benzodiazepine receptor/chloride ion channel complex with ethyl beta-carboline-3-carboxylate: Presence of independent binding site for ethyl beta-carboline-3-carboxylate

    Taguchi, J.; Kuriyama, K. (Kyoto Prefectural Univ. of Medicine (Japan))

    1990-05-01

    Effect of ethyl beta-carboline-3-carboxylate (beta-CCE) on the function of gamma-aminobutyric acid (GABA)A receptor/benzodiazepine receptor/chloride ion channel complex was studied. Beta-CCE noncompetitively and competitively inhibited (3H)flunitrazepam binding to benzodiazepine receptor, but not (3H)muscimol binding to GABAA receptor as well as t-(3H)butylbicycloorthobenzoate (( 3H) TBOB) binding to chloride ion channel, in particulate fraction of the mouse brain. Ro15-1788 also inhibited competitively (3H) flunitrazepam binding. On the other hand, the binding of beta-(3H)CCE was inhibited noncompetitively and competitively by clonazepam and competitively by Ro15-1788. In agreement with these results, benzodiazepines-stimulated (3H)muscimol binding was antagonized by beta-CCE and Ro15-1788. Gel column chromatography for the solubilized fraction from cerebral particulate fraction by 0.2% sodium deoxycholate (DOC-Na) in the presence of 1 M KCl indicated that beta-(3H)CCE binding site was eluted in the same fraction (molecular weight, 250,000) as the binding sites for (3H)flunitrazepam, (3H)muscimol and (3H)TBOB. GABA-stimulated 36Cl- influx into membrane vesicles prepared from the bovine cerebral cortex was stimulated and attenuated by flunitrazepam and beta-CCE, respectively. These effects of flunitrazepam and beta-CCE on the GABA-stimulated 36Cl- influx were antagonized by Ro15-1788. The present results suggest that the binding site for beta-CCE, which resides on GABAA receptor/benzodiazepine receptor/chloride ion channel complex, may be different from that for benzodiazepine. Possible roles of beta-CCE binding site in the allosteric inhibitions on benzodiazepine binding site as well as on the functional coupling between chloride ion channel and GABAA receptor are also suggested.

  15. S100A4 and BMP-2 Co-Dependently Induce Vascular Smooth Muscle Cell Migration via pERK and Chloride Intracellular Channel 4 (CLIC4)

    Spiekerkoetter, Edda; Guignabert, Christophe; de Jesus Perez, Vinicio; Alastalo, Tero-Pekka; Powers, Janine M; Wang, Lingli; Lawrie, Allan; Ambartsumian, Noona; Schmidt, Ann-Marie; Berryman, Mark; Ashley, Richard H; Rabinovitch, Marlene

    2009-01-01

    Rationale S100A4/Mts1 is implicated in motility of human pulmonary artery smooth muscle cells (hPASMC), through an interaction with the receptor for advanced glycation end products (RAGE). Objective We hypothesized that S100A4/Mts1-mediated hPASMC motility might be enhanced by loss of function of bone morphogenetic protein (BMP) receptor (R) II, observed in pulmonary arterial hypertension (PAH). Methods and Results Both S100A4/Mts1 (500ng/ml) and BMP-2 (10ng/ml) induce migration of hPASMCS in a novel co-dependent manner, in that the response to either ligand is lost with anti-RAGE or BMPRII siRNA. Phosphorylation of ERK is induced by both ligands and is required for motility by inducing MMP2 activity, but phosphoERK1/2 is blocked by anti-RAGE and not by BMPRII siRNA. In contrast, BMPRII siRNA, but not anti-RAGE, reduces expression of intracellular chloride channel 4 (CLIC4), a scaffolding molecule necessary for motility in response to S100A4/Mts1 or BMP-2. Reduced CLIC4 expression does not interfere with S100A4/Mts1 internalization or its interaction with myosin heavy chain IIA (MHCIIA), but does alter alignment of MHCIIA and actin filaments creating the appearance of vacuoles. This abnormality is associated with reduced peripheral distribution and/or delayed activation of RhoA and Rac1, small GTPases required for retraction and extension of lamellipodiae in motile cells. Conclusions Our studies demonstrate how a single ligand (BMP-2 or S100A4/Mts1) can recruit multiple cell surface receptors to relay signals that coordinate events culminating in a functional response, i.e., cell motility. We speculate that this carefully controlled process limits signals from multiple ligands, but could be subverted in disease. PMID:19713532

  16. Structures of a minimal human CFTR first nucleotide-binding domain as a monomer, head-to-tail homodimer, and pathogenic mutant

    Atwell, Shane; Brouillette, Christie G.; Conners, Kris; Emtage, Spencer; Gheyi, Tarun; Guggino, William B.; Hendle, Jorg; Hunt, John F.; Lewis, Hal A.; Lu, Frances; Protasevich, Irina I.; Rodgers, Logan A.; Romero, Rich; Wasserman, Stephen R.; Weber, Patricia C.; Wetmore, Diana; Zhang, Feiyu F.; Zhao, Xun (Cystic); (UAB); (JHU); (Columbia); (Lilly)

    2010-04-26

    Upon removal of the regulatory insert (RI), the first nucleotide binding domain (NBD1) of human cystic fibrosis transmembrane conductance regulator (CFTR) can be heterologously expressed and purified in a form that remains stable without solubilizing mutations, stabilizing agents or the regulatory extension (RE). This protein, NBD1 387-646({Delta}405-436), crystallizes as a homodimer with a head-to-tail association equivalent to the active conformation observed for NBDs from symmetric ATP transporters. The 1.7-{angstrom} resolution X-ray structure shows how ATP occupies the signature LSGGQ half-site in CFTR NBD1. The {Delta}F508 version of this protein also crystallizes as a homodimer and differs from the wild-type structure only in the vicinity of the disease-causing F508 deletion. A slightly longer construct crystallizes as a monomer. Comparisons of the homodimer structure with this and previously published monomeric structures show that the main effect of ATP binding at the signature site is to order the residues immediately preceding the signature sequence, residues 542-547, in a conformation compatible with nucleotide binding. These residues likely interact with a transmembrane domain intracellular loop in the full-length CFTR channel. The experiments described here show that removing the RI from NBD1 converts it into a well-behaved protein amenable to biophysical studies yielding deeper insights into CFTR function.

  17. Chloride channels in toad skin

    Larsen, Erik Hviid; Rasmussen, B E

    1982-01-01

    A study of the voltage and time dependence of a transepithelial Cl- current in toad skin (Bufo bufo) by the voltage-clamp method leads to the conclusion that potential has a dual role for Cl- transport. One is to control the permeability of an apical membrane Cl-pathway, the other is to drive Cl-...

  18. Sphingosine-1-Phosphate Is a Novel Regulator of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR Activity.

    Firhan A Malik

    Full Text Available The cystic fibrosis transmembrane conductance regulator (CFTR attenuates sphingosine-1-phosphate (S1P signaling in resistance arteries and has emerged as a prominent regulator of myogenic vasoconstriction. This investigation demonstrates that S1P inhibits CFTR activity via adenosine monophosphate-activated kinase (AMPK, establishing a potential feedback link. In Baby Hamster Kidney (BHK cells expressing wild-type human CFTR, S1P (1μmol/L attenuates forskolin-stimulated, CFTR-dependent iodide efflux. S1P's inhibitory effect is rapid (within 30 seconds, transient and correlates with CFTR serine residue 737 (S737 phosphorylation. Both S1P receptor antagonism (4μmol/L VPC 23019 and AMPK inhibition (80μmol/L Compound C or AMPK siRNA attenuate S1P-stimluated (i AMPK phosphorylation, (ii CFTR S737 phosphorylation and (iii CFTR activity inhibition. In BHK cells expressing the ΔF508 CFTR mutant (CFTRΔF508, the most common mutation causing cystic fibrosis, both S1P receptor antagonism and AMPK inhibition enhance CFTR activity, without instigating discernable correction. In summary, we demonstrate that S1P/AMPK signaling transiently attenuates CFTR activity. Since our previous work positions CFTR as a negative S1P signaling regulator, this signaling link may positively reinforce S1P signals. This discovery has clinical ramifications for the treatment of disease states associated with enhanced S1P signaling and/or deficient CFTR activity (e.g. cystic fibrosis, heart failure. S1P receptor/AMPK inhibition could synergistically enhance the efficacy of therapeutic strategies aiming to correct aberrant CFTR trafficking.

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

    Wang, Guoshun; Nauseef, William M.

    2015-01-01

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

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

    Wang, Guoshun; Nauseef, William M

    2015-08-01

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

  1. Frequency of common CFTR gene mutations in Venezuelan patients with cystic fibrosis

    Sánchez, Karen; Arcia, Orlando; Matute, Xiorama; Mindiola, Luz; Chaustre, Ismenia; Takiff, Howard

    2014-01-01

    Mutations in the CFTR gene in Cystic Fibrosis (CF) patients have geographic differences and there is scant data on their prevalence in Venezuelan patients. This study determined the frequency of common CFTR gene mutations in these patients. We amplified and sequenced exons 7, 10, 11, 19, 20 and 21, which contain the most common CFTR mutations, from 105 Venezuelan patients in the National CF Program. Eleven different mutations were identified, four with frequencies greater than 1%: p.Phe508del...

  2. Adrenomedullin increased the short-circuit current in the pig oviduct through chloride channels via the CGRP receptor: mediation by cAMP and calcium ions but not by nitric oxide.

    Liao, S B; Cheung, K H; Cheung, M P L; To, Y T; O, W S; Tang, F

    2013-10-01

    The oviduct serves as a site for the fertilization of the ovum and the transport of the conceptus down to the uterus for implantation. In this study, we investigated the presence of adrenomedullin (ADM) and its receptor component proteins in the pig oviduct. The effect of ADM on oviductal secretion, the specific receptor, and the mechanisms involved were also investigated. The presence of ADM and its receptor component proteins in the pig oviduct were confirmed using immunostaining. Short-circuit current (I(sc)) technique was employed to study chloride ion secretion in the oviductal epithelium. ADM increased I(sc) through cAMP- and calcium-activated chloride channels, and this effect could be inhibited by the CGRP receptor antagonist, hCGRP8-37. In contrast, the nitric oxide synthase inhibitor, L-NG-nitroarginine methyl ester (L-NAME), could not block the effect of ADM on I(sc). In summary, ADM may increase oviductal fluid secretion via chloride secretion independent of the nitric oxide pathway for the transport of sperm and the conceptus.

  3. Assessment of Correlation between Sweat Chloride Levels and Clinical Features of Cystic Fibrosis Patients.

    Raina, Manzoor A; Khan, Mosin S; Malik, Showkat A; Raina, Ab Hameed; Makhdoomi, Mudassir J; Bhat, Javed I; Mudassar, Syed

    2016-12-01

    Cystic Fibrosis (CF) is an autosomal recessive disorder and the incidence of this disease is undermined in Northern India. The distinguishable salty character of the sweat belonging to individuals suffering from CF makes sweat chloride estimation essential for diagnosis of CF disease. The aim of this prospective study was to elucidate the relationship of sweat chloride levels with clinical features and pattern of CF. A total of 182 patients, with clinical features of CF were included in this study for quantitative measurement of sweat chloride. Sweat stimulation and collection involved pilocarpine iontophoresis based on the Gibson and Cooks methodology. The quantitative estimation of chloride was done by Schales and Schales method with some modifications. Cystic Fibrosis Trans Membrane Conductance Regulator (CFTR) mutation status was recorded in case of patients with borderline sweat chloride levels to correlate the results and for follow-up. Out of 182 patients having clinical features consistent with CF, borderline and elevated sweat chloride levels were present in 9 (5%) and 41 (22.5%) subjects respectively. Elevated sweat chloride levels were significantly associated with wheeze, Failure To Thrive (FTT), history of CF in Siblings, product of Consanguineous Marriage (CM), digital clubbing and steatorrhoea on univariate analysis. On multivariate analysis only wheeze, FTT and steatorrhoea were found to be significantly associated with elevated sweat chloride levels (p<0.05). Among the nine borderline cases six cases were positive for at least two CFTR mutations and rest of the three cases were not having any mutation in CFTR gene. The diagnosis is often delayed and the disease is advanced in most patients at the time of diagnosis. Sweat testing is a gold standard for diagnosis of CF patients as genetic mutation profile being heterozygous and unlikely to become diagnostic test.

  4. Osteoblast CFTR inactivation reduces differentiation and osteoprotegerin expression in a mouse model of cystic fibrosis-related bone disease.

    Michael S Stalvey

    Full Text Available Low bone mass and increased fracture risk are recognized complications of cystic fibrosis (CF. CF-related bone disease (CFBD is characterized by uncoupled bone turnover--impaired osteoblastic bone formation and enhanced osteoclastic bone resorption. Intestinal malabsorption, vitamin D deficiency and inflammatory cytokines contribute to CFBD. However, epidemiological investigations and animal models also support a direct causal link between inactivation of skeletal cystic fibrosis transmembrane regulator (CFTR, the gene that when mutated causes CF, and CFBD. The objective of this study was to examine the direct actions of CFTR on bone. Expression analyses revealed that CFTR mRNA and protein were expressed in murine osteoblasts, but not in osteoclasts. Functional studies were then performed to investigate the direct actions of CFTR on osteoblasts using a CFTR knockout (Cftr-/- mouse model. In the murine calvarial organ culture assay, Cftr-/- calvariae displayed significantly less bone formation and osteoblast numbers than calvariae harvested from wildtype (Cftr+/+ littermates. CFTR inactivation also reduced alkaline phosphatase expression in cultured murine calvarial osteoblasts. Although CFTR was not expressed in murine osteoclasts, significantly more osteoclasts formed in Cftr-/- compared to Cftr+/+ bone marrow cultures. Indirect regulation of osteoclastogenesis by the osteoblast through RANK/RANKL/OPG signaling was next examined. Although no difference in receptor activator of NF-κB ligand (Rankl mRNA was detected, significantly less osteoprotegerin (Opg was expressed in Cftr-/- compared to Cftr+/+ osteoblasts. Together, the Rankl:Opg ratio was significantly higher in Cftr-/- murine calvarial osteoblasts contributing to a higher osteoclastogenesis potential. The combined findings of reduced osteoblast differentiation and lower Opg expression suggested a possible defect in canonical Wnt signaling. In fact, Wnt3a and PTH-stimulated canonical Wnt

  5. CFTR mutation analysis and haplotype associations in CF patients☆

    Cordovado, S.K.; Hendrix, M.; Greene, C.N.; Mochal, S.; Earley, M.C.; Farrell, P.M.; Kharrazi, M.; Hannon, W.H.; Mueller, P.W.

    2011-01-01

    Most newborn screening (NBS) laboratories use second-tier molecular tests for cystic fibrosis (CF) using dried blood spots (DBS). The Centers for Disease Control and Prevention’s NBS Quality Assurance Program offers proficiency testing (PT) in DBS for CF transmembrane conductance regulator (CFTR) gene mutation detection. Extensive molecular characterization on 76 CF patients, family members or screen positive newborns was performed for quality assurance. The coding, regulatory regions and por...

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

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

    2016-09-01

    ClC-K2, a member of the ClC family of Cl(-) channels and transporters, forms the major basolateral Cl(-) conductance in distal nephron epithelial cells and therefore plays a central role in renal Cl(-) absorption. However, its regulation remains largely unknown because of the fact that recombinant ClC-K2 has not yet been studied at the single-channel level. In the present study, we investigate the effects of voltage, pH, Cl(-), and Ca(2+) on native ClC-K2 in the basolateral membrane of intercalated cells from the mouse connecting tubule. The ∼10-pS channel shows a steep voltage dependence such that channel activity increases with membrane depolarization. Intracellular pH (pHi) and extracellular pH (pHo) differentially modulate the voltage dependence curve: alkaline pHi flattens the curve by causing an increase in activity at negative voltages, whereas alkaline pHo shifts the curve toward negative voltages. In addition, pHi, pHo, and extracellular Ca(2+) strongly increase activity, mainly because of an increase in the number of active channels with a comparatively minor effect on channel open probability. Furthermore, voltage alters both the number of active channels and their open probability, whereas intracellular Cl(-) has little influence. We propose that changes in the number of active channels correspond to them entering or leaving an inactivated state, whereas modulation of open probability corresponds to common gating by these channels. We suggest that pH, through the combined effects of pHi and pHo on ClC-K2, might be a key regulator of NaCl absorption and Cl(-)/HCO3 (-) exchange in type B intercalated cells. © 2016 Pinelli et al.

  7. Cftr Modulates Wnt/β-Catenin Signaling and Stem Cell Proliferation in Murine Intestine

    Ashlee M. Strubberg

    2018-01-01

    Conclusions: CF intestine shows increased ISC proliferation and Wnt/β-catenin signaling. Loss of Cftr increases pHi in ISCs, which stabilizes the plasma membrane association of the Wnt transducer Dvl, likely facilitating Wnt/β-catenin signaling. Absence of Cftr-dependent suppression of ISC proliferation in the CF intestine may contribute to increased risk for intestinal tumors.

  8. Rescuing mutant CFTR: a multi-task approach to a better outcome in treating cystic fibrosis.

    Amaral, Margarida D; Farinha, Carlos M

    2013-01-01

    Correcting multiple defects of mutant CFTR with small molecule compounds has been the goal of an increasing number of recent Cystic Fibrosis (CF) drug discovery programmes. However, the mechanism of action (MoA) by which these molecules restore mutant CFTR is still poorly understood, in particular of CFTR correctors, i.e., compounds rescuing to the cells surface the most prevalent mutant in CF patients--F508del-CFTR. However, there is increasing evidence that to fully restore the multiple defects associated with F508del-CFTR, different small molecules with distinct corrective properties may be required. Towards this goal, a better insight into MoA of correctors is needed and several constraints should be addressed. The methodological approaches to achieve this include: 1) testing the combined effect of compounds with that of other (non-pharmacological) rescuing strategies (e.g., revertants or low temperature); 2) assessing effects in multiple cellular models (non-epithelial vs epithelial, non-human vs human, immortalized vs primary cultures, polarized vs non polarized, cells vs tissues); 3) assessing compound effects on isolated CFTR domains (e.g., compound binding by surface plasmon resonance, assessing effects on domain folding and aggregation); and finally 4) assessing compounds specificity in rescuing different CFTR mutants and other mutant proteins. These topics are reviewed and discussed here so as to provide a state-of-the art review on how to combine multiple ways of rescuing mutant CFTR to the ultimate benefit of CF patients.

  9. Evaluation of potential regulatory elements identified as DNase I hypersensitive sites in the CFTR gene

    Phylactides, M.; Rowntree, R.; Nuthall, H.

    2002-01-01

    hypersensitive sites (DHS) within the locus. We previously identified at least 12 clusters of DHS across the CFTR gene and here further evaluate DHS in introns 2,3,10,16,17a, 18, 20 and 21 to assess their functional importance in regulation of CFTR gene expression. Transient transfections of enhancer/reporter...

  10. Calcium Channels, Rho-Kinase, Protein Kinase-C, and Phospholipase-C Pathways Mediate Mercury Chloride-Induced Myometrial Contractions in Rats.

    Koli, Swati; Prakash, Atul; Choudhury, Soumen; Mandil, Rajesh; Garg, Satish K

    2018-05-21

    Adverse effects of mercury on female reproduction are reported; however, its effect on myogenic activity of uterus and mechanism thereof is obscure. Present study was undertaken to unravel the mechanistic pathways of mercuric chloride (HgCl 2 )-induced myometrial contraction in rats. Isometric tension in myometrial strips of rats following in vitro exposure to HgCl 2 was recorded using data acquisition system-based physiograph. HgCl 2 produced concentration-dependent (10 nM-100 μM) uterotonic effect which was significantly (p Graphical Abstract Graphical abstract depicting the mechanism of mercury-induced myometrial contraction in rats. M receptor: Muscarinic receptor; PIP2: phospho-inositol bisphosphate; PLC: phospholipase-C; DAG: diacyl glycerol; IP3: inositol triphosphate; IP3R: inositol triphosphate receptor; PKC; protein kinase-C; MLCP: myosin light chain phosphatise; MYPT: myosin phosphatase; SR: sarco-endoplasmic reticulum.

  11. Phosphatidylinositol 4,5-bisphosphate, cholesterol, and fatty acids modulate the calcium-activated chloride channel TMEM16A (ANO1).

    De Jesús-Pérez, José J; Cruz-Rangel, Silvia; Espino-Saldaña, Ángeles E; Martínez-Torres, Ataúlfo; Qu, Zhiqiang; Hartzell, H Criss; Corral-Fernandez, Nancy E; Pérez-Cornejo, Patricia; Arreola, Jorge

    2018-03-01

    The TMEM16A-mediated Ca 2+ -activated Cl - current drives several important physiological functions. Membrane lipids regulate ion channels and transporters but their influence on members of the TMEM16 family is poorly understood. Here we have studied the regulation of TMEM16A by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), cholesterol, and fatty acids using patch clamp, biochemistry and fluorescence microscopy. We found that depletion of membrane PI(4,5)P2 causes a decline in TMEM16A current that is independent of cytoskeleton, but is partially prevented by removing intracellular Ca 2+ . On the other hand, supplying PI(4,5)P2 to inside-out patches attenuated channel rundown and/or partially rescued activity after channel rundown. Also, depletion (with methyl-β-cyclodextrin M-βCD) or restoration (with M-βCD+cholesterol) of membrane cholesterol slows down the current decay observed after reduction of PI(4,5)P2. Neither depletion nor restoration of cholesterol change PI(4,5)P2 content. However, M-βCD alone transiently increases TMEM16A activity and dampens rundown whereas M-βCD+cholesterol increases channel rundown. Thus, PI(4,5)P2 is required for TMEM16A function while cholesterol directly and indirectly via a PI(4,5)P2-independent mechanism regulate channel function. Stearic, arachidonic, oleic, docosahexaenoic, and eicosapentaenoic fatty acids as well as methyl stearate inhibit TMEM16A in a dose- and voltage-dependent manner. Phosphatidylserine, a phospholipid whose hydrocarbon tails contain stearic and oleic acids also inhibits TMEM16A. Finally, we show that TMEM16A remains in the plasma membrane after treatment with M-βCD, M-βCD+cholesterol, oleic, or docosahexaenoic acids. Thus, we propose that lipids and fatty acids regulate TMEM16A channels through a membrane-delimited protein-lipid interaction. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. CFTR Genotype and Maximal Exercise Capacity in Cystic Fibrosis: A Cross-sectional Study.

    Radtke, Thomas; Hebestreit, Helge; Gallati, Sabina; Schneiderman, Jane E; Braun, Julia; Stevens, Daniel; Hulzebos, Erik Hj; Takken, Tim; Boas, Steven R; Urquhart, Don S; Lands, Larry C; Tejero, Sergio; Sovtic, Aleksandar; Dwyer, Tiffany; Petrovic, Milos; Harris, Ryan A; Karila, Chantal; Savi, Daniela; Usemann, Jakob; Mei-Zahav, Meir; Hatziagorou, Elpis; Ratjen, Felix; Kriemler, Susi

    2018-02-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in human skeletal muscle cells. Variations of CFTR dysfunction among patients with cystic fibrosis may be an important determinant of maximal exercise capacity in cystic fibrosis. Previous studies on the relationship between CFTR genotype and maximal exercise capacity are scarce and contradictory. This study was designed to explore factors influencing maximal exercise capacity, expressed as peak oxygen uptake (V.O2peak), with a specific focus on CFTR genotype in children and adults with cystic fibrosis. In an international, multicenter, cross-sectional study, we collected data on CFTR genotype and cardiopulmonary exercise tests in patients with cystic fibrosis who were ages 8 years and older. CFTR mutations were classified into functional classes I–V. The final analysis included 726 patients (45% females; age range, 8–61 yr; forced expiratory volume in 1 s, 16 to 123% predicted) from 17 cystic fibrosis centers in North America, Europe, Australia, and Asia, all of whom had both valid maximal cardiopulmonary exercise tests and complete CFTR genotype data. Overall, patients exhibited exercise intolerance (V.O2peak, 77.3 ± 19.1% predicted), but values were comparable among different CFTR classes. We did not detect an association between CFTR genotype functional classes I–III and either V.O2peak (percent predicted) (adjusted β = −0.95; 95% CI, −4.18 to 2.29; P = 0.57) or maximum work rate (Wattmax) (adjusted β = −1.38; 95% CI, −5.04 to 2.27; P = 0.46) compared with classes IV–V. Those with at least one copy of a F508del-CFTR mutation and one copy of a class V mutation had a significantly lower V.O2peak (β = −8.24%; 95% CI, −14.53 to −2.99; P = 0.003) and lower Wattmax (adjusted β = −7.59%; 95% CI, −14.21 to −0.95; P = 0.025) than those with two copies of a class II mutation. On the basis of linear regression analysis adjusted for

  13. Polyvinyl chloride resin

    Kim, Hong Jae

    1976-06-01

    This book contains polyvinyl chloride resin industry with present condition such as plastic industry and polyvinyl chloride in the world and Japan, manufacture of polyvinyl chloride resin ; suspension polymerization and solution polymerization, extruding, injection process, hollow molding vinyl record, vacuum forming, polymer powders process, vinyl chloride varnish, vinyl chloride latex, safety and construction on vinyl chloride. Each chapter has descriptions on of process and kinds of polyvinyl chloride resin.

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

    Alzamora, Rodrigo

    2011-01-01

    Berberine is a plant alkaloid with multiple pharmacological actions, including antidiarrhoeal activity and has been shown to inhibit Cl(-) secretion in distal colon. The aims of this study were to determine the molecular signaling mechanisms of action of berberine on Cl(-) secretion and the ion transporter targets. Monolayers of T84 human colonic carcinoma cells grown in permeable supports were placed in Ussing chambers and short-circuit current measured in response to secretagogues and berberine. Whole-cell current recordings were performed in T84 cells using the patch-clamp technique. Berberine decreased forskolin-induced short-circuit current in a concentration-dependent manner (IC(50) 80 ± 8 μM). In apically permeabilized monolayers and whole-cell current recordings, berberine inhibited a cAMP-dependent and chromanol 293B-sensitive basolateral membrane K(+) current by 88%, suggesting inhibition of KCNQ1 K(+) channels. Berberine did not affect either apical Cl(-) conductance or basolateral Na(+)-K(+)-ATPase activity. Berberine stimulated p38 MAPK, PKCα and PKA, but had no effect on p42\\/p44 MAPK and PKCδ. However, berberine pre-treatment prevented stimulation of p42\\/p44 MAPK by epidermal growth factor. The inhibitory effect of berberine on Cl(-) secretion was partially blocked by HBDDE (∼65%), an inhibitor of PKCα and to a smaller extent by inhibition of p38 MAPK with SB202190 (∼15%). Berberine treatment induced an increase in association between PKCα and PKA with KCNQ1 and produced phosphorylation of the channel. We conclude that berberine exerts its inhibitory effect on colonic Cl(-) secretion through inhibition of basolateral KCNQ1 channels responsible for K(+) recycling via a PKCα-dependent pathway.

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

    Alzamora, Rodrigo

    2012-02-01

    Berberine is a plant alkaloid with multiple pharmacological actions, including antidiarrhoeal activity and has been shown to inhibit Cl(-) secretion in distal colon. The aims of this study were to determine the molecular signaling mechanisms of action of berberine on Cl(-) secretion and the ion transporter targets. Monolayers of T84 human colonic carcinoma cells grown in permeable supports were placed in Ussing chambers and short-circuit current measured in response to secretagogues and berberine. Whole-cell current recordings were performed in T84 cells using the patch-clamp technique. Berberine decreased forskolin-induced short-circuit current in a concentration-dependent manner (IC(50) 80 +\\/- 8 muM). In apically permeabilized monolayers and whole-cell current recordings, berberine inhibited a cAMP-dependent and chromanol 293B-sensitive basolateral membrane K(+) current by 88%, suggesting inhibition of KCNQ1 K(+) channels. Berberine did not affect either apical Cl(-) conductance or basolateral Na(+)-K(+)-ATPase activity. Berberine stimulated p38 MAPK, PKCalpha and PKA, but had no effect on p42\\/p44 MAPK and PKCdelta. However, berberine pre-treatment prevented stimulation of p42\\/p44 MAPK by epidermal growth factor. The inhibitory effect of berberine on Cl(-) secretion was partially blocked by HBDDE ( approximately 65%), an inhibitor of PKCalpha and to a smaller extent by inhibition of p38 MAPK with SB202190 ( approximately 15%). Berberine treatment induced an increase in association between PKCalpha and PKA with KCNQ1 and produced phosphorylation of the channel. We conclude that berberine exerts its inhibitory effect on colonic Cl(-) secretion through inhibition of basolateral KCNQ1 channels responsible for K(+) recycling via a PKCalpha-dependent pathway.

  16. Potassium and ANO1/TMEM16A chloride channel profiles distinguish atypical and typical smooth muscle cells from interstitial cells in the mouse renal pelvis

    Iqbal, Javed; Tonta, Mary A; Mitsui, Retsu; Li, Qun; Kett, Michelle; Li, Jinhua; Parkington, Helena C; Hashitani, Hikaru; Lang, Richard J

    2012-01-01

    BACKGROUND AND PURPOSE Although atypical smooth muscle cells (SMCs) in the proximal renal pelvis are thought to generate the pacemaker signals that drive pyeloureteric peristalsis, their location and electrical properties remain obscure. EXPERIMENTAL APPROACH Standard patch clamp, intracellular microelectrode and immunohistochemistry techniques were used. To unequivocally identify SMCs, transgenic mice with enhanced yellow fluorescent protein (eYFP) expressed in cells containing α-smooth muscle actin (α-SMA) were sometimes used. KEY RESULTS Atypical SMCs were distinguished from typical SMCs by the absence of both a transient 4-aminopyridine-sensitive K+ current (IKA) and spontaneous transient outward currents (STOCs) upon the opening of large-conductance Ca2+-activated K+ (BK) channels. Many typical SMCs displayed a slowly activating, slowly decaying Cl- current blocked by niflumic acid (NFA). Immunostaining for KV4.3 and ANO1/ TMEM16A Cl- channel subunits co-localized with α-SMA immunoreactive product predominately in the distal renal pelvis. Atypical SMCs fired spontaneous inward currents that were either selective for Cl- and blocked by NFA, or cation-selective and blocked by La3+. α-SMA- interstitial cells (ICs) were distinguished by the presence of a Xe991-sensitive KV7 current, BK channel STOCs and Cl- selective, NFA-sensitive spontaneous transient inward currents (STICs). Intense ANO1/ TMEM16A and KV7.5 immunostaining was present in Kit-α-SMA- ICs in the suburothelial and adventitial regions of the renal pelvis. CONCLUSIONS AND IMPLICATIONS We conclude that KV4.3+α-SMA+ SMCs are typical SMCs that facilitate muscle wall contraction, that ANO1/ TMEM16A and KV7.5 immunoreactivity may be selective markers of Kit- ICs and that atypical SMCs which discharge spontaneous inward currents are the pelviureteric pacemakers. PMID:22014103

  17. A survey of detergents for the purification of stable, active human cystic fibrosis transmembrane conductance regulator (CFTR).

    Hildebrandt, Ellen; Zhang, Qinghai; Cant, Natasha; Ding, Haitao; Dai, Qun; Peng, Lingling; Fu, Yu; DeLucas, Lawrence J; Ford, Robert; Kappes, John C; Urbatsch, Ina L

    2014-11-01

    Structural knowledge of the cystic fibrosis transmembrane conductance regulator (CFTR) requires developing methods to purify and stabilize this aggregation-prone membrane protein above 1mg/ml. Starting with green fluorescent protein- and epitope-tagged human CFTR produced in mammalian cells known to properly fold and process CFTR, we devised a rapid tandem affinity purification scheme to minimize CFTR exposure to detergent in order to preserve its ATPase function. We compared a panel of detergents, including widely used detergents (maltosides, neopentyl glycols (MNG), C12E8, lysolipids, Chaps) and innovative detergents (branched alkylmaltosides, facial amphiphiles) for CFTR purification, function, monodispersity and stability. ATPase activity after reconstitution into proteoliposomes was 2-3 times higher when CFTR was purified using facial amphiphiles. ATPase activity was also demonstrated in purified CFTR samples without detergent removal using a novel lipid supplementation assay. By electron microscopy, negatively stained CFTR samples were monodisperse at low concentration, and size exclusion chromatography showed a predominance of monomer even after CFTR concentration above 1mg/ml. Rates of CFTR aggregation quantified in an electrophoretic mobility shift assay showed that detergents which best preserved reconstituted ATPase activity also supported the greatest stability, with CFTR monomer half-lives of 6-9days in MNG or Chaps, and 12-17days in facial amphiphile. Cryoelectron microscopy of concentrated CFTR in MNG or facial amphiphile confirmed mostly monomeric protein, producing low resolution reconstructions in conformity with similar proteins. These protocols can be used to generate samples of pure, functional, stable CFTR at concentrations amenable to biophysical characterization. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Side chain and backbone contributions of Phe508 to CFTR folding

    Thibodeau, Patrick H.; Brautigam, Chad A.; Machius, Mischa; Thomas, Philip J. (U. of Texas-SMED)

    2010-12-07

    Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an integral membrane protein, cause cystic fibrosis (CF). The most common CF-causing mutant, deletion of Phe508, fails to properly fold. To elucidate the role Phe508 plays in the folding of CFTR, missense mutations at this position were generated. Only one missense mutation had a pronounced effect on the stability and folding of the isolated domain in vitro. In contrast, many substitutions, including those of charged and bulky residues, disrupted folding of full-length CFTR in cells. Structures of two mutant nucleotide-binding domains (NBDs) reveal only local alterations of the surface near position 508. These results suggest that the peptide backbone plays a role in the proper folding of the domain, whereas the side chain plays a role in defining a surface of NBD1 that potentially interacts with other domains during the maturation of intact CFTR.

  19. AMP-activated protein kinase and adenosine are both metabolic modulators that regulate chloride secretion in the shark rectal gland ( Squalus acanthias).

    Neuman, Rugina I; van Kalmthout, Juliette A M; Pfau, Daniel J; Menendez, Dhariyat M; Young, Lawrence H; Forrest, John N

    2018-04-01

    The production of endogenous adenosine during secretagogue stimulation of CFTR leads to feedback inhibition limiting further chloride secretion in the rectal gland of the dogfish shark (Squalus acanthias). In the present study, we examined the role of AMP-kinase (AMPK) as an energy sensor also modulating chloride secretion through CFTR. We found that glands perfused with forskolin and isobutylmethylxanthine (F + I), potent stimulators of chloride secretion in this ancient model, caused significant phosphorylation of the catalytic subunit Thr 172 of AMPK. These findings indicate that AMPK is activated during energy-requiring stimulated chloride secretion. In molecular studies, we confirmed that the activating Thr 172 site is indeed present in the α-catalytic subunit of AMPK in this ancient gland, which reveals striking homology to AMPKα subunits sequenced in other vertebrates. When perfused rectal glands stimulated with F + I were subjected to severe hypoxic stress or perfused with pharmacologic inhibitors of metabolism (FCCP or oligomycin), phosphorylation of AMPK Thr 172 was further increased and chloride secretion was dramatically diminished. The pharmacologic activation of AMPK with AICAR-inhibited chloride secretion, as measured by short-circuit current, when applied to the apical side of shark rectal gland monolayers in primary culture. These results indicate that that activated AMPK, similar to adenosine, transmits an inhibitory signal from metabolism, that limits chloride secretion in the shark rectal gland.

  20. A European regulatory perspective on cystic fibrosis: current treatments, trends in drug development and translational challenges for CFTR modulators.

    Ponzano, Stefano; Nigrelli, Giulia; Fregonese, Laura; Eichler, Irmgard; Bertozzi, Fabio; Bandiera, Tiziano; Galietta, Luis J V; Papaluca, Marisa

    2018-06-30

    In this article we analyse the current authorised treatments and trends in early drug development for cystic fibrosis (CF) in the European Union for the time period 2000-2016. The analysis indicates a significant improvement in the innovation and development of new potential medicines for CF, shifting from products that act on the symptoms of the disease towards new therapies targeting the cause of CF. However, within these new innovative medicines, results for CF transmembrane conductance regulator (CFTR) modulators indicate that one major challenge for turning a CF concept product into an actual medicine for the benefit of patients resides in the fact that, although pre-clinical models have shown good predictability for certain mutations, a good correlation to clinical end-points or biomarkers ( e.g. forced expiratory volume in 1 s and sweat chloride) for all mutations has not yet been achieved. In this respect, the use of alternative end-points and innovative nonclinical models could be helpful for the understanding of those translational discrepancies. Collaborative endeavours to promote further research and development in these areas as well as early dialogue with the regulatory bodies available at the European competent authorities are recommended. Copyright ©ERS 2018.

  1. Genotype-phenotype correlation and functional studies in patients with cystic fibrosis bearing CFTR complex alleles.

    Terlizzi, Vito; Castaldo, Giuseppe; Salvatore, Donatello; Lucarelli, Marco; Raia, Valeria; Angioni, Adriano; Carnovale, Vincenzo; Cirilli, Natalia; Casciaro, Rosaria; Colombo, Carla; Di Lullo, Antonella Miriam; Elce, Ausilia; Iacotucci, Paola; Comegna, Marika; Scorza, Manuela; Lucidi, Vincenzina; Perfetti, Anna; Cimino, Roberta; Quattrucci, Serena; Seia, Manuela; Sofia, Valentina Maria; Zarrilli, Federica; Amato, Felice

    2017-04-01

    The effect of complex alleles in cystic fibrosis (CF) is poorly defined for the lack of functional studies. To describe the genotype-phenotype correlation and the results of either in vitro and ex vivo studies performed on nasal epithelial cells (NEC) in a cohort of patients with CF carrying cystic fibrosis transmembrane conductance regulator ( CFTR ) complex alleles. We studied 70 homozygous, compound heterozygous or heterozygous for CFTR mutations: p.[Arg74Trp;Val201Met;Asp1270Asn], n=8; p.[Ile148Thr;Ile1023_Val1024del], n=5; p.[Arg117Leu;Leu997Phe], n=6; c.[1210-34TG[12];1210-12T[5];2930C>T], n=3; p.[Arg74Trp;Asp1270Asn], n=4; p.Asp1270Asn, n=2; p.Ile148Thr, n=6; p.Leu997Phe, n=36. In 39 patients, we analysed the CFTR gating activity on NEC in comparison with patients with CF (n=8) and carriers (n=4). Finally, we analysed in vitro the p.[Arg74Trp;Val201Met;Asp1270Asn] complex allele. The p.[Ile148Thr;Ile1023_Val1024del] caused severe CF in five compound heterozygous with a class I-II mutation. Their CFTR activity on NEC was comparable with patients with two class I-II mutations (mean 7.3% vs 6.9%). The p.[Arg74Trp;Asp1270Asn] and the p.Asp1270Asn have scarce functional effects, while p.[Arg74Trp;Val201Met;Asp1270Asn] caused mild CF in four of five subjects carrying a class I-II mutation in trans , or CFTR-related disorders (CFTR-RD) in three having in trans a class IV-V mutation. The p.[Arg74Trp;Val201Met;Asp1270Asn] causes significantly (pT] and a class I-II mutation had mild CF or CFTR-RD (gating activity: 18.5-19.0%). The effect of complex alleles partially depends on the mutation in trans . Although larger studies are necessary, the CFTR activity on NEC is a rapid contributory tool to classify patients with CFTR dysfunction. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  2. Altered intestinal bile salt biotransformation in a cystic fibrosis (Cftr-/-) mouse model with hepato-biliary pathology.

    Bodewes, Frank A J A; van der Wulp, Mariëtte Y M; Beharry, Satti; Doktorova, Marcela; Havinga, Rick; Boverhof, Renze; James Phillips, M; Durie, Peter R; Verkade, Henkjan J

    2015-07-01

    Cftr(-/-tm1Unc) mice develop progressive hepato-biliary pathology. We hypothesize that this liver pathology is related to alterations in biliary bile hydrophobicity and bile salt metabolism in Cftr(-/-tm1Unc) mice. We determined bile production, biliary and fecal bile salt- and lipid compositions and fecal bacterial composition of C57BL/6J Cftr(-/-tm1Unc) and control mice. We found no differences between the total biliary bile salt or lipid concentrations of Cftr(-/-) and controls. Compared to controls, Cftr(-/-) mice had a ~30% higher bile production and a low bile hydrophobicity, related to a ~7 fold higher concentration of the choleretic and hydrophilic bile salt ursocholate. These findings coexisted with a significantly smaller quantity of fecal Bacteroides bacteria. Liver pathology in Cftr(-/-tm1Unc) is not related to increased bile hydrophobicity. Cftr(-/-) mice do however display a biliary phenotype characterized by increased bile production and decreased biliary hydrophobicity. Our findings suggest Cftr dependent, alterations in intestinal bacterial biotransformation of bile salts. Copyright © 2014. Published by Elsevier B.V.

  3. First functional polymorphism in CFTR promoter that results in decreased transcriptional activity and Sp1/USF binding

    Taulan, M.; Lopez, E.; Guittard, C.; Rene, C.; Baux, D.; Altieri, J.P.; DesGeorges, M.; Claustres, M.; Romey, M.C.

    2007-01-01

    Growing evidences show that functionally relevant polymorphisms in various promoters alter both transcriptional activity and affinities of existing protein-DNA interactions, and thus influence disease progression in humans. We previously reported the -94G>T CFTR promoter variant in a female CF patient in whom any known disease-causing mutation has been detected. To investigate whether the -94G>T could be a regulatory variant, we have proceeded to in silico analyses and functional studies including EMSA and reporter gene assays. Our data indicate that the promoter variant decreases basal CFTR transcriptional activity in different epithelial cells and alters binding affinities of both Sp1 and USF nuclear proteins to the CFTR promoter. The present report provides evidence for the first functional polymorphism that negatively affects the CFTR transcriptional activity and demonstrates a cooperative role of Sp1 and USF transcription factors in transactivation of the CFTR gene promoter

  4. Mutations in CFTR gene and clinical correlation in Argentine patients with congenital bilateral absence of the vas deferens Correlación de las características clínicas con mutaciones del gen CFTR en pacientes argentinos con ausencia bilateral congénita de vasos deferentes

    Estrella M Levy

    2004-06-01

    Full Text Available Congenital bilateral absence of the vas deferens (CBAVD is a form of male infertility in which mutations in the cystic fibrosis transmembrane conductance regulator (CFTR gene have been identified. Here we identify different mutations of CFTR and the poly-T variant of intron 8 (IVS8 in Argentine patients and analyze sweat test values and clinical characteristic related to Cystic Fibrosis (CF. For counseling purposes the two most frequent mutations in Argentine CF population: DF508 and G542X were screened in wives. In all cases, it was possible to reduce the risk of CF/CBAVD descendants in these couples because none of the mutation were found in the 36 samples. Eight patients (23% showed abnormal chloride values (> 60 mmol/l. A second group of 6 patients (18% had borderline values of sweat chloride (40-59 mmol/l. We defined another group with 6 patients (18%, with normal sweat chloride levels (30-39 mmo/l and a fourth group of 14 (41% patients with sweat chloride below 30 mmol/l. DF508, the most frequent CF mutation in the Argentine population, was found on 15 of the 72 chromosomes (21%, R117H mutation was detected on 2 of 62 chromosomes (3%. Only one R347P allele was found on 28 chromosomes analyzed (2%. On a sample of 27 patients, IVS8 analysis showed a frequency of 6/56 chromosomes (11% of 5T allele. Even though these findings present an improvement in the detection of mutations related to clinical correlations in Argentine CBAVD population, the search for other common and uncommon mutations should be continued.La ausencia bilateral congénita de vasos deferentes (CBAVD es una forma de infertilidad masculina en la que se han identificado mutaciones en el gen de la conductancia transmembrana de la fibrosis quística (CFTR. Hemos estudiado en pacientes argentinos diferentes mutaciones en el CFTR y la variante poli T del intron 8 (IVS8 y analizado los valores de test del sudor y las características clínicas relacionadas a la Fibrosis Qu

  5. Resveratrol increases F508del-CFTR dependent salivary secretion in cystic fibrosis mice

    Barbara Dhooghe

    2015-07-01

    Full Text Available Cystic fibrosis (CF is a fatal genetic disease associated with widespread exocrine gland dysfunction. Studies have suggested activating effects of resveratrol, a naturally-occurring polyphenol compound with antioxidant and anti-inflammatory properties, on CF transmembrane conductance regulator (CFTR protein function. We assayed, in F508del-CFTR homozygous (CF and in wild-type mice, the effect of resveratrol on salivary secretion in basal conditions, in response to inhibition by atropine (basal β-adrenergic-dependent component and to stimulation by isoprenaline (CFTR-dependent component. Both components of the salivary secretion were smaller in CF mice than in controls. Two hours after intraperitoneal administration of resveratrol (50 mg/kg dissolved in DMSO, the compound was detected in salivary glands. As in both CF and in wild-type mice, DMSO alone increased the response to isoprenaline in males but not in females, the effect of resveratrol was only measured in females. In wild-type mice, isoprenaline increased secretion by more than half. In CF mice, resveratrol rescued the response to isoprenaline, eliciting a 2.5-fold increase of β-adrenergic-stimulated secretion. We conclude that the salivary secretion assay is suitable to test DMSO-soluble CFTR modulators in female mice. We show that resveratrol applied in vivo to mice reaches salivary glands and increases β-adrenergic secretion. Immunolabelling of CFTR in human bronchial epithelial cells suggests that the effect is associated with increased CFTR protein expression. Our data support the view that resveratrol is beneficial for treating CF. The salivary secretion assay has a potential application to test efficacy of novel CF therapies.

  6. Osmoregulation of chloride channels in epithelial cells

    C.H. Lim (Christina)

    2008-01-01

    markdownabstract__Abstract__ The plasma membrane of mammalian cells is formed by two layers of lipids (lipid bilayer), primarily phospholipids, glycolipids and cholesterol, in which many different proteins are embedded. Phospholipid consists of a glycerol backbone esterified to fatty acids

  7. Chloride test - blood

    Serum chloride test ... A greater-than-normal level of chloride is called hyperchloremia. It may be due to: Carbonic anhydrase inhibitors (used to treat glaucoma) Diarrhea Metabolic acidosis Respiratory alkalosis (compensated) Renal ...

  8. Chloride in diet

    ... this page: //medlineplus.gov/ency/article/002417.htm Chloride in diet To use the sharing features on this page, please enable JavaScript. Chloride is found in many chemicals and other substances ...

  9. Mercuric chloride poisoning

    ... page: //medlineplus.gov/ency/article/002474.htm Mercuric chloride poisoning To use the sharing features on this page, please enable JavaScript. Mercuric chloride is a very poisonous form of mercury. It ...

  10. Chloride regulates afferent arteriolar contraction in response to depolarization

    Hansen, P B; Jensen, B L; Skott, O

    1998-01-01

    -Renal vascular reactivity is influenced by the level of dietary salt intake. Recent in vitro data suggest that afferent arteriolar contractility is modulated by extracellular chloride. In the present study, we assessed the influence of chloride on K+-induced contraction in isolated perfused rabbit...... afferent arterioles. In 70% of vessels examined, K+-induced contraction was abolished by acute substitution of bath chloride. Consecutive addition of Cl- (30, 60, 80, 100, 110, and 117 mmol/L) restored the sensitivity to K+, and half-maximal response was observed at 82 mmol/L chloride. The calcium channel...... antagonist diltiazem (10(-6) mol/L) abolished K+-induced contractions. Bicarbonate did not modify the sensitivity to chloride. Norepinephrine (10(-6) mol/L) induced full contraction in depolarized vessels even in the absence of chloride. Iodide and nitrate were substituted for chloride with no inhibitory...

  11. Analysis of Y chromosome microdeletions and CFTR gene mutations as genetic markers of infertility in Serbian men

    Dinić Jelena

    2007-01-01

    Full Text Available Background/Aim. Impaired fertility of a male partner is the main cause of infertility in up to one half of all infertile couples. At the genetic level, male infertility can be caused by chromosome aberrations or gene mutations. The presence and types of Y chromosome microdeletions and cystic fybrosis transmembrane conductance regulator (CFTR gene mutations as genetic cause of male infertility was tested in Serbian men. The aim of this study was to analyze CFTR gene mutations and Y chromosome microdelations as potential causes of male infertility in Serbian patients, as well as to test the hypothesis that CFTR mutations in infertile men are predominantly located in the several last exons of the gene. Methods. This study has encompassed 33 men with oligo- or azoospermia. The screening for Y chromosome microdeletions in the azoospermia factor (AZF region was performed by multiplex PCR analysis. The screening of the CFTR gene was performed by denaturing gradient gel electrophoresis (DGGE method. Results. Deletions on Y chromosome were detected in four patients, predominantly in AZFc region (four of total six deletions. Mutations in the CFTR gene were detected on eight out of 66 analyzed chromosomes of infertile men. The most common mutation was F508del (six of total eight mutations. Conclusion. This study confirmed that both Y chromosome microdeletions and CFTR gene mutations played important role in etiology of male infertility in Serbian infertile men. Genetic testing for Y chromosome microdeletions and CFTR gene mutations has been introduced in routine diagnostics and offered to couples undergoing assisted reproduction techniques. Considering that both the type of Y chromosome microdeletion and the type of CFTR mutation have a prognostic value, it is recommended that AZF and CFTR genotyping should not only be performed in patients with reduced sperm quality before undergoing assisted reproduction, but also for the purpose of preimplantation and

  12. A New Targeted CFTR Mutation Panel Based on Next-Generation Sequencing Technology.

    Lucarelli, Marco; Porcaro, Luigi; Biffignandi, Alice; Costantino, Lucy; Giannone, Valentina; Alberti, Luisella; Bruno, Sabina Maria; Corbetta, Carlo; Torresani, Erminio; Colombo, Carla; Seia, Manuela

    2017-09-01

    Searching for mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) is a key step in the diagnosis of and neonatal and carrier screening for cystic fibrosis (CF), and it has implications for prognosis and personalized therapy. The large number of mutations and genetic and phenotypic variability make this search a complex task. Herein, we developed, validated, and tested a laboratory assay for an extended search for mutations in CFTR using a next-generation sequencing-based method, with a panel of 188 CFTR mutations customized for the Italian population. Overall, 1426 dried blood spots from neonatal screening, 402 genomic DNA samples from various origins, and 1138 genomic DNA samples from patients with CF were analyzed. The assay showed excellent analytical and diagnostic operative characteristics. We identified and experimentally validated 159 (of 188) CFTR mutations. The assay achieved detection rates of 95.0% and 95.6% in two large-scale case series of CF patients from central and northern Italy, respectively. These detection rates are among the highest reported so far with a genetic test for CF based on a mutation panel. This assay appears to be well suited for diagnostics, neonatal and carrier screening, and assisted reproduction, and it represents a considerable advantage in CF genetic counseling. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  13. Important role of platelets in modulating endotoxin-induced lung inflammation in CFTR-deficient mice.

    Caiqi Zhao

    Full Text Available Mutation of CFTR (cystic fibrosis transmembrane conductance regulator leads to cystic fibrosis (CF. Patients with CF develop abnormalities of blood platelets and recurrent lung inflammation. However, whether CFTR-mutated platelets play a role in the development of lung inflammation is elusive. Therefore, we intratracheally challenged wildtype and F508del (a common type of CFTR mutation mice with LPS to observe changes of F508del platelets in the peripheral blood and indexes of lung inflammation (BAL neutrophils and protein levels. Furthermore, we investigated whether or not and how F508del platelets modulate the LPS-induced acute lung inflammation by targeting anti-platelet aggregation, depletion of neutrophils, reconstitution of bone marrow or neutrophils, blockade of P-selectin glycoprotein ligand-1 (PSGL-1, platelet activating factor (PAF, and correction of mutated CFTR trafficking. We found that LPS-challenged F508del mice developed severe thrombocytopenia and had higher levels of plasma TXB2 coincided with neutrophilic lung inflammation relative to wildtype control. Inhibition of F508del platelet aggregation or depletion of F508del neutrophils diminished the LPS-induced lung inflammation in the F508del mice. Moreover, wildtype mice reconstituted with either F508del bone marrow or neutrophils developed worse thrombocytopenia. Blocking PSGL-1, platelet activating factor (PAF, or rectifying trafficking of mutated CFTR in F508del mice diminished and alveolar neutrophil transmigration in the LPS-challenged F508del mice. These findings suggest that F508del platelets and their interaction with neutrophils are requisite for the development of LPS-induced lung inflammation and injury. As such, targeting platelets might be an emerging strategy for dampening recurrent lung inflammation in cystic fibrosis patients.

  14. Chloride equilibrium potential in salamander cones

    Bryson Eric J

    2004-12-01

    Full Text Available Abstract Background GABAergic inhibition and effects of intracellular chloride ions on calcium channel activity have been proposed to regulate neurotransmission from photoreceptors. To assess the impact of these and other chloride-dependent mechanisms on release from cones, the chloride equilibrium potential (ECl was determined in red-sensitive, large single cones from the tiger salamander retinal slice. Results Whole cell recordings were done using gramicidin perforated patch techniques to maintain endogenous Cl- levels. Membrane potentials were corrected for liquid junction potentials. Cone resting potentials were found to average -46 mV. To measure ECl, we applied long depolarizing steps to activate the calcium-activated chloride current (ICl(Ca and then determined the reversal potential for the current component that was inhibited by the Cl- channel blocker, niflumic acid. With this method, ECl was found to average -46 mV. In a complementary approach, we used a Cl-sensitive dye, MEQ, to measure the Cl- flux produced by depolarization with elevated concentrations of K+. The membrane potentials produced by the various high K+ solutions were measured in separate current clamp experiments. Consistent with electrophysiological experiments, MEQ fluorescence measurements indicated that ECl was below -36 mV. Conclusions The results of this study indicate that ECl is close to the dark resting potential. This will minimize the impact of chloride-dependent presynaptic mechanisms in cone terminals involving GABAa receptors, glutamate transporters and ICl(Ca.

  15. Analysis of cystic fibrosis gener product (CFTR) function in patients with pancreas divisum and recurrent acute pancreatitis.

    Gelrud, Andres; Sheth, Sunil; Banerjee, Subhas; Weed, Deborah; Shea, Julie; Chuttani, Ram; Howell, Douglas A; Telford, Jennifer J; Carr-Locke, David L; Regan, Meredith M; Ellis, Lynda; Durie, Peter R; Freedman, Steven D

    2004-08-01

    The mechanism by which pancreas divisum may lead to recurrent episodes of acute pancreatitis in a subset of individuals is unknown. Abnormalities of the cystic fibrosis gene product (CFTR) have been implicated in the genesis of idiopathic chronic pancreatitis. The aim of this study was to determine if CFTR function is abnormal in patients with pancreas divisum and recurrent acute pancreatitis (PD/RAP). A total of 69 healthy control subjects, 12 patients with PD/RAP, 16 obligate heterozygotes with a single CFTR mutation, and 95 patients with cystic fibrosis were enrolled. CFTR function was analyzed by nasal transepithelial potential difference testing in vivo. The outcomes of the PD/RAP patients following endoscopic and surgical treatments were concomitantly analyzed. Direct measurement of CFTR function in nasal epithelium in response to isoproterenol demonstrated that the values for PD/RAP were intermediate between those observed for healthy controls and cystic fibrosis patients. The median value was 13 mV for PD/RAP subjects, which was statistically different from healthy controls (22 mV, p= 0.001) and cystic fibrosis pancreatic sufficient (-1 mV, p < 0.0001) and pancreatic insufficient (-3 mV, p < 0.0001) patients. These results suggest a link between CFTR dysfunction and recurrent acute pancreatitis in patients with pancreas divisum and may explain why a subset of patients with pancreas divisum develops recurrent acute pancreatitis. Copyright 2004 American College of Gastroenterology

  16. CFTR mutations spectrum and the efficiency of molecular diagnostics in Polish cystic fibrosis patients.

    Ewa Ziętkiewicz

    Full Text Available Cystic fibrosis (CF is caused by mutations in the cystic fibrosis transmembrane regulator gene (CFTR. In light of the strong allelic heterogeneity and regional specificity of the mutation spectrum, the strategy of molecular diagnostics and counseling in CF requires genetic tests to reflect the frequency profile characteristic for a given population. The goal of the study was to provide an updated comprehensive estimation of the distribution of CFTR mutations in Polish CF patients and to assess the effectiveness of INNOLiPA_CFTR tests in Polish population. The analyzed cohort consisted of 738 patients with the clinically confirmed CF diagnosis, prescreened for molecular defects using INNOLiPA_CFTR panels from Innogenetics. A combined efficiency of INNOLiPA CFTR_19 and CFTR_17_TnUpdate tests was 75.5%; both mutations were detected in 68.2%, and one mutation in 14.8% of the affected individuals. The group composed of all the patients with only one or with no mutation detected (109 and 126 individuals, respectively was analyzed further using a mutation screening approach, i.e. SSCP/HD (single strand conformational polymorphism/heteroduplex analysis of PCR products followed by sequencing of the coding sequence. As a result, 53 more mutations were found in 97 patients. The overall efficiency of the CF allele detection was 82.5% (7.0% increase compared to INNOLiPA tests alone. The distribution of the most frequent mutations in Poland was assessed. Most of the mutations repetitively found in Polish patients had been previously described in other European populations. The most frequent mutated allele, F508del, represented 54.5% of Polish CF chromosomes. Another eight mutations had frequencies over 1%, 24 had frequencies between 1 and 0.1%; c.2052-2053insA and c.3468+2_3468+3insT were the most frequent non-INNOLiPA mutations. Mutation distribution described herein is also relevant to the Polish diaspora. Our study also demonstrates that the reported

  17. Chloride ingress prediction

    Frederiksen, Jens Mejer; Geiker, Mette Rica

    2008-01-01

    Prediction of chloride ingress into concrete is an important part of durability design of reinforced concrete structures exposed to chloride containing environment. This paper presents experimentally based design parameters for Portland cement concretes with and without silica fume and fly ash...... in marine atmospheric and submersed South Scandinavian environment. The design parameters are based on sequential measurements of 86 chloride profiles taken over ten years from 13 different types of concrete. The design parameters provide the input for an analytical model for chloride profiles as function...... of depth and time, when both the surface chloride concentration and the diffusion coefficient are allowed to vary in time. The model is presented in a companion paper....

  18. CFTR depletion results in changes in fatty acid composition and promotes lipogenesis in intestinal Caco 2/15 cells.

    Geneviève Mailhot

    2010-05-01

    Full Text Available Abnormal fatty acid composition (FA in plasma and tissue lipids frequently occurs in homozygous and even in heterozygous carriers of cystic fibrosis transmembrane conductance regulator (CFTR mutations. The mechanism(s underlying these abnormalities remained, however, poorly understood despite the potentially CFTR contributing role.The aim of the present study was to investigate the impact of CFTR depletion on FA uptake, composition and metabolism using the intestinal Caco-2/15 cell line. shRNA-mediated cftr gene silencing induced qualitative and quantitative modifications in FA composition in differentiated enterocytes as determined by gas-liquid chromatography. With the cftr gene disruption, there was a 1,5 fold increase in the total FA amount, largely attributable to monounsaturated and saturated FA compared to controls. The activity of delta-7 desaturase, estimated by the 16:1(n-7/16:0, was significantly higher in knockdown cells and consistent with the striking elevation of the n-7 FA family. When incubated with [14C]-oleic acid, CFTR-depleted cells were capable of quick incorporation and export to the medium concomitantly with the high protein expression of L-FABP known to promote intracellular FA trafficking. Accordingly, lipoprotein vehicles (CM, VLDL, LDL and HDL, isolated from CFTR knockdown cells, exhibited higher levels of radiolabeled FA. Moreover, in the presence of [14C]-acetate, knockdown cells exhibited enhanced secretion of newly synthesized phospholipids, triglycerides, cholesteryl esters and free FA, thereby suggesting a stimulation of the lipogenic pathway. Conformably, gene expression of SREBP-1c, a key lipogenic transcription factor, was increased while protein expression of the phosphorylated and inactive form of acetylCoA carboxylase was reduced, confirming lipogenesis induction. Finally, CFTR-depleted cells exhibited lower gene expression of transcription factors (PPARalpha, LXRalpha, LXRbeta and RXRalpha

  19. F508del-CFTR rescue: a matter of cell stress response.

    Nieddu, Erika; Pollarolo, Benedetta; Merello, Luisa; Schenone, Silvia; Mazzei, Mauro

    2013-01-01

    Cystic fibrosis (CF) is a common inherited fatal disease affecting 70,000 people worldwide, with a median predicted age of survival of approximately 38 years. The deletion of Phenylalanine in position 508 of the Cystic Fibrosis Transmembrane conductance Regulator (F508del-CFTR) is the most common mutation in CF patients: the deleted protein, not properly folded, is degraded. To date no commercial drugs are available. Low temperature, some osmolytes and conditions able to induce heat shock protein 70 (Hsp70) expression and heat shock cognate 70 (Hsc70) inhibition result in F508del-CFTR rescue, hence restoring its physiological function: this review sheds light on the correlation between these several evidences. Interestingly, all these approaches have a role in the cell stress response (CSR), a set of cell reactions to stress. In addition, unpredictably, F508del-CFTR rescue has to be considered in the frame of CSR: entities that induce - or are induced during - the CSR are, in general, also able to correct trafficking defect of CFTR. Specifically, the low temperature induces, by definition, a CSR; osmolytes, such as glycerol and trimethylamine N-oxide (TMAO), are products of the CSR; pharmacological correctors, such as Matrine and 4-phenylbutirric acid (4PBA), down-regulate the constitutive Hsc70 in favor of an up-regulation of the inducible chaperone Hsp70, another component of the CSR. The identification of a common mechanism of action for different types of correctors could drive the discovery of new active molecules in CF, overcoming methods clinically inapplicable, such as the low temperature.

  20. Stimulation of Intestinal Cl- Secretion Through CFTR by Caffeine Intake in Salt-Sensitive Hypertensive Rats

    Xiao Wei

    2018-03-01

    Full Text Available Background/Aims: High salt consumption is a major risk factor for hypertension, and sodium homeostasis is regulated by both intestinal sodium absorption and urinary sodium excretion. Chronic caffeine intake has been reported to attenuate salt-sensitive hypertension by promoting urinary sodium excretion; however, its exact role in intestinal sodium absorption remains unknown. Here, we investigated whether and how chronic caffeine consumption antagonizes salt-sensitive hypertension by inhibiting intestinal sodium absorption. Methods: Dahl salt-sensitive rats were fed 8% NaCl chow and 0.1% caffeine in their drinking water for 15 days. The blood pressure and fecal sodium content were measured. The effect of caffeine on the movement of Cl- in enterocyte cells was determined with the Ussing chamber assay. Results: Rats that were treated with caffeine displayed significantly lower mean blood pressure and higher fecal sodium content than the controls. Consistent with these findings, caffeine intake decreased fluid absorption by the intestine in the fluid perfusion experiment. Further, the results from the Ussing chamber assay indicated that caffeine promoted Cl- secretion through enterocyte apical cystic fibrosis transmembrane conductance regulator (CFTR, and thus inhibited sodium absorption. Moreover, depletion of cAMP or inhibition of CFTR completely abolished the effect of caffeine on Cl- secretion. Conclusion: The results indicate that chronic caffeine consumption reduces sodium absorption by promoting CFTR-mediated Cl- secretion in the intestine, which contributes to the anti-hypertensive effect of caffeine in salt-sensitive rats.

  1. CFTR-dependent defect in alternatively-activated macrophages in cystic fibrosis.

    Tarique, Abdullah A; Sly, Peter D; Holt, Patrick G; Bosco, Anthony; Ware, Robert S; Logan, Jayden; Bell, Scott C; Wainwright, Claire E; Fantino, Emmanuelle

    2017-07-01

    The role of the macrophages in cystic fibrosis (CF) lung disease has been poorly studied. We hypothesized that alternatively activated M2 macrophages are abnormal in CF lung disease. Blood samples were collected from adults (n=13) children (n=27) with CF on admission for acute pulmonary exacerbation and when clinically stable. Monocytes were differentiated into macrophages and polarized into classical (M1) and alternatively-activated (M2) phenotypes, function determined ex-vivo and compared with healthy controls. In the absence of functional cystic fibrosis trans-membrane conductance regulator (CFTR), either naturally in patients with CF or induced with CFTR inhibitors, monocyte-derived macrophages do not respond to IL-13/IL-4, fail to polarize into M2s associated with a post-transcriptional failure to produce and express IL-13Rα1 on the macrophage surface Polarization to the M1 phenotype was unaffected. CFTR-dependent imbalance of macrophage phenotypes and functions could contribute to the exaggerated inflammatory response seen in CF lung disease. Copyright © 2017 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

  2. A new compound heterozygous CFTR mutation in a Chinese family with cystic fibrosis.

    Xie, Yingjun; Huang, Xueqiong; Liang, Yujian; Xu, Lingling; Pei, Yuxin; Cheng, Yucai; Zhang, Lidan; Tang, Wen

    2017-11-01

    Cystic fibrosis (CF) is the most common autosomal recessive disease among Caucasians but is rarer in the Chinese population, because mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. To elucidate the causative role of a novel compound heterozygous mutation of CF. In this study, clinical samples were obtained from two siblings with recurrent airway infections, clubbed fingers, salt-sweat and failure to gain weight in a non-consanguineous Chinese family. Next-generation sequencing was performed on the 27 coding exons of CFTR in both children, with confirmation by Sanger sequencing. Next-generation sequencing showed the same compound heterozygous CFTR mutation (c.865A>T p.Arg289X and c.3651_3652insAAAT p.Tyr1219X) in both children. As this mutation is consistent with the clinical manifestations of CF and no other mutations were detected after scanning the gene sequence, we suggest that the CF phenotype is caused by compound heterozygosity for c.865A>T and c.3651_3652insAAAT. As c865A>T is not currently listed in the "Cystic Fibrosis Mutation Database", this information about CF in a Chinese population is of interest. © 2015 John Wiley & Sons Ltd.

  3. Longevity and plasticity of CFTR provide an argument for noncanonical SNP organization in hominid DNA.

    Aubrey E Hill

    Full Text Available Like many other ancient genes, the cystic fibrosis transmembrane conductance regulator (CFTR has survived for hundreds of millions of years. In this report, we consider whether such prodigious longevity of an individual gene--as opposed to an entire genome or species--should be considered surprising in the face of eons of relentless DNA replication errors, mutagenesis, and other causes of sequence polymorphism. The conventions that modern human SNP patterns result either from purifying selection or random (neutral drift were not well supported, since extant models account rather poorly for the known plasticity and function (or the established SNP distributions found in a multitude of genes such as CFTR. Instead, our analysis can be taken as a polemic indicating that SNPs in CFTR and many other mammalian genes may have been generated--and continue to accrue--in a fundamentally more organized manner than would otherwise have been expected. The resulting viewpoint contradicts earlier claims of 'directional' or 'intelligent design-type' SNP formation, and has important implications regarding the pace of DNA adaptation, the genesis of conserved non-coding DNA, and the extent to which eukaryotic SNP formation should be viewed as adaptive.

  4. Chloride flux in phagocytes.

    Wang, Guoshun

    2016-09-01

    Phagocytes, such as neutrophils and macrophages, engulf microbes into phagosomes and launch chemical attacks to kill and degrade them. Such a critical innate immune function necessitates ion participation. Chloride, the most abundant anion in the human body, is an indispensable constituent of the myeloperoxidase (MPO)-H2 O2 -halide system that produces the potent microbicide hypochlorous acid (HOCl). It also serves as a balancing ion to set membrane potentials, optimize cytosolic and phagosomal pH, and regulate phagosomal enzymatic activities. Deficient supply of this anion to or defective attainment of this anion by phagocytes is linked to innate immune defects. However, how phagocytes acquire chloride from their residing environment especially when they are deployed to epithelium-lined lumens, and how chloride is intracellularly transported to phagosomes remain largely unknown. This review article will provide an overview of chloride protein carriers, potential mechanisms for phagocytic chloride preservation and acquisition, intracellular chloride supply to phagosomes for oxidant production, and methods to measure chloride levels in phagocytes and their phagosomes. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. Cellular mechanisms underlying the inhibitory effect of flufenamic acid on chloride secretion in human intestinal epithelial cells

    Pawin Pongkorpsakol

    2017-06-01

    Full Text Available Intestinal Cl− secretion is involved in the pathogenesis of secretory diarrheas including cholera. We recently demonstrated that flufenamic acid (FFA suppressed Vibrio cholerae El Tor variant-induced intestinal fluid secretion via mechanisms involving AMPK activation and NF-κB-suppression. The present study aimed to investigate the effect of FFA on transepithelial Cl− secretion in human intestinal epithelial (T84 cells. FFA inhibited cAMP-dependent Cl− secretion in T84 cell monolayers with IC50 of ∼8 μM. Other fenamate drugs including tolfenamic acid, meclofenamic acid and mefenamic acid exhibited the same effect albeit with lower potency. FFA also inhibited activities of CFTR, a cAMP-activated apical Cl− channel, and KCNQ1/KCNE3, a cAMP-activated basolateral K+ channel. Mechanisms of CFTR inhibition by FFA did not involve activation of its negative regulators. Interestingly, FFA inhibited Ca2+-dependent Cl− secretion with IC50 of ∼10 μM. FFA inhibited activities of Ca2+-activated Cl− channels and KCa3.1, a Ca2+-activated basolateral K+ channels, but had no effect on activities of Na+–K+–Cl− cotransporters and Na+–K+ ATPases. These results indicate that FFA inhibits both cAMP and Ca2+-dependent Cl− secretion by suppressing activities of both apical Cl− channels and basolateral K+ channels. FFA and other fenamate drugs may be useful in the treatment of secretory diarrheas.

  6. Adenosine receptors in rat and human pancreatic ducts stimulate chloride transport

    Novak, Ivana; Hede, Susanne; Hansen, Mette

    2007-01-01

    , it was found that 58% of PANC-1 cells responded to adenosine, whereas only 9% of CFPAC-1 cells responded. Adenosine elicited Ca(2+) signals only in a few rat and human duct cells, which did not seem to correlate with Cl(-) signals. A(2A) receptors were localized in the luminal membranes of rat pancreatic ducts......, plasma membrane of many PANC-1 cells, but only a few CFPAC-1 cells. Taken together, our data indicate that A(2A) receptors open Cl(-) channels in pancreatic ducts cells with functional CFTR. We propose that adenosine can stimulate pancreatic secretion and, thereby, is an active player in the acini...

  7. Osmoregulated Chloride Currents in Hemocytes from Mytilus galloprovincialis.

    Monica Bregante

    Full Text Available We investigated the biophysical properties of the transport mediated by ion channels in hemocytes from the hemolymph of the bivalve Mytilus galloprovincialis. Besides other transporters, mytilus hemocytes possess a specialized channel sensitive to the osmotic pressure with functional properties similar to those of other transport proteins present in vertebrates. As chloride fluxes may play an important role in the regulation of cell volume in case of modifications of the ionic composition of the external medium, we focused our attention on an inwardly-rectifying voltage-dependent, chloride-selective channel activated by negative membrane potentials and potentiated by the low osmolality of the external solution. The chloride channel was slightly inhibited by micromolar concentrations of zinc chloride in the bath solution, while the antifouling agent zinc pyrithione did not affect the channel conductance at all. This is the first direct electrophysiological characterization of a functional ion channel in ancestral immunocytes of mytilus, which may bring a contribution to the understanding of the response of bivalves to salt and contaminant stresses.

  8. Increased cystic fibrosis transmembrane conductance regulators expression and decreased epithelial sodium channel alpha subunits expression in early abortion: findings from a mouse model and clinical cases of abortion.

    Min Zhou

    Full Text Available The status of the maternal endometrium is vital in regulating humoral homeostasis and for ensuring embryo implantation. Cystic fibrosis transmembrane conductance regulators (CFTR and epithelial sodium channel alpha subunits (ENaC-α play an important role in female reproduction by maintaining humoral and cell homeostasis. However, it is not clear whether the expression levels of CFTR and ENaC-α in the decidual component during early pregnancy are related with early miscarriage. CBA×DBA/2 mouse mating has been widely accepted as a classical model of early miscarriage. The abortion rate associated with this mating was 33.33% in our study. The decidua of abortion-prone CBA female mice (DBA/2 mated had higher CFTR mRNA and protein expression and lower ENaC-α mRNA and protein expression, compared to normal pregnant CBA mice (BLAB/C mated. Furthermore, increased CFTR expression and decreased ENaC-α expression were observed in the uterine tissue from women with early miscarriage, as compared to those with successful pregnancy. In conclusion, increased CFTR expression and decreased ENaC-α expression in the decidua of early abortion may relate with failure of early pregnancy.

  9. Resveratrol enhances airway surface liquid depth in sinonasal epithelium by increasing cystic fibrosis transmembrane conductance regulator open probability.

    Shaoyan Zhang

    Full Text Available Chronic rhinosinusitis engenders enormous morbidity in the general population, and is often refractory to medical intervention. Compounds that augment mucociliary clearance in airway epithelia represent a novel treatment strategy for diseases of mucus stasis. A dominant fluid and electrolyte secretory pathway in the nasal airways is governed by the cystic fibrosis transmembrane conductance regulator (CFTR. The objectives of the present study were to test resveratrol, a strong potentiator of CFTR channel open probability, in preparation for a clinical trial of mucociliary activators in human sinus disease.Primary sinonasal epithelial cells, immortalized bronchoepithelial cells (wild type and F508del CFTR, and HEK293 cells expressing exogenous human CFTR were investigated by Ussing chamber as well as patch clamp technique under non-phosphorylating conditions. Effects on airway surface liquid depth were measured using confocal laser scanning microscopy. Impact on CFTR gene expression was measured by quantitative reverse transcriptase polymerase chain reaction.Resveratrol is a robust CFTR channel potentiator in numerous mammalian species. The compound also activated temperature corrected F508del CFTR and enhanced CFTR-dependent chloride secretion in human sinus epithelium ex vivo to an extent comparable to the recently approved CFTR potentiator, ivacaftor. Using inside out patches from apical membranes of murine cells, resveratrol stimulated an ~8 picosiemens chloride channel consistent with CFTR. This observation was confirmed in HEK293 cells expressing exogenous CFTR. Treatment of sinonasal epithelium resulted in a significant increase in airway surface liquid depth (in µm: 8.08+/-1.68 vs. 6.11+/-0.47,control,p<0.05. There was no increase CFTR mRNA.Resveratrol is a potent chloride secretagogue from the mucosal surface of sinonasal epithelium, and hydrates airway surface liquid by increasing CFTR channel open probability. The foundation for a

  10. An immortal cell line to study the role of endogenous CFTR in electrolyte absorption.

    Bell, C L; Quinton, P M

    1995-01-01

    The intact human reabsorptive sweat duct (RD) has been a reliable model for investigations of the functional role of "endogenous" CFTR (cystic fibrosis transmembrane conductance regulator) in normal and abnormal electrolyte absorptive function. But to overcome the limitations imposed by the use of fresh, intact tissue, we transformed cultured RD cells using the chimeric virus Ad5/SV40 1613 ori-. The resultant cell line, RD2(NL), has remained differentiated forming a polarized epithelium that expressed two fundamental components of absorption, a cAMP activated Cl- conductance (GCl) and an amiloride-sensitive Na+ conductance (GNa). In the unstimulated state, there was a low level of transport activity; however, addition of forskolin (10(-5) M) significantly increased the Cl- diffusion potential (Vt) generated by a luminally directed Cl- gradient from -15.3 +/- 0.7 mV to -23.9 +/- 1.1 mV, n = 39; and decreased the transepithelial resistance (Rt) from 814.8 +/- 56.3 omega.cm2 to 750.5 +/- 47.5 omega.cm2, n = 39, (n = number of cultures). cAMP activation, anion selectivity (Cl- > I- > gluconate), and a dependence upon metabolic energy (metabolic poisoning inhibited GCl), all indicate that the GCl expressed in RD2(NL) is in fact CFTR-GCl. The presence of an apical amiloride-sensitive GNa was shown by the amiloride (10(-5) M) inhibition of GNa as indicated by a reduction of Vt and equivalent short circuit current by 78.0 +/- 3.1% and 77.9 +/- 2.6%, respectively, and an increase in Rt by 7.2 +/- 0.8%, n = 36. In conclusion, the RD2(NL) cell line presents the first model system in which CFTR-GCl is expressed in a purely absorptive tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Association between F508 deletion in CFTR and chronic pancreatitis risk.

    Zhao, Dong; Xu, Yanzhen; Li, Jiatong; Fu, Shien; Xiao, Feifan; Song, Xiaowei; Xie, Zhibin; Jiang, Min; He, Yan; Liu, Chengwu; Wen, Qiongxian; Yang, Xiaoli

    2017-09-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) has been reported to influence individual susceptibility to chronic pancreatitis (CP), but the results of previous studies are controversial. We performed a study to demonstrate the relationship between CFTR and CP. We searched PubMed, Scopus, and Embase for studies of patients with CP. Seven studies from 1995 to 2016 were identified, and included 64,832 patients. Pooled prevalence and 95% confidence intervals (CIs) were calculated. F508 deletion in CFTR was significantly positively associated with CP risk in the overall analysis (odds ratio [OR]=3.20, 95% CI: 2.30-4.44, I 2 =31.7%). In subgroup analysis stratified by ethnicity, F508 deletion was significantly associated with CP risk in Indian populations, using a fixed effects model (ORs=5.45, 95% CI: 2.52-11.79, I 2 =0.0%), and in non-Indian populations, using a random effects model (ORs=3.59, 95% CI: 1.73-7.48, I 2 =60.9%). At the same time, we found that Indians with F508 deletion had much higher CP prevalence than non-Indians. Interestingly, F508 deletion was also associated with CP and idiopathic CP risk in subgroup analysis stratified by aeitiology, using the fixed effects model. Based on current evidence, F508 deletion is a risk factor for CP, and Indians with F508 deletion have much higher CP morbidity. Copyright © 2017 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

  12. Ion channels in glioblastoma.

    Molenaar, Remco J

    2011-01-01

    Glioblastoma is the most common primary brain tumor with the most dismal prognosis. It is characterized by extensive invasion, migration, and angiogenesis. Median survival is only 15 months due to this behavior, rendering focal surgical resection ineffective and adequate radiotherapy impossible. At this moment, several ion channels have been implicated in glioblastoma proliferation, migration, and invasion. This paper summarizes studies on potassium, sodium, chloride, and calcium channels of glioblastoma. It provides an up-to-date overview of the literature that could ultimately lead to new therapeutic targets.

  13. Chloride removal from vitrification offgas

    Slaathaug, E.J. [Westinghouse Hanford Co., Richland, WA (United States)

    1995-06-01

    This study identified and investigated techniques of selectively purging chlorides from the low-level waste (LLW) vitrification process with the purge stream acceptable for burial on the Hanford Site. Chlorides will be present in high concentration in several individual feeds to the LLW Vitrification Plant. The chlorides are highly volatile in combustion type melters and are readily absorbed by wet scrubbing of the melter offgas. The Tank Waste Remediation System (TWRS) process flow sheets show that the resulting chloride rich scrub solution is recycled back to the melter. The chlorides must be purged from the recycle loop to prevent the buildup of excessively high chloride concentrations.

  14. Chloride removal from vitrification offgas

    Slaathaug, E.J.

    1995-01-01

    This study identified and investigated techniques of selectively purging chlorides from the low-level waste (LLW) vitrification process with the purge stream acceptable for burial on the Hanford Site. Chlorides will be present in high concentration in several individual feeds to the LLW Vitrification Plant. The chlorides are highly volatile in combustion type melters and are readily absorbed by wet scrubbing of the melter offgas. The Tank Waste Remediation System (TWRS) process flow sheets show that the resulting chloride rich scrub solution is recycled back to the melter. The chlorides must be purged from the recycle loop to prevent the buildup of excessively high chloride concentrations

  15. Is sweat chloride predictive of severity of cystic fibrosis lung disease assessed by chest computed tomography?

    Caudri, Daan; Zitter, David; Bronsveld, Inez; Tiddens, Harm

    2017-09-01

    Cystic Fibrosis (CF) lung disease is characterized by a marked heterogeneity. Sweat chloride-level is a functional marker of the CF Transmembrane Regulator (CFTR) protein and could be an important predictor of later disease severity. In this retrospective analysis children from the Rotterdam CF clinic with available sweat chloride level at diagnosis and at least one routine spirometry-controlled volumetric chest CT scan in follow-up were included. CT scans were scored using the CF-CT scoring system (% of maximum). Associations between sweat chloride-levels and CF-CT scores were calculated using linear regression models, adjusting for age at sweat test and age at follow-up. Because structural lung damage develops over the course of many years, effect modification by the age at follow-up CT-scan was tested for by age-stratification. In 59 children (30 male) sweat chloride was measured at diagnosis (median age 0.5 years, range 0-13) and later chest CT performed (median age 14 years, range 6-18). Sweat chloride was associated with significantly higher CT-CT total score, bronchiectasis score, and mucus plugging score. Stratification for age at follow-up in tertiles showed this association remained only in the oldest age group (range 15-18 years). In that subgroup associations were found with all but one of the CF-CT subscores, as well as with all tested lung functions parameters. Sweat chloride-level is a significant predictor of CF lung disease severity as determined by chest CT and lung function. This association could only be demonstrated in children with follow-up to age 15 years and above. © 2017 Wiley Periodicals, Inc.

  16. Lithium thionyl chloride battery

    Saathoff, D.J.; Venkatasetty, H.V.

    1982-10-19

    The discharge rate and internal conductivity of electrochemical cell including a lithium anode, and a cathode and an electrolyte including LiAlCl4 and SOC2 is improved by the addition of an amount of a mixture containing AlCl3 and butyl pyridinium chloride.

  17. The medical sodium chloride

    Mirsaidov, U.M.

    2002-01-01

    In the institute was investigated the chemical composition of rock salt of some deposits of Tajikistan and was show the presence in it admixture of ions of Ca 2 + , Mg 2 + a nd SO 2 - a nd absence of heavy metals, ammonium salts, iron, potassium and arsenic. Was elaborated the fundamental instrument-technologic scheme of sodium chloride receiving

  18. Is congenital bilateral absence of vas deferens a primary form of cystic fibrosis? Analyses of the CFTR gene in 67 patients

    Mercier, B.; Verlingue, C.; Audrezet, M.P.; Ferec, C. [Centre de Biogenetique C.D.T.S., Brest (France); Lissens, W.; Bonduelle, M. [University Hospital VUB, Brussels (United Kingdom); Silber, S.J. [St. Luke`s Hospital, St. Louis, MO (United States); Novelli, G. [Catholic Univ. of Rome (Italy)

    1995-01-01

    Congenital bilateral absence of the vas deferens (CBAVD) is an important cause of sterility in men. Although the genetic basis of this condition is still unclear, it has been shown recently that some of these patients carry mutations in their cystic fibrosis transmembrane conductance regulator (CFTR) genes. To extend this observation, we have analyzed the entire coding sequence of the CFTR gene in a cohort of 67 men with CBAVD, who are otherwise healthy. We have identified four novel missense mutations (A800G, G149R, R258G, and E193K). We have shown that 42% of subjects were carriers of one CFTR allele and that 24% are compound heterozygous for CFTR alleles. Thus, we have been unable to identify 76% of these patients as carrying two CFTR mutations. Furthermore, we have described the segregation of CFTR haplotypes in the family of one CBAVD male; in this family are two male siblings, with identical CFTR loci but displaying different phenotypes, one of them being fertile and the other sterile. The data presented in this family, indicating a discordance between the CBAVD phenotype and a marked carrier ({delta}F508) chromosome, support the involvement of another gene(s), in the etiology of CBAVD. 35 refs., 2 figs., 1 tab.

  19. Renin release from permeabilized juxtaglomerular cells is stimulated by chloride but not by low calcium

    Jensen, B L; Skøtt, O

    1994-01-01

    of chloride channels followed by a drop in the intracellular chloride concentration. The stimulation caused by the high calcium concentration may be a toxic effect or may be due to stimulation of the fusion between granules and cell membrane in a way analogous to other secretory cells....

  20. Development of allele-specific multiplex PCR to determine the length of poly-T in intron 8 of CFTR

    Neng Chen

    2014-07-01

    Full Text Available Cystic fibrosis transmembrane conductance regulator (CFTR gene mutation analysis has been implemented for Cystic Fibrosis (CF carrier screening, and molecular diagnosis of CF and congenital bilateral absence of the vas deferens (CBAVD. Although poly-T allele analysis in intron 8 of CFTR is required when a patient is positive for R117H, it is not recommended for routine carrier screening. Therefore, commercial kits for CFTR mutation analysis were designed either to mask the poly-T allele results, unless a patient is R117H positive, or to have the poly-T analysis as a standalone reflex test using the same commercial platform. There are other standalone assays developed to detect poly-T alleles, such as heteroduplex analysis, High Resolution Melting (HRM curve analysis, allele-specific PCR (AS-PCR and Sanger sequencing. In this report, we developed a simple and easy-to-implement multiplex AS-PCR assay using unlabeled standard length primers, which can be used as a reflex or standalone test for CFTR poly-T track analysis. Out of 115 human gDNA samples tested, results from our new AS-PCR matched to the previous known poly-T results or results from Sanger sequencing.

  1. Interaction between a novel TGFB1 haplotype and CFTR genotype is associated with improved lung function in cystic fibrosis.

    Bremer, Lindsay A; Blackman, Scott M; Vanscoy, Lori L; McDougal, Kathryn E; Bowers, Amanda; Naughton, Kathleen M; Cutler, David J; Cutting, Garry R

    2008-07-15

    Cystic fibrosis (CF), the most common lethal single gene disorder in Caucasians, is due to mutations in the CFTR gene. Twin and sibling analysis indicates that modifier genes, rather than allelic variation in CFTR, are responsible for most of the variability in severity of lung disease, the major cause of mortality in CF patients. We used a family-based approach to test for association between lung function and two functional SNPs (rs1800469, '-509' and rs1982073, 'codon 10') in the 5' region of transforming growth factor-beta1 (TGFB1), a putative CF modifier gene. Quantitative transmission disequilibrium testing of 472 CF patient-parent-parent trios revealed that both TGFB1 SNPs showed significant transmission distortion when patients were stratified by CFTR genotype. Although lung function and nutritional status are correlated in CF patients, there was no evidence of association between the TGFB1 SNPs and variation in nutritional status. Additional tagging SNPs (rs8179181, rs2278422, rs8110090, rs4803455 and rs1982072) that capture most of the diversity in TGFB1 were also typed but none showed association with variation in lung function. However, a haplotype composed of the -509 C and codon 10 T alleles along with the C allele of the 3' SNP rs8179181 was highly associated with increased lung function in patients grouped by CFTR genotype. These results demonstrate that TGFB1 is a modifier of CF lung disease and reveal a previously unrecognized beneficial effect of TGFB1 variants upon the pulmonary phenotype.

  2. Inhibition of cAMP-activated intestinal chloride secretion by diclofenac: cellular mechanism and potential application in cholera.

    Pongkorpsakol, Pawin; Pathomthongtaweechai, Nutthapoom; Srimanote, Potjanee; Soodvilai, Sunhapas; Chatsudthipong, Varanuj; Muanprasat, Chatchai

    2014-09-01

    Cyclic AMP-activated intestinal Cl- secretion plays an important role in pathogenesis of cholera. This study aimed to investigate the effect of diclofenac on cAMP-activated Cl- secretion, its underlying mechanisms, and possible application in the treatment of cholera. Diclofenac inhibited cAMP-activated Cl- secretion in human intestinal epithelial (T84) cells with IC50 of ∼ 20 µM. The effect required no cytochrome P450 enzyme-mediated metabolic activation. Interestingly, exposures of T84 cell monolayers to diclofenac, either in apical or basolateral solutions, produced similar degree of inhibitions. Analyses of the apical Cl- current showed that diclofenac reversibly inhibited CFTR Cl- channel activity (IC50 ∼ 10 µM) via mechanisms not involving either changes in intracellular cAMP levels or CFTR channel inactivation by AMP-activated protein kinase and protein phosphatase. Of interest, diclofenac had no effect on Na(+)-K(+) ATPases and Na(+)-K(+)-Cl- cotransporters, but inhibited cAMP-activated basolateral K(+) channels with IC50 of ∼ 3 µM. In addition, diclofenac suppressed Ca(2+)-activated Cl- channels, inwardly rectifying Cl- channels, and Ca(2+)-activated basolateral K(+) channels. Furthermore, diclofenac (up to 200 µM; 24 h of treatment) had no effect on cell viability and barrier function in T84 cells. Importantly, cholera toxin (CT)-induced Cl- secretion across T84 cell monolayers was effectively suppressed by diclofenac. Intraperitoneal administration of diclofenac (30 mg/kg) reduced both CT and Vibrio cholerae-induced intestinal fluid secretion by ∼ 70% without affecting intestinal fluid absorption in mice. Collectively, our results indicate that diclofenac inhibits both cAMP-activated and Ca(2+)-activated Cl- secretion by inhibiting both apical Cl- channels and basolateral K+ channels in intestinal epithelial cells. Diclofenac may be useful in the treatment of cholera and other types of secretory diarrheas resulting from intestinal

  3. Inhibition of cAMP-activated intestinal chloride secretion by diclofenac: cellular mechanism and potential application in cholera.

    Pawin Pongkorpsakol

    2014-09-01

    Full Text Available Cyclic AMP-activated intestinal Cl- secretion plays an important role in pathogenesis of cholera. This study aimed to investigate the effect of diclofenac on cAMP-activated Cl- secretion, its underlying mechanisms, and possible application in the treatment of cholera. Diclofenac inhibited cAMP-activated Cl- secretion in human intestinal epithelial (T84 cells with IC50 of ∼ 20 µM. The effect required no cytochrome P450 enzyme-mediated metabolic activation. Interestingly, exposures of T84 cell monolayers to diclofenac, either in apical or basolateral solutions, produced similar degree of inhibitions. Analyses of the apical Cl- current showed that diclofenac reversibly inhibited CFTR Cl- channel activity (IC50 ∼ 10 µM via mechanisms not involving either changes in intracellular cAMP levels or CFTR channel inactivation by AMP-activated protein kinase and protein phosphatase. Of interest, diclofenac had no effect on Na(+-K(+ ATPases and Na(+-K(+-Cl- cotransporters, but inhibited cAMP-activated basolateral K(+ channels with IC50 of ∼ 3 µM. In addition, diclofenac suppressed Ca(2+-activated Cl- channels, inwardly rectifying Cl- channels, and Ca(2+-activated basolateral K(+ channels. Furthermore, diclofenac (up to 200 µM; 24 h of treatment had no effect on cell viability and barrier function in T84 cells. Importantly, cholera toxin (CT-induced Cl- secretion across T84 cell monolayers was effectively suppressed by diclofenac. Intraperitoneal administration of diclofenac (30 mg/kg reduced both CT and Vibrio cholerae-induced intestinal fluid secretion by ∼ 70% without affecting intestinal fluid absorption in mice. Collectively, our results indicate that diclofenac inhibits both cAMP-activated and Ca(2+-activated Cl- secretion by inhibiting both apical Cl- channels and basolateral K+ channels in intestinal epithelial cells. Diclofenac may be useful in the treatment of cholera and other types of secretory diarrheas resulting from intestinal

  4. Incidence and Carrier Frequency of CFTR Gene Mutations in Pregnancies With Echogenic Bowel in Nova Scotia and Prince Edward Island.

    Miller, Michelle E; Allen, Victoria M; Brock, Jo-Ann K

    2018-03-01

    Fetal echogenic bowel (echogenic bowel) is associated with cystic fibrosis (CF), with a reported incidence ranging from 1% to 13%. Prenatal testing for CF in the setting of echogenic bowel can be done by screening parental or fetal samples for pathogenic CFTR variants. If only one pathogenic variant is identified, sequencing of the CFTR gene can be undertaken, to identify a second pathogenic variant not covered in the standard screening panel. Full gene sequencing, however, also introduces the potential to identify variants of uncertain significance (VUSs) that can create counselling challenges and cause parental anxiety. To provide accurate counselling for families in the study population, the incidence of CF associated with echogenic bowel and the carrier frequency of CFTR variants were investigated. All pregnancies for which CF testing was undertaken for the indication of echogenic bowel (from Nova Scotia and Prince Edward Island) were identified (January 2007-July 2017). The CFTR screening and sequencing results were reviewed, and fetal outcomes related to CF were assessed. A total of 463 pregnancies with echogenic bowel were tested. Four were confirmed to be affected with CF, giving an incidence of 0.9% in this cohort. The carrier frequency of CF among all parents in the cohort was 5.0% (1 in 20); however, when excluding parents of affected fetuses, the carrier frequency for the population was estimated at 4.1% (1 in 25). CFTR gene sequencing identified an additional VUS in two samples. The incidence of CF in pregnancies with echogenic bowel in Nova Scotia and Prince Edward Island is 0.9%, with an estimated population carrier frequency of 4.1%. These results provide the basis for improved counselling to assess the risk of CF in the pregnancy, after parental carrier screening, using Bayesian probability. Counselling regarding VUSs should be undertaken before gene sequencing. Copyright © 2017 Society of Obstetricians and Gynaecologists of Canada. Published by

  5. Airway epithelial cell exposure to distinct e-cigarette liquid flavorings reveals toxicity thresholds and activation of CFTR by the chocolate flavoring 2,5-dimethypyrazine.

    Sherwood, Cara L; Boitano, Scott

    2016-05-17

    that the increases in ion conductance evoked by 2,5-dimethylpyrazine were largely attributed to a protein kinase A-dependent (PKA) activation of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel. Data from our high-capacity screening assays demonstrates that individual e-cigarette liquid flavoring chemicals vary in their cytotoxicity profiles and that some constituents evoke a cellular physiological response on their own independent of cell death. The activation of CFTR by 2,5-dimethylpyrazine may have detrimental consequences for airway surface liquid homeostasis in individuals that use e-cigarettes habitually.

  6. The importance of functional tests to assess the effect of a new CFTR variant when genotype-phenotype correlation is not possible.

    Hinzpeter, Alexandre; Reboul, Marie-Pierre; Callebaut, Isabelle; Zordan, Cécile; Costes, Bruno; Guichoux, Julie; Iron, Albert; Lacombe, Didier; Martin, Natacha; Arveiler, Benoit; Fanen, Pascale; Fergelot, Patricia; Girodon, Emmanuelle

    2017-05-01

    In vitro functional tests aimed to investigate CFTR dysfunction appear critical to help elucidate the functional impact of new variants of uncertain clinical significance and solve inconclusive cases, especially in early deceased newborns.

  7. Lipoxin A4 and platelet activating factor are involved in E. coli or LPS-induced lung inflammation in CFTR-deficient mice.

    Haiya Wu

    Full Text Available CFTR (cystic fibrosis transmembrane conductance regulator is expressed by both neutrophils and platelets. Lack of functional CFTR could lead to severe lung infection and inflammation. Here, we found that mutation of CFTR (F508del or inhibition of CFTR in mice led to more severe thrombocytopenia, alveolar neutrocytosis and bacteriosis, and lower lipoxin A4/MIP-2 (macrophage inhibitory protein-2 or lipoxin A4/neutrophil ratios in the BAL (bronchoalveolar lavage during acute E. coli pneumonia. In vitro, inhibition of CFTR promotes MIP-2 production in LPS-stimulated neutrophils; however, lipoxin A4 could dose-dependently suppress this effect. In LPS-induced acute lung inflammation, blockade of PSGL-1 (P-selectin glycoprotein ligand-1 or P-selectin, antagonism of PAF by WEB2086, or correction of mutated CFTR trafficking by KM11060 could significantly increase plasma lipoxin A4 levels in F508del relevant to wildtype mice. Concurrently, F508del mice had higher plasma platelet activating factor (PAF levels and PAF-AH activity compared to wildtype under LPS challenge. Inhibiting hydrolysis of PAF by a specific PAF-AH (PAF-acetylhydrolase inhibitor, MAFP, could worsen LPS-induced lung inflammation in F508del mice compared to vehicle treated F508del group. Particularly, depletion of platelets in F508del mice could significantly decrease plasma lipoxin A4 and PAF-AH activity and deteriorate LPS-induced lung inflammation compared to control F508del mice. Taken together, lipoxin A4 and PAF are involved in E. coli or LPS-induced lung inflammation in CFTR-deficient mice, suggesting that lipoxin A4 and PAF might be therapeutic targets for ameliorating CFTR-deficiency deteriorated lung inflammation.

  8. Nobiletin Stimulates Chloride Secretion in Human Bronchial Epithelia via a cAMP/PKA-Dependent Pathway

    Yuan Hao

    2015-08-01

    Full Text Available Background/Aims: Nobiletin, a citrus flavonoid isolated from tangerines, alters ion transport functions in intestinal epithelia, and has antagonistic effects on eosinophilic airway inflammation of asthmatic rats. The present study examined the effects of nobiletin on basal short-circuit current (ISC in a human bronchial epithelial cell line (16HBE14o-, and characterized the signal transduction pathways that allowed nobiletin to regulate electrolyte transport. Methods: The ISC measurement technique was used for transepithelial electrical measurements. Intracellular calcium ([Ca2+]i and cAMP were also quantified. Results: Nobiletin stimulated a concentration-dependent increase in ISC, which was due to Cl- secretion. The increase in ISC was inhibited by a cystic fibrosis transmembrane conductance regulator inhibitor (CFTRinh-172, but not by 4,4'-diisothiocyano-stilbene-2,2'-disulphonic acid (DIDS, Chromanol 293B, clotrimazole, or TRAM-34. Nobiletin-stimulated ISC was also sensitive to a protein kinase A (PKA inhibitor, H89, and an adenylate cyclase inhibitor, MDL-12330A. Nobiletin could not stimulate any increase in ISC in a cystic fibrosis (CF cell line, CFBE41o-, which lacked a functional CFTR. Nobiletin stimulated a real-time increase in cAMP, but not [Ca2+]i. Conclusion: Nobiletin stimulated transepithelial Cl- secretion across human bronchial epithelia. The mechanisms involved activation of adenylate cyclase- and cAMP/PKA-dependent pathways, leading to activation of apical CFTR Cl- channels.

  9. Tb3O2Cl[SeO3]2 and Tb5O4Cl3[SeO3]2: Oxide Chloride Oxoselenates(IV) of Trivalent Terbium with ''Lone-Pair'' Channel or Layer Structures

    Wontcheu, Joseph; Schleid, Thomas

    2005-01-01

    Orthorhombic Tb 3 O 2 Cl[SeO 3 ] 2 (Pnma; a = 535.16(4), b = 1530.51(9), c = 1081.72(7) pm; Z = 4) is formed by reacting a stoichiometric mixture of Tb 4 O 7 , Tb, TbCl 3 , and SeO 2 in a suitable molar ratio (12: 8: 7: 42) within seven days in an evacuated sealed silica tube at 850 C. The needle-shaped, colourless single crystals (light, water and air stable) exhibit one-dimensional strands [(Tb1) 3/3 (Tb2) 2/1 O 4/2 ] 5+ [O 2 Tb 3 ] 5+ along [100] formed by two parallel chains [OTb 4/2 ] 4+ of trans-edge connected [OTb 4 ] 10+ tetrahedra (d(O-Tb) = 220 - 231 pm) which share an extra edge per chain link. The crystal structure contains two crystallographically different Tb 3+ cations: Tb1 is coordinated as bicapped trigonal prism, while Tb2 resides in square antiprismatic coordination. The Se 4+ coordination is best described as Ψ 1 tetrahedral ([SeO 3 E] 2- ; E: non-binding electron pair). The non-binding ''lone-pair'' electrons of four [SeO 3 ] 2- groups and two Cl - anions form pseudo-hexagonal empty channels along [100] between four cationic double chains. Tb 5 O 4 Cl 3 [SeO 3 ] 2 was prepared likewise as plate-like, colourless single crystals by solid-state reaction of an admixture of Tb 4 O 7 , Tb, TbOCl, TbCl 3 , and SeO 2 (molar ratio: 9: 6: 21: 7: 28) in an evacuated sealed silica tube during seven days at 850 C. This compound crystallizes in the monoclinic system (C2/m; a = 1229.13(9), b = 546.17(4), c = 978.79(7) pm, β = 90.485(6) ; Z = 2) and contains three crystallographically different Tb 3+ cations in seven- and eightfold coordination of O 2- and Cl - anions, respectively. The crystal structure of Tb 5 O 4 Cl 3 [SeO 3 ] 2 is layered and built up of corrugated terbium-oxygen sheets [O 4 Tb 5 ] 7+ formed by edge- and vertex-shared [OTb 4 ] 10+ tetrahedra (d(O-Tb) = 226-232 pm) spreading parallel (001). The structure is strongly related to the ''lone-pair'' channel structures of Tb 2 O[SeO 3 ] 2 and Tb 3 O 2 Cl[SeO 3 ] 2 , where single ([OTb 2 ] 4

  10. Chloride is essential for contraction of afferent arterioles after agonists and potassium

    Jensen, B L; Ellekvist, Peter; Skøtt, O

    1997-01-01

    to norepinephrine, angiotensin II (ANG II), and potassium were measured after chloride depletion and compared with controls. Chloride depletion did not change arteriolar diameters, but the response to norepinephrine was markedly reduced when chloride was substituted with gluconate (n = 6) or isethionate (n = 6......). Reintroduction of chloride fully restored the sensitivity to norepinephrine. Contractions after ANG II and potassium were totally abolished in the absence of chloride (n = 6). In additional experiments (n = 7), the arteriolar contraction to 100 mM potassium was abolished only 1 min after removal of extracellular......A depolarizing chloride efflux has been suggested to activate voltage-dependent calcium channels in renal afferent arteriolar smooth muscle cells in response to vasoconstrictors. To test this proposal, rabbit afferent arterioles were microperfused, and the contractile dose responses...

  11. Chloride Transport in Heterogeneous Formation

    Mukherjee, A.; Holt, R. M.

    2017-12-01

    The chloride mass balance (CMB) is a commonly-used method for estimating groundwater recharge. Observations of the vertical distribution of pore-water chloride are related to the groundwater infiltration rates (i.e. recharge rates). In CMB method, the chloride distribution is attributed mainly to the assumption of one dimensional piston flow. In many places, however, the vertical distribution of chloride will be influenced by heterogeneity, leading to horizontal movement of infiltrating waters. The impact of heterogeneity will be particularly important when recharge is locally focused. When recharge is focused in an area, horizontal movement of chloride-bearing waters, coupled with upward movement driven by evapotranspiration, may lead to chloride bulges that could be misinterpreted if the CMB method is used to estimate recharge. We numerically simulate chloride transport and evaluate the validity of the CMB method in highly heterogeneous systems. This simulation is conducted for the unsaturated zone of Ogallala, Antlers, and Gatuna (OAG) formations in Andrews County, Texas. A two dimensional finite element model will show the movement of chloride through heterogeneous systems. We expect to see chloride bulges not only close to the surface but also at depths characterized by horizontal or upward movement. A comparative study of focused recharge estimates in this study with available recharge data will be presented.

  12. Les vésicules extracellulaires comme vecteurs de macromolécules bioactives : modèle du transporteur ABCC7 (CFTR) et application à la biothérapie de la mucoviscidose

    Vituret , Cyrielle

    2015-01-01

    Cystic fibrosis is a genetic disease in which its prognosis depends on the lung damage. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR), resulting in a dysfunctional CFTR protein normally located at the plasma membrane of epithelial cells. This thesis is a study of a novel therapeutic approach to use extracellular vesicles (EVs), microvesicles and exosomes, as transfer vectors for CFTR mRNA and protein to target cells. The proof of concept for ...

  13. Analysis of the CFTR gene in Venezuelan cystic fibrosis patients, identification of six novel cystic fibrosis-causing genetic variants

    Sánchez, Karen; De Mendonca,Elizabeth; Matute,Xiorama; Chaustre,Ismenia; Villalon,Marlene; Takiff,Howard

    2016-01-01

    Karen Sánchez,1 Elizabeth de Mendonca,1 Xiorama Matute,2 Ismenia Chaustre,2 Marlene Villalón,3 Howard Takiff4 1Unit of Genetic and Forensic Studies, Venezuelan Institute for Scientific Research (IVIC), 2Hospital JM de los Ríos, 3Hospital José Ignacio Baldo, Algodonal, National Reference Unit, 4Laboratory of Molecular Genetics, Venezuelan Institute for Scientific Research (IVIC), Caracas, Venezuela. Abstract: The mutations in the CFTR gene found in ...

  14. Analysis of the CFTR gene in Venezuelan cystic fibrosis patients, identification of six novel cystic fibrosis-causing genetic variants.

    Sánchez, Karen; de Mendonca, Elizabeth; Matute, Xiorama; Chaustre, Ismenia; Villalón, Marlene; Takiff, Howard

    2016-01-01

    The mutations in the CFTR gene found in patients with cystic fibrosis (CF) have geographic differences, but there are scant data on their prevalence in Venezuelan patients. This study determined the frequency of common CFTR gene mutations in a group of Venezuelan patients with CF. The 27 exons of the CFTR gene from 110 Venezuelan patients in the National CF Program were amplified and sequenced. A total of 36 different mutations were identified, seven with frequencies greater than 1%: p.Phe508del (27.27%), p.Gly542* (3.18%), c.2988+1G>A (3.18%), p.Arg334Trp (1.36%), p.Arg1162* (1.36%), c.1-8G>C (1.36%), and p.[Gly628Arg;Ser1235Arg](1.36). In 40% of patients, all with a clinical diagnosis of CF, no mutations were found. This report represents the largest cohort of Venezuelan patients with CF ever examined, and includes a wider mutation panel than has been previously studied in this population. Mutations common in Southern European populations predominate, and several new mutations were discovered, but no mutations were found in 40% of the cohort.

  15. Valyl benzyl ester chloride

    Grzegorz Dutkiewicz

    2010-02-01

    Full Text Available In the title compound (systematic name: 1-benzyloxy-3-methyl-1-oxobutan-2-aminium chloride, C12H18NO2+·Cl−, the ester group is approximately planar, with a maximum deviation of 0.040 (2 Å from the least-squares plane, and makes a dihedral angle of 28.92 (16° with the phenyl ring. The crystal structure is organized by N—H...Cl hydrogen bonds which join the two components into a chain along the b axis. Pairs of chains arranged antiparallel are interconnected by further N—H...Cl hydrogen bonds, forming eight-membered rings. Similar packing modes have been observed in a number of amino acid ester halides with a short unit-cell parameter of ca 5.5 Å along the direction in which the chains run.

  16. Chloride on the Move

    Li, Bo

    2017-01-09

    Chloride (Cl−) is an essential plant nutrient but under saline conditions it can accumulate to toxic levels in leaves; limiting this accumulation improves the salt tolerance of some crops. The rate-limiting step for this process – the transfer of Cl− from root symplast to xylem apoplast, which can antagonize delivery of the macronutrient nitrate (NO3−) to shoots – is regulated by abscisic acid (ABA) and is multigenic. Until recently the molecular mechanisms underpinning this salt-tolerance trait were poorly defined. We discuss here how recent advances highlight the role of newly identified transport proteins, some that directly transfer Cl− into the xylem, and others that act on endomembranes in ‘gatekeeper’ cell types in the root stele to control root-to-shoot delivery of Cl−.

  17. Novel CFTR missense mutations in Brazilian patients with congenital absence of vas deferens: counseling issues Mutações novas no gene CFTR de pacientes brasileiros portadores de agenesia dos vasos deferentes: dificuldades no aconselhamento

    Patricia de Campos Pieri

    2007-01-01

    Full Text Available PURPOSE: Screening for mutations in the entire Cystic Fibrosis gene (CFTR of Brazilian infertile men with congenital absence of vas deferens, in order to prevent transmission of CFTR mutations to offspring with the use of assisted reproductive technologies. METHOD: Specific polymerase chain reaction (PCR primers were designed to each of the 27 exons and splicing sites of interest followed by single strand conformational polymorphism and Heteroduplex Analysis (SSCP-HA in precast 12.5% polyacrylamide gels at 7ºC and 20ºC. Fragments with abnormal SSCP migration pattern were sequenced. RESULTS: Two novel missense mutations (S753R and G149W were found in three patients (two brothers together with the IVS8-5T allele in hetrozygosis. CONCLUSION: The available screenings for CF mutations do not include the atypical mutations associated to absence of vas deferens and thus, when these tests fail to find mutations, there is still a genetic risk of affected children with the help of assisted reproduction. We recommend the screening of the whole CFTR gene for these infertile couples, as part of the work-up before assisted reproduction.OBJETIVO: Pesquisar mutações em toda a extensão do gene que causa a Fibrose Cística (CFTR de homens brasileiros inférteis por agenesia congênita dos vasos deferentes, com a finalidade de prevenir a transmissão de mutações em CFTR à prole com o uso das tecnologias de reprodução assistida. MÉTODOS: Foram desenhados oligonucleotídeos específicos para realização de reação de polimerização em cadeia (PCR para cada um dos 27 exons e sítios de processamento de interesse no gene CFTR. O PCR foi seguido pela técnica de SSCP-HA (polimorfismos de conformação no DNA de fita simples e na formação de heteroduplexes em géis pré-fabricados de poliacrilamida a 12,5% em duas temperaturas, 7ºC e 20ºC. Os fragmentos com padrão alterado na migração do SSCP foram submetidos a seqüenciamento automatizado

  18. Funciones de los canales iónicos CFTR y ENAC en la fibrosis quística

    Alejandra G. Palma

    2014-04-01

    Full Text Available La fibrosis quística se debe a la ausencia o defecto del canal transmembrana regulador de la fibrosis quística (CFTR, un canal de cloruro codificado en el gen cftr que juega un papel clave en la homeostasis del agua e iones. El CFTR es activado por el AMPc y se localiza en las membranas apicales y basolaterales de las vías aéreas, intestino y glándulas exocrinas. Una de sus funciones primarias en los pulmones es mantener la capa de líquido superficial a través de su función de canal y regular el canal epitelial de sodio sensible al amiloride (ENaC. Se han identificado más de 1900 mutaciones en el gen cftr. La enfermedad se caracteriza por secreciones viscosas en las glándulas exocrinas y por niveles elevados de cloruro de sodio en el sudor. En la fibrosis quística el CFTR no funciona y el ENaC está desregulado; el resultado es un aumento en la reabsorción de sodio y agua con la formación de un líquido viscoso. En las glándulas sudoríparas tanto el Na+ como el Cl- se retienen en el lumen causando una pérdida de electrolitos durante la sudoración y el NaCl se elimina al sudor. Así, los niveles elevados de NaCl son la base del test del sudor inducido por pilocarpina, un método de diagnóstico para la enfermedad. En esta revisión se discuten los movimientos de Cl- y Na+ en las glándulas sudoríparas y pulmón así como el papel del ENaC en la patogénesis de la enfermedad.

  19. Cerium(terbium, erbium)chloride-choline chloride aqueous systems

    Gajfutdinova, R.K.; Zhuravlev, E.F.; Bikbaeva, G.G.; Domrachev, V.N.; Vanskova, G.I.

    1985-01-01

    To clarify the effect of rare earth nature on mutual solubility of rare earth salts and amines the solubility of solid phases in the systems, consisting of choline chloride, water and cerium, terbium, erbium chlorides, has been studied. It is established, that solubility isotherms of all the systems, testify to the formation of new solid phases of the composition: Ce(Tb, Er)xCl 3 x2C 5 H 14 ONClx3H 2 O. Individuality of new solid phases is proved by DTA method, the composition is confirmed by chemical analysis and data of PMR spectra, for choline chloride and its complexes with rare earth chlorides of the given composition PMR and IR spectra are studied

  20. Clinical expression of patients with the D1152H CFTR mutation.

    Terlizzi, Vito; Carnovale, Vincenzo; Castaldo, Giuseppe; Castellani, Carlo; Cirilli, Natalia; Colombo, Carla; Corti, Fabiola; Cresta, Federico; D'Adda, Alice; Lucarelli, Marco; Lucidi, Vincenzina; Macchiaroli, Annamaria; Madarena, Elisa; Padoan, Rita; Quattrucci, Serena; Salvatore, Donatello; Zarrilli, Federica; Raia, Valeria

    2015-07-01

    Discordant results were reported on the clinical expression of subjects bearing the D1152H CFTR mutation, and also for the small number of cases reported so far. A retrospective review of clinical, genetic and biochemical data was performed from individuals homozygous or compound heterozygous for the D1152H mutation followed in 12 Italian cystic fibrosis (CF) centers. 89 subjects carrying at least D1152H on one allele were identified. 7 homozygous patients had very mild clinical expression. Over half of the 74 subjects compound heterozygous for D1152H and a I-II-III class mutation had borderline or pathological sweat test and respiratory or gastrointestinal symptoms; one third had pulmonary bacteria colonization and 10/74 cases had complications (i.e. diabetes, allergic bronchopulmonary aspergillosis, and hemoptysis). However, their clinical expression was less severe as compared to a group of CF patients homozygous for the F508del mutation. Finally, 8 subjects compound heterozygous for D1152H and a IV-V class mutation showed very mild disease. The natural history of subjects bearing the D1152H mutation is widely heterogeneous and is influenced by the mutation in trans. Copyright © 2014 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

  1. Chloride removal from plutonium alloy

    Holcomb, H.P.

    1983-01-01

    SRP is evaluating a program to recover plutonium from a metallic alloy that will contain chloride salt impurities. Removal of chloride to sufficiently low levels to prevent damaging corrosion to canyon equipment is feasible as a head-end step following dissolution. Silver nitrate and mercurous nitrate were each successfully used in laboratory tests to remove chloride from simulated alloy dissolver solution containing plutonium. Levels less than 10 ppM chloride were achieved in the supernates over the precipitated and centrifuged insoluble salts. Also, less than 0.05% loss of plutonium in the +3, +4, or +6 oxidation states was incurred via precipitate carrying. These results provide impetus for further study and development of a plant-scale process to recover plutonium from metal alloy at SRP

  2. Dynamic electrochemical measurement of chloride ions

    Abbas, Yawar; de Graaf, Derk B.; Olthuis, Wouter; van den Berg, Albert

    2016-01-01

    This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver chloride electrode is used extensively for potentiometric measurement of chloride ions concentration in electrolyte. In this measurement,

  3. Producing ammonium chloride from coal or shale

    Christenson, O L

    1921-02-25

    Process of producing ammonium chloride consists of mixing the substance to be treated with a chloride of an alkali or alkaline earth metal, free silica, water and free hydrochloric acid, heating the mixture until ammonium chloride distills off and collecting the ammonium chloride.

  4. 21 CFR 184.1138 - Ammonium chloride.

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Ammonium chloride. 184.1138 Section 184.1138 Food... Specific Substances Affirmed as GRAS § 184.1138 Ammonium chloride. (a) Ammonium chloride (NH4Cl, CAS Reg. No. 12125-02-9) is produced by the reaction of sodium chloride and an ammonium salt in solution. The...

  5. 21 CFR 173.375 - Cetylpyridinium chloride.

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Cetylpyridinium chloride. 173.375 Section 173.375... CONSUMPTION Specific Usage Additives § 173.375 Cetylpyridinium chloride. Cetylpyridinium chloride (CAS Reg. No....1666 of this chapter, at a concentration of 1.5 times that of cetylpyridinium chloride. (c) The...

  6. 21 CFR 184.1622 - Potassium chloride.

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Potassium chloride. 184.1622 Section 184.1622 Food... Specific Substances Affirmed as GRAS § 184.1622 Potassium chloride. (a) Potassium chloride (KCl, CAS Reg... levels not to exceed current good manufacturing practice. Potassium chloride may be used in infant...

  7. 21 CFR 184.1426 - Magnesium chloride.

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Magnesium chloride. 184.1426 Section 184.1426 Food... Specific Substances Affirmed as GRAS § 184.1426 Magnesium chloride. (a) Magnesium chloride (MgC12·6H2O, CAS... hydrochloric acid solution and crystallizing out magnesium chloride hexahydrate. (b) The ingredient meets the...

  8. Adrenomedullin increases the short-circuit current in the mouse seminal vesicle: actions on chloride secretion.

    Liao, S B; Cheung, K H; O, W S; Tang, Fai

    2014-08-01

    Adrenomedullin (ADM) may regulate seminal vesicle fluid secretion, and this may affect sperm quality. In this study, we investigated the effect of ADM on chloride secretion in the mouse seminal vesicle. The presence of ADM in mouse seminal vesicle was confirmed using immunostaining, and the molecular species was determined using gel filtration chromatography coupled with enzyme-linked assay for ADM. The effects of ADM on chloride secretion were studied by short-circuit current technique in a whole-mount preparation of mouse seminal vesicle in an Ussing chamber. The effects of specific ADM and calcitonin gene-related peptide (CGRP) receptor antagonists were investigated. Whether the ADM effect depended on the cAMP- and/or calcium-activated chloride channel was also studied using specific chloride channel blockers. The results showed that ADM was present in seminal vesicle epithelial cells. The major molecular species was precursor in the mouse seminal vesicle. ADM increased short-circuit current through the calcium-activated chloride channel in mouse seminal vesicle, and CGRP receptor was involved. We conclude that ADM may regulate chloride and fluid secretion from the seminal vesicle, which may affect the composition of the seminal plasma bathing the sperm and, hence, fertility. © 2014 by the Society for the Study of Reproduction, Inc.

  9. The role of the UPS in cystic fibrosis

    Cyr Douglas M

    2007-11-01

    Full Text Available Abstract CF is an inherited autosomal recessive disease whose lethality arises from malfunction of CFTR, a single chloride (Cl- ion channel protein. CF patients harbor mutations in the CFTR gene that lead to misfolding of the resulting CFTR protein, rendering it inactive and mislocalized. Hundreds of CF-related mutations have been identified, many of which abrogate CFTR folding in the endoplasmic reticulum (ER. More than 70% of patients harbor the ΔF508 CFTR mutation that causes misfolding of the CFTR proteins. Consequently, mutant CFTR is unable to reach the apical plasma membrane of epithelial cells that line the lungs and gut, and is instead targeted for degradation by the UPS. Proteins located in both the cytoplasm and ER membrane are believed to identify misfolded CFTR for UPS-mediated degradation. The aberrantly folded CFTR protein then undergoes polyubiquitylation, carried out by an E1-E2-E3 ubiquitin ligase system, leading to degradation by the 26S proteasome. This ubiquitin-dependent loss of misfolded CFTR protein can be inhibited by the application of ‘corrector’ drugs that aid CFTR folding, shielding it from the UPS machinery. Corrector molecules elevate cellular CFTR protein levels by protecting the protein from degradation and aiding folding, promoting its maturation and localization to the apical plasma membrane. Combinatory application of corrector drugs with activator molecules that enhance CFTR Cl- ion channel activity offers significant potential for treatment of CF patients. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com.

  10. 46 CFR 151.50-34 - Vinyl chloride (vinyl chloride monomer).

    2010-10-01

    ... 46 Shipping 5 2010-10-01 2010-10-01 false Vinyl chloride (vinyl chloride monomer). 151.50-34... chloride (vinyl chloride monomer). (a) Copper, aluminum, magnesium, mercury, silver, and their alloys shall... equipment that may come in contact with vinyl chloride liquid or vapor. (b) Valves, flanges, and pipe...

  11. 40 CFR 61.65 - Emission standard for ethylene dichloride, vinyl chloride and polyvinyl chloride plants.

    2010-07-01

    ... dichloride, vinyl chloride and polyvinyl chloride plants. 61.65 Section 61.65 Protection of Environment... AIR POLLUTANTS National Emission Standard for Vinyl Chloride § 61.65 Emission standard for ethylene dichloride, vinyl chloride and polyvinyl chloride plants. An owner or operator of an ethylene dichloride...

  12. Role of ATP binding and hydrolysis in the gating of the cystic fibrosis transmembrane conductance regulator

    Taras Gout

    2012-01-01

    Full Text Available The CFTR gene is unique within the ATP-binding cassette (ABC protein family, predominantly of transporters, by coding a chloride channel. The gating mechanism of ABC proteins has been characterized by the ATP Switch model in terms cycles of dimer formation and dissociation linked to ATP binding and hydrolysis, respectively. It would be of interest to assess the extent that Cystic Fibrosis Transmembrane Conductance Regulator (CFTR, a functional channel, fits the ATP Switch model for ABC transporters. Additional transporter mechanisms, namely those of Pgp and HlyB, are discussed for perspective. Literature search of databases selected key references in comparing and contrasting the gating mechanism. CFTR is a functional chloride channel facilitating transmembrane anion flow down electrochemical gradients. A dysfunctional CFTR protein results in cystic fibrosis, a fatal pleiotropic disease currently managed symptomatically. Understanding the gating mechanism will help target drug development aimed at alleviating and curing the disease.

  13. 21 CFR 184.1297 - Ferric chloride.

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Ferric chloride. 184.1297 Section 184.1297 Food and... Substances Affirmed as GRAS § 184.1297 Ferric chloride. (a) Ferric chloride (iron (III) chloride, FeC13, CAS Reg. No. 7705-08-0) may be prepared from iron and chlorine or from ferric oxide and hydrogen chloride...

  14. Preparation of pure anhydrous rare earth chlorides

    Bel'kova, N.L.; Slastenova, N.M.; Batyaev, I.M.; Solov'ev, M.A.

    1979-01-01

    A method has been suggested for obtaining extra-pure anhydrous REE chlorides by chloridizing corresponding oxalates by chlorine in a fluid bed, the chloridizing agents being diluted by an inert gas in a ratio of 2-to-1. The method is applicable to the manufacture of quality chlorides not only of light, but also of heavy REE. Neodymium chloride has an excited life of tau=30 μs, this evidencing the absence of the damping impurities

  15. Dendrimer-based selective autophagy-induction rescues ΔF508-CFTR and inhibits Pseudomonas aeruginosa infection in cystic fibrosis.

    Scott Mackenzie Brockman

    Full Text Available Cystic Fibrosis (CF is a genetic disorder caused by mutation(s in the CF-transmembrane conductance regulator (Cftr gene. The most common mutation, ΔF508, leads to accumulation of defective-CFTR protein in aggresome-bodies. Additionally, Pseudomonas aeruginosa (Pa, a common CF pathogen, exacerbates obstructive CF lung pathology. In the present study, we aimed to develop and test a novel strategy to improve the bioavailability and potentially achieve targeted drug delivery of cysteamine, a potent autophagy-inducing drug with anti-bacterial properties, by developing a dendrimer (PAMAM-DEN-based cysteamine analogue.We first evaluated the effect of dendrimer-based cysteamine analogue (PAMAM-DENCYS on the intrinsic autophagy response in IB3-1 cells and observed a significant reduction in Ub-RFP and LC3-GFP co-localization (aggresome-bodies by PAMAM-DENCYS treatment as compared to plain dendrimer (PAMAM-DEN control. Next, we observed that PAMAM-DENCYS treatment shows a modest rescue of ΔF508-CFTR as the C-form. Moreover, immunofluorescence microscopy of HEK-293 cells transfected with ΔF508-CFTR-GFP showed that PAMAM-DENCYS is able to rescue the misfolded-ΔF508-CFTR from aggresome-bodies by inducing its trafficking to the plasma membrane. We further verified these results by flow cytometry and observed significant (p<0.05; PAMAM-DEN vs. PAMAM-DENCYS rescue of membrane-ΔF508-CFTR with PAMAM-DENCYS treatment using non-permeabilized IB3-1 cells immunostained for CFTR. Finally, we assessed the autophagy-mediated bacterial clearance potential of PAMAM-DENCYS by treating IB3-1 cells infected with PA01-GFP, and observed a significant (p<0.01; PAMAM-DEN vs. PAMAM-DENCYS decrease in intracellular bacterial counts by immunofluorescence microscopy and flow cytometry. Also, PAMAM-DENCYS treatment significantly inhibits the growth of PA01-GFP bacteria and demonstrates potent mucolytic properties.We demonstrate here the efficacy of dendrimer-based autophagy

  16. Reaction of calcium chloride with alkali metal chlorides in melts

    Savin, V.D.; Mikhajlova, N.P.

    1984-01-01

    Thermochemical characteristics of CaCl 2 reaction with sodium, potassium, rubidium and cesium chlorides in melts at 890 deg C are determined. The values of formation enthalpies of infinitely diluted by CaCl 2 solutions (ΔH) in the chloride row increase from -22 in NaCl to -47 kJ/mol of CaCl 2 in CsCl. With increasing the concentration of calcium chloride in the solution the ΔH values decrease. The regularities of separation from the solution of the CaCl 2 -CsCl system at 890 deg C of the CaCl 2 x CsCl in solid are studied. Formation enthalpies under the given conditions constitutes -70+-3 kJ/mol

  17. Genomic sequencing in cystic fibrosis newborn screening: what works best, two-tier predefined CFTR mutation panels or second-tier CFTR panel followed by third-tier sequencing?

    Currier, Robert J; Sciortino, Stan; Liu, Ruiling; Bishop, Tracey; Alikhani Koupaei, Rasoul; Feuchtbaum, Lisa

    2017-10-01

    PurposeThe purpose of this study was to model the performance of several known two-tier, predefined mutation panels and three-tier algorithms for cystic fibrosis (CF) screening utilizing the ethnically diverse California population.MethodsThe cystic fibrosis transmembrane conductance regulator (CFTR) mutations identified among the 317 CF cases in California screened between 12 August 2008 and 18 December 2012 were used to compare the expected CF detection rates for several two- and three-tier screening approaches, including the current California approach, which consists of a population-specific 40-mutation panel followed by third-tier sequencing when indicated.ResultsThe data show that the strategy of using third-tier sequencing improves CF detection following an initial elevated immunoreactive trypsinogen and detection of only one mutation on a second-tier panel.ConclusionIn a diverse population, the use of a second-tier panel followed by third-tier CFTR gene sequencing provides a better detection rate for CF, compared with the use of a second-tier approach alone, and is an effective way to minimize the referrals of CF carriers for sweat testing. Restricting screening to a second-tier testing to predefined mutation panels, even broad ones, results in some missed CF cases and demonstrates the limited utility of this approach in states that have diverse multiethnic populations.

  18. Electrochemical Chloride extraction using external electrodes?

    Ottosen, Lisbeth M.; Pedersen, Anne Juul

    2006-01-01

    Electrochemical methods for the removal of chloride from concrete have been developed and the methods are primarily designed for situations where corrosion has started due to an increased chloride concentration in the vicinity of the reinforcement. In these methods the reinforcement is used...... as the cathode. However, some unwanted side effects can occur, including alkali-silica reaction and in some cases hydrogen embrittlement. It is also suggested also to use electrochemical chloride extraction in a preventive way in constructions where chloride induced corrosion is likely to be a problem after...... a period of time, i.e. remove the chlorides before the chloride front reaches the reinforcement. If the chlorides are removed from outer few centimetres from the surface, the chloride will not reach the reinforcement and cause damage. By using the electrochemical chloride removal in this preventive way...

  19. Lumacaftor/ivacaftor, a novel agent for the treatment of cystic fibrosis patients who are homozygous for the F580del CFTR mutation.

    Bulloch, Marilyn N; Hanna, Cameron; Giovane, Richard

    2017-10-01

    Cystic Fibrosis (CF) is an autosomal recessive disease affecting up to 90,000 people worldwide. Approximately 73% of patients are homozygous for the F508del cystic fibrosis transmembrane conductance regulator [CFTR] mutation. Traditionally treatment has only included supportive care. Therefore, there is a need for safe and effective novel therapies targeting the underlying molecular defects seen with CF. Areas covered: In 2016, the Food and Drug Administration and the European Commission approved LUM/IVA (Orkambi), a CFTR modulator that includes both a CFTR corrector and potentiator, for CF patients homozygous for the F508del CFTR mutation. This article reviews the pharmacologic features, clinical efficacy, and safety of LUM/IVA and summarize the available pre-clinical and clinical data of LUM/IVA use. Expert commentary: LUM/IVA showed modest, but significant improvements from baseline in percent predicted FEV 1 (ppFEV 1 ) as well as a reduction in pulmonary exacerbations by 35% It was shown to be safe for short- and long-term use. Currently, LUM/IVA is the only oral agent in its class available and represents a milestone the development of therapies for the management of CF. Nonetheless, pharmacoeconomic data are necessary to justify its high cost before is use becomes standard of care.

  20. Ion channeling

    Erramli, H.; Blondiaux, G.

    1994-01-01

    Channeling phenomenon was predicted, many years ago, by stark. The first channeling experiments were performed in 1963 by Davies and his coworkers. Parallely Robinson and Oen have investigated this process by simulating trajectories of ions in monocrystals. This technique has been combined with many methods like Rutherford Backscattering Spectrometry (R.B.S.), Particles Induced X-rays Emission (P.I.X.E) and online Nuclear Reaction (N.R.A.) to localize trace elements in the crystal or to determine crystalline quality. To use channeling for material characterization we need data about the stopping power of the incident particle in the channeled direction. The ratios of channeled to random stopping powers of silicon for irradiation in the direction have been investigated and compared to the available theoretical results. We describe few applications of ion channeling in the field of materials characterization. Special attention is given to ion channeling combined with Charged Particle Activation Analysis (C.P.A.A.) for studying the behaviour of oxygen atoms in Czochralski silicon lattices under the influence of internal gettering and in different gaseous atmospheres. Association between ion channeling and C.P.A.A was also utilised for studying the influence of the growing conditions on concentration and position of carbon atoms at trace levels in the MOVPE Ga sub (1-x) Al sub x lattice. 6 figs., 1 tab., 32 refs. (author)

  1. Rhenium corrosion in chloride melts

    Stepanov, A.D.; Shkol'nikov, S.N.; Vetyukov, M.M.

    1989-01-01

    The results investigating rhenium corrosion in chloride melts containing sodium, potassium and chromium ions by a gravimetry potentials in argon atmosphere in a sealing quarth cell are described. Rhenium corrosion is shown to be rather considerable in melts containing CrCl 2 . The value of corrosion rate depending on temperature is determined

  2. Elicitation threshold of cobalt chloride

    Fischer, Louise A; Johansen, Jeanne D; Voelund, Aage

    2016-01-01

    : On the basis of five included studies, the ED10 values of aqueous cobalt chloride ranged between 0.0663 and 1.95 µg cobalt/cm(2), corresponding to 30.8-259 ppm. CONCLUSIONS: Our analysis provides an overview of the doses of cobalt that are required to elicit allergic cobalt contactdermatitis in sensitized...

  3. Surface Chloride Levels in Colorado Structural Concrete

    2018-01-01

    This project focused on the chloride-induced corrosion of reinforcing steel in structural concrete. The primary goal of this project is to analyze the surface chloride concentration level of the concrete bridge decks throughout Colorado. The study in...

  4. Pharmacokinetics of vinyl chloride in the rat

    Bolt, H.M.; Laib, R.J.; Kappus, H.; Buchter, A.

    1977-01-01

    When rats are exposed to [ 14 C]vinyl chloride in a closed system, the vinyl chloride present in the atmosphere equilibrates with the animals' organism within 15 min. The course of equilibration could be determined using rats which had been given 6-nitro-1,2,3-benzothiadiazole. This compound completely blocks metabolism of vinyl chloride. The enzymes responsible for metabolism of vinyl chloride are saturated at an atmospheric concentration of vinyl chloride of 250 ppm. Pharmacokinetic analysis shows that no significant cumulation of vinyl chloride or its major metabolites is to be expected on repeated administration of vinyl chlorides. This may be consistent with the theory that a reactive, shortly living metabolite which occurs in low concentration only, may be responsible for the toxic effects of vinyl chloride

  5. Compounds that correct F508del-CFTR trafficking can also correct other protein trafficking diseases: an in vitro study using cell lines

    Sampson Heidi M

    2013-01-01

    Full Text Available Abstract Background Many genetic diseases are due to defects in protein trafficking where the mutant protein is recognized by the quality control systems, retained in the endoplasmic reticulum (ER, and degraded by the proteasome. In many cases, the mutant protein retains function if it can be trafficked to its proper cellular location. We have identified structurally diverse correctors that restore the trafficking and function of the most common mutation causing cystic fibrosis, F508del-CFTR. Most of these correctors do not act directly as ligands of CFTR, but indirectly on other pathways to promote folding and correction. We hypothesize that these proteostasis regulators may also correct other protein trafficking diseases. Methods To test our hypothesis, we used stable cell lines or transient transfection to express 2 well-studied trafficking disease mutations in each of 3 different proteins: the arginine-vasopressin receptor 2 (AVPR2, also known as V2R, the human ether-a-go-go-related gene (KCNH2, also known as hERG, and finally the sulfonylurea receptor 1 (ABCC8, also known as SUR1. We treated cells expressing these mutant proteins with 9 structurally diverse F508del-CFTR correctors that function through different cellular mechanisms and assessed whether correction occurred via immunoblotting and functional assays. Results were deemed significantly different from controls by a one-way ANOVA (p  Results Here we show that F508del-CFTR correctors RDR1, KM60 and KM57 also correct some mutant alleles of other protein trafficking diseases. We also show that one corrector, the cardiac glycoside ouabain, was found to alter the glycosylation of all mutant alleles tested. Conclusions Correctors of F508del-CFTR trafficking might have broader applications to other protein trafficking diseases.

  6. 21 CFR 184.1193 - Calcium chloride.

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Calcium chloride. 184.1193 Section 184.1193 Food... Specific Substances Affirmed as GRAS § 184.1193 Calcium chloride. (a) Calcium chloride (CaCl2·2H2O, CAS Reg. No. 10035-04-8) or anhydrous calcium chloride (CaCl2, CAS Reg. No. 10043-52-4) may be commercially...

  7. Microbial reductive dehalogenation of vinyl chloride

    Spormann, Alfred M [Stanford, CA; Muller, Jochen A [Baltimore, MD; Rosner, Bettina M [Berlin, DE; Von Abendroth, Gregory [Nannhein, DE; Meshulam-Simon, Galit [Los Altos, CA; McCarty, Perry L [Stanford, CA

    2011-11-22

    Compositions and methods are provided that relate to the bioremediation of chlorinated ethenes, particularly the bioremediation of vinyl chloride by Dehalococcoides-like organisms. An isolated strain of bacteria, Dehalococcoides sp. strain VS, that metabolizes vinyl chloride is provided; the genetic sequence of the enzyme responsible for vinyl chloride dehalogenation; methods of assessing the capability of endogenous organisms at an environmental site to metabolize vinyl chloride; and a method of using the strains of the invention for bioremediation.

  8. Channel box

    Tanabe, Akira.

    1993-01-01

    In a channel box of a BWR type reactor, protruding pads are disposed in axial position on the lateral side of a channel box opposing to a control rod and facing the outer side portion of the control rod in a reactor core loaded state. In the initial loading stage of fuel assemblies, channel fasteners and spacer pads are abutted against each other in the upper portion between the channel boxes sandwiching the control rod therebetween. Further, in the lower portion, a gap as a channel for the movement of the control rod is ensured by the support of fuel support metals. If the channel box is bent toward the control rod along with reactor operation, the pads are abutted against each other to always ensure the gap through which the control rod can move easily. Further, when the pads are brought into contact with each other, the bending deformation of the channel box is corrected by urging to each other. Thus, the control rod can always be moved smoothly to attain reactor safety operation. (N.H.)

  9. 21 CFR 173.255 - Methylene chloride.

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Methylene chloride. 173.255 Section 173.255 Food... Solvents, Lubricants, Release Agents and Related Substances § 173.255 Methylene chloride. Methylene chloride may be present in food under the following conditions: (a) In spice oleoresins as a residue from...

  10. 21 CFR 182.8252 - Choline chloride.

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Choline chloride. 182.8252 Section 182.8252 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... chloride. (a) Product. Choline chloride. (b) Conditions of use. This substance is generally recognized as...

  11. 21 CFR 184.1446 - Manganese chloride.

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Manganese chloride. 184.1446 Section 184.1446 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... Specific Substances Affirmed as GRAS § 184.1446 Manganese chloride. (a) Manganese chloride (MnCl2·4H2O, CAS...

  12. 21 CFR 582.5985 - Zinc chloride.

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Zinc chloride. 582.5985 Section 582.5985 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS... 1 § 582.5985 Zinc chloride. (a) Product. Zinc chloride. (b) Conditions of use. This substance is...

  13. 21 CFR 582.3845 - Stannous chloride.

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Stannous chloride. 582.3845 Section 582.3845 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL....3845 Stannous chloride. (a) Product. Stannous chloride. (b) Tolerance. This substance is generally...

  14. 21 CFR 582.6193 - Calcium chloride.

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Calcium chloride. 582.6193 Section 582.6193 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Calcium chloride. (a) Product. Calcium chloride. (b) Conditions of use. This substance is generally...

  15. 21 CFR 582.5446 - Manganese chloride.

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Manganese chloride. 582.5446 Section 582.5446 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Supplements 1 § 582.5446 Manganese chloride. (a) Product. Manganese chloride. (b) Conditions of use. This...

  16. 21 CFR 182.8985 - Zinc chloride.

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Zinc chloride. 182.8985 Section 182.8985 Food and... CONSUMPTION (CONTINUED) SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients § 182.8985 Zinc chloride. (a) Product. Zinc chloride. (b) Conditions of use. This substance is generally recognized as safe when used in...

  17. 21 CFR 172.180 - Stannous chloride.

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Stannous chloride. 172.180 Section 172.180 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN... Preservatives § 172.180 Stannous chloride. The food additive stannous chloride may be safely used for color...

  18. 49 CFR 173.322 - Ethyl chloride.

    2010-10-01

    ... 49 Transportation 2 2010-10-01 2010-10-01 false Ethyl chloride. 173.322 Section 173.322 Transportation Other Regulations Relating to Transportation PIPELINE AND HAZARDOUS MATERIALS SAFETY... SHIPMENTS AND PACKAGINGS Gases; Preparation and Packaging § 173.322 Ethyl chloride. Ethyl chloride must be...

  19. 21 CFR 582.5252 - Choline chloride.

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Choline chloride. 582.5252 Section 582.5252 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Supplements 1 § 582.5252 Choline chloride. (a) Product. Choline chloride. (b) Conditions of use. This...

  20. 21 CFR 582.5622 - Potassium chloride.

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Potassium chloride. 582.5622 Section 582.5622 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Supplements 1 § 582.5622 Potassium chloride. (a) Product. Potassium chloride. (b) Conditions of use. This...

  1. 21 CFR 582.1193 - Calcium chloride.

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Calcium chloride. 582.1193 Section 582.1193 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1193 Calcium chloride. (a) Product. Calcium chloride. (b) Conditions of use. This substance...

  2. 7 CFR 58.434 - Calcium chloride.

    2010-01-01

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

  3. 21 CFR 173.400 - Dimethyldialkylammonium chloride.

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Dimethyldialkylammonium chloride. 173.400 Section... HUMAN CONSUMPTION Specific Usage Additives § 173.400 Dimethyldialkylammonium chloride. Dimethyldialkylammonium chloride may be safely used in food in accordance with the following prescribed conditions: (a...

  4. Structure and Dynamics of NBD1 from CFTR Characterized Using Crystallography and Hydrogen/Deuterium Exchange Mass Spectrometry

    Lewis, H.A.; Wang, C.; Zhao, X.; Hamuro, Y.; Conners, K.; Kearins, M.C.; Lu, F.; Sauder, J.M.; Molnar, K.S.; Coales, S.J.; Maloney, P.C.; Guggino, W.B.; Wetmore, D.R.; Weber, P.C.; Hunt, J.F. (SGX); (ExSAR); (Cystic); (JHU-MED); (Columbia)

    2012-04-30

    The {Delta}F508 mutation in nucleotide-binding domain 1 (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the predominant cause of cystic fibrosis. Previous biophysical studies on human F508 and {Delta}F508 domains showed only local structural changes restricted to residues 509-511 and only minor differences in folding rate and stability. These results were remarkable because {Delta}F508 was widely assumed to perturb domain folding based on the fact that it prevents trafficking of CFTR out of the endoplasmic reticulum. However, the previously reported crystal structures did not come from matched F508 and {Delta}F508 constructs, and the {Delta}F508 structure contained additional mutations that were required to obtain sufficient protein solubility. In this article, we present additional biophysical studies of NBD1 designed to address these ambiguities. Mass spectral measurements of backbone amide {sup 1}H/{sup 2}H exchange rates in matched F508 and {Delta}F508 constructs reveal that {Delta}F508 increases backbone dynamics at residues 509-511 and the adjacent protein segments but not elsewhere in NBD1. These measurements also confirm a high level of flexibility in the protein segments exhibiting variable conformations in the crystal structures. We additionally present crystal structures of a broader set of human NBD1 constructs, including one harboring the native F508 residue and others harboring the {Delta}F508 mutation in the presence of fewer and different solubilizing mutations. The only consistent conformational difference is observed at residues 509-511. The side chain of residue V510 in this loop is mostly buried in all non-{Delta}F508 structures but completely solvent exposed in all {Delta}F508 structures. These results reinforce the importance of the perturbation {Delta}F508 causes in the surface topography of NBD1 in a region likely to mediate contact with the transmembrane domains of CFTR. However, they also suggest that increased

  5. Surface channeling

    Sizmann, R.; Varelas, C.

    1976-01-01

    There is experimental evidence that swift light ions incident at small angles towards single crystalline surfaces can lose an appreciable fraction of their kinetic energy during reflection. It is shown that these projectiles penetrate into the bulk surface region of the crystal. They can travel as channeled particles along long paths through the solid (surface channeling). The angular distribution and the depth history of the re-emerged projectiles are investigated by computer simulations. A considerable fraction of the penetrating projectiles re-emerges from the crystal with constant transverse energy if the angle of incidence is smaller than the critical angle for axial channeling. Analytical formulae are derived based on a diffusion model for surface channeling. A comparison with experimental data exhibits the relevance of the analytical solutions. (Auth.)

  6. Targeting a genetic defect: cystic fibrosis transmembrane conductance regulator modulators in cystic fibrosis

    Nico Derichs

    2013-03-01

    Full Text Available Cystic fibrosis (CF is caused by genetic mutations that affect the cystic fibrosis transmembrane conductance regulator (CFTR protein. These mutations can impact the synthesis and transfer of the CFTR protein to the apical membrane of epithelial cells, as well as influencing the gating or conductance of chloride and bicarbonate ions through the channel. CFTR dysfunction results in ionic imbalance of epithelial secretions in several organ systems, such as the pancreas, gastrointestinal tract, liver and the respiratory system. Since discovery of the CFTR gene in 1989, research has focussed on targeting the underlying genetic defect to identify a disease-modifying treatment for CF. Investigated management strategies have included gene therapy and the development of small molecules that target CFTR mutations, known as CFTR modulators. CFTR modulators are typically identified by high-throughput screening assays, followed by preclinical validation using cell culture systems. Recently, one such modulator, the CFTR potentiator ivacaftor, was approved as an oral therapy for CF patients with the G551D-CFTR mutation. The clinical development of ivacaftor not only represents a breakthrough in CF care but also serves as a noteworthy example of personalised medicine.

  7. [Channels: a new way to revisit pathology].

    Fournier, Emmanuel

    2014-02-01

    Many "essential" diseases that manifest themselves in the form of crises or fits (epilepsies, episodic ataxia, periodic paralyses, myotonia, heart rhythm disorders, etc.) are due to ionic channel dysfunction and are thus referred to as "channelopathies". Some of these disorders are congenital, due to mutations of genes encoding channel subunits, while others result from toxic, immune or hormonal disturbances affecting channelfunction. Channelopathies take on a wide variety of clinical forms, depending on the type of channel (sodium, potassium, calcium, chloride...) and the type of dysfunction (loss or gain of function). Some apparently unrelated diseases affecting distinct organs are due to a similar dysfunction of the same channel, revealing unsuspected relationships between organs and between medical specialties. In addition, a given syndrome can be caused by distinct channel dysfunctions. This provides new opportunities for diferential diagnosis and specific correction of the causal defects, although some treatments find applications across multiple medical specialties.

  8. Spark Channels

    Haydon, S. C. [Department of Physics, University of New England, Armidale, NSW (Australia)

    1968-04-15

    A brief summary is given of the principal methods used for initiating spark channels and the various highly time-resolved techniques developed recently for studies with nanosecond resolution. The importance of the percentage overvoltage in determining the early history and subsequent development of the various phases of the growth of the spark channel is discussed. An account is then given of the recent photographic, oscillographic and spectroscopic investigations of spark channels initiated by co-axial cable discharges of spark gaps at low [{approx} 1%] overvoltages. The phenomena observed in the development of the immediate post-breakdown phase, the diffuse glow structure, the growth of the luminous filament and the final formation of the spark channel in hydrogen are described. A brief account is also given of the salient features emerging from corresponding studies of highly overvolted spark gaps in which the spark channel develops from single avalanche conditions. The essential differences between the two types of channel formation are summarized and possible explanations of the general features are indicated. (author)

  9. A large-scale study of the random variability of a coding sequence: a study on the CFTR gene.

    Modiano, Guido; Bombieri, Cristina; Ciminelli, Bianca Maria; Belpinati, Francesca; Giorgi, Silvia; Georges, Marie des; Scotet, Virginie; Pompei, Fiorenza; Ciccacci, Cinzia; Guittard, Caroline; Audrézet, Marie Pierre; Begnini, Angela; Toepfer, Michael; Macek, Milan; Ferec, Claude; Claustres, Mireille; Pignatti, Pier Franco

    2005-02-01

    Coding single nucleotide substitutions (cSNSs) have been studied on hundreds of genes using small samples (n(g) approximately 100-150 genes). In the present investigation, a large random European population sample (average n(g) approximately 1500) was studied for a single gene, the CFTR (Cystic Fibrosis Transmembrane conductance Regulator). The nonsynonymous (NS) substitutions exhibited, in accordance with previous reports, a mean probability of being polymorphic (q > 0.005), much lower than that of the synonymous (S) substitutions, but they showed a similar rate of subpolymorphic (q < 0.005) variability. This indicates that, in autosomal genes that may have harmful recessive alleles (nonduplicated genes with important functions), genetic drift overwhelms selection in the subpolymorphic range of variability, making disadvantageous alleles behave as neutral. These results imply that the majority of the subpolymorphic nonsynonymous alleles of these genes are selectively negative or even pathogenic.

  10. Removal of chloride from MSWI fly ash.

    Chen, Wei-Sheng; Chang, Fang-Chih; Shen, Yun-Hwei; Tsai, Min-Shing; Ko, Chun-Han

    2012-10-30

    The high levels of alkali chloride and soluble metal salts present in MSWI fly ash is worth noting for their impact on the environment. In addition, the recycling or reuse of fly ash has become an issue because of limited landfill space. The chloride content in fly ash limits its application as basis for construction materials. Water-soluble chlorides such as potassium chloride (KCl), sodium chloride (NaCl), and calcium chloride hydrate (CaCl(2) · 2H(2)O) in fly ash are easily washed away. However, calcium chloride hydroxide (Ca(OH)Cl) might not be easy to leach away at room temperature. The roasting and washing-flushing processes were applied to remove chloride content in this study. Additionally, air and CO(2) were introduced into the washing process to neutralize the hazardous nature of chlorides. In comparison with the water flushing process, the roasting process is more efficient in reducing the process of solid-liquid separation and drying for the reuse of Cl-removed fly ash particles. In several roasting experiments, the removal of chloride content from fly ash at 1050°C for 3h showed the best results (83% chloride removal efficiency). At a solid to liquid ratio of 1:10 the water-flushing process can almost totally remove water-soluble chloride (97% chloride removal efficiency). Analyses of mineralogical change also prove the efficiency of the fly ash roasting and washing mechanisms for chloride removal. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Impact of the CFTR-potentiator ivacaftor on airway microbiota in cystic fibrosis patients carrying a G551D mutation.

    Cédric Bernarde

    Full Text Available Airway microbiota composition has been clearly correlated with many pulmonary diseases, and notably with cystic fibrosis (CF, an autosomal genetic disorder caused by mutation in the CF transmembrane conductance regulator (CFTR. Recently, a new molecule, ivacaftor, has been shown to re-establish the functionality of the G551D-mutated CFTR, allowing significant improvement in lung function.The purpose of this study was to follow the evolution of the airway microbiota in CF patients treated with ivacaftor, using quantitative PCR and pyrosequencing of 16S rRNA amplicons, in order to identify quantitative and qualitative changes in bacterial communities. Three G551D children were followed up longitudinally over a mean period of more than one year covering several months before and after initiation of ivacaftor treatment.129 operational taxonomy units (OTUs, representing 64 genera, were identified. There was no significant difference in total bacterial load before and after treatment. Comparison of global community composition found no significant changes in microbiota. Two OTUs, however, showed contrasting dynamics: after initiation of ivacaftor, the relative abundance of the anaerobe Porphyromonas 1 increased (p<0.01 and that of Streptococcus 1 (S. mitis group decreased (p<0.05, possibly in relation to the anti-Gram-positive properties of ivacaftor. The anaerobe Prevotella 2 correlated positively with the pulmonary function test FEV-1 (r=0.73, p<0.05. The study confirmed the presumed positive role of anaerobes in lung function.Several airway microbiota components, notably anaerobes (obligate or facultative anaerobes, could be valuable biomarkers of lung function improvement under ivacaftor, and could shed light on the pathophysiology of lung disease in CF patients.

  12. The CFTR Met 470 allele is associated with lower birth rates in fertile men from a population isolate.

    Gülüm Kosova

    2010-06-01

    Full Text Available Although little is known about the role of the cystic fibrosis transmembrane regulator (CFTR gene in reproductive physiology, numerous variants in this gene have been implicated in etiology of male infertility due to congenital bilateral absence of the vas deferens (CBAVD. Here, we studied the fertility effects of three CBAVD-associated CFTR polymorphisms, the (TGm and polyT repeat polymorphisms in intron 8 and Met470Val in exon 10, in healthy men of European descent. Homozygosity for the Met470 allele was associated with lower birth rates, defined as the number of births per year of marriage (P = 0.0029. The Met470Val locus explained 4.36% of the phenotypic variance in birth rate, and men homozygous for the Met470 allele had 0.56 fewer children on average compared to Val470 carrier men. The derived Val470 allele occurs at high frequencies in non-African populations (allele frequency = 0.51 in HapMap CEU, whereas it is very rare in African population (Fst = 0.43 between HapMap CEU and YRI. In addition, haplotypes bearing Val470 show a lack of genetic diversity and are thus longer than haplotypes bearing Met470 (measured by an integrated haplotype score [iHS] of -1.93 in HapMap CEU. The fraction of SNPs in the HapMap Phase2 data set with more extreme Fst and iHS measures is 0.003, consistent with a selective sweep outside of Africa. The fertility advantage conferred by Val470 relative to Met470 may provide a selective mechanism for these population genetic observations.

  13. Invariant TAD Boundaries Constrain Cell-Type-Specific Looping Interactions between Promoters and Distal Elements around the CFTR Locus.

    Smith, Emily M; Lajoie, Bryan R; Jain, Gaurav; Dekker, Job

    2016-01-07

    Three-dimensional genome structure plays an important role in gene regulation. Globally, chromosomes are organized into active and inactive compartments while, at the gene level, looping interactions connect promoters to regulatory elements. Topologically associating domains (TADs), typically several hundred kilobases in size, form an intermediate level of organization. Major questions include how TADs are formed and how they are related to looping interactions between genes and regulatory elements. Here we performed a focused 5C analysis of a 2.8 Mb chromosome 7 region surrounding CFTR in a panel of cell types. We find that the same TAD boundaries are present in all cell types, indicating that TADs represent a universal chromosome architecture. Furthermore, we find that these TAD boundaries are present irrespective of the expression and looping of genes located between them. In contrast, looping interactions between promoters and regulatory elements are cell-type specific and occur mostly within TADs. This is exemplified by the CFTR promoter that in different cell types interacts with distinct sets of distal cell-type-specific regulatory elements that are all located within the same TAD. Finally, we find that long-range associations between loci located in different TADs are also detected, but these display much lower interaction frequencies than looping interactions within TADs. Interestingly, interactions between TADs are also highly cell-type-specific and often involve loci clustered around TAD boundaries. These data point to key roles of invariant TAD boundaries in constraining as well as mediating cell-type-specific long-range interactions and gene regulation. Copyright © 2016 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  14. Ultrastructural Observation of the Skin Chloride Cells of Japanese Flounder Paralichthys olivaceus and Turbot Scophthamus maximus Larvae

    2003-01-01

    The ultrastructures of skin chloride cells in cultured Japanese flounder and turbot larvae in metamorphosis, which grow in the same feeding conditions, are examined with a transmission electron microscope. These developed skin chloride cells were shaped like flattened ellipsoids and similar in morphology and ultrastructure to typical chloride cells of euryhaline fish gill. They locate in the epidermis and contract with the extra and interior environment through the apical pit and narrow channels. The cytoplasm of cell is full of numerous mitochondria and a ramifying network of tubules. The degeneration of skin chloride cells is observed with development of Japanese flounder larvae. Skin chloride cells of turbot are less developmental than those of Japanese flounder in the same developmental stage.

  15. Role of Interaction and Nucleoside Diphosphate Kinase B in Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Function by cAMP-Dependent Protein Kinase A.

    Lee A Borthwick

    Full Text Available Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR, a cAMP-dependent protein kinase A (PKA and ATP-regulated chloride channel. Here, we demonstrate that nucleoside diphosphate kinase B (NDPK-B, NM23-H2 forms a functional complex with CFTR. In airway epithelia forskolin/IBMX significantly increases NDPK-B co-localisation with CFTR whereas PKA inhibitors attenuate complex formation. Furthermore, an NDPK-B derived peptide (but not its NDPK-A equivalent disrupts the NDPK-B/CFTR complex in vitro (19-mers comprising amino acids 36-54 from NDPK-B or NDPK-A. Overlay (Far-Western and Surface Plasmon Resonance (SPR analysis both demonstrate that NDPK-B binds CFTR within its first nucleotide binding domain (NBD1, CFTR amino acids 351-727. Analysis of chloride currents reflective of CFTR or outwardly rectifying chloride channels (ORCC, DIDS-sensitive showed that the 19-mer NDPK-B peptide (but not its NDPK-A equivalent reduced both chloride conductances. Additionally, the NDPK-B (but not NDPK-A peptide also attenuated acetylcholine-induced intestinal short circuit currents. In silico analysis of the NBD1/NDPK-B complex reveals an extended interaction surface between the two proteins. This binding zone is also target of the 19-mer NDPK-B peptide, thus confirming its capability to disrupt NDPK-B/CFTR complex. We propose that NDPK-B forms part of the complex that controls chloride currents in epithelia.

  16. Renal tubular NHE3 is required in the maintenance of water and sodium chloride homeostasis.

    Fenton, Robert A; Poulsen, Søren B; de la Mora Chavez, Samantha; Soleimani, Manoocher; Dominguez Rieg, Jessica A; Rieg, Timo

    2017-08-01

    The sodium/proton exchanger isoform 3 (NHE3) is expressed in the intestine and the kidney, where it facilitates sodium (re)absorption and proton secretion. The importance of NHE3 in the kidney for sodium chloride homeostasis, relative to the intestine, is unknown. Constitutive tubule-specific NHE3 knockout mice (NHE3 loxloxCre) did not show significant differences compared to control mice in body weight, blood pH or bicarbonate and plasma sodium, potassium, or aldosterone levels. Fluid intake, urinary flow rate, urinary sodium/creatinine, and pH were significantly elevated in NHE3 loxloxCre mice, while urine osmolality and GFR were significantly lower. Water deprivation revealed a small urinary concentrating defect in NHE3 loxloxCre mice on a control diet, exaggerated on low sodium chloride. Ten days of low or high sodium chloride diet did not affect plasma sodium in control mice; however, NHE3 loxloxCre mice were susceptible to low sodium chloride (about -4 mM) or high sodium chloride intake (about +2 mM) versus baseline, effects without differences in plasma aldosterone between groups. Blood pressure was significantly lower in NHE3 loxloxCre mice and was sodium chloride sensitive. In control mice, the expression of the sodium/phosphate co-transporter Npt2c was sodium chloride sensitive. However, lack of tubular NHE3 blunted Npt2c expression. Alterations in the abundances of sodium/chloride cotransporter and its phosphorylation at threonine 58 as well as the abundances of the α-subunit of the epithelial sodium channel, and its cleaved form, were also apparent in NHE3 loxloxCre mice. Thus, renal NHE3 is required to maintain blood pressure and steady-state plasma sodium levels when dietary sodium chloride intake is modified. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  17. Hydrolysis of cupric chloride in aqueous ammoniacal ammonium chloride solutions

    Limpo, J. L.

    1995-06-01

    Full Text Available Cupric solubility in the CuCl2-NH4Cl-NH3-H2O system for chloride concentrations lower than 4 molal in the temperature range 25-60 °C was studied. The experimental results show that for chloride concentration between 3.0 and 1.0 molal the cupric solubility is determined by the solubility of the cupric hydroxychloride Cu(OH1.5Cl0.5. For a chloride concentration value of 4.0 molal, there are two cupric compounds, the hydroxychloride Cu(OH1.5Cl0.5 or the diammine chloride Cu(NH32Cl2, on which the solubility of Cu(II depends, according to the temperature and the value of the ratio [NH3]Total/[Cu]Total.

    Se estudia la solubilidad del Cu(II en el sistema CuCl2-NH4Cl-NH3-H2O para concentraciones de cloruro inferiores a 4 molal en el intervalo de temperaturas 25-60 °C. Los resultados experimentales muestran que, para concentraciones de cloruros comprendidas entre 3,0 y 1,0 molal, la solubilidad cúprica viene determinada por la solubilidad del hidroxicloruro cúprico, Cu(OH1.5Cl0.5. Para concentraciones de cloruro 4,0 molal, existen dos compuestos cúpricos, el hidroxicloruro, Cu(OH1.5Cl0.5 o el cloruro de diamina, Cu(NH32Cl2, de los que, de acuerdo con la temperatura y con el valor de la relación [NH3]Total/[Cu]Total depende la solubilidad del Cu(II.

  18. Prompt and delayed Coulomb explosion of doubly ionized hydrogen chloride molecules in intense femtosecond laser fields

    Ma, Junyang; Li, Hui; Lin, Kang; Song, Qiying; Ji, Qinying; Zhang, Wenbin; Li, Hanxiao; Sun, Fenghao; Qiang, Junjie; Lu, Peifen; Gong, Xiaochun; Zeng, Heping; Wu, Jian

    2018-06-01

    We experimentally investigate the dissociative double ionization of hydrogen chloride (HCl) molecules in intense femtosecond laser pulses. In addition to the prompt dissociation channels which occur on femtosecond timescales, long-lived hydrogen chloride dications which Coulomb-explode in flight towards the detector are clearly identified in the photoion-photoion coincidence spectrum. Different pathways leading to these prompt and delayed dissociation channels involving various bound and repulsive states of the HCl dication are discussed based on the observed kinetic energy release and momentum distributions. Our results indicate that the specific features of the HCl dication potential energy curves are responsible for the generation of the delayed fragmentation channels, which are expected to be general processes for the hydrogen halides.

  19. MARKETING CHANNELS

    Ljiljana Stošić Mihajlović

    2014-07-01

    Full Text Available Marketing channel is a set of entities and institutions, completion of distribution and marketing activities, attend the efficient and effective networking of producers and consumers. Marketing channels include the total flows of goods, money and information taking place between the institutions in the system of marketing, establishing a connection between them. The functions of the exchange, the physical supply and service activities, inherent in the system of marketing and trade. They represent paths which products and services are moving after the production, which will ultimately end up buying and eating by the user.

  20. Synthesis of Zirconium Lower Chlorides

    Gaviria, Juan P.

    2002-01-01

    This research is accurately related to the Halox concept of research reactor spent fuel element treatment.The aim of this project is to work the conditioning through selected chlorination of the element that make the spent fuel element. This research studied the physical chemistry conditions which produce formation of the lower zirconium chlorides through the reaction between metallic Zr and gaseous ZrCl 4 in a silica reactor.This work focused special attention in the analysis and confrontation of the published results among the different authors in order to reveal coincidences and contradictions.Experimental section consisted in a set of synthesis with different reaction conditions and reactor design. After reaction were analyzed the products on Zr shavings and the deposit growth on wall reactor.The products were strongly dependent of reactor design. It was observed that as the distance between Zr and wall reactor increased greater was tendency to lower chlorides formation.In reactors with small distance the reaction follows other way without formation of lower chlorides.Analysis on deposit growth on reactor showed that may be formed to a mixture of Si x Zr y intermetallics and zirconium oxides.Presence of oxygen in Zr and Zr-Si compounds on wall reactor reveals that there is an interaction between quartz and reactants.This interaction is in gaseous phase because contamination is observed in experiences where Zr was not in contact with reactor.Finally, it was made a global analysis of all experiences and a possible mechanism that interprets reaction ways is proposed

  1. Melting in trivalent metal chlorides

    Saboungi, M.L.; Price, D.L.; Scamehorn, C.; Tosi, M.P.

    1990-11-01

    We report a neutron diffraction study of the liquid structure of YCl 3 and combine the structural data with macroscopic melting and transport data to contrast the behaviour of this molten salt with those of SrCl 2 , ZnCl 2 and AlCl 3 as prototypes of different melting mechanisms for ionic materials. A novel melting mechanism for trivalent metal chlorides, leading to a loose disordered network of edge-sharing octahedral units in the liquid phase, is thereby established. The various melting behaviours are related to bonding character with the help of Pettifor's phenomenological chemical scale. (author). 25 refs, 4 figs, 3 tabs

  2. Haplotype block structure study of the CFTR gene. Most variants are associated with the M470 allele in several European populations.

    Pompei, Fiorenza; Ciminelli, Bianca Maria; Bombieri, Cristina; Ciccacci, Cinzia; Koudova, Monika; Giorgi, Silvia; Belpinati, Francesca; Begnini, Angela; Cerny, Milos; Des Georges, Marie; Claustres, Mireille; Ferec, Claude; Macek, Milan; Modiano, Guido; Pignatti, Pier Franco

    2006-01-01

    An average of about 1700 CFTR (cystic fibrosis transmembrane conductance regulator) alleles from normal individuals from different European populations were extensively screened for DNA sequence variation. A total of 80 variants were observed: 61 coding SNSs (results already published), 13 noncoding SNSs, three STRs, two short deletions, and one nucleotide insertion. Eight DNA variants were classified as non-CF causing due to their high frequency of occurrence. Through this survey the CFTR has become the most exhaustively studied gene for its coding sequence variability and, though to a lesser extent, for its noncoding sequence variability as well. Interestingly, most variation was associated with the M470 allele, while the V470 allele showed an 'extended haplotype homozygosity' (EHH). These findings make us suggest a role for selection acting either on the M470V itself or through an hitchhiking mechanism involving a second site. The possible ancient origin of the V allele in an 'out of Africa' time frame is discussed.

  3. Compounds that correct F508del-CFTR trafficking can also correct other protein trafficking diseases: an in vitro study using cell lines

    Sampson Heidi M; Lam Hung; Chen Pei-Chun; Zhang Donglei; Mottillo Cristina; Mirza Myriam; Qasim Karim; Shrier Alvin; Shyng Show-Ling; Hanrahan John W; Thomas David Y

    2013-01-01

    Abstract Background Many genetic diseases are due to defects in protein trafficking where the mutant protein is recognized by the quality control systems, retained in the endoplasmic reticulum (ER), and degraded by the proteasome. In many cases, the mutant protein retains function if it can be trafficked to its proper cellular location. We have identified structurally diverse correctors that restore the trafficking and function of the most common mutation causing cystic fibrosis, F508del-CFTR...

  4. Renal abnormalities in congenital chloride diarrhea

    Al-Hamad, Nadia M.; Al-Eisa, Amal A.

    2004-01-01

    Congenital chloride diarrhea CLD is a rare autosomal recessive disorder caused by a defect in the chloride/ bicarbonate exchange in the ileum and colon. It is characterized by watery diarrhea, abdominal distension, hypochloremic hypokalemic metabolic alkalosis with high fecal content of chloride >90 mmol/l. We report 3 patients with CLD associated with various renal abnormalities including chronic renal failure secondary to renal hypoplasia, nephrocalcinosis and congenital nephrotic syndrome. (author)

  5. Time-resolved energy transfer from single chloride-terminated nanocrystals to graphene

    Ajayi, O. A.; Wong, C. W.; Anderson, N. C.; Wolcott, A.; Owen, J. S.; Cotlet, M.; Petrone, N.; Hone, J.; Gu, T.; Gesuele, F.

    2014-01-01

    We examine the time-resolved resonance energy transfer of excitons from single n-butyl amine-bound, chloride-terminated nanocrystals to two-dimensional graphene through time-correlated single photon counting. The radiative biexponential lifetime kinetics and blinking statistics of the individual surface-modified nanocrystal elucidate the non-radiative decay channels. Blinking modification as well as a 4× reduction in spontaneous emission were observed with the short chloride and n-butylamine ligands, probing the energy transfer pathways for the development of graphene-nanocrystal nanophotonic devices

  6. Time-resolved energy transfer from single chloride-terminated nanocrystals to graphene

    Ajayi, O. A., E-mail: oaa2114@columbia.edu, E-mail: cww2104@columbia.edu; Wong, C. W., E-mail: oaa2114@columbia.edu, E-mail: cww2104@columbia.edu [Optical Nanostructures Laboratory, Center for Integrated Science and Engineering, Solid-State Science and Engineering, Columbia University, New York, New York 10027 (United States); Department of Mechanical Engineering, Columbia University, New York, New York 10027 (United States); Anderson, N. C.; Wolcott, A.; Owen, J. S. [Department of Chemistry, Columbia University, New York, New York 10027 (United States); Cotlet, M. [Brookhaven National Laboratory, Upton, New York, New York 11973 (United States); Petrone, N.; Hone, J. [Department of Mechanical Engineering, Columbia University, New York, New York 10027 (United States); Gu, T.; Gesuele, F. [Optical Nanostructures Laboratory, Center for Integrated Science and Engineering, Solid-State Science and Engineering, Columbia University, New York, New York 10027 (United States)

    2014-04-28

    We examine the time-resolved resonance energy transfer of excitons from single n-butyl amine-bound, chloride-terminated nanocrystals to two-dimensional graphene through time-correlated single photon counting. The radiative biexponential lifetime kinetics and blinking statistics of the individual surface-modified nanocrystal elucidate the non-radiative decay channels. Blinking modification as well as a 4× reduction in spontaneous emission were observed with the short chloride and n-butylamine ligands, probing the energy transfer pathways for the development of graphene-nanocrystal nanophotonic devices.

  7. Synthesis of carbon-14 labelled ethyl chloride

    Kanski, R.

    1976-01-01

    A new efficient method of synthesis of ethyl chloride (1,2- 14 C), based on the Ba 14 CO 3 and dry hydrogen chloride as starting materials has been developed and described. Addition of the hydrogen chloride to ethylene (1,2- 14 C), obtained from Ba 14 CO 3 , has been carried out in the presence of the AlCl 3 as catalyst. The outlined method leads to ethyl chloride (1,2- 14 C) of high specific activity. The radiochemical yield of the reaction based on the activity of barium carbonate used was 72%. (author)

  8. Spectrum of CFTR gene mutations in Ecuadorian cystic fibrosis patients: the second report of the p.H609R mutation.

    Ortiz, Sofía C; Aguirre, Santiago J; Flores, Sofía; Maldonado, Claudio; Mejía, Juan; Salinas, Lilian

    2017-11-01

    High heterogeneity in the CFTR gene mutations disturbs the molecular diagnosis of cystic fibrosis (CF). In order to improve the diagnosis of CF in our country, the present study aims to define a panel of common CFTR gene mutations by sequencing 27 exons of the gene in Ecuadorian Cystic Fibrosis patients. Forty-eight Ecuadorian individuals with suspected/confirmed CF diagnosis were included. Twenty-seven exons of CFTR gene were sequenced to find sequence variations. Prevalence of pathogenic variations were determined and compared with other countries' data. We found 70 sequence variations. Eight of these are CF-causing mutations: p.F508del, p.G85E, p.G330E, p.A455E, p.G970S, W1098X, R1162X, and N1303K. Also this study is the second report of p.H609R in Ecuadorian population. Mutation prevalence differences between Ecuadorian population and other Latin America countries were found. The panel of mutations suggested as an initial screening for the Ecuadorian population with cystic fibrosis should contain the mutations: p.F508del, p.G85E, p.G330E, p.A455E, p.G970S, W1098X, R1162X, and N1303K. © 2017 NETLAB Laboratorios Especializados. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.

  9. [Frequency of the most common mutations of the CFTR gene in peruvian patients with cystic fibrosis using the ARMS-PCR technique].

    Aquino, Ruth; Protzel, Ana; Rivera, Juan; Abarca, Hugo; Dueñas, Milagros; Nestarez, Cecilia; Purizaga, Nestor; Diringer, Benoit

    2017-01-01

    To determine the frequency of the ten most common mutations of the CFTR gene reported in Latin Americausing amplification-refractory mutation system-polymerase chain reaction (ARMS-PCR) in patients with cystic fibrosis (CF) in two referral hospitals in Peru during the year 2014. The frequency of the ten most common mutations of the CFTR gene was assessed in patients of the Hospital Nacional Edgardo Rebagliati Martins and the Instituto Nacional de Salud del Niño, both located in Lima, Peru. Blood samples were collected from 36 patients with CF, and the ARMS-PCR technique was used to determine the presence of these mutations. The study group included 73.5% of patients with a known diagnosis of CF in the country when the study was carried out. ARMS-PCR allowed three of the mutations to be identified in a combined 30.6% of the alleles from patients with CF, and 64.9% of the mutated alleles were not identified. The mutations found were p.Phe508del (22,2%), p.Gly542* (6,9%), and p.Arg1162* (1,4%). There is significant variability in both the frequency and type of mutations present in our study population and in what has been reported in other Latin American countries. It is necessary to perform studies that use complete sequencing technology for the CFTR gene to identify other mutations present in our population.

  10. Method of processing chloride waste

    Tokiwai, Moriyasu; Tsunashima, Mikiyasu; Horie, Masaaki; Koyama, Masafumi; Sudo, Minoru; Kitagawa, Masatoshi; Ogasawara, Tadashi.

    1991-01-01

    In a method of applying molten salt electrolysis to chloride wastes discharged from a electrolytic refining step of a dry reprocessing step for spent fuels, and removed with transuranium elements of long half-decaying time, metals capable of alloying with alkali and alkaline earth metals under melting by electrolysis are used as a cathode material, and an electrolytic temperature is made higher than the melting point of salts in a molten salt electrolysis bath, to recover Li, Ca and Na as alloys with the cathode material in a first electrolysis step. Then, the electrolytic temperature is made higher than the melting point of the chloride salts remained in the bath after the electrolysis step described above by using the cathode material, to recover Ba, Rb, Sr and Cs of nuclear fission products also as alloys with the cathode material in a second electrolysis step. Accordingly, the amount of wastes formed can be reduced, and the wastes contain no heat generating nuclear fission elements. (T.M.)

  11. Electrochemical chloride extraction of a beam polluted by chlorides after 40 years in the sea

    BOUTEILLER, Véronique; LAPLAUD, André; MALOULA, Aurélie; MORELLE, René Stéphane; DUCHESNE, Béatrice; MORIN, Mathieu

    2006-01-01

    A beam element, naturally polluted by chlorides after 40 years of a marine tidal exposure, has been treated by electrochemical chloride extraction. The chloride profiles, before and after treatment, show that free chlorides are extrated with an efficiency of 70 % close to the steel, 50 % in the intermediate cover and only 5 % at the concrete surface. From the electrochemical characterizations (before, after, 1, 2 and 17 months after treatment), the steel potential values can, semehow, indicat...

  12. Channel Power in Multi-Channel Environments

    M.G. Dekimpe (Marnik); B. Skiera (Bernd)

    2004-01-01

    textabstractIn the literature, little attention has been paid to instances where companies add an Internet channel to their direct channel portfolio. However, actively managing multiple sales channels requires knowing the customers’ channel preferences and the resulting channel power. Two key

  13. Thermochemistry of alkali chloride - lanthanoide(III) chlorides

    Blachnik, R.; Selle, D.

    1979-01-01

    The phase diagrams of the mixtures KCl + GdCl 3 resp. DyCl 3 and of CsCl + PrCl 3 (DyCl 3 , ErCl 3 , and YbCl 3 ) were investigated by differential thermal analysis. In the mixtures of lanthanoide(III) chlorides with CsCl resp. KCl three different stoichiometries of the compounds were found, namely A 3 MCl 6 , A 2 MCl 5 , and AM 2 Cl 7 . Debyeograms of the compounds A 3 MCl 6 and AM 2 Cl 7 reveal, that in the case of the latter type all compounds with the same alkali halide have identical structure, whereas in the A 3 MCl 6 compounds three different types of X-ray patterns were observed. The stabilities of the congruently melting compounds can be estimated by comparing the melting point of the compound with the temperature of an extrapolated eutectic point. (author)

  14. Diminished self-chaperoning activity of the DeltaF508 mutant of CFTR results in protein misfolding.

    Adrian W R Serohijos

    2008-02-01

    Full Text Available The absence of a functional ATP Binding Cassette (ABC protein called the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR from apical membranes of epithelial cells is responsible for cystic fibrosis (CF. Over 90% of CF patients carry at least one mutant allele with deletion of phenylalanine at position 508 located in the N-terminal nucleotide binding domain (NBD1. Biochemical and cell biological studies show that the DeltaF508 mutant exhibits inefficient biosynthetic maturation and susceptibility to degradation probably due to misfolding of NBD1 and the resultant misassembly of other domains. However, little is known about the direct effect of the Phe508 deletion on the NBD1 folding, which is essential for rational design strategies of cystic fibrosis treatment. Here we show that the deletion of Phe508 alters the folding dynamics and kinetics of NBD1, thus possibly affecting the assembly of the complete CFTR. Using molecular dynamics simulations, we find that meta-stable intermediate states appearing on wild type and mutant folding pathways are populated differently and that their kinetic accessibilities are distinct. The structural basis of the increased misfolding propensity of the DeltaF508 NBD1 mutant is the perturbation of interactions in residue pairs Q493/P574 and F575/F578 found in loop S7-H6. As a proof-of-principle that the S7-H6 loop conformation can modulate the folding kinetics of NBD1, we virtually design rescue mutations in the identified critical interactions to force the S7-H6 loop into the wild type conformation. Two redesigned NBD1-DeltaF508 variants exhibited significantly higher folding probabilities than the original NBD1-DeltaF508, thereby partially rescuing folding ability of the NBD1-DeltaF508 mutant. We propose that these observed defects in folding kinetics of mutant NBD1 may also be modulated by structures separate from the 508 site. The identified structural determinants of increased misfolding propensity of

  15. Chloride ingress in cement paste and mortar

    Jensen, Ole Mejlhede; Hansen, Per Freiesleben; Coats, Alison M.

    1999-01-01

    In this paper chloride ingress in cement paste and mortar is followed by electron probe microanalysis. The influence of several paste and exposure parameters on chloride ingress are examined (e.g., water-cement ratio, silica fume addition, exposure time, and temperature), The measurements...

  16. Chloride binding site of neurotransmitter sodium symporters

    Kantcheva, Adriana Krassimirova; Quick, Matthias; Shi, Lei

    2013-01-01

    Neurotransmitter:sodium symporters (NSSs) play a critical role in signaling by reuptake of neurotransmitters. Eukaryotic NSSs are chloride-dependent, whereas prokaryotic NSS homologs like LeuT are chloride-independent but contain an acidic residue (Glu290 in LeuT) at a site where eukaryotic NSSs...

  17. Synthesis of 14C-dehydrocorydaline chloride

    Zhang Rui; Wang Ding

    1988-01-01

    A method for synthesis of 14 C-dehydrocorydaline chloride is described. In the presence of sodium hydroxide, acetonylpalmatine is reacted with 14 C-methyl iodide in sealed glass ampoule to give 14 C-13-methylpalmatine iodide which is then converted to chloride. The radiochemical purity of 14 C-dehydrocorydaline determined by TLC is over 98% and the labelling efficiency is 54%

  18. Chronopotentiometric chloride sensing using transition time measurement

    Abbas, Yawar; de Graaf, D.B.; Olthuis, Wouter; van den Berg, Albert

    2013-01-01

    Detection of chloride ions is crucial to accurately access the concrete structure durability[1]. The existing electrochemical method of chloride ions detection in concrete, potentiometry[1], is not suitable for in-situ measurement due to the long term stability issue of conventional reference

  19. 29 CFR 1915.1017 - Vinyl chloride.

    2010-07-01

    ... 29 Labor 7 2010-07-01 2010-07-01 false Vinyl chloride. 1915.1017 Section 1915.1017 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR... § 1915.1017 Vinyl chloride. Note: The requirements applicable to shipyard employment under this section...

  20. 29 CFR 1926.1152 - Methylene chloride.

    2010-07-01

    ... 29 Labor 8 2010-07-01 2010-07-01 false Methylene chloride. 1926.1152 Section 1926.1152 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR... Methylene chloride. Note: The requirements applicable to construction employment under this section are...

  1. 29 CFR 1915.1052 - Methylene chloride.

    2010-07-01

    ... 29 Labor 7 2010-07-01 2010-07-01 false Methylene chloride. 1915.1052 Section 1915.1052 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR... § 1915.1052 Methylene chloride. Note: The requirements applicable to shipyard employment under this...

  2. 29 CFR 1926.1117 - Vinyl chloride.

    2010-07-01

    ... 29 Labor 8 2010-07-01 2010-07-01 false Vinyl chloride. 1926.1117 Section 1926.1117 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR... chloride. Note: The requirements applicable to construction work under this section are identical to those...

  3. Properties of silver chloride track detectors

    Dmitriev, V.D.; Kocherov, N.P.; Novikova, N.R.; Perfilov, N.A.

    1976-01-01

    The experiments on preparation of silver chloride track detectors and their properties are described. The results of X-ray structural analysis and data on sensitivity to charged particles and actinic light of silver chloride crystals, doped with several elements, are presented. (orig.) [de

  4. Dechlorinating reaction of organic chlorides

    Yahata, Taneaki; Kihara, Shinji [Japan Atomic Energy Research Inst., Oarai, Ibaraki (Japan). Oarai Research Establishment; Ohuchi, Misao

    1996-06-01

    Dechlorination has been examined by the reaction between iron, aluminum powder or CaO and organic chlorides such as C{sub 2}HCl{sub 3} and CH{sub 2}Cl{sub 2}. Progress of the reaction was analyzed with mass spectrometer. The reaction between iron and organic chloride was rapidly occurred at the temperature between 350 and 440degC in an atmosphere of argon. Above 380degC, more than 99.5% of C{sub 2}HCl{sub 3} was decomposed within approximately 100 minutes. At 440degC, approximately 60% of C{sub 2}HCl{sub 3} was decomposed by the reaction with aluminium powder within approximately 100 minutes. At 440degC, reaction between C{sub 2}HCl{sub 3} and CaO powder were occurred rapidly in an atmosphere of argon to form CaCl{sub 2} and free carbon. Also in an atmosphere of air, nearly the same result was obtained. In this reaction, CaCl{sub 2}, CO and CO{sub 2} were formed. CH{sub 2}Cl{sub 2} was also decomposed by the reaction with iron at the temperature between 380 and 440degC. In the reaction, FeCl{sub 2}, carbon and hydrogen were formed. CH{sub 3}{sup +} and CH{sub 4} were observed during the dechlorinating reaction of CH{sub 2}Cl{sub 2}. Variation in particle size of iron powder such as 100, 150 and 250 mesh did not affect the reaction rate. (author)

  5. Cystic fibrosis transmembrane conductance regulator intracellular processing, trafficking, and opportunities for mutation-specific treatment.

    Rogan, Mark P

    2012-02-01

    Recent advances in basic science have greatly expanded our understanding of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), the chloride and bicarbonate channel that is encoded by the gene, which is mutated in patients with CF. We review the structure, function, biosynthetic processing, and intracellular trafficking of CFTR and discuss the five classes of mutations and their impact on the CF phenotype. The therapeutic discussion is focused on the significant progress toward CFTR mutation-specific therapies. We review the results of encouraging clinical trials examining orally administered therapeutics, including agents that promote read-through of class I mutations (premature termination codons); correctors, which overcome the CFTR misfolding that characterizes the common class II mutation F508del; and potentiators, which enhance the function of class III or IV mutated CFTR at the plasma membrane. Long-term outcomes from successful mutation-specific treatments could finally answer the question that has been lingering since and even before the CFTR gene discovery: Will therapies that specifically restore CFTR-mediated chloride secretion slow or arrest the deleterious cascade of events leading to chronic infection, bronchiectasis, and end-stage lung disease?

  6. Chloride Blood Test: MedlinePlus Lab Test Information

    ... this page: https://medlineplus.gov/labtests/chloridebloodtest.html Chloride Blood Test To use the sharing features on this page, please enable JavaScript. What is a Chloride Blood Test? A chloride blood test measures the ...

  7. Effects of platinic chloride on Tetrahymena pyrifromis GL

    Nilsson, Jytte R.

    1992-01-01

    Cellebiologi, platinum(IV)chloride, endocytosis, detoxification, cell proliferation, fine structure, cisplatin......Cellebiologi, platinum(IV)chloride, endocytosis, detoxification, cell proliferation, fine structure, cisplatin...

  8. Chloride Transport in Undersea Concrete Tunnel

    Yuanzhu Zhang

    2016-01-01

    Full Text Available Based on water penetration in unsaturated concrete of underwater tunnel, a diffusion-advection theoretical model of chloride in undersea concrete tunnel was proposed. The basic parameters including porosity, saturated hydraulic conductivity, chloride diffusion coefficient, initial water saturation, and moisture retention function of concrete specimens with two water-binder ratios were determined through lab-scale experiments. The variation of chloride concentration with pressuring time, location, solution concentration, initial saturation, hydraulic pressure, and water-binder ratio was investigated through chloride transport tests under external water pressure. In addition, the change and distribution of chloride concentration of isothermal horizontal flow were numerically analyzed using TOUGH2 software. The results show that chloride transport in unsaturated concrete under external water pressure is a combined effect of diffusion and advection instead of diffusion. Chloride concentration increased with increasing solution concentration for diffusion and increased with an increase in water pressure and a decrease in initial saturation for advection. The dominant driving force converted with time and saturation. When predicting the service life of undersea concrete tunnel, it is suggested that advection is taken into consideration; otherwise the durability tends to be unsafe.

  9. Channelling and electromagnetic radiation of channelling particles

    Kalashnikov, N.

    1983-01-01

    A brief description is presented of the channelling of charged particles between atoms in the crystal lattice. The specificities are discussed of the transverse motion of channelling particles as are the origin and properties of quasi-characteristic radiation of channelling particles which accompany transfers from one band of permissible energies of the transverse motion of channelling particles to the other. (B.S.)

  10. Synthesis of aminocarbonyl N-acylhydrazones by a three-component reaction of isocyanides, hydrazonoyl chlorides, and carboxylic acids.

    Giustiniano, Mariateresa; Meneghetti, Fiorella; Mercalli, Valentina; Varese, Monica; Giustiniano, Francesco; Novellino, Ettore; Tron, Gian Cesare

    2014-10-17

    A novel one-pot multicomponent synthesis of α-aminocarbonyl N-acylhydrazones starting from readily available hydrazonoyl chlorides, isocyanides, and carboxylic acids is reported. The strategy exploits the ability of the carboxylic acid as a third component to suppress all competing reactions between nitrile imines and isocyanides, channeling the course of the reaction toward the formation of this novel class of compounds.

  11. Factors influencing chloride deposition in a coastal hilly area and application to chloride deposition mapping

    H. Guan

    2010-05-01

    Full Text Available Chloride is commonly used as an environmental tracer for studying water flow and solute transport in the environment. It is especially useful for estimating groundwater recharge based on the commonly used chloride mass balance (CMB method. Strong spatial variability in chloride deposition in coastal areas is one difficulty encountered in appropriately applying the method. A high-resolution bulk chloride deposition map in the coastal region is thus needed. The aim of this study is to construct a chloride deposition map in the Mount Lofty Ranges (MLR, a coastal hilly area of approximately 9000 km2 spatial extent in South Australia. We examined geographic (related to coastal distance, orographic, and atmospheric factors that may influence chloride deposition, using partial correlation and regression analyses. The results indicate that coastal distance, elevation, as well as terrain aspect and slope, appear to be significant factors controlling chloride deposition in the study area. Coastal distance accounts for 70% of spatial variability in bulk chloride deposition, with elevation, terrain aspect and slope an additional 15%. The results are incorporated into a de-trended residual kriging model (ASOADeK to produce a 1 km×1 km resolution bulk chloride deposition and concentration maps. The average uncertainty of the deposition map is about 20–30% in the western MLR, and 40–50% in the eastern MLR. The maps will form a useful basis for examining catchment chloride balance for the CMB application in the study area.

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

    Peipei Wei

    2016-07-01

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

  13. Mechanisms underlying KCNQ1channel cell volume sensitivity

    Hammami, Sofia

    Cells are constantly exposed to changes in cell volume during cell metabolism, nutrient uptake, cell proliferation, cell migration and salt and water transport. In order to cope with these perturbations, potassium channels in line with chloride channels have been shown to be likely contributors...... to the process of cell volume adjustments. A great diversity of potassium channels being members of either the 6TM, 4 TM or 2 TM K+ channel gene family have been shown to be strictly regulated by small, fast changes in cell volume. However, the precise mechanism underlying the K+ channel sensitivity to cell...... volume alterations is not yet fully understood. The KCNQ1 channel belonging to the voltage gated KCNQ family is considered a precise sensor of volume changes. The goal of this thesis was to elucidate the mechanism that induces cell volume sensitivity. Until now, a number of investigators have implicitly...

  14. Channel Modeling

    Schmitz, Arne; Schinnenburg, Marc; Gross, James; Aguiar, Ana

    For any communication system the Signal-to-Interference-plus-Noise-Ratio of the link is a fundamental metric. Recall (cf. Chapter 9) that the SINR is defined as the ratio between the received power of the signal of interest and the sum of all "disturbing" power sources (i.e. interference and noise). From information theory it is known that a higher SINR increases the maximum possible error-free transmission rate (referred to as Shannon capacity [417] of any communication system and vice versa). Conversely, the higher the SINR, the lower will be the bit error rate in practical systems. While one aspect of the SINR is the sum of all distracting power sources, another issue is the received power. This depends on the transmitted power, the used antennas, possibly on signal processing techniques and ultimately on the channel gain between transmitter and receiver.

  15. Channeling experiment

    Abelin, H.; Birgersson, L.; Widen, H.; Aagren, T.; Moreno, L.; Neretnieks, I.

    1990-07-01

    Channeling of water flow and tracer transport in real fractures in a granite body at Stripa have been investigated experimentally. The experimental site was located 360 m below the ground level. Two kinds of experiments were performed. In the single hole experiments, 20 cm diameter holes were drilled about 2.5 m into the rock in the plane of the fracture. Specially designed packers were used to inject water into the fracture in 5 cm intervals all along the fracture trace in the hole. The variation of the injection flowrates along the fracture were used to determine the transmissivity variations in the fracture plane. Detailed photographs were taken from inside the hole and the visual fracture aperture was compared with the injection flowrates in the same locations. Geostatistical methods were used to evaluate the results. Five holes were measured in great detail. In addition 7 holes were drilled and scanned by simpler packer systems. A double hole experiment was performed where two parallel holes were drilled in the same fracture plane at nearly 2 m distance. Pressure pulse tests were made between the holes in both directions. Tracers were injected in 5 locations in one hole and monitored for in many locations in the other hole. The single hole experiment and the double hole experiment show that most of the fracture planes are tight but that there are open sections which form connected channels over distances of at least 2 meters. It was also found in the double hole experiment that the investigated fracture was intersected by at least one fracture between the two holes which diverted a large amount of the injected tracers to several distant locations at the tunnel wall. (authours)

  16. Lithium-thionyl chloride battery

    Wong, D.; Bowden, W.; Hamilton, N.; Cubbison, D.; Dey, A. N.

    1981-04-01

    The main objective is to develop, fabricate, test, and deliver safe high rate lithium-thionyl chloride batteries for various U.S. Army applications such as manpack ratios and GLLD Laser Designators. We have devoted our efforts in the following major areas: (1) Optimization of the spirally wound D cell for high rate applications, (2) Development of a 3 inch diameter flat cylindrical cell for the GLLD laser designator application, and (3) Investigation of the reduction mechanism of SOCl2. The rate capability of the spirally wound D cell previously developed by us has been optimized for both the manpack radio (BA5590) battery and GLLD laser designator battery application in this program. A flat cylindrical cell has also been developed for the GLLD laser designator application. It is 3 inches in diameter and 0.9 inch in height with extremely low internal cell impedance that minimizes cell heating and polarization on the GLLD load. Typical cell capacity was found to be 18.0-19.0 Ahr with a few cells delivering up to about 21.0 Ahr on the GLLD test load. Study of the reduction mechanism of SOCl2 using electrochemical and spectroscopic techniques has also been carried out in this program which may be directly relevant to the intrinsic safety of the system.

  17. Spontaneous and α-adrenoceptor-induced contractility in human collecting lymphatic vessels require chloride

    Mohanakumar, Sheyanth; Majgaard, Jens; Telinius, Niklas

    2018-01-01

    - with the impermeant anion aspartate and inhibition of Cl- transport and channels with the clinical diuretics furosemide and bendroflumethiazide, as well as DIDS and NPPB. The molecular expression of calcium-activated chloride channels was investigated by RT-PCR and proteins localized using immunoreactivity....... Spontaneous and norepinephrine-induced contractility in human lymphatic vessels was highly abrogated after Cl- substitution with aspartate. 100‒300µM DIDS or NPPB inhibited spontaneous contractile behavior. Norepinephrine-stimulated tone was furthermore markedly abrogated by 200µM DIDS. Furosemide lowered...

  18. Corneal Neurotoxicity Due to Topical Benzalkonium Chloride

    Sarkar, Joy; Chaudhary, Shweta; Namavari, Abed; Ozturk, Okan; Chang, Jin-Hong; Yco, Lisette; Sonawane, Snehal; Khanolkar, Vishakha; Hallak, Joelle; Jain, Sandeep

    2012-01-01

    Topical application of benzalkonium chloride (BAK) to the eye causes dose-related corneal neurotoxicity. Corneal inflammation and reduction in aqueous tear production accompany neurotoxicity. Cessation of BAK treatment leads to recovery of corneal nerve density.

  19. Catastrophic event modeling. [lithium thionyl chloride batteries

    Frank, H. A.

    1981-01-01

    A mathematical model for the catastrophic failures (venting or explosion of the cell) in lithium thionyl chloride batteries is presented. The phenomenology of the various processes leading to cell failure is reviewed.

  20. Lithium thionyl chloride high rate discharge

    Klinedinst, K. A.

    1980-04-01

    Improvements in high rate lithium thionyl chloride power technology achieved by varying the electrolyte composition, operating temperature, cathode design, and cathode composition are discussed. Discharge capacities are plotted as a function of current density, cell voltage, and temperature.

  1. Chloride Ingress into Concrete under Water Pressure

    Lund, Mia Schou; Sander, Lotte Braad; Grelk, Bent

    2011-01-01

    The chloride ingress into concrete under water pressures of 100 kPa and 800 kPa have been investigated by experiments. The specimens were exposed to a 10% NaCl solution and water mixture. For the concrete having w/c = 0.35 the experimental results show the chloride diffusion coefficient at 800 k......Pa (~8 atm.) is 12 times greater than at 100 kPa (~1 atm.). For w/c = 0.45 and w/c = 0.55 the chloride diffusion coefficients are 7 and 3 times greater. This means that a change in pressure highly influences the chloride ingress into the concrete and thereby the life length models for concrete structures....

  2. Inert Reassessment Document for Cerous Chloride

    The rare earth chlorides have a wide variety of scientific applications. They a re used in superconductors, lasers, magnets, catalytic converters, fertilizes, supper alloys, cigarette lighters and as catalysts in the production of petroleum products.

  3. Process for making rare earth metal chlorides

    Kruesi, P.R.

    1981-01-01

    An uncombined metal or a metal compound such as a sulfide, oxide, carbonate or sulfate is converted in a liquid salt bath to the corresponding metal chloride by reacting it with chlorine gas or a chlorine donor. The process applies to metals of groups 1b, 2a, 2b, 3a, 3b, 4a, 5a and 8 of the periodic table and to the rare earth metals. The chlorine donor may be ferric or sulfur chloride. The liquid fused salt bath is made up of chlorides of alkali metals, alkaline earth metals, ammonia, zinc and ferric iron. Because the formed metal chlorides are soluble in the liquid fused salt bath, they can be recovered by various conventional means

  4. The molecular analysis of mutations in exons 4, 11 and 21 of the cystic fibrosis transmembrane conductance regulator (CFTR gene in cystic fibrosis patients in Kermanshah, Iran

    Nasibe Karimi

    2017-03-01

    Full Text Available Introduction: Cystic fibrosis (CF is a common genetic disorder in white populations with an autosomal recessive pattern, caused by mutations in the CFTR gene. The frequency of more than 1950 various mutations reported in the CFTR gene significantly varies in different populations. ∆F508 is a common mutation in exon 10, which is first addressed in the molecular analysis of the disease. Other exons are required to be investigated owing to failing to identify mutations in the patients. The present study was conducted to investigate mutations in exons 4, 11 and 21 of the CFTR gene using the sequencing method in CF patients in Kermanshah province, Iran. Methods: The present descriptive study was conducted on all patients with CF presenting to the medical genetics center in Kermanshah in 2010-2011. After taking blood samples and extracting DNA using saturated NaCl solution, sequences of exons were amplified using PCR and sequenced for identifying mutations. Results: The frequency of mutations was found to be respectively 0, 0 and 5.5% in exon 11, 21 and 4. The D110H mutation was found to be homozygous in one subject and heterozygous in another. Moreover, the 4029A>G polymorphism (12.9% was found to be homozygous in two subjects and heterozygous in three others. Conclusion: The D110H mutation is recommended to be included in the screening programs of the study population. The results obtained support the effects of ethnic and geographical factors on the distribution of CF mutations.

  5. Applicability and Efficiency of NGS in Routine Diagnosis: In-Depth Performance Analysis of a Complete Workflow for CFTR Mutation Analysis.

    Adrien Pagin

    Full Text Available Actually, about 2000 sequence variations have been documented in the CFTR gene requiring extensive and multi-step genetic testing in the diagnosis of cystic fibrosis and CFTR-related disorders. We present a two phases study, with validation and performance monitoring, of a single experiment methodology based on multiplex PCR and high throughput sequencing that allows detection of all variants, including large rearrangements, affecting the coding regions plus three deep intronic loci.A total of 340 samples, including 257 patients and 83 previously characterized control samples, were sequenced in 17 MiSeq runs and analyzed with two bioinformatic pipelines in routine diagnostic conditions. We obtained 100% coverage for all the target regions in every tested sample.We correctly identified all the 87 known variants in the control samples and successfully confirmed the 62 variants identified among the patients without observing false positive results. Large rearrangements were identified in 18/18 control samples. Only 17 patient samples showed false positive signals (6.6%, 12 of which showed a borderline result for a single amplicon. We also demonstrated the ability of the assay to detect allele specific dropout of amplicons when a sequence variation occurs at a primer binding site thus limiting the risk for false negative results.We described here the first NGS workflow for CFTR routine analysis that demonstrated equivalent diagnostic performances compared to Sanger sequencing and multiplex ligation-dependent probe amplification. This study illustrates the advantages of NGS in term of scalability, workload reduction and cost-effectiveness in combination with an improvement of the overall data quality due to the simultaneous detection of SNVs and large rearrangements.

  6. Molecular mechanisms of reduced glutathione transport: role of the MRP/CFTR/ABCC and OATP/SLC21A families of membrane proteins

    Ballatori, Nazzareno; Hammond, Christine L.; Cunningham, Jennifer B.; Krance, Suzanne M.; Marchan, Rosemarie

    2005-01-01

    The initial step in reduced glutathione (GSH) turnover in all mammalian cells is its transport across the plasma membrane into the extracellular space; however, the mechanisms of GSH transport are not clearly defined. GSH export is required for the delivery of its constituent amino acids to other tissues, detoxification of drugs, metals, and other reactive compounds of both endogenous and exogenous origin, protection against oxidant stress, and secretion of hepatic bile. Recent studies indicate that some members of the multidrug resistance-associated protein (MRP/CFTR or ABCC) family of ATP-binding cassette (ABC) proteins, as well as some members of the organic anion transporting polypeptide (OATP or SLC21A) family of transporters contribute to this process. In particular, five of the 12 members of the MRP/CFTR family appear to mediate GSH export from cells namely, MRP1, MRP2, MRP4, MRP5, and CFTR. Additionally, two members of the OATP family, rat Oatp1 and Oatp2, have been identified as GSH transporters. For the Oatp1 transporter, efflux of GSH may provide the driving force for the uptake of extracellular substrates. In humans, OATP-B and OATP8 do not appear to transport GSH; however, other members of this family have yet to be characterized in regards to GSH transport. In yeast, the ABC proteins Ycf1p and Bpt1p transport GSH from the cytosol into the vacuole, whereas Hgt1p mediates GSH uptake across the plasma membrane. Because transport is a key step in GSH homeostasis and is intimately linked to its biological functions, GSH export proteins are likely to modulate essential cellular functions

  7. Frecuencia de las mutaciones más comunes del gen CFTR en pacientes peruanos con fibrosis quística mediante la técnica ARMS-PCR

    Aquino, Ruth; Protzel, Ana; Rivera, Juan; Abarca, Hugo; Dueñas, Milagros; Nestarez, Cecilia; Purizaga, Nestor; Diringer, Benoit

    2017-01-01

    Objetivos. Determinar la frecuencia de las diez mutaciones más comúnmente reportadas en América Latina del gen CFTR mediante Sistema de Mutación Refractario a la amplificación por PCR (ARMS-PCR) en los pacientes con fibrosis quística (FQ) de dos instituciones hospitalarias de referencia en el Perú durante el año 2014. Materiales y métodos. Se evaluó la frecuencia de las diez comúnmente reportadas más comúnmente reportadas del gen CFTR en los pacientes del Hospital Nacional Edgardo Rebagliati ...

  8. Influence of Chloride-Ion Adsorption Agent on Chloride Ions in Concrete and Mortar

    Gai-Fei Peng

    2014-04-01

    Full Text Available The influence of a chloride-ion adsorption agent (Cl agent in short, composed of zeolite, calcium aluminate hydrate and calcium nitrite, on the ingress of chloride ions into concrete and mortar has been experimentally studied. The permeability of concrete was measured, and the chloride ion content in mortar was tested. The experimental results reveal that the Cl agent could adsorb chloride ions effectively, which had penetrated into concrete and mortar. When the Cl agent was used at a dosage of 6% by mass of cementitious materials in mortar, the resistance to the penetration of chloride ions could be improved greatly, which was more pronounced when a combination of the Cl agent and fly ash or slag was employed. Such an effect is not the result of the low permeability of the mortar, but might be a result of the interaction between the Cl agent and the chloride ions penetrated into the mortar. There are two possible mechanisms for the interaction between the Cl agent and chloride ion ingress. One is the reaction between calcium aluminate hydrate in the Cl agent and chloride ions to form Friedel’s salt, and the other one is that calcium aluminate hydrate reacts with calcium nitrite to form AFm during the early-age hydration of mortar and later the NO2− in AFm is replaced by chloride ions, which then penetrate into the mortar, also forming Friedel’s salt. More research is needed to confirm the mechanisms.

  9. 21 CFR 522.1862 - Sterile pralidoxime chloride.

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Sterile pralidoxime chloride. 522.1862 Section 522....1862 Sterile pralidoxime chloride. (a) Chemical name. 2-Formyl-1-methylpyridinium chloride oxime. (b) Specifications. Sterile pralidoxime chloride is packaged in vials. Each vial contains 1 gram of sterile...

  10. Plant ion channels: gene families, physiology, and functional genomics analyses.

    Ward, John M; Mäser, Pascal; Schroeder, Julian I

    2009-01-01

    Distinct potassium, anion, and calcium channels in the plasma membrane and vacuolar membrane of plant cells have been identified and characterized by patch clamping. Primarily owing to advances in Arabidopsis genetics and genomics, and yeast functional complementation, many of the corresponding genes have been identified. Recent advances in our understanding of ion channel genes that mediate signal transduction and ion transport are discussed here. Some plant ion channels, for example, ALMT and SLAC anion channel subunits, are unique. The majority of plant ion channel families exhibit homology to animal genes; such families include both hyperpolarization- and depolarization-activated Shaker-type potassium channels, CLC chloride transporters/channels, cyclic nucleotide-gated channels, and ionotropic glutamate receptor homologs. These plant ion channels offer unique opportunities to analyze the structural mechanisms and functions of ion channels. Here we review gene families of selected plant ion channel classes and discuss unique structure-function aspects and their physiological roles in plant cell signaling and transport.

  11. Deciphering the mode of action of clinically relevant next generation c2 corrector compounds GLPG2737 and GLPG3221

    Peters, F.; Sahasrabudhe, P.; Gross-Wilde, H.; Kleizen, Bertrand; Conrath, K.; Braakman, I.

    2017-01-01

    The current therapeutic strategy to repair cystic fibrosis-causing defects in the chloride channel CFTR is to develop novel and better correctors (to improve folding) and potentiators (to improve function). Galapagos- AbbVie identified C2 correctors by high-throughput compound screening and Med Chem

  12. Atmospheric chloride: Its implication for foliar uptake and damage

    McWilliams, E. L.; Sealy, R. L.

    Atmospheric chloride is inversely related to distance from the Texas coast; r2 = 0.86. Levels of atmospheric chloride are higher in the early summer than in the winter because of salt storms. Leaf chloride l'evels of Tillandsia usneoides L. (Spanish moss) reflect the atmospheric chloride levels; r2 = 0.78. The importance of considering the effect of atmospheric chloride on leaf damage to horticultural crops is discussed.

  13. Determination of chloride in MOX samples using chloride ion selective electrode

    Govindan, R; Das, D K; Mallik, G K; Sumathi, A; Patil, Sangeeta; Raul, Seema; Bhargava, V K; Kamath, H S [Bhabha Atomic Research Centre, Tarapur (India). Advanced Fuel Fabrication Facility

    1997-09-01

    The chloride present in the MOX fuel is separated from the matrix by pyrohydrolysis at a temperature of 950 {+-} 50 degC and is then analyzed by chloride ion selective electrode (Cl-ISE). The range covered is 0.4-4 ppm with a precision of better than {+-}5% R.S.D. (author). 4 refs., 1 tab.

  14. Study on the chloride migration coefficient obtained following different Rapid Chloride Migration (RCM) test guidelines

    Spiesz, P.R.; Brouwers, H.J.H.; Uzoegbo, H.C.; Schmidt, W.

    2013-01-01

    This work presents the differences in the available Rapid Chloride Migration (RCM) test guidelines, and their influence on the values of the chloride migration coefficients DRCM, obtained following these guidelines. It is shown that the differences between the guidelines are significant and concern

  15. Laboratory investigation of electro-chemical chloride extraction from concrete with penetrated chloride

    Polder, R.B.; Hondel, A.W.M. van den

    2002-01-01

    Chloride extraction of concrete is a short-term electrochemical treatment against corrosion of reinforcing steel. The aim is to remove chloride ions from the concrete cover in order to reinstate passive behaviour. Physically sound concrete is left in place. To make this method more predictable and

  16. Amine and Titanium (IV Chloride, Boron (III Chloride or Zirconium (IV Chloride-Promoted Baylis-Hillman Reactions

    Shi-Cong Cui

    2001-10-01

    Full Text Available The Baylis-Hillman reactions of various aryl aldehydes with methyl vinyl ketone at temperatures below -20oC using Lewis acids such as titanium (IV chloride, boron (III chloride or zirconium (IV chloride in the presence of a catalytic amount of selected amines used as a Lewis bases afford the chlorinated compounds 1 as the major product in very high yields. Acrylonitrile can also undergo the same reaction to give the corresponding chlorinated product in moderate yield. A plausible reaction mechanism is proposed. However, if the reaction was carried out at room temperature (ca. 20oC, then the Z-configuration of the elimination product 3, derived from 1, was formed as the major product.

  17. Identification of eight novel mutations in a collaborative analysis of a part of the second transmembrane domain of the CFTR gene

    Mercier, B.; Audrezet, M.P.; Guillermit, H.; Quere, I.; Verlingue, C.; Ferec, C. (CDTS, Brest (France)); Lissens, W.; Bonduelle, M.; Liebaers, I. (University Hospital VUB, Brussels (Belgium)); Novelli, G.; Sangiuolo, F.; Dallapiccola, B. (IRCCS, Rotondo (Italy)); Kalaydjieva, L. (Inst. of Obstetrics, Sofia (Bulgaria)); Arce, M. De; Cashman, S. (Trinity College, Dublin (Ireland)); Kapranov, N. (NRC of medical Genetics, Moscow (Russian Federation)); Canki Klain, N. (Tozd Univerzitetna Ginekoloska Klinika, Ljubljana (Yugoslavia)); Lenoir, G. (Hopital des Enfants Malades Necker, Paris (France)); Chauveau, P. (Centre Hospitalier General, Le Havre (France)); Lanaerts, C. (Centre Hospitalier Regional et Universitaire, Amiens (France)); Rault, G. (Centre Helio-Marin, Roscoff (France))

    1993-04-01

    Cystic fibrosis transmembrane conductance regulator (CFTR), the gene responsible, when mutated, for cystic fibrosis (CF), spans over 230 kb on the long arm of chromosome 7 and is composed of 27 exons. The most common mutation responsible for CF worldwide is the deletion of a phenylalanine amino acid at codon 508 in the first nucleotide-binding fold and accounts for approximately 70% of CF chromosomes studied. More than 250 other mutations have been reported through the CF Genetic Analysis Consortium. The majority of the mutations previously described lie in the two nucleotide-binding folds. To explore exhaustively other regions of the gene, particularly exons coding for transmembrane domains, the authors have initiated a collaborative study between different laboratories to screen 369 non-[Delta]F508 CF chromosomes of seven ethnic European populations (Belgian, French, Breton, Irish, Italian, Yugoslavian, Russian). Among these chromosomes carrying an unidentified mutation, 63 were from Brittany, 50 of various French origin, 45 of Irish origin, 56 of Italian origin, 41 of Belgian origin, 2 of Turkish origin, 38 of Yugoslavian origin, 22 of Russian origin, and 52 of Bulgarian origin. Diagnostic criteria for CF included at least one positive sweat test and pulmonary disease with or without pancreatic disease. Using a denaturing gradient gel electrophoresis (DGGE) assay, they have identified eight novel mutations in exon 17b coding for part of the second transmembrane domain of the CFTR and they describe them in this report. 8 refs., 1 fig., 1 tab.

  18. Temperature dependence of diffusion coefficients of trivalent uranium ions in chloride and chloride-fluoride melts

    Komarov, V.E.; Borodina, N.P.

    1981-01-01

    Diffusion coefficients of U 3+ ions are measured by chronopotentiometric method in chloride 3LiCl-2KCl and in mixed chloride fluoride 3LiCl(LiF)-2KCl melts in the temperature range 633-1235 K. It is shown It is shown that experimental values of diffusion-coefficients are approximated in a direct line in lg D-1/T coordinate in chloride melt in the whole temperature range and in chloride-fluoride melt in the range of 644-1040 K. Experimental values of diffusion coefficients diviate from Arrhenius equation in the direction of large values in chloride-fluoride melt at further increase of temperature up to 1235 K. Possible causes of such a diviation are considered [ru

  19. CLC channel function and dysfunction in health and disease

    Gabriel eStölting

    2014-10-01

    Full Text Available CLC channels and transporters are expressed in most tissues and fulfill diverse functions. There are four human CLC channels, ClC-1, ClC-2, ClC-Ka and ClC-Kb, and five CLC transporters, ClC-3 through -7. Some of the CLC channels additionally associate with accessory subunits. Whereas barttin is mandatory for the functional expression of CLC-K, GlialCam is a facultative subunit of ClC-2 which modifies gating and thus increases the functional variability within the CLC family. Isoform-specific ion conduction and gating properties optimize distinct CLC channels for their cellular tasks. ClC-1 preferentially conducts at negative voltages, and the resulting inward rectification provides a large resting chloride conductance without interference with the muscle action potential. Exclusive opening at voltages negative to the chloride reversal potential allows for ClC-2 to regulate intracellular chloride concentrations. ClC-Ka and ClC-Kb are equally suited for inward and outward currents to support transcellular chloride fluxes. Every human CLC channel gene has been linked to a genetic disease, and studying these mutations has provided much information about the physiological roles and the molecular basis of CLC channel function. Mutations in the gene encoding ClC-1 cause myotonia congenita, a disease characterized by sarcolemmal hyperexcitability and muscle stiffness. Loss-of-function of ClC-Kb/barttin channels in patients suffering from Bartter syndrome identified the determinants of chloride conductances in the limb of Henle. Mutations in CLCN2 were found in patients with CNS disorders but the functional role of this isoform is still not understood. Recent links between ClC-1 and epilepsy and ClC-Ka and heart failure suggested novel cellular functions of these proteins. This review aims to survey the knowledge about physiological and pathophysiological functions of human CLC channels in the light of recent discoveries from biophysical, physiological

  20. Hydrolysis of ferric chloride in solution

    Lussiez, G.; Beckstead, L.

    1996-11-01

    The Detox trademark process uses concentrated ferric chloride and small amounts of catalysts to oxidize organic compounds. It is under consideration for oxidizing transuranic organic wastes. Although the solution is reused extensively, at some point it will reach the acceptable limit of radioactivity or maximum solubility of the radioisotopes. This solution could be cemented, but the volume would be increased substantially because of the poor compatibility of chlorides and cement. A process has been developed that recovers the chloride ions as HCl and either minimizes the volume of radioactive waste or permits recycling of the radioactive chlorides. The process involves a two-step hydrolysis at atmospheric pressure, or preferably under a slight vacuum, and relatively low temperature, about 200 degrees C. During the first step of the process, hydrolysis occurs according to the reaction below: FeCl 3 liquid + H 2 O → FeOCl solid + 2 HCl gas During the second step, the hot, solid, iron oxychloride is sprayed with water or placed in contact with steam, and hydrolysis proceeds to the iron oxide according to the following reaction: 2 FeOCl solid + H 2 O → Fe 2 O 3 solid + 2 HCl gas . The iron oxide, which contains radioisotopes, can then be disposed of by cementation or encapsulation. Alternately, these chlorides can be washed off of the solids and can then either be recycled or disposed of in some other way

  1. Advanced intermediate temperature sodium copper chloride battery

    Yang, Li-Ping; Liu, Xiao-Min; Zhang, Yi-Wei; Yang, Hui; Shen, Xiao-Dong

    2014-12-01

    Sodium metal chloride batteries, also called as ZEBRA batteries, possess many merits such as low cost, high energy density and high safety, but their high operation temperature (270-350 °C) may cause several issues and limit their applications. Therefore, decreasing the operation temperature is of great importance in order to broaden their usage. Using a room temperature ionic liquid (RTIL) catholyte composed of sodium chloride buffered 1-ethyl-3-methylimidazolium chloride-aluminum chloride and a dense β″-aluminates solid electrolyte film with 500 micron thickness, we report an intermediate temperature sodium copper chloride battery which can be operated at only 150 °C, therefore alleviating the corrosion issues, improving the material compatibilities and reducing the operating complexities associated with the conventional ZEBRA batteries. The RTIL presents a high ionic conductivity (0.247 S cm-1) at 150 °C and a wide electrochemical window (-2.6 to 2.18 vs. Al3+/Al). With the discharge plateau at 2.64 V toward sodium and the specific capacity of 285 mAh g-1, this intermediate temperature battery exhibits an energy density (750 mWh g-1) comparable to the conventional ZEBRA batteries (728-785 mWh g-1) and superior to commercialized Li-ion batteries (550-680 mWh g-1), making it very attractive for renewable energy integration and other grid related applications.

  2. Simple chloride sensors for continuous groundwater monitoring

    Thorn, Paul; Mortensen, John

    2012-01-01

    The development of chloride sensors which can be used for continuous, on-line monitoring of groundwater could be very valuable in the management of our coastal water resources. However, sensor stability, drift, and durability all need to be addressed in order for the sensors to be used in continu......The development of chloride sensors which can be used for continuous, on-line monitoring of groundwater could be very valuable in the management of our coastal water resources. However, sensor stability, drift, and durability all need to be addressed in order for the sensors to be used...... in continuous application. This study looks at the development of a simple, inexpensive chloride electrode, and evaluates its performance under continuous use, both in the laboratory and in a field test in a monitoring well. The results from the study showed a consistent response to changing chloride...... concentrations over longer periods. The signal was seen to be stable, with regular drift in both laboratory and field test. In the field application, the sensor signal was corrected for drift, and errors were observed to be under 7% of that of conductivity measurements. The study also found that the chloride...

  3. Curcumin/poly(2-methyl-2-oxazoline-b-tetrahydrofuran-b-2-methyl-2-oxazoline) formulation: An improved penetration and biological effect of curcumin in F508del-CFTR cell lines.

    Gonçalves, Cristine; Gomez, Jean-Pierre; Même, William; Rasolonjatovo, Bazoly; Gosset, David; Nedellec, Steven; Hulin, Philippe; Huin, Cécile; Le Gall, Tony; Montier, Tristan; Lehn, Pierre; Pichon, Chantal; Guégan, Philippe; Cheradame, Hervé; Midoux, Patrick

    2017-08-01

    Neutral amphiphilic triblock ABA copolymers are of great interest to solubilize hydrophobic drugs. We reported that a triblock ABA copolymer consisting of methyl-2-oxazoline (MeOx) and tetrahydrofuran (THF) (MeOx 6 -THF 19 -MeOx 6 ) (TBCP2) can solubilize curcumin (Cur) a very hydrophobic molecule exhibiting multiple therapeutic effects but whose insolubility and low stability in water is a major drawback for clinical applications. Here, we provide evidences by flow cytometry and confocal microscopy that Cur penetration in normal and ΔF508-CFTR human airway epithelial cell lines is facilitated by TBCP2. When used on ΔF508-CFTR cell lines, the Cur/TBCP2 formulation promotes the restoration of the expression of the CFTR protein in the plasma membrane. Furthermore, patch-clamp and MQAE fluorescence experiments show that this effect is associated with a correction of a Cl - selective current at the membrane surface of F508del-CFTR cells. The results show the great potential of the neutral amphiphilic triblock copolymer MeOx 6 -THF 19 -MeOx 6 as carrier for curcumin in a Cystic Fibrosis context. We anticipate that other MeOx n -THF m -MeOx n copolymers could have similar behaviours for other highly insoluble therapeutic drugs or cosmetic active ingredients. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Combinatorial effects of genistein and sex-steroids on the level of cystic fibrosis transmembrane regulator (CFTR), adenylate cyclase (AC) and cAMP in the cervix of ovariectomised rats.

    Salleh, Naguib; Ismail, Nurain; Muniandy, Sekaran; Korla, Praveen Kumar; Giribabu, Nelli

    2015-12-01

    The combinatorial effects of genistein and estrogen (E) or estrogen plus progesterone (E+P) on CFTR, AC and cAMP levels in cervix were investigated. Ovariectomised adult female rats received 50 or 100mg/kg/day genistein with E or E followed by E+P [E+(E+P)] for seven consecutive days. Cervixes were harvested and analyzed for CFTR mRNA levels by Real-time PCR. Distribution of AC and CFTR proteins in endocervix were observed by immunohistochemistry. Levels of cAMP were measured by enzyme-immunoassay. Molecular docking predicted interaction between genistein and AC. Our results indicate that levels of CFTR, AC and cAMP in cervix of rats receiving genistein plus E were higher than E-only treatment (pcervix of E and E+(E+P)-treated rats by genistein could affect the cervical secretory function which could influence the female reproductive processes. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Determination of chloride content in crystalline silicotitanate

    Wilmarth, W.R.

    1999-01-01

    Crystalline Silicotitanate (CST) is one of three options under evaluation to replace the In-Tank Precipitation process. This Salt Disposition Alternatives team identified three options for pretreatment of High Level Waste supernate: non-elutable ion exchange, precipitation with sodium tetraphenylborate or direct disposal in grout. The ion exchange option would use crystalline silicotitanate (CST). Researchers at Texas A and M and Sandia National Laboratory developed CST. The engineered form of CST was procured from UOP LLC under the trade name IONSIVreg s ign IE-911. Review of vendor literature and discussions with UOP personnel led to speculation concerning the fate of chloride ion during the manufacture process of IE-911. Walker proposed tests to examine the chloride content of CST and removal methods. This report describes the results of tests to determine the chloride levels in as received CST and washed CST

  6. Mutagenicity of vinyl chloride after metabolic activation

    Rannug, U; Johansson, A; Ramel, C; Wachtmeister, C A

    1974-01-01

    Vinyl chloride has recently been shown to cause a malignant liver tumor disease in man after occupational exposure in PVC plants. This actualizes the problem of whether such hazards could be avoided or at least diminished in the future by a screening for mutagenicity of chemicals used in industries. The basis for such a screening procedure is the close correlation between carcinogenic and mutagenic effects of chemicals. Experiments with Salmonella bacteria showed that the carcinogenic hazard of vinyl chloride could have been traced by means of mutagenicity tests. The data indicate that vinyl chloride is not mutagenic per se but becomes mutagenic after a metabolic activation in the liver. 24 references, 1 figure, 4 tables.

  7. Chloride migration in concrete with superabsorbent polymers

    Hasholt, Marianne Tange; Jensen, Ole Mejlhede

    2015-01-01

    Superabsorbent polymers (SAP) can be used as a means for internal curing of concrete. In the present study, the development of transport properties of concrete with SAP is investigated. The chloride migration coefficient according to NT BUILD 492 is used as a measure of this. Twenty concrete...... contribute to increase the degree of hydration. No matter if SAP is added with or without extra water, it appears that the so-called gel space ratio can be used as a key parameter to link age and mixture proportions (water-to-cement ratio and SAP dosage) to the resulting chloride migration coefficient......; the higher the volume of gel solid relative to the space available for it, the lower the chloride migration coefficient, because the pore system becomes more tortuous and the porosity becomes less....

  8. Alkali metal and ammonium chlorides in water and heavy water (binary systems)

    Cohen-Adad, R

    1991-01-01

    This volume surveys the data available in the literature for solid-fluid solubility equilibria plus selected solid-liquid-vapour equilibria, for binary systems containing alkali and ammonium chlorides in water or heavy water. Solubilities covered are lithium chloride, sodium chloride, potassium chloride, rubidium chloride, caesium chloride and ammonium chloride in water and heavy water.

  9. Hazards of lithium thionyl chloride batteries

    Parry, J. M.

    1978-01-01

    Two different topics which only relate in that they are pertinent to lithium thionyl chloride battery safety are discussed. The first topic is a hazards analysis of a system (risk assessment), a formal approach that is used in nuclear engineering, predicting oil spills, etc. It is a formalized approach for obtaining assessment of the degree of risk associated with the use of any particular system. The second topic is a small piece of chemistry related to the explosions that can occur with lithium thionyl chloride systems. After the two topics are presented, a discussion is generated among the Workshop participants.

  10. Surface adsorption in strontium chloride ammines

    Ammitzbøll, Andreas L.; Lysgaard, Steen; Klukowska, Agata

    2013-01-01

    An adsorbed state and its implications on the ab- and desorption kinetics of ammonia in strontium chloride ammine is identified using a combination of ammonia absorption measurements, thermogravimetric analysis, and density functional theory calculations. During thermogravimetric analysis, ammonia...... desorption originating from the adsorbed state is directly observed below the bulk desorption temperature, as confirmed by density functional theory calculations. The desorption enthalpy of the adsorbed state of strontium chloride octa-ammine is determined with both techniques to be around 37-39 k...

  11. Citizens and service channels: channel choice and channel management implications

    Pieterson, Willem Jan

    2010-01-01

    The arrival of electronic channels in the 1990s has had a huge impact on governmental service delivery. The new channels have led to many new opportunities to improve public service delivery, not only in terms of citizen satisfaction, but also in cost reduction for governmental agencies. However,

  12. Crystal field influence on vibration spectra: anhydrous uranyl chloride and dihydroxodiuranyl chloride tetrahydrate

    Perrin, Andre; Caillet, Paul

    1976-01-01

    Vibrational spectra of anhydrous uranyl chloride UO 2 Cl 2 and so called basic uranyl chloride: dihydroxodiuranyl chloride tetrahydrate /UO 2 (OH) 2 UO 2 /Cl 2 (H 2 O) 4 are reported. Factor group method analysis leads for the first time to complete and comprehensive interpretation of their spectra. Two extreme examples of crystal field influence on vibrational spectra are pointed out: for UO 2 Cl 2 , one is unable to explain spectra without taking into account all the elements of primitive crystalline cell, whilst for dihydroxodiuranyl dichloride tetrahydrate the crystal packing has very little effect on vibrational spectra [fr

  13. Chloride Ingress in Concrete with Different Age at Time of First Chloride Exposure

    Hansen, Esben Østergaard; Iskau, Martin Riis; Hasholt, Marianne Tange

    2016-01-01

    Concrete structures cast in spring have longer time to hydrate and are therefore denser and more resistant to chloride ingress when first subjected to deicing salts in winter than structures cast in autumn. Consequently, it is expected that a spring casting will have a longer service life....... This hypothesis is investigated in the present study by testing drilled cores from concrete cast in 2012 and 2013 on the Svendborgsund Bridge. The cores are subject to petrographic examination and mapping of chloride profiles. Moreover, chloride migration coefficients have been measured. The study shows...

  14. Method for preparation of melts of alkali metal chlorides with highly volatile polyvalent metal chlorides

    Salyulev, A.B.; Kudyakov, V.Ya.

    1990-01-01

    A method for production of alkali metal (Cs, Rb, K) chloride melts with highly volatile polyvalent metal chlorides is suggested. The method consists, in saturation of alkali metal chlorides, preheated to the melting point, by volatile component vapours (titanium tetrachloride, molybdenum or tantalum pentachloride) in proportion, corresponding to the composition reguired. The saturation is realized in an evacuated vessel with two heating areas for 1-1.5 h. After gradual levelling of temperature in both areas the product is rapidly cooled. 1 fig.; 1 tab

  15. Absorption media for irreversibly gettering thionyl chloride

    Buffleben, George; Goods, Steven H.; Shepodd, Timothy; Wheeler, David R.; Whinnery, Jr., LeRoy

    2002-01-01

    Thionyl chloride is a hazardous and reactive chemical used as the liquid cathode in commercial primary batteries. Contrary to previous thinking, ASZM-TEDA.RTM. carbon (Calgon Corporation) reversibly absorbs thionyl chloride. Thus, several candidate materials were examined as irreversible getters for thionyl chloride. The capacity, rate and effect of temperature were also explored. A wide variety of likely materials were investigated through screening experiments focusing on the degree of heat generated by the reaction as well as the material absorption capacity and irreversibility, in order to help narrow the group of possible getter choices. More thorough, quantitative measurements were performed on promising materials. The best performing getter was a mixture of ZnO and ASZM-TEDA.RTM. carbon. In this example, the ZnO reacts with thionyl chloride to form ZnCl.sub.2 and SO.sub.2. The SO.sub.2 is then irreversibly gettered by ASZM-TEDA.RTM. carbon. This combination of ZnO and carbon has a high capacity, is irreversible and functions effectively above -20.degree. C.

  16. Detection of chloride ion concentration using chronopotentiometry

    Abbas, Yawar; Olthuis, Wouter; van den Berg, Albert

    2013-01-01

    In this paper, a novel approach is reported for the electrochemical measurement of chloride ions using chronopotentiometry. A current pulse is applied at the Ag/AgCl working electrode and the potential change is measured with respect to another identical Ag/AgCl electrode in the bulk electrolyte.

  17. 75 FR 19657 - Barium Chloride From China

    2010-04-15

    ... China AGENCY: United States International Trade Commission. ACTION: Notice of Commission determination... China. SUMMARY: The Commission hereby gives notice that it will proceed with a full review pursuant to... antidumping duty order on barium chloride from China would be likely to lead to continuation or recurrence of...

  18. Thermal Decomposition of Aluminium Chloride Hexahydrate

    Hartman, Miloslav; Trnka, Otakar; Šolcová, Olga

    2005-01-01

    Roč. 44, č. 17 (2005), s. 6591-6598 ISSN 0888-5885 R&D Projects: GA ČR(CZ) GA203/02/0002 Institutional research plan: CEZ:AV0Z40720504 Keywords : aluminum chloride hexahydrate * thermal decomposition * reaction kinetics Subject RIV: CI - Industrial Chemistry, Chemical Engineering Impact factor: 1.504, year: 2005

  19. Reliability-Based Planning of Chloride Measurements

    Sørensen, John Dalsgaard; Engelund, S.

    1996-01-01

    In reinforced concrete structures corrosion is initiated when the chloride concentration around the reinforcement exceeds a threshold value. If corrosion starts then expensive repairs can be necessary. The estimation of the probability that corrosion has been initiated in a given structure is bas...

  20. Commercial production of thallium-201 chloride

    Sokolov, S.V.; Volkova, N.M.; Skokov, V.S.

    1989-01-01

    Thallium-201 chloride pharmaceuticals production practice at the Medradiopreparat factory under USSR Ministry of Public Health is described. The factory is carried out series-produced supplies of the compound prepared according to a new practice from September, 1985. Thallium-201 extraction from cyclotron targets irradiated is carried out by the extraction method

  1. 75 FR 20625 - Barium Chloride From China

    2010-04-20

    ... INTERNATIONAL TRADE COMMISSION [Investigation No. 731-TA-149 (Third Review)] Barium Chloride From China AGENCY: United States International Trade Commission. ACTION: Revised schedule for the subject review. DATES: Effective Date: April 9, 2010. FOR FURTHER INFORMATION CONTACT: Amy Sherman (202-205-3289...

  2. 75 FR 33824 - Barium Chloride From China

    2010-06-15

    ... China Determination On the basis of the record\\1\\ developed in the subject five-year review, the United... China would be likely to lead to continuation or recurrence of material injury to an industry in the... contained in USITC Publication 4157 (June 2010), entitled Barium Chloride from China: Investigation No. 731...

  3. 29 CFR 1910.1017 - Vinyl chloride.

    2010-07-01

    ... employee exposure to vinyl chloride (chloroethene), Chemical Abstracts Service Registry No. 75014. (2) This section applies to the manufacture, reaction, packaging, repackaging, storage, handling or use of vinyl... this section by engineering, work practice, and personal protective controls as follows: (1) Feasible...

  4. Niobium interaction with chloride-carbonate melts

    Kuznetsov, S.A.; Kuznetsova, S.V.

    1996-01-01

    Niobium interaction with chloride-carbonate melt NaCl-KCl-K 2 CO 3 (5 mass %) in the temperature range of 973-1123 K has been studied. The products and niobium corrosion rate have been ascertained, depending on the temperature of melt and time of allowance. Potentials of niobium corrosion have been measured. Refs. 11, figs. 3, tabs. 2

  5. Amperometric Sensor for Detection of Chloride Ions

    Rene Kizek

    2008-09-01

    Full Text Available Chloride ion sensing is important in many fields such as clinical diagnosis, environmental monitoring and industrial applications. We have measured chloride ions at a carbon paste electrode (CPE and at a CPE modified with solid AgNO3, a solution of AgNO3 and/or solid silver particles. Detection limits (3 S/N for chloride ions were 100 μM, 100 μM and 10 μM for solid AgNO3, solution of AgNO3 and/or solid silver particles, respectively. The CPE modified with silver particles is the most sensitive to the presence chloride ions. After that we approached to the miniaturization of the whole electrochemical instrument. Measurements were carried out on miniaturized instrument consisting of a potentiostat with dimensions 35 × 166 × 125 mm, screen printed electrodes, a peristaltic pump and a PC with control software. Under the most suitable experimental conditions (Britton-Robinson buffer, pH 1.8 and working electrode potential 550 mV we estimated the limit of detection (3 S/N as 500 nM.

  6. Chloride concentration affects soil microbial community

    Gryndler, Milan; Rohlenová, Jana; Kopecký, Jan; Matucha, Miroslav

    2008-01-01

    Roč. 71, č. 7 (2008), s. 1401-1408 ISSN 0045-6535 R&D Projects: GA ČR GA526/05/0636 Institutional research plan: CEZ:AV0Z50200510; CEZ:AV0Z50380511 Keywords : soil chloride * terminal restriction fragments * soil microorganisms Subject RIV: EE - Microbiology, Virology Impact factor: 3.054, year: 2008

  7. An improved calcium chloride method preparation and ...

    Transformation is one of the fundamental and essential molecular cloning techniques. In this paper, we have reported a modified method for preparation and transformation of competent cells. This modified method, improved from a classical protocol, has made some modifications on the concentration of calcium chloride ...

  8. Analysis of the Rapid Chloride Migration test

    Spiesz, P.R.; Ballari, M.; Brouwers, H.J.H.; Ferreira, R. M.; Gulikers, J.; Andrade, C.

    2009-01-01

    In this study the Rapid Chloride Migration test (RCM) standardized as NT Build 492 and BAW-Merkblatt is reviewed. Since the traditional natural diffusion tests are laborious, time consuming and costly, they are not always preferred from a practical point of view. To overcome these disadvantages,

  9. Oral cadmium chloride intoxication in mice

    Andersen, O; Nielsen, J B; Svendsen, P

    1988-01-01

    Diethyldithiocarbamate (DDC) is known to alleviate acute toxicity due to injection of cadmium salts. However, when cadmium chloride was administered by the oral route, DDC enhanced rather than alleviated the acute toxicity; both oral and intraperitoneal (i.p.) administration of DDC had this effect...

  10. Chloride diffusion in partially saturated cementitious material

    Nielsen, Erik Pram; Geiker, Mette Rica

    2003-01-01

    The paper proposes a combined application of composite theory and Powers' model for microstructural development for the estimation of the diffusion coefficient as a function of the moisture content of a defect-free cementitious material. Measurements of chloride diffusion in mortar samples (440 kg...

  11. Amperometric Sensor for Detection of Chloride Ions†

    Trnkova, Libuse; Adam, Vojtech; Hubalek, Jaromir; Babula, Petr; Kizek, Rene

    2008-01-01

    Chloride ion sensing is important in many fields such as clinical diagnosis, environmental monitoring and industrial applications. We have measured chloride ions at a carbon paste electrode (CPE) and at a CPE modified with solid AgNO3, a solution of AgNO3 and/or solid silver particles. Detection limits (3 S/N) for chloride ions were 100 μM, 100 μM and 10 μM for solid AgNO3, solution of AgNO3 and/or solid silver particles, respectively. The CPE modified with silver particles is the most sensitive to the presence chloride ions. After that we approached to the miniaturization of the whole electrochemical instrument. Measurements were carried out on miniaturized instrument consisting of a potentiostat with dimensions 35 × 166 × 125 mm, screen printed electrodes, a peristaltic pump and a PC with control software. Under the most suitable experimental conditions (Britton-Robinson buffer, pH 1.8 and working electrode potential 550 mV) we estimated the limit of detection (3 S/N) as 500 nM. PMID:27873832

  12. Amperometric Sensor for Detection of Chloride Ions.

    Trnkova, Libuse; Adam, Vojtech; Hubalek, Jaromir; Babula, Petr; Kizek, Rene

    2008-09-15

    Chloride ion sensing is important in many fields such as clinical diagnosis, environmental monitoring and industrial applications. We have measured chloride ions at a carbon paste electrode (CPE) and at a CPE modified with solid AgNO₃, a solution of AgNO₃ and/or solid silver particles. Detection limits (3 S/N) for chloride ions were 100 μM, 100 μM and 10 μM for solid AgNO₃, solution of AgNO₃ and/or solid silver particles, respectively. The CPE modified with silver particles is the most sensitive to the presence chloride ions. After that we approached to the miniaturization of the whole electrochemical instrument. Measurements were carried out on miniaturized instrument consisting of a potentiostat with dimensions 35 × 166 × 125 mm, screen printed electrodes, a peristaltic pump and a PC with control software. Under the most suitable experimental conditions (Britton-Robinson buffer, pH 1.8 and working electrode potential 550 mV) we estimated the limit of detection (3 S/N) as 500 nM.

  13. Binary nucleation of water and sodium chloride

    Němec, Tomáš; Maršík, František; Palmer, A.

    2006-01-01

    Roč. 124, č. 4 (2006), 0445091-0445096 ISSN 0021-9606 R&D Projects: GA ČR(CZ) GA101/05/2536 Institutional research plan: CEZ:AV0Z20760514 Keywords : binary nucleation * sodium chloride * water Subject RIV: BJ - Thermodynamics Impact factor: 3.166, year: 2006

  14. Repeated nebulisation of non-viral CFTR gene therapy in patients with cystic fibrosis: a randomised, double-blind, placebo-controlled, phase 2b trial.

    Alton, Eric W F W; Armstrong, David K; Ashby, Deborah; Bayfield, Katie J; Bilton, Diana; Bloomfield, Emily V; Boyd, A Christopher; Brand, June; Buchan, Ruaridh; Calcedo, Roberto; Carvelli, Paula; Chan, Mario; Cheng, Seng H; Collie, D David S; Cunningham, Steve; Davidson, Heather E; Davies, Gwyneth; Davies, Jane C; Davies, Lee A; Dewar, Maria H; Doherty, Ann; Donovan, Jackie; Dwyer, Natalie S; Elgmati, Hala I; Featherstone, Rosanna F; Gavino, Jemyr; Gea-Sorli, Sabrina; Geddes, Duncan M; Gibson, James S R; Gill, Deborah R; Greening, Andrew P; Griesenbach, Uta; Hansell, David M; Harman, Katharine; Higgins, Tracy E; Hodges, Samantha L; Hyde, Stephen C; Hyndman, Laura; Innes, J Alastair; Jacob, Joseph; Jones, Nancy; Keogh, Brian F; Limberis, Maria P; Lloyd-Evans, Paul; Maclean, Alan W; Manvell, Michelle C; McCormick, Dominique; McGovern, Michael; McLachlan, Gerry; Meng, Cuixiang; Montero, M Angeles; Milligan, Hazel; Moyce, Laura J; Murray, Gordon D; Nicholson, Andrew G; Osadolor, Tina; Parra-Leiton, Javier; Porteous, David J; Pringle, Ian A; Punch, Emma K; Pytel, Kamila M; Quittner, Alexandra L; Rivellini, Gina; Saunders, Clare J; Scheule, Ronald K; Sheard, Sarah; Simmonds, Nicholas J; Smith, Keith; Smith, Stephen N; Soussi, Najwa; Soussi, Samia; Spearing, Emma J; Stevenson, Barbara J; Sumner-Jones, Stephanie G; Turkkila, Minna; Ureta, Rosa P; Waller, Michael D; Wasowicz, Marguerite Y; Wilson, James M; Wolstenholme-Hogg, Paul

    2015-09-01

    Lung delivery of plasmid DNA encoding the CFTR gene complexed with a cationic liposome is a potential treatment option for patients with cystic fibrosis. We aimed to assess the efficacy of non-viral CFTR gene therapy in patients with cystic fibrosis. We did this randomised, double-blind, placebo-controlled, phase 2b trial in two cystic fibrosis centres with patients recruited from 18 sites in the UK. Patients (aged ≥12 years) with a forced expiratory volume in 1 s (FEV1) of 50-90% predicted and any combination of CFTR mutations, were randomly assigned, via a computer-based randomisation system, to receive 5 mL of either nebulised pGM169/GL67A gene-liposome complex or 0.9% saline (placebo) every 28 days (plus or minus 5 days) for 1 year. Randomisation was stratified by % predicted FEV1 (<70 vs ≥70%), age (<18 vs ≥18 years), inclusion in the mechanistic substudy, and dosing site (London or Edinburgh). Participants and investigators were masked to treatment allocation. The primary endpoint was the relative change in % predicted FEV1. The primary analysis was per protocol. This trial is registered with ClinicalTrials.gov, number NCT01621867. Between June 12, 2012, and June 24, 2013, we randomly assigned 140 patients to receive placebo (n=62) or pGM169/GL67A (n=78), of whom 116 (83%) patients comprised the per-protocol population. We noted a significant, albeit modest, treatment effect in the pGM169/GL67A group versus placebo at 12 months' follow-up (3.7%, 95% CI 0.1-7.3; p=0.046). This outcome was associated with a stabilisation of lung function in the pGM169/GL67A group compared with a decline in the placebo group. We recorded no significant difference in treatment-attributable adverse events between groups. Monthly application of the pGM169/GL67A gene therapy formulation was associated with a significant, albeit modest, benefit in FEV1 compared with placebo at 1 year, indicating a stabilisation of lung function in the treatment group. Further improvements in

  15. Viscosity and density tables of sodium chloride solutions

    Fair, J.A.; Ozbek, H. (comps.)

    1977-04-01

    A file is presented containing tabulated data extracted from the scientific literature on the density and viscosity of aqueous sodium chloride solutions. Also included is a bibliography of the properties of aqueous sodium chloride solutions. (MHR)

  16. Improved electrolyte for lithium-thionyl chloride battery. [Patent application

    Shipman, W.H.; McCartney, J.F.

    1980-12-17

    A lithium, thionyl chloride battery is provided with an electrolyte which makes it safe under a reverse voltage condition. The electrolyte is niobium pentachloride which is dissolved in the thionyl chloride.

  17. IRIS Toxicological Review of Vinyl Chloride (Final Report, 2000)

    EPA is announcing the release of the final report, Toxicological Review of Vinyl Chloride: in support of the Integrated Risk Information System (IRIS). The updated Summary for Vinyl Chloride and accompanying Quickview have also been added to the IRIS Database.

  18. Thallium-201 chloride dynamic analysis using thallium-201 chloride and sodium iodide-131 thyroid subtraction scintigraphy

    Morimoto, Setsuo; Hiraki, Yoshio; Togami, Izumi [Okayama Univ. (Japan). School of Medicine

    1984-10-01

    The mechanism of /sup 201/Tl chloride accumulation is unclear in thyroid gland and thyroid tumor. This report examines 108 patients that received thyroid scintigraphy examinations with both /sup 201/Tl chloride and sodium /sup 131/I. The patients were diagnosed clinically and histologically whenever possible. The ROI were obtained by subtraction imaging with both isotopes and by subtraction positive and negative areas of imaging. Dynamic curves were obtained for /sup 201/Tl chloride per square unit of each ROI. The dynamic curve in the radioiodide-accumulated area was examined. The data indicate that the clearance rate of /sup 201/Tl chloride (T/sub 15/) was correlated with the sodium /sup 131/I uptake rate at 24 h (r=0.70).

  19. Durability of cracked fibre reinforced concrete structures exposed to chlorides

    Hansen, Ernst Jan De Place; Ekman, Tom; Hansen, Kurt Kielsgaard

    1999-01-01

    is used as environmental load. The chloride penetration is characterized both qualitatively (UV-test) and quantitatively (chloride profile) and by microscopy. The test programme involves three different concrete qualities. Both steel fibres and polypropylene fibres are used in the concrete beams as well...... as main reinforcement. The effect of the cracks, the fibres and the concrete quality on the chloride penetration is studied....

  20. 21 CFR 177.1950 - Vinyl chloride-ethylene copolymers.

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Vinyl chloride-ethylene copolymers. 177.1950... Basic Components of Single and Repeated Use Food Contact Surfaces § 177.1950 Vinyl chloride-ethylene copolymers. The vinyl chloride-ethylene copolymers identified in paragraph (a) of this section may be safely...

  1. Accelerated testing for chloride threshold of reinforcing steel in concrete

    Polder, R.B.; Put, M. van; Peelen, W.H.A.

    2017-01-01

    Testing for the chloride threshold (also called critical chloride content) for corrosion initiation of steel in concrete has been found difficult and, at best, time consuming. Nevertheless, the chloride threshold is an important parameter in service life design of new structures and for evaluation

  2. Potentiometric Determination of Free Chloride in Cement Paste – an ...

    ... cement paste.16 The accuracy and reliability of this analytical technique has been checked against a certified reference material, Merck sodium chloride solution. Confidence levels (CL0.95), of 0.03 and relative standard deviations of 0.2 % for chloride were determined for ordinary Portland cement (OPC) chloride binding ...

  3. Removal of iron contaminant from zirconium chloride solution

    Voit, D.O.

    1992-01-01

    This patent describes a process for eliminating iron contaminant from an aqueous zirconium chloride solution that has been contaminated with FeCl 3 in a plant in which zirconium and hafnium chloride solutions are separated by a main MINK solvent extraction system and the FeCl 3 is normally removed from the zirconium chloride solution by a secondary MINK solvent extraction system

  4. Chloride sensing by WNK1 kinase involves inhibition of autophosphorylation

    Piala, Alexander T.; Moon, Thomas M.; Akella, Radha; He, Haixia; Cobb, Melanie H.; Goldsmith, Elizabeth J.

    2014-01-01

    WNK1 [with no lysine (K)] is a serine-threonine kinase associated with a form of familial hypertension. WNK1 is at the top of a kinase cascade leading to phosphorylation of several cotransporters, in particular those transporting sodium, potassium, and chloride (NKCC), sodium and chloride (NCC), and potassium and chloride (KCC). The responsiveness of NKCC, NCC, and KCC to changes in extracellular chloride parallels their phosphorylation state, provoking the proposal that these transporters are controlled by a chloride-sensitive protein kinase. Here, we found that chloride stabilizes the inactive conformation of WNK1, preventing kinase autophosphorylation and activation. Crystallographic studies of inactive WNK1 in the presence of chloride revealed that chloride binds directly to the catalytic site, providing a basis for the unique position of the catalytic lysine. Mutagenesis of the chloride binding site rendered the kinase less sensitive to inhibition of autophosphorylation by chloride, validating the binding site. Thus, these data suggest that WNK1 functions as a chloride sensor through direct binding of a regulatory chloride ion to the active site, which inhibits autophosphorylation. PMID:24803536

  5. CLICs-dependent chloride efflux is an essential and proximal upstream event for NLRP3 inflammasome activation.

    Tang, Tiantian; Lang, Xueting; Xu, Congfei; Wang, Xiaqiong; Gong, Tao; Yang, Yanqing; Cui, Jun; Bai, Li; Wang, Jun; Jiang, Wei; Zhou, Rongbin

    2017-08-04

    The NLRP3 inflammasome can sense different pathogens or danger signals, and has been reported to be involved in the development of many human diseases. Potassium efflux and mitochondrial damage are both reported to mediate NLRP3 inflammasome activation, but the underlying, orchestrating signaling events are still unclear. Here we show that chloride intracellular channels (CLIC) act downstream of the potassium efflux-mitochondrial reactive oxygen species (ROS) axis to promote NLRP3 inflammasome activation. NLRP3 agonists induce potassium efflux, which causes mitochondrial damage and ROS production. Mitochondrial ROS then induces the translocation of CLICs to the plasma membrane for the induction of chloride efflux to promote NEK7-NLRP3 interaction, inflammasome assembly, caspase-1 activation, and IL-1β secretion. Thus, our results identify CLICs-dependent chloride efflux as an essential and proximal upstream event for NLRP3 activation.The NLRP3 inflammasome is key to the regulation of innate immunity against pathogens or stress, but the underlying signaling regulation is still unclear. Here the authors show that chloride intracellular channels (CLIC) interface between mitochondria stress and inflammasome activation to modulate inflammatory responses.

  6. Calculated Third Order Rate Constants for Interpreting the Mechanisms of Hydrolyses of Chloroformates, Carboxylic Acid Halides, Sulfonyl Chlorides and Phosphorochloridates

    T. William Bentley

    2015-05-01

    Full Text Available Hydrolyses of acid derivatives (e.g., carboxylic acid chlorides and fluorides, fluoro- and chloroformates, sulfonyl chlorides, phosphorochloridates, anhydrides exhibit pseudo-first order kinetics. Reaction mechanisms vary from those involving a cationic intermediate (SN1 to concerted SN2 processes, and further to third order reactions, in which one solvent molecule acts as the attacking nucleophile and a second molecule acts as a general base catalyst. A unified framework is discussed, in which there are two reaction channels—an SN1-SN2 spectrum and an SN2-SN3 spectrum. Third order rate constants (k3 are calculated for solvolytic reactions in a wide range of compositions of acetone-water mixtures, and are shown to be either approximately constant or correlated with the Grunwald-Winstein Y parameter. These data and kinetic solvent isotope effects, provide the experimental evidence for the SN2-SN3 spectrum (e.g., for chloro- and fluoroformates, chloroacetyl chloride, p-nitrobenzoyl p-toluenesulfonate, sulfonyl chlorides. Deviations from linearity lead to U- or V-shaped plots, which assist in the identification of the point at which the reaction channel changes from SN2-SN3 to SN1-SN2 (e.g., for benzoyl chloride.

  7. Relation between chloride exchange diffusion and a conductive chloride pathway across the isolated skin of the toad (Bufo bufo)

    Kristensen, P; Larsen, Erik Hviid

    1978-01-01

    Substitution of chloride in the outside bathing medium of the toad skin with bromide, iodide, nitrate and sulphate leads to a reduction in the apparent exchange diffusion of chloride across this tissue, and also to a reduction of the chloride current recorded during hyperpolarization. A series...

  8. Determination of Chloride Content in Cementitious Materials : From Fundamental Aspects to Application of Ag/AgCl Chloride Sensors

    Pargar, F.; Koleva, D.A.; van Breugel, K.

    2017-01-01

    This paper reports on the advantages and drawbacks of available test methods for the determination of chloride content in cementitious materials in general, and the application of Ag/AgCl chloride sensors in particular. The main factors that affect the reliability of a chloride sensor are presented.

  9. USACE Navigation Channels 2012

    California Natural Resource Agency — This dataset represents both San Francisco and Los Angeles District navigation channel lines. All San Francisco District channel lines were digitized from CAD files...

  10. Calcium channel blocker overdose

    ... this page: //medlineplus.gov/ency/article/002580.htm Calcium-channel blocker overdose To use the sharing features on this page, please enable JavaScript. Calcium-channel blockers are a type of medicine used ...

  11. Influence of chloride admixtures on cement matrix durability

    Sheikh, I.A.; Zamorani, E.; Serrini, G.

    1989-01-01

    The influence of various inorganic salts, as chloride admixtures to Portland cement, on the mechanical properties and the durability of the matrix has been studied. The salts used in this study are chromium, nickel and cadmium chlorides. Improved compressive strength values are obtained which have been correlated to the stable metal hydroxide formation in high pH environment. Under static water conditions at 50 0 C, hydrolyzed chloride ions exhibit adverse effects on the matrix durability through rapid release of calcium as calcium chloride in the initial period of leaching. On the contrary, enhanced matrix durability is obtained on long term leaching in the case of cement containing chromium chloride

  12. Thermochemistry of certain rare earth and ammonium double chlorides

    Usubaliev, D.U.; Abramtsev, V.A.; Kydynov, M.K.; Vilyaev, A.N.

    1987-01-01

    In a calorimeter with isothermal casing at 25 deg C dissolution enthalpies of double chlorides of rare earths and ammonium LnCl 3 x2NH 4 Cl (Ln=La, Sm) and LnCl 3 x3NH 4 Cl (Ln=Gd, Tb, Ho) in water, as well as dissolution enthalpy of rare earth chlorides in solution of ammonium chloride and NH 4 Cl in solution of rare earth chloride, have been measured. Formation enthalpies, standard formation enthalpies, dissociation enthalpies of the above-mentioned double chlorides are calculated

  13. STABILISATION OF SILTY CLAY SOIL USING CHLORIDE

    TAMADHER T. ABOOD

    2007-04-01

    Full Text Available The object of this paper is to investigate the effect of adding different chloride compounds including (NaCl, MgCl2, CaCl2 on the engineering properties of silty clay soil. Various amounts of salts (2%, 4%, and 8% were added to the soil to study the effect of salts on the compaction characteristics, consistency limits and compressive strength. The main findings of this study were that the increase in the percentage of each of the chloride compounds increased the maximum dry density and decrease the optimum moisture content. The liquid limit, plastic limit and plasticity index decreased with the increase in salt content. The unconfinedcompressive strength increased as the salt content increased.

  14. Precipitation of metal nitrides from chloride melts

    Slater, S.A.; Miller, W.E.; Willit, J.L.

    1996-01-01

    Precipitation of actinides, lanthanides, and fission products as nitrides from molten chloride melts is being investigated for use as a final cleanup step in treating radioactive salt wastes generated by electrometallurgical processing of spent nuclear fuel. The radioactive components (eg, fission products) need to be removed to reduce the volume of high-level waste that requires disposal. To extract the fission products from the salt, a nitride precipitation process is being developed. The salt waste is first contacted with a molten metal; after equilibrium is reached, a nitride is added to the metal phase. The insoluble nitrides can be recovered and converted to a borosilicate glass after air oxidation. For a bench-scale experimental setup, a crucible was designed to contact the salt and metal phases. Solubility tests were performed with candidate nitrides and metal nitrides for which there are no solubility data. Experiments were performed to assess feasibility of precipitation of metal nitrides from chloride melts

  15. Quantum Channels With Memory

    Rybar, T.

    2012-01-01

    Quantum memory channels represent a very general, yet simple and comprehensible model for causal processes. As such they have attracted considerable research interest, mostly aimed on their transfer capabilities and structure properties. Most notably it was shown that memory channels can be implemented via physically naturally motivated collision models. We also define the concept of repeatable channels and show that only unital channels can be implemented repeat ably with pure memory channels. In the special case of qubit channels we also show that every unital qubit channel has a repeatable implementation. We also briefly explore the possibilities of stroboscopical simulation of channels and show that all random unitary channels can be stroboscopically simulated. Particularly in qubit case, all indivisible qubit channels are also random unitary, hence for qubit all indivisible channels can be stroboscopically simulated. Memory channels also naturally capture the framework of correlated experiments. We develop methods to gather and interpret data obtained in such setting and in detail examine the two qubit case. We also show that for control unitary interactions the measured data will never contradict a simple unitary evolution. Thus no memory effects can be spotted then. (author)

  16. Eight channel fast scalar

    Waddoup, W D; Stubbs, R J [Durham Univ. (UK)

    1977-11-01

    An eight channel 64-bit scaler has been constructed with a static CMOS memory. Scaling frequencies are independently variable, at each channel, as are the number of bits/channel. The scaler, when used in conjunction with a multichannel charge to time converter results in a very flexible, gated multichannel ADC.

  17. KV7 potassium channels

    Stott, Jennifer B; Jepps, Thomas Andrew; Greenwood, Iain A

    2014-01-01

    Potassium channels are key regulators of smooth muscle tone, with increases in activity resulting in hyperpolarisation of the cell membrane, which acts to oppose vasoconstriction. Several potassium channels exist within smooth muscle, but the KV7 family of voltage-gated potassium channels have been...

  18. Crystallization and Preliminary Diffraction Analysis of the CAL PDZ Domain in Complex with a Selective Peptide Inhibitor

    J Amacher; P Cushing; J Weiner; D Madden

    2011-12-31

    Cystic fibrosis (CF) is associated with loss-of-function mutations in the CF transmembrane conductance regulator (CFTR), which regulates epithelial fluid and ion homeostasis. The CFTR cytoplasmic C-terminus interacts with a number of PDZ (PSD-95/Dlg/ZO-1) proteins that modulate its intracellular trafficking and chloride-channel activity. Among these, the CFTR-associated ligand (CAL) has a negative effect on apical-membrane expression levels of the most common disease-associated mutant {Delta}F508-CFTR, making CAL a candidate target for the treatment of CF. A selective peptide inhibitor of the CAL PDZ domain (iCAL36) has recently been developed and shown to stabilize apical expression of {Delta}F508-CFTR, enhancing net chloride-channel activity, both alone and in combination with the folding corrector corr-4a. As a basis for structural studies of the CAL-iCAL36 interaction, a purification protocol has been developed that increases the oligomeric homogeneity of the protein. Here, the cocrystallization of the complex in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 35.9, b = 47.7, c = 97.3 {angstrom}, is reported. The crystals diffracted to 1.4 {angstrom} resolution. Based on the calculated Matthews coefficient (1.96 {angstrom}{sup 3} Da{sup -1}), it appears that the asymmetric unit contains two complexes.

  19. Magnetic interactions in iron (III) porphyrin chlorides

    Ernst, J.; Subramanian, Japyesan; Fuhrhop, J.H.

    1977-01-01

    Intermolecular exchange interactions in iron(III) porphyrin chlorides (porphyrin = OEP, proto, TPP) have been studied by X-ray structure, EPR and magnetic susceptibility studies. The crystal structure of Fe(III)OEP-Cl was found to be different from that of the other two. Different types of exchange broadened EPR-spectra are obtained which are attributable to the arrangement in the crystals. The EPR results correlate well with magnetic susceptibility data. (orig.) [de

  20. Potassium chloride production by microcline chlorination

    Orosco, Pablo, E-mail: porosco@unsl.edu.ar [Instituto de Investigaciones en Tecnología Química (INTEQUI), Chacabuco y Pedernera, San Luis (Argentina); Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis, Chacabuco y Pedernera, San Luis (Argentina); Ruiz, María del Carmen [Instituto de Investigaciones en Tecnología Química (INTEQUI), Chacabuco y Pedernera, San Luis (Argentina)

    2015-08-10

    Highlights: • Use of chlorination for the KCl production. • The reagents used were microcline, hydromagnesite and chlorine. • Isothermal and non-isothermal assays were performed in Cl{sub 2}–N{sub 2} mixture. • The chlorination generated KCl at 700 °C. • The chlorination products promote KCl formation. - Abstract: The potassium chloride is one of the most important fertilizers used in agriculture. The current demand of this salt makes interesting the study of potassium chloride production from unconventional potassium resources. In this work the potassium chloride production by chlorination of microcline was investigated. The starting reagents were microcline, hydromagnesite and chlorine. Non-isothermal and isothermal chlorination assays were carried out in a thermogravimetric device adapted to work in corrosive atmospheres. The temperature effect on potassium extraction and the phase transformations produced during chlorination of microcline were studied. The reagents and reaction products were analyzed by X-ray fluorescence (XRF) and X-ray diffraction (XRD). The experimental results indicated that by chlorination of microcline an important extraction of potassium in the temperature range from 800 to 900 °C was produced. Moreover, at 800 °C the forsterite, enstatite and magnesium aluminate spinel phases were generated.

  1. Radiochemical determination of methylmercury chloride Part 1

    Stary, J.; Prasilova, J.

    1976-01-01

    The isotope exchange between methylmercury species and an excess of inorganic radiomercury in sulphuric acid medium has been used for the simple determination of methylmercury chloride down to 0.01 ppm. The determination is not influenced by the presence of a great excess of other metals, however, chlorides, bromides and iodides interfere in higher concentrations. It has been found that the isotope exchange between CH 3 HgCl and 203 HgCl 4 2- (or 203 HgCl 2 ) in 0.01-3M hydrochloric acid is extremely slow, for the bimolecular reaction the rate constant is lower than 10 -3 mol -1 s -1 at 25 deg C. The isotope exchange rate between methylmercury chloride and mercuric-nitrate 0n on 0.5M sulphuric acid is higher. The isotope exchange is a bimolecular reaction with a rate constant k=0.050+-0.004 mol -1 s -1 at 25 deg C. (T.I.)

  2. Analysis of lithium/thionyl chloride batteries

    Jain, Mukul

    The lithium/thionyl chloride battery (Li/SOClsb2) has received considerable attention as a primary energy source due to its high energy density, high operating cell voltage, voltage stability over 95% of the discharge, large operating temperature range (-55sp°C to 70sp°C), long storage life, and low cost of materials. In this dissertation, a one-dimensional mathematical model of a spirally wound lithium/thionyl chloride primary battery has been developed. Mathematical models can be used to tailor a battery design to a specific application, perform accelerated testing, and reduce the amount of experimental data required to yield efficient, yet safe cells. The Model was used in conjunction with the experimental data for parameter estimation and to obtain insights into the fundamental processes occurring in the battery. The diffusion coefficient and the kinetic parameters for the reactions at the anode and the cathode are obtained as a function of temperature by fitting the simulated capacity and average cell voltage to experimental data over a wide range of temperatures (-55 to 49sp°C) and discharge loads (10 to 250 ohms). The experiments were performed on D-sized, cathode-limited, spirally wound lithium/thionyl chloride cells at Sandia National Laboratories. The model is also used to study the effect of cathode thickness and current and temperature pulsing on the cell capacity. Thionyl chloride reduction in the porous cathode is accompanied with a volume reduction. The material balance used previously in one-dimensional mathematical models of porous electrodes is invalid when the volume occupied by the reactants and the products is not equal. It is shown here how the material balance has to be modified to either account for the loss in volume, or to account for the inflow of electrolyte from the header into the active pores. The one-dimensional mathematical model of lithium/thionyl chloride primary battery is used to illustrate the effect of this material balance

  3. Fine Channel Networks

    1997-01-01

    A color image of fine channel networks on Mars; north toward top. The scene shows heavily cratered highlands dissected by dendritic open channel networks that dissect steep slopes of impact crater walls. This image is a composite of Viking high-resolution images in black and white and low-resolution images in color. The image extends from latitude 9 degrees S. to 5 degrees S. and from longitude 312 degrees to 320 degrees; Mercator projection. The dendritic pattern of the fine channels and their location on steep slopes leads to the interpretation that these are runoff channels. The restriction of these types of channels to ancient highland rocks suggests that these channels are old and date from a time on Mars when conditions existed for precipitation to actively erode rocks. After the channels reach a low plain, they appear to end. Termination may have resulted from burial by younger deposits or perhaps the flows percolated into the surface materials and continued underground.

  4. Ion channels in plants.

    Hedrich, Rainer

    2012-10-01

    Since the first recordings of single potassium channel activities in the plasma membrane of guard cells more than 25 years ago, patch-clamp studies discovered a variety of ion channels in all cell types and plant species under inspection. Their properties differed in a cell type- and cell membrane-dependent manner. Guard cells, for which the existence of plant potassium channels was initially documented, advanced to a versatile model system for studying plant ion channel structure, function, and physiology. Interestingly, one of the first identified potassium-channel genes encoding the Shaker-type channel KAT1 was shown to be highly expressed in guard cells. KAT1-type channels from Arabidopsis thaliana and its homologs from other species were found to encode the K(+)-selective inward rectifiers that had already been recorded in early patch-clamp studies with guard cells. Within the genome era, additional Arabidopsis Shaker-type channels appeared. All nine members of the Arabidopsis Shaker family are localized at the plasma membrane, where they either operate as inward rectifiers, outward rectifiers, weak voltage-dependent channels, or electrically silent, but modulatory subunits. The vacuole membrane, in contrast, harbors a set of two-pore K(+) channels. Just very recently, two plant anion channel families of the SLAC/SLAH and ALMT/QUAC type were identified. SLAC1/SLAH3 and QUAC1 are expressed in guard cells and mediate Slow- and Rapid-type anion currents, respectively, that are involved in volume and turgor regulation. Anion channels in guard cells and other plant cells are key targets within often complex signaling networks. Here, the present knowledge is reviewed for the plant ion channel biology. Special emphasis is drawn to the molecular mechanisms of channel regulation, in the context of model systems and in the light of evolution.

  5. Crystal structures of salicylideneguanylhydrazinium chloride and its copper(II) and cobalt(III) chloride complexes

    Chumakov, Yu. M.; Tsapkov, V. I.; Bocelli, G.; Antosyak, B. Ya.; Shova, S. G.; Gulea, A. P.

    2006-01-01

    The crystal structures of salicylideneguanylhydrazinium chloride hydrate hemiethanol solvate (I), salicylideneguanylhydrazinium trichloroaquacuprate(II) (II), and bis(salicylideneguanylhydrazino)cobalt(III) chloride trihydrate (III) are determined using X-ray diffraction. The structures of compounds I, II, and III are solved by direct methods and refined using the least-squares procedure in the anisotropic approximation for the non-hydrogen atoms to the final factors R = 0.0597, 0.0212, and 0.0283, respectively. In the structure of compound I, the monoprotonated molecules and chlorine ions linked by hydrogen bonds form layers aligned parallel to the (010) plane. In the structure of compound II, the salicylaldehyde guanylhydrazone cations and polymer chains consisting of trichloroaquacuprate(II) anions are joined by an extended three-dimensional network of hydrogen bonds. In the structure of compound III, the [Co(LH) 2 ] + cations, chloride ions, and molecules of crystallization water are linked together by a similar network

  6. Growth and characterization of magnesium chloride and lanthanum chloride doped strontium tartrate crystals - gel method

    Kalaiarasi, S.; Jaikumar, D.

    2014-01-01

    Growth of single crystals of doped strontium tartrate by controlled diffusion of strontium chloride into the silica gel charged with tartaric acid at room temperature is narrated. In this study, we synthesized magnesium chloride (5% and 10%) doped strontium tartrate crystals and Lanthanum chloride (5%, 10% and 15%) doped strontium tartrate crystals are grown. The crystal structure of the compound crystals was confirmed by single crystal X-ray diffraction. The Fourier transform infrared spectrum of pure and doped crystals are recorded and analyzed. The UV-Vis-NIR spectrum analysis reveals that the optical study of the grown crystals. The second harmonic generation efficiency was measured by using Kurtz powder technique with Nd:YAG laser of wavelength 1064 nm. (author)

  7. Endogenous chloride channels of insect sf9 cells. Evidence for coordinated activity of small elementary channel units

    Larsen, Erik Hviid; Gabriel, S. E.; Stutts, M. J.

    1996-01-01

    The endogenous Cl- conductance of Spodoptera frugiperda (Sf9) cells was studied 20-35 h after plating out of either uninfected cells or cells infected by a baculovirus vector carrying the cloned beta-galactosidase gene (beta-Gal cells). With the cation Tris+ in the pipette and Na+ in the bath...

  8. An increase in [Ca2+]i activates basolateral chloride channels and inhibits apical sodium channels in frog skin epithelium

    Brodin, Birger; Rytved, K A; Nielsen, R

    1996-01-01

    The aim of this study was to investigate the mechanisms by which increases in free cytosolic calcium ([Ca2+]i) cause a decrease in macroscopic sodium absorption across principal cells of the frog skin epithelium. [Ca2+]i was measured with fura-2 in an epifluorescence microscope set-up, sodium abs...

  9. Genetic disorders of transporters/channels in the inner ear and their relation to the kidney.

    Peters, T.A.; Monnens, L.A.H.; Cremers, C.W.R.J.; Curfs, J.H.A.J.

    2004-01-01

    Inner ear physiology is reviewed with emphasis on features common to renal physiology. Genetic disorders in transporters/channels for chloride (ClC-K), bicarbonate (Cl(-)/HCO(3)(-) exchanger), protons (H(+)-ATPase), sodium (ENaC, NKKC1, NBC3, NHE3), potassium (KCNQ1/KCNE1, Kcc4), and water (AQP4) in

  10. Endocochlear potential depends on Cl- channels: Mechanism underlying deafness in Bartter syndrome IV

    G. Rickheit (Gesa); H. Maier (Hannes); N. Strenzke (Nicola); C.E. Andreescu (Corina); C.I. de Zeeuw (Chris); A. Muenscher (Adrian); A.A. Zdebik (Anselm); T.J. Jentsch (Thomas)

    2008-01-01

    textabstractHuman Bartter syndrome IV is an autosomal recessive disorder characterized by congenital deafness and severe renal salt and fluid loss. It is caused by mutations in BSND, which encodes barttin, a β-subunit of ClC-Ka and ClC-Kb chloride channels. Inner-ear-specific disruption of Bsnd in

  11. Sorption of sulphur dioxide in calcium chloride and nitrate chloride liquids

    Trzepierczynska, I.; Gostomczyk, M.A.

    1989-01-01

    Flue gas desulphurization via application of suspensions has one inherent disadvantage: fixation of sulphur dioxide is very poor. This should be attributed to the low content of calcium ions which results from the solubility of the sorbing species. The solubility of sparingly soluble salts (CaO, CaCO 3 ) may be increased by decreasing the pH of the solution; yet, there is a serious limitation in this method: the corrosivity of the scrubber. The objective of this paper was to assess the sorbing capacity of two soluble calcium salts, calcium chloride and calcium nitrate, as a function of calcium ion concentration in the range of 20 to 82 kg/m 3 . It has been found that sorbing capacity increases with the increasing calcium ion concentration until the calcium concentration in the calcium chloride solution reaches the level of 60 kg/m 3 which is equivalent to the chloride ion content of ∼ 110 kg/m 3 . Addition of calcium hydroxide to the solutions brings about an increase in the sorbing capacity up to 1.6 kg/m 3 and 2.2 kg/m 3 for calcium chloride and calcium nitrate, respectively, as a result of the increased sorbent alkalinity. The sorption capacity of the solutions is considerably enhanced by supplementing them by acetate ions (2.8 to 13.9 kg/m 3 ). Increase in the sorption capacity of calcium nitrate solutions enriched with calcium acetate was approximately 30% as high as that of the chloride solutions enriched with calcium acetate was approximately 30% as high as that of the chloride solutions supplemented in the same way. (author). 12 refs, 7 refs, 4 tabs

  12. NuLYTELY (PEG 3350, sodium chloride, sodium bicarbonate and potassium chloride for oral solution).

    Swartz, M L

    1992-02-01

    NuLYTELY (PEG 3350, Sodium Chloride, Sodium Bicarbonate, and Potassium Chloride for Oral Solution), a product from Braintree Laboratories, Inc. is a modification of GoLYTELY (PEG 3350 and Electrolytes for Oral Solution) that has been found to have the same therapeutic advantages in terms of safety, efficacy, speed and patient acceptance. This product was developed to improve upon the taste of GoLYTELY. NuLYTELY represents an effective alternative for bowel cleansing prior to colonoscopy that may be more acceptable to some patients.

  13. Compound Wiretap Channels

    Shlomo Shamai (Shitz

    2009-01-01

    Full Text Available This paper considers the compound wiretap channel, which generalizes Wyner's wiretap model to allow the channels to the (legitimate receiver and to the eavesdropper to take a number of possible states. No matter which states occur, the transmitter guarantees that the receiver decodes its message and that the eavesdropper is kept in full ignorance about the message. The compound wiretap channel can also be viewed as a multicast channel with multiple eavesdroppers, in which the transmitter sends information to all receivers and keeps the information secret from all eavesdroppers. For the discrete memoryless channel, lower and upper bounds on the secrecy capacity are derived. The secrecy capacity is established for the degraded channel and the semideterministic channel with one receiver. The parallel Gaussian channel is further studied. The secrecy capacity and the secrecy degree of freedom (s.d.o.f. are derived for the degraded case with one receiver. Schemes to achieve the s.d.o.f. for the case with two receivers and two eavesdroppers are constructed to demonstrate the necessity of a prefix channel in encoder design. Finally, the multi-antenna (i.e., MIMO compound wiretap channel is studied. The secrecy capacity is established for the degraded case and an achievable s.d.o.f. is given for the general case.

  14. Compound Wiretap Channels

    Kramer Gerhard

    2009-01-01

    Full Text Available Abstract This paper considers the compound wiretap channel, which generalizes Wyner's wiretap model to allow the channels to the (legitimate receiver and to the eavesdropper to take a number of possible states. No matter which states occur, the transmitter guarantees that the receiver decodes its message and that the eavesdropper is kept in full ignorance about the message. The compound wiretap channel can also be viewed as a multicast channel with multiple eavesdroppers, in which the transmitter sends information to all receivers and keeps the information secret from all eavesdroppers. For the discrete memoryless channel, lower and upper bounds on the secrecy capacity are derived. The secrecy capacity is established for the degraded channel and the semideterministic channel with one receiver. The parallel Gaussian channel is further studied. The secrecy capacity and the secrecy degree of freedom ( are derived for the degraded case with one receiver. Schemes to achieve the for the case with two receivers and two eavesdroppers are constructed to demonstrate the necessity of a prefix channel in encoder design. Finally, the multi-antenna (i.e., MIMO compound wiretap channel is studied. The secrecy capacity is established for the degraded case and an achievable is given for the general case.

  15. ATP Release Channels

    Akiyuki Taruno

    2018-03-01

    Full Text Available Adenosine triphosphate (ATP has been well established as an important extracellular ligand of autocrine signaling, intercellular communication, and neurotransmission with numerous physiological and pathophysiological roles. In addition to the classical exocytosis, non-vesicular mechanisms of cellular ATP release have been demonstrated in many cell types. Although large and negatively charged ATP molecules cannot diffuse across the lipid bilayer of the plasma membrane, conductive ATP release from the cytosol into the extracellular space is possible through ATP-permeable channels. Such channels must possess two minimum qualifications for ATP permeation: anion permeability and a large ion-conducting pore. Currently, five groups of channels are acknowledged as ATP-release channels: connexin hemichannels, pannexin 1, calcium homeostasis modulator 1 (CALHM1, volume-regulated anion channels (VRACs, also known as volume-sensitive outwardly rectifying (VSOR anion channels, and maxi-anion channels (MACs. Recently, major breakthroughs have been made in the field by molecular identification of CALHM1 as the action potential-dependent ATP-release channel in taste bud cells, LRRC8s as components of VRACs, and SLCO2A1 as a core subunit of MACs. Here, the function and physiological roles of these five groups of ATP-release channels are summarized, along with a discussion on the future implications of understanding these channels.

  16. Mapping the spatial distribution of chloride deposition across Australia

    Davies, P. J.; Crosbie, R. S.

    2018-06-01

    The high solubility and conservative behaviour of chloride make it ideal for use as an environmental tracer of water and salt movement through the hydrologic cycle. For such use the spatial distribution of chloride deposition in rainfall at a suitable scale must be known. A number of authors have used point data acquired from field studies of chloride deposition around Australia to construct relationships to characterise chloride deposition as a function of distance from the coast; these relationships have allowed chloride deposition to be interpolated in different regions around Australia. In this paper we took this a step further and developed a chloride deposition map for all of Australia which includes a quantification of uncertainty. A previously developed four parameter model of chloride deposition as a function of distance from the coast for Australia was used as the basis for producing a continental scale chloride deposition map. Each of the four model parameters were made spatially variable by creating parameter surfaces that were interpolated using a pilot point regularisation approach within a parameter estimation software. The observations of chloride deposition were drawn from a literature review that identified 291 point measurements of chloride deposition over a period of 80 years spread unevenly across all Australian States and Territories. A best estimate chloride deposition map was developed from the resulting surfaces on a 0.05 degree grid. The uncertainty in the chloride deposition map was quantified as the 5th and 95th percentile of 1000 calibrated models produced via Null Space Monte Carlo analysis and the spatial variability of chloride deposition across the continent was consistent with landscape morphology. The temporal variability in chloride deposition on a decadal scale was investigated in the Murray-Darling Basin, this highlighted the need for long-term monitoring of chloride deposition if the uncertainty of the continental scale map is

  17. Volume Regulated Channels

    Klausen, Thomas Kjær

    of volume perturbations evolution have developed system of channels and transporters to tightly control volume homeostasis. In the past decades evidence has been mounting, that the importance of these volume regulated channels and transporters are not restricted to the defense of cellular volume...... but are also essential for a number of physiological processes such as proliferation, controlled cell death, migration and endocrinology. The thesis have been focusing on two Channels, namely the swelling activated Cl- channel (ICl, swell) and the transient receptor potential Vanilloid (TRPV4) channel. I: Cl......- serves a multitude of functions in the mammalian cell, regulating the membrane potential (Em), cell volume, protein activity and the driving force for facilitated transporters giving Cl- and Cl- channels a major potential of regulating cellular function. These functions include control of the cell cycle...

  18. Properties of Single K+ and Cl− Channels in Asclepias tuberosa Protoplasts 1

    Schauf, Charles L.; Wilson, Kathryn J.

    1987-01-01

    Potassium and chloride channels were characterized in Asclepias tuberosa suspension cell derived protoplasts by patch voltage-clamp. Whole-cell currents and single channels in excised patches had linear instantaneous current-voltage relations, reversing at the Nernst potentials for K+ and Cl−, respectively. Whole cell K+ currents activated exponentially during step depolarizations, while voltage-dependent Cl− channels were activated by hyperpolarizations. Single K+ channel conductance was 40 ± 5 pS with a mean open time of 4.5 milliseconds at 100 millivolts. Potassium channels were blocked by Cs+ and tetraethylammonium, but were insensitive to 4-aminopyridine. Chloride channels had a single-channel conductance of 100 ± 17 picosiemens, mean open time of 8.8 milliseconds, and were blocked by Zn2+ and ethacrynic acid. Whole-cell Cl− currents were inhibited by abscisic acid, and were unaffected by indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid. Since internal and external composition can be controlled, patch-clamped protoplasts are ideal systems for studying the role of ion channels in plant physiology and development. Images Fig. 5 PMID:16665712

  19. Buried chloride stereochemistry in the Protein Data Bank.

    Carugo, Oliviero

    2014-09-23

    Despite the chloride anion is involved in fundamental biological processes, its interactions with proteins are little known. In particular, we lack a systematic survey of its coordination spheres. The analysis of a non-redundant set (pairwise sequence identity chloride anion shows that the first coordination spheres of the chlorides are essentially constituted by hydrogen bond donors. Amongst the side-chains positively charged, arginine interacts with chlorides much more frequently than lysine. Although the most common coordination number is 4, the coordination stereochemistry is closer to the expected geometry when the coordination number is 5, suggesting that this is the coordination number towards which the chlorides tend when they interact with proteins. The results of these analyses are useful in interpreting, describing, and validating new protein crystal structures that contain chloride anions.

  20. HIPPI and Fibre Channel

    Tolmie, D.E.

    1992-01-01

    The High-Performance Parallel Interface (HIPPI) and Fibre Channel are near-gigabit per second data communications interfaces being developed in ANSI standards Task Group X3T9.3. HIPPI is the current interface of choice in the high-end and supercomputer arena, and Fibre Channel is a follow-on effort. HIPPI came from a local area network background, and Fibre Channel came from a mainframe to peripheral interface background

  1. Nuclear reactor coolant channels

    Macbeth, R.V.

    1978-01-01

    Reference is made to coolant channels for pressurised water and boiling water reactors and the arrangement described aims to improve heat transfer between the fuel rods and the coolant. Baffle means extending axially within the channel are provided and disposed relative to the fuel rods so as to restrict flow oscillations occurring within the coolant from being propagated transversely to the axis of the channel. (UK)

  2. New Channels, New Possibilities

    Pieterson, Willem; Ebbers, Wolfgang; Østergaard Madsen, Christian

    2017-01-01

    In this contribution we discuss the characteristics of what we call the fourth generation of public sector service channels: social robots. Based on a review of relevant literature we discuss their characteristics and place into multi-channel models of service delivery. We argue that social robots......-channel models of service delivery. This is especially relevant given the current lack of evaluations of such models, the broad range of channels available, and their different stages of deployment at governments around the world. Nevertheless, social robots offer an potentially very relevant addition...

  3. Calcium Channel Blockers

    ... Certain calcium channel blockers interact with grapefruit products. Kaplan NM, et al. Treatment of hypertension: Drug therapy. In: Kaplan's Clinical Hypertension. 11th ed. Philadelphia, Pa.: Wolters Kluwer ...

  4. A channel profile analyser

    Gobbur, S.G.

    1983-01-01

    It is well understood that due to the wide band noise present in a nuclear analog-to-digital converter, events at the boundaries of adjacent channels are shared. It is a difficult and laborious process to exactly find out the shape of the channels at the boundaries. A simple scheme has been developed for the direct display of channel shape of any type of ADC on a cathode ray oscilliscope display. This has been accomplished by sequentially incrementing the reference voltage of a precision pulse generator by a fraction of a channel and storing ADC data in alternative memory locations of a multichannel pulse height analyser. Alternative channels are needed due to the sharing at the boundaries of channels. In the flat region of the profile alternate memory locations are channels with zero counts and channels with the full scale counts. At the boundaries all memory locations will have counts. The shape of this is a direct display of the channel boundaries. (orig.)

  5. Anodic Behavior of Alloy 22 in Calcium Chloride and in Calcium Chloride Plus Calcium Nitrate Brines

    Evans, K.J.; Day, S.D.; Ilevbare, G.O.; Whalen, M.T.; King, K.J.; Hust, G.A.; Wong, L.L.; Estill, J.C.; Rebak, R.B.

    2003-01-01

    Alloy 22 (UNS N60622) is a nickel-based alloy, which is extensively used in aggressive industrial applications, especially due to its resistance to localized corrosion and stress corrosion cracking in high chloride environments. The purpose of this work was to characterize the anodic behavior of Alloy 22 in concentrated calcium chloride (CaCl 2 ) brines and to evaluate the inhibitive effect of nitrate, especially to localized corrosion. Standard electrochemical tests such as polarization resistance and cyclic polarization were used. Results show that the corrosion potential of Alloy 22 was approximately -360 mV in the silver-silver chloride (SSC) scale and independent of the tested temperature. Cyclic polarization tests showed that Alloy 22 was mainly susceptible to localized attack in 5 M CaCl 2 at 75 C and higher temperatures. The addition of nitrate in a molar ratio of chloride to nitrate equal to 10 increased the onset of localized corrosion to approximately 105 C. The addition of nitrate to the solution also decreased the uniform corrosion rate and the passive current of the alloy

  6. Structure and function of the cystic fibrosis transmembrane conductance regulator

    M.M. Morales

    1999-08-01

    Full Text Available Cystic fibrosis (CF is a lethal autosomal recessive genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR. Mutations in the CFTR gene may result in a defective processing of its protein and alter the function and regulation of this channel. Mutations are associated with different symptoms, including pancreatic insufficiency, bile duct obstruction, infertility in males, high sweat Cl-, intestinal obstruction, nasal polyp formation, chronic sinusitis, mucus dehydration, and chronic Pseudomonas aeruginosa and Staphylococcus aureus lung infection, responsible for 90% of the mortality of CF patients. The gene responsible for the cellular defect in CF was cloned in 1989 and its protein product CFTR is activated by an increase of intracellular cAMP. The CFTR contains two membrane domains, each with six transmembrane domain segments, two nucleotide-binding domains (NBDs, and a cytoplasmic domain. In this review we discuss the studies that have correlated the role of each CFTR domain in the protein function as a chloride channel and as a regulator of the outwardly rectifying Cl- channels (ORCCs.

  7. Catalytic Conversion of Cellulose to Levulinic Acid by Metal Chlorides

    Beixiao Zhang

    2010-08-01

    Full Text Available The catalytic performance of various metal chlorides in the conversion of cellulose to levulinic acid in liquid water at high temperatures was investigated. The effects of reaction parameters on the yield of levulinic acid were also explored. The results showed that alkali and alkaline earth metal chlorides were not effective in conversion of cellulose, while transition metal chlorides, especially CrCl3, FeCl3 and CuCl2 and a group IIIA metal chloride (AlCl3, exhibited high catalytic activity. The catalytic performance was correlated with the acidity of the reaction system due to the addition of the metal chlorides, but more dependent on the type of metal chloride. Among those metal chlorides, chromium chloride was found to be exceptionally effective for the conversion of cellulose to levulinic acid, affording an optimum yield of 67 mol % after a reaction time of 180 min, at 200 °C, with a catalyst dosage of 0.02 M and substrate concentration of 50 wt %. Chromium metal, most of which was present in its oxide form in the solid sample and only a small part in solution as Cr3+ ion, can be easily separated from the resulting product mixture and recycled. Finally, a plausible reaction scheme for the chromium chloride catalyzed conversion of cellulose in water was proposed.

  8. On barium oxide solubility in barium-containing chloride melts

    Nikolaeva, Elena V.; Zakiryanova, Irina D.; Bovet, Andrey L.; Korzun, Iraida V.

    2016-01-01

    Oxide solubility in chloride melts depends on temperature and composition of molten solvent. The solubility of barium oxide in the solvents with barium chloride content is essentially higher than that in molten alkali chlorides. Spectral data demonstrate the existence of oxychloride ionic groupings in such melts. This work presents the results of the BaO solubility in two molten BaCl 2 -NaCl systems with different barium chloride content. The received data together with earlier published results revealed the main regularities of BaO solubility in molten BaO-BaCl 2 -MCl systems.

  9. Interpretation of postmortem vitreous concentrations of sodium and chloride.

    Zilg, B; Alkass, K; Berg, S; Druid, H

    2016-06-01

    Vitreous fluid can be used to analyze sodium and chloride levels in deceased persons, but it remains unclear to what extent such results can be used to diagnose antemortem sodium or chloride imbalances. In this study we present vitreous sodium and chloride levels from more than 3000 cases. We show that vitreous sodium and chloride levels both decrease with approximately 2.2mmol/L per day after death. Since potassium is a well-established marker for postmortem interval (PMI) and easily can be analyzed along with sodium and chloride, we have correlated sodium and chloride levels with the potassium levels and present postmortem reference ranges relative the potassium levels. We found that virtually all cases outside the reference range show signs of antemortem hypo- or hypernatremia. Vitreous sodium or chloride levels can be the only means to diagnose cases of water or salt intoxication, beer potomania or dehydration. We further show that postmortem vitreous sodium and chloride strongly correlate and in practice can be used interchangeably if analysis of one of the ions fails. It has been suggested that vitreous sodium and chloride levels can be used to diagnose drowning or to distinguish saltwater from freshwater drowning. Our results show that in cases of freshwater drowning, vitreous sodium levels are decreased, but that this mainly is an effect of postmortem diffusion between the eye and surrounding water rather than due to the drowning process, since the decrease in sodium levels correlates with immersion time. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  10. VOLATILE CHLORIDE PROCESS FOR THE RECOVERY OF METAL VALUES

    Hanley, W.R.

    1959-01-01

    A process is presented for recovering uranium, iron, and aluminum from centain shale type ores which contain uranium in minute quantities. The ore is heated wiih a chlorinating agent. such as chlorine, to form a volatilized stream of metal chlorides. The chloride stream is then passed through granular alumina which preferentially absorbs the volatile uranium chloride and from which the uranium may later be recovered. The remaining volatilized chlorides, chiefly those of iron and aluminum, are further treated to recover chlorine gas for recycle, and to recover ferric oxide and aluminum oxide as valuable by-products.

  11. Fault locator of an allyl chloride plant

    Savković-Stevanović Jelenka B.

    2004-01-01

    Full Text Available Process safety analysis, which includes qualitative fault event identification, the relative frequency and event probability functions, as well as consequence analysis, was performed on an allye chloride plant. An event tree for fault diagnosis and cognitive reliability analysis, as well as a troubleshooting system, were developed. Fuzzy inductive reasoning illustrated the advantages compared to crisp inductive reasoning. A qualitative model forecast the future behavior of the system in the case of accident detection and then compared it with the actual measured data. A cognitive model including qualitative and quantitative information by fuzzy logic of the incident scenario was derived as a fault locator for an ally! chloride plant. The obtained results showed the successful application of cognitive dispersion modeling to process safety analysis. A fuzzy inductive reasoner illustrated good performance to discriminate between different types of malfunctions. This fault locator allowed risk analysis and the construction of a fault tolerant system. This study is the first report in the literature showing the cognitive reliability analysis method.

  12. Thermodynamic properties of potassium chloride aqueous solutions

    Zezin, Denis; Driesner, Thomas

    2017-04-01

    Potassium chloride is a ubiquitous salt in natural fluids, being the second most abundant dissolved salt in many geological aqueous solutions after sodium chloride. It is a simple solute and strong electrolyte easily dissociating in water, however the thermodynamic properties of KCl aqueous solutions were never correlated with sufficient accuracy for a wide range of physicochemical conditions. In this communication we propose a set of parameters for a Pitzer-type model which allows calculation of all necessary thermodynamic properties of KCl solution, namely excess Gibbs free energy and derived activity coefficient, apparent molar enthalpy, heat capacity and volume, as well as osmotic coefficient and activity of water in solutions. The system KCl-water is one of the best studied aqueous systems containing electrolytes. Although extensive experimental data were collected for thermodynamic properties of these solutions over the years, the accurate volumetric data became available only recently, thus making possible a complete thermodynamic formulation including a pressure dependence of excess Gibbs free energy and derived properties of the KCl-water liquids. Our proposed model is intended for calculation of major thermodynamic properties of KCl aqueous solutions at temperatures ranging from freezing point of a solution to 623 K, pressures ranging from saturated water vapor up to 150 MPa, and concentrations up to the salt saturation. This parameterized model will be further implemented in geochemical software packages and can facilitate the calculation of aqueous equilibrium for reactive transport codes.

  13. The DELTA 181 lithium thionyl chloride battery

    Sullivan, Ralph M.; Brown, Lawrence E.; Leigh, A. P.

    In 1986, the Johns Hopkins University/Applied Physics Laboratory (JHU/APL) undertook the development of a sensor module for the DELTA 181 spacecraft, a low earth orbit (LEO) mission of less than two months duration. A large lithium thionyl chloride battery was developed as the spacecraft's primary power source, the first known such use for this technology. The exceptionally high energy density of the lithium thionyl chloride cell was the primary driver for its use, resulting in a completed battery with a specific energy density of 120 Wh/lb. Safety requirements became the primary driver shaping all aspects of the power system design and development due to concerns about the potential hazards of this relatively new, high-energy technology. However, the program was completed without incident. The spacecraft was launched on February 8, 1988, from Kennedy Space Center (KSC) with over 60,000 Wh of battery energy. It reentered on April 2, 1988, still operating after 55 days, providing a successful, practical, and visible demonstration of the use of this technology for spacecraft applications.

  14. Different Methods for Conditioning Chloride Salt Wastes

    De Angelis, G.; Fedeli, C.; Capone, M.; Marzo, G.A.; Mariani, M.; Da Ros, M.; Giacobbo, F.; Macerata, E.; Giola, M.

    2015-01-01

    Three different methods have been used to condition chloride salt wastes coming from pyro-processes. Two of them allow to synthesise sodalite, a naturally occurring mineral containing chlorine: the former, starting from Zeolite 4A, which transforms the zeolite into sodalite; the latter, which starts from kaolinite, giving sodalite as well. In addition, a new matrix, termed SAP (SiO 2 -Al 2 O 3 -P 2 O 5 ), has been synthesised. It is able to form different mineral phases which occlude fission metals. The products from the different processes have been fully characterised. In particular the chemical durability of the final waste forms has been determined using the standard product consistency test. According to the results obtained, SAP seems to be a promising matrix for the incorporation of chloride salt wastes from pyro-processes. Financial support from the Nuclear Fission Safety Programme of the European Union (projects ACSEPT, contract FP7-CP-2007- 211 267, and SACSESS, Collaborative Project 323282), as well as from Italian Ministry for Economic Development (Accordo di Programma: Piano Annuale di Realizzazione 2008-2009) is gratefully acknowledged. (authors)

  15. Total gastrectomy due to ferric chloride intoxication.

    Menéndez, A Mesut; Abramson, Leonardo; Vera, Raúl A; Duza, Guillermo E; Palermo, Mariano

    2015-09-01

    The ferric chloride intoxication is frequently caused by accident. Its toxicity is generally underrated, which can lead to fatal evolution or irreversible consequences. In this case, the caustic condition of the substance is related to the toxic properties of iron. A 36-year-old male patient arrives by ambulance indicating sensory deterioration. He presents erosive injuries in the buccal cavity and in the oropharynx, brownish teeth and metabolic acidosis. Toxicology tests and ferritin blood dosage are requested, which show a result from 1400 mg/dl. The symptoms are interpreted as acute iron intoxication. Due to the unfavorable evolution of his condition, an abdominal and pelvic CT scan are performed, which show extensive pneumoperitoneum and free fluid in the abdominal cavity. An exploratory laparotomy, a total gastrectomy with esophagostomy and feeding jejunostomy, washing and drainage due to perforated gastric necrosis caused by caustic ingestion are performed. In our country, there is a high rate of intoxication caused by iron compounds, although it is not statistically measured. Nevertheless, the ferric chloride intoxication is extremely infrequent. The ingestion of this product leads to complications, which are associated with the iron concentration and its condition as a caustic agent. The surgical indications in the presence of intoxication caused by iron compounds are: stomach evacuation of iron, gastric necrosis, perforation or peritonitis and stenosis. Early or prophylactic gastrectomy is contraindicated. However, if complications that require immediate surgical intervention arise, there should be no hesitation and the corresponding procedure should be performed.

  16. CORK Study in Cystic Fibrosis: Sustained Improvements in Ultra-Low-Dose Chest CT Scores After CFTR Modulation With Ivacaftor.

    Ronan, Nicola J; Einarsson, Gisli G; Twomey, Maria; Mooney, Denver; Mullane, David; NiChroinin, Muireann; O'Callaghan, Grace; Shanahan, Fergus; Murphy, Desmond M; O'Connor, Owen J; Shortt, Cathy A; Tunney, Michael M; Eustace, Joseph A; Maher, Michael M; Elborn, J Stuart; Plant, Barry J

    2018-02-01

    Ivacaftor produces significant clinical benefit in patients with cystic fibrosis (CF) with the G551D mutation. Prevalence of this mutation at the Cork CF Centre is 23%. This study assessed the impact of cystic fibrosis transmembrane conductance regulator modulation on multiple modalities of patient assessment. Thirty-three patients with the G551D mutation were assessed at baseline and prospectively every 3 months for 1 year after initiation of ivacaftor. Change in ultra-low-dose chest CT scans, blood inflammatory mediators, and the sputum microbiome were assessed. Significant improvements in FEV 1 , BMI, and sweat chloride levels were observed post-ivacaftor treatment. Improvement in ultra-low-dose CT imaging scores were observed after treatment, with significant mean reductions in total Bhalla score (P < .01), peribronchial thickening (P = .035), and extent of mucous plugging (P < .001). Reductions in circulating inflammatory markers, including interleukin (IL)-1β, IL-6, and IL-8 were demonstrated. There was a 30% reduction in the relative abundance of Pseudomonas species and an increase in the relative abundance of bacteria associated with more stable community structures. Posttreatment community richness increased significantly (P = .03). Early and sustained improvements on ultra-low-dose CT scores suggest it may be a useful method of evaluating treatment response. It paralleled improvement in symptoms, circulating inflammatory markers, and changes in the lung microbiota. Copyright © 2017 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.

  17. Opening up of plasmalemma type-1 VDAC to form apoptotic "find me signal" pathways is essential in early apoptosis - evidence from the pathogenesis of cystic fibrosis resulting from failure of apoptotic cell clearance followed by sterile inflammation.

    Thinnes, Friedrich P

    2014-04-01

    Cell membrane-standing type-1 VDAC is involved in cell volume regulation and thus apoptosis. The channel has been shown to figure as a pathway for osmolytes of varying classes, ATP included. An early event in apoptotic cell death is the release of "find me signals" by cells that enter the apoptotic process. ATP is one of those signals. Apoptotic cells this way attract phagocytes for an immunologically silent cell clearance. Thus, whenever apoptosis fails by a blockade of plasmalemma type-1 VDAC processes of sterile inflammation must be assumed for cell elimination. This is evident from a close look on the pathogenetic process of cystic fibrosis (CF). However, in normal airway epithelia two different anion channels cooperate to guarantee an appropriate volume of airway surface liquid (ASL) necessary for surface clearing: the cystic fibrosis conductance regulator (CFTR) and the outwardly rectifying chloride channel (ORCC) complex also called "alternate chloride channel" and under the control of the CFTR. There are arguments, that type-1 VDAC forms the channel part of the ORCC complex, and it has been shown that CFTR and type-1 VDAC co-localize in the apical membranes of human surface respiratory epithelium. In cystic fibrosis, the central cAMP-dependent regulation of ion and water transport via functional CFTR is lost. Here, CFTR molecules do not reach the apical membranes of airway epithelia anymore or work in an insufficient way, respectively. In addition, type-1 VDAC is no longer available to work as a "find me signal" pathway. In consequence, clearing away of apoptotic cells is blocked. There are experimental data on the channel characteristics of type-1 VDAC under the anion channel blocker DIDS (4,4-diisothiocyanato-stilbenedisulphonic acid) that argue in favor of this hypothesis. Together, type-1 VDAC should be kept as a "find me signal" pathway, which may give way to several classes of such signals. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. A linearization of quantum channels

    Crowder, Tanner

    2015-06-01

    Because the quantum channels form a compact, convex set, we can express any quantum channel as a convex combination of extremal channels. We give a Euclidean representation for the channels whose inverses are also valid channels; these are a subset of the extreme points. They form a compact, connected Lie group, and we calculate its Lie algebra. Lastly, we calculate a maximal torus for the group and provide a constructive approach to decomposing any invertible channel into a product of elementary channels.

  19. Degradation of fly ash concrete under the coupled effect of carbonation and chloride aerosol ingress

    Liu, Jun; Qiu, Qiwen; Chen, Xiaochi; Wang, Xiaodong; Xing, Feng; Han, Ningxu; He, Yijian

    2016-01-01

    Highlights: • Carbonation affects the chloride profile in concrete under chloride aerosol attack. • The chloride binding capacity can be reduced by the presence of carbonation. • Carbonation increases the rate of chloride diffusion for chloride aerosol ingress. • Chloride aerosol ingress reduces the carbonation depth and increases the pH value. • The use of fly ash in concrete enhances the resistance of chloride aerosol ingress. - Abstract: This paper presents an experimental investigation regarding the coupled effect of carbonation and chloride aerosol ingress on the durability performance of fly ash concrete. Test results demonstrate that carbonation significantly affects the chloride ingress profile, reduces the chloride binding capacity, and accelerates the rate of chloride ion diffusion. On the other hand, the carbonation rate of fly ash concrete is reduced by the presence of chlorides aerosol. The interaction nature between concrete carbonation and chloride aerosol ingress is also demonstrated by the microscopic analysis results obtained from scanning electron microscope and mercury intrusion porosimetry.

  20. Omni channel fashion shopping

    Kemperman, A.D.A.M.; van Delft, L.; Borgers, A.W.J.; Pantano, E.

    2015-01-01

    This chapter gives insight into consumers' online and offline fashion shopping behavior, consumers' omni-channel usage during the shopping process, and consumer fashion shopper segments. Based on a literature review, omni-channel shopping behavior during the shopping process was operationalized.

  1. An improved ivermectin-activated chloride channel receptor for inhibiting electrical activity in defined neuronal populations

    Lynagh, Timothy Peter; Lynch, Joseph W

    2010-01-01

    The ability to silence the electrical activity of defined neuronal populations in vivo is dramatically advancing our understanding of brain function. This technology may eventually be useful clinically for treating a variety of neuropathological disorders caused by excessive neuronal activity...... conductance, homomeric expression, and human origin may render the F207A/A288G alpha1 glycine receptor an improved silencing receptor for neuroscientific and clinical purposes. As all known highly ivermectin-sensitive GluClRs contain an endogenous glycine residue at the corresponding location, this residue...

  2. Identification of Chloride Intracellular Channel Protein 3 as a Novel Gene Affecting Human Bone Formation

    Brum, A M; Leije, M; J, Schreuders-Koedam

    2017-01-01

    is diminished and more adipocytes are seen in the bone marrow, suggesting a shift in MSC lineage commitment. Identification of specific factors that stimulate osteoblast differentiation from human MSCs may deliver therapeutic targets to treat osteoporosis. The aim of this study was to identify novel genes...... an in vivo human bone formation model in which hMSCs lentivirally transduced with the CLIC3 overexpression construct were loaded onto a scaffold (hydroxyapatite-tricalcium-phosphate), implanted under the skin of NOD-SCID mice, and analyzed for bone formation 8 weeks later. CLIC3 overexpression led to a 15...

  3. Mechanism of action of the insecticides, lindane and fipronil, on glycine receptor chloride channels.

    Islam, Robiul; Lynch, Joseph W

    2012-04-01

    Docking studies predict that the insecticides, lindane and fipronil, block GABA(A) receptors by binding to 6' pore-lining residues. However, this has never been tested at any Cys-loop receptor. The neurotoxic effects of these insecticides are also thought to be mediated by GABA(A) receptors, although a recent morphological study suggested glycine receptors mediated fipronil toxicity in zebrafish. Here we investigated whether human α1, α1β, α2 and α3 glycine receptors were sufficiently sensitive to block by either compound as to represent possible neurotoxicity targets. We also investigated the mechanisms by which lindane and fipronil inhibit α1 glycine receptors. Glycine receptors were recombinantly expressed in HEK293 cells and insecticide effects were studied using patch-clamp electrophysiology. Both compounds completely inhibited all tested glycine receptor subtypes with IC(50) values ranging from 0.2-2 µM, similar to their potencies at vertebrate GABA(A) receptors. Consistent with molecular docking predictions, both lindane and fipronil interacted with 6' threonine residues via hydrophobic interactions and hydrogen bonds. In contrast with predictions, we found no evidence for lindane interacting at the 2' level. We present evidence for fipronil binding in a non-blocking mode in the anaesthetic binding pocket, and for lindane as an excellent pharmacological tool for identifying the presence of β subunits in αβ heteromeric glycine receptors. This study implicates glycine receptors as novel vertebrate toxicity targets for fipronil and lindane. Furthermore, lindane interacted with pore-lining 6' threonine residues, whereas fipronil may have both pore and non-pore binding sites. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

  4. Channel electron multipliers

    Seidman, A.; Avrahami, Z.; Sheinfux, B.; Grinberg, J.

    1976-01-01

    A channel electron multiplier is described having a tubular wall coated with a secondary-electron emitting material and including an electric field for accelerating the electrons, the electric field comprising a plurality of low-resistive conductive rings each alternating with a high-resistive insulating ring. The thickness of the low-resistive rings is many times larger than that of the high-resistive rings, being in the order of tens of microns for the low-resistive rings and at least one order of magnitude lower for the high-resistive rings; and the diameter of the channel tubular walls is also many times larger than the thickness of the high-resistive rings. Both single-channel and multiple-channel electron multipliers are described. A very important advantage, particularly in making multiple-channel multipliers, is the simplicity of the procedure that may be used in constructing such multipliers. Other operational advantages are described

  5. Cardiac potassium channel subtypes

    Schmitt, Nicole; Grunnet, Morten; Olesen, Søren-Peter

    2014-01-01

    About 10 distinct potassium channels in the heart are involved in shaping the action potential. Some of the K(+) channels are primarily responsible for early repolarization, whereas others drive late repolarization and still others are open throughout the cardiac cycle. Three main K(+) channels...... drive the late repolarization of the ventricle with some redundancy, and in atria this repolarization reserve is supplemented by the fairly atrial-specific KV1.5, Kir3, KCa, and K2P channels. The role of the latter two subtypes in atria is currently being clarified, and several findings indicate...... that they could constitute targets for new pharmacological treatment of atrial fibrillation. The interplay between the different K(+) channel subtypes in both atria and ventricle is dynamic, and a significant up- and downregulation occurs in disease states such as atrial fibrillation or heart failure...

  6. CHANNEL ESTIMATION TECHNIQUE

    2015-01-01

    A method includes determining a sequence of first coefficient estimates of a communication channel based on a sequence of pilots arranged according to a known pilot pattern and based on a receive signal, wherein the receive signal is based on the sequence of pilots transmitted over the communicat......A method includes determining a sequence of first coefficient estimates of a communication channel based on a sequence of pilots arranged according to a known pilot pattern and based on a receive signal, wherein the receive signal is based on the sequence of pilots transmitted over...... the communication channel. The method further includes determining a sequence of second coefficient estimates of the communication channel based on a decomposition of the first coefficient estimates in a dictionary matrix and a sparse vector of the second coefficient estimates, the dictionary matrix including...... filter characteristics of at least one known transceiver filter arranged in the communication channel....

  7. Simple Ion Channels: From Structure to Electrophysiology and Back

    Pohorille, Andrzej

    2018-01-01

    A reliable way to establish whether our understanding of a channel is satisfactory is to reproduce its measured ionic conductance over a broad range of applied voltages in computer simulations. In molecular dynamics (MD), this can be done by way of applying an external electric field to the system and counting the number of ions that traverse the channel per unit time. Since this approach is computationally very expensive, we have developed a markedly more efficient alternative in which MD is combined with the electrodiffusion (ED) equation. In this approach, the assumptions of the ED equation can be rigorously tested, and the precision and consistency of the calculated conductance can be determined. We have demonstrated that the full current/voltage dependence and the underlying free energy profile for a simple channel can be reliably calculated from equilibrium or non-equilibrium MD simulations at a single voltage. To carry out MD simulations, a structural model of a channel has to be assumed, which is an important constraint, considering that high-resolution structures are available for only very few simple channels. If the comparison of calculated ionic conductance with electrophysiological data is satisfactory, it greatly increases our confidence that the structure and the function are described sufficiently accurately. We examined the validity of the ED for several channels embedded in phospholipid membranes - four naturally occurring channels: trichotoxin, alamethicin, p7 from hepatitis C virus (HCV) and Vpu from the HIV-1 virus, and a synthetic, hexameric channel, formed by a 21-residue peptide that contains only leucine and serine. All these channels mediate transport of potassium and chloride ions. It was found that the ED equation is satisfactory for these systems. In some of them experimental and calculated electrophysiological properties are in good agreement, whereas in others there are strong indications that the structural models are incorrect.

  8. Effects of ammonium nitrate, cesium chloride and ...

    Jane

    2011-10-12

    Oct 12, 2011 ... Full Length Research Paper. Effects of ... constitutes 2 to 10% of plant dry weight (Leigh et al.,. 1984 ... membrane proteins has been reported, including K+ channels ... have functions in the plasma membrane and tonoplast. (Senn et al. .... analysis of K+ uptake in a solution containing 50 µM K+ showed a ...

  9. Chlorides behavior in raw fly ash washing experiments

    Zhu Fenfen; Takaoka, Masaki; Oshita, Kazuyuki; Kitajima, Yoshinori; Inada, Yasuhiro; Morisawa, Shinsuke; Tsuno, Hiroshi

    2010-01-01

    Chloride in fly ash from municipal solid waste incinerators (MSWIs) is one of the obstructive substances in recycling fly ash as building materials. As a result, we have to understand the behavior of chlorides in recycling process, such as washing. In this study, we used X-ray absorption near edge structure (XANES) and X-ray diffraction (XRD) to study the chloride behavior in washed residue of raw fly ash (RFA). We found that a combination of XRD and XANES, which is to use XRD to identify the situation of some compounds first and then process XANES data, was an effective way to explain the chlorides behavior in washing process. Approximately 15% of the chlorine in RFA was in the form of NaCl, 10% was in the form of KCl, 51% was CaCl 2 , and the remainder was in the form of Friedel's salt. In washing experiments not only the mole percentage but also the amount of soluble chlorides including NaCl, KCl and CaCl 2 decreases quickly with the increase of liquid to solid (L/S) ratio or washing frequency. However, those of insoluble chlorides decrease slower. Moreover, Friedel's salt and its related compound (11CaO.7Al 2 O 3 .CaCl 2 ) were reliable standards for the insoluble chlorides in RFA, which are strongly related to CaCl 2 . Washing of RFA promoted the release of insoluble chlorides, most of which were in the form of CaCl 2 .

  10. Interaction of calcium oxide with molten alkali metal chlorides

    Volkovich, A.V.; Zhuravlev, V.I.; Ermakov, D.S.; Magurina, M.V.

    1999-01-01

    Calcium oxide solubility in molten lithium, sodium, potassium, cesium chlorides and their binary mixtures is determined in a temperature range of 973-1173 K by the method of isothermal saturation. Mechanisms of calcium oxide interaction with molten alkali metal chlorides are proposed

  11. Hydrophobic treatment of concrete as protection against chloride penetration

    Vries, J. de; Polder, R.B.; Borsje, H.

    1996-01-01

    Hydrophobic treatment makes a concrete surface absorb less water and less chloride. Hydrophobic treatment was studied as a protection agninst chloride penetration from deicing salts. Test methods were designed. Nine hydrophobic products were tested, of which three complied to the requirements on

  12. Aerobic biodegradation of vinyl chloride in groundwater samples

    Davis, J.W.; Carpenter, C.L.

    1990-01-01

    Studies were conducted to examine the biodegradation of 14 C-labeled vinyl chloride in samples taken from a shallow aquifer. Under aerobic conditions, vinyl chloride was readily degraded, with greater than 99% of the labeled material being degraded after 108 days and approximately 65% being mineralized to 14 CO 2

  13. Influence of granular strontium chloride as additives on some ...

    Influence of granular strontium chloride as additives on some electrical and mechanical properties for pure polyvinyl alcohol. A B Elaydy M Hafez ... Keywords. Polyvinyl-alcohol (PVA); granular strontium chloride, SrCl2; a.c. electrical conductivity; dielectric constant; dielectric loss; Young's modulus; creep relaxation curve.

  14. Chloride penetration into cementitious mortar at early age

    Caballero, J.; Polder, R.B.; Leegwater, G.A.; Fraaij, A.L.A.

    2012-01-01

    Modern service life design methods for concrete structures use chloride diffusion data as an input parameter. Abundant data exist for concrete at 28 days and, to a lesser extent, at later ages. This paper presents chloride diffusion data for mortar at ages between 1 day and 28 days age. Rapid

  15. Local impermeant anions establish the neuronal chloride concentration

    Glykys, J; Dzhala, V; Egawa, K

    2014-01-01

    Neuronal intracellular chloride concentration [Cl(-)](i) is an important determinant of γ-aminobutyric acid type A (GABA(A)) receptor (GABA(A)R)-mediated inhibition and cytoplasmic volume regulation. Equilibrative cation-chloride cotransporters (CCCs) move Cl(-) across the membrane, but accumulat...

  16. Determination of the chloride diffusion coefficient in blended cement mortars

    Elfmarkova, V.; Spiesz, P.R.; Brouwers, H.J.H.

    2015-01-01

    The rapid chloride migration test (RCM) is a commonly used accelerated test for the determination of the chloride diffusion coefficient in concrete. Nevertheless, the initial development and further experience with the RCM test concern mainly the ordinary Portland cement system. Therefore, the

  17. [Forensic Analysis for 54 Cases of Suxamethonium Chloride Poisoning].

    Zhao, Y F; Zhao, B Q; Ma, K J; Zhang, J; Chen, F Y

    2017-08-01

    To observe and analyze the performance of forensic science in the cases of suxa- methonium chloride poisoning, and to improve the identification of suxamethonium chloride poisoning. Fifty-four cases of suxamethonium chloride poisoning were collected. The rules of determination of suxamethonium chloride poisoning were observed by the retrospective analysis of pathological and toxicological changes as well as case features. The pathological features of suxamethonium chloride poisoning were similar to the general changes of sudden death, which mainly included acute pulmonary congestion and edema, and partly showed myocardial disarray and fracture. Suxamethonium chloride could be detected in the heart blood of all cases and in skin tissue of part cases. Suxa-methonium chloride poisoning has the characteristics with fast death and covert means, which are difficult to rescue and easily miss inspection. For the cases of sudden death or suspicious death, determination of suxamethonium chloride should be taken as a routine detection index to prevent missing inspection. Copyright© by the Editorial Department of Journal of Forensic Medicine

  18. Microwave Mapping Demonstration Using the Thermochromic Cobalt Chloride Equilibrium

    Nguyen, Vu D.; Birdwhistell, Kurt R.

    2014-01-01

    An update to the thermochromic cobalt(II) chloride equilibrium demonstration is described. Filter paper that has been saturated with aqueous cobalt(II) chloride is heated for seconds in a microwave oven, producing a color change. The resulting pink and blue map is used to colorfully demonstrate Le Châtelier's principle and to illuminate the…

  19. Stability constants of the Europium complexes with the chloride ions

    Jimenez R, M.; Solache R, M.; Rojas H, A.

    2000-01-01

    The stability constants of lanthanides complexes with chloride ions which were determined at the same ionic force but in different media, are significantly different. It does not exist a systematic study over these stability constants. The purpose of this work is to determine the stability constants of the europium complexes with chloride ions at 303 K, by the solvents extraction method. (Author)

  20. Free and bound chloride contents in cementitious materials

    Marinescu, M.V.A.; Brouwers, H.J.H.; Fischer, G.; Geiker, M.; Hededal, O.; Ottoson, L.; Stang, H.

    2010-01-01

    Chloride attack is the main cause of structural damage in reinforced concrete buildings exposed to marine environments. When a certain threshold concentration of chlorides is reached at the concrete-reinforcement interface, the corrosion of the steel rebars is initiated. A part of the intruding