scholarly journals Hypotonicity activates a native chloride current in Xenopus oocytes.

1994 ◽  
Vol 103 (2) ◽  
pp. 153-179 ◽  
Author(s):  
M J Ackerman ◽  
K D Wickman ◽  
D E Clapham
Keyword(s):  
Xenopus Oocytes ◽  
Critical Role ◽  
Chloride Current ◽  
Signaling Cascades ◽  
Channel Blockers ◽  
Chloride Currents ◽  
Channel Proteins ◽  
Ion Channel Proteins

Xenopus oocytes are frequently utilized for in vivo expression of cellular proteins, especially ion channel proteins. A thorough understanding of the endogenous conductances and their regulation is paramount for proper characterization of expressed channel proteins. Here we detail a novel chloride current (ICl.swell) responsive to hypotonicity in Xenopus oocytes using the two-electrode voltage clamp technique. Reducing the extracellular osmolarity by 50% elicited a calcium-independent chloride current having an anion conductivity sequence identical with swelling-induced chloride currents observed in epithelial cells. The hypotonicity-activated current was blocked by chloride channel blockers, trivalent lanthanides, and nucleotides. G-protein, cAMP-PKA, and arachidonic acid signaling cascades were not involved in ICl.swell activation. ICl.swell is distinct from both stretch-activated nonselective cation channels and the calcium-activated chloride current in oocytes and may play a critical role in volume regulation in Xenopus oocytes.

ClC-3 is a candidate of the channel proteins mediating acid-activated chloride currents in nasopharyngeal carcinoma cells

2012 ◽  
Vol 303 (1) ◽  
pp. C14-C23 ◽  
Author(s):  
Liwei Wang ◽  
Wenbo Ma ◽  
Linyan Zhu ◽  
Dong Ye ◽  
Yuan Li ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Chloride Channel ◽  
Cell Function ◽  
Extracellular Ph ◽  
Carcinoma Cells ◽  
Chloride Current ◽  
Disulfonic Acid ◽  
Channel Blockers ◽  
Chloride Currents ◽  
Channel Proteins

Acid-activated chloride currents have been reported in several cell types and may play important roles in regulation of cell function. However, the molecular identities of the channels that mediate the currents are not defined. In this study, activation of the acid-induced chloride current and the possible candidates of the acid-activated chloride channel were investigated in human nasopharyngeal carcinoma cells (CNE-2Z). A chloride current was activated when extracellular pH was reduced to 6.6 from 7.4. However, a further decrease of extracellular pH to 5.8 inhibited the current. The current was weakly outward-rectified and was suppressed by hypertonicity-induced cell shrinkage and by the chloride channel blockers 5-nitro-2–3-phenylpropylamino benzoic acid (NPPB), tamoxifen, and 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid disodium salt hydrate (DIDS). The permeability sequence of the channel to anions was I− > Br− > Cl− > gluconate−. Among the ClC chloride channels, ClC-3 and ClC-7 were strongly expressed in CNE-2Z cells. Knockdown of ClC-3 expression with ClC-3 small interfering (si)RNA prevented the activation of the acid-induced current, but silence of ClC-7 expression with ClC-7 siRNA did not significantly affect the current. The results suggest that the chloride channel mediating the acid-induced chloride current was volume sensitive. ClC-3 is a candidate of the channel proteins that mediate or regulate the acid-activated chloride current in nasopharyngeal carcinoma cells.

Culture of murine nasal epithelia: model for cystic fibrosis

2006 ◽  
Vol 290 (2) ◽  
pp. L270-L277 ◽  
Author(s):  
B. R. Grubb ◽  
T. D. Rogers ◽  
P. C. Diggs ◽  
R. C. Boucher ◽  
L. E. Ostrowski
Keyword(s):  
Cystic Fibrosis ◽  
Ion Transport ◽  
Ussing Chamber ◽  
Channel Blockers ◽  
Culture Model ◽  
Δf508 Cftr ◽  
Human Airways ◽  
Columnar Cells

The ion transport defects reported for human cystic fibrosis (CF) airways are reproduced in nasal epithelia of the CF mouse. Although this tissue has been studied in vivo using the nasal potential difference technique and as a native tissue mounted in the Ussing chamber, little information is available on cultured murine nasal epithelia. We have developed a polarized cell culture model of primary murine nasal epithelia in which the CF tissue exhibits not only a defect in cAMP-mediated Cl−secretion but also the Na+hyperabsorption and upregulation of the Ca2+-activated Cl−conductance observed in human airways. Both the wild-type and CF cultures were constituted predominantly of undifferentiated cuboidal columnar cells, with most cultures exhibiting a small number of ciliated cells. Although no goblet cells were observed, RT-PCR demonstrated the expression of Muc5ac RNA after ∼22 days in culture. The CF tissue exhibited an adherent layer of mucus similar to the mucus plaques reported in the distal airways of human CF patients. Furthermore, we found that treatment of CF preparations with a Na+channel blocker for 7 days prevented formation of mucus adherent to epithelial surfaces. The cultured murine nasal epithelial preparation should be an excellent model tissue for gene transfer studies and pharmacological studies of Na+channel blockers and mucolytic agents as well as for further characterization of CF ion transport defects. Culture of nasal epithelia from ΔF508 mice will be particularly useful in testing drugs that allow ΔF508 CFTR to traffic to the membrane.

scholarly journals A Ral GAP complex links PI 3-kinase/Akt signaling to RalA activation in insulin action

2011 ◽  
Vol 22 (1) ◽  
pp. 141-152 ◽  
Author(s):  
Xiao-Wei Chen ◽  
Dara Leto ◽  
Tingting Xiong ◽  
Genggeng Yu ◽  
Alan Cheng ◽  
...  
Keyword(s):  
Glucose Transporter ◽  
Critical Role ◽  
Inhibitory Effect ◽  
Small Gtpase ◽  
Regulatory Subunit ◽  
Hydrolysis Of ◽  
Identification And Characterization

Insulin stimulates glucose transport in muscle  and adipose tissue by translocation of glucose transporter 4 (GLUT4) to the plasma membrane. We previously reported that activation of the small GTPase RalA downstream of PI 3-kinase plays a critical role in this process by mobilizing the exocyst complex for GLUT4 vesicle targeting in adipocytes. Here we report the identification and characterization of a Ral GAP complex (RGC) that mediates the activation of RalA downstream of the PI 3-kinase/Akt pathway. The complex is composed of an RGC1 regulatory subunit and an RGC2 catalytic subunit (previously identified as AS250) that directly stimulates the guanosine triphosphate hydrolysis of RalA. Knockdown of RGC proteins leads to increased RalA activity and glucose uptake in adipocytes. Insulin inhibits the GAP complex through Akt2-catalyzed phosphorylation of RGC2 in vitro and in vivo, while activated Akt relieves the inhibitory effect of RGC proteins on RalA activity. The RGC complex thus connects PI 3-kinase/Akt activity to the transport machineries responsible for GLUT4 translocation.

scholarly journals Full in vivo characterization of carbonate chemistry at the site of calcification in corals

2019 ◽  
Vol 5 (1) ◽  
pp. eaau7447 ◽  
Author(s):  
Duygu S. Sevilgen ◽  
Alexander A. Venn ◽  
Marian Y. Hu ◽  
Eric Tambutté ◽  
Dirk de Beer ◽  
...  
Keyword(s):  
Critical Role ◽  
Carbonate Chemistry ◽  
Saturation State ◽  
Stylophora Pistillata ◽  
Coral Calcification ◽  
Carbon Concentrating Mechanisms ◽  
In Vivo Characterization

Reef-building corals form their calcium carbonate skeletons within an extracellular calcifying medium (ECM). Despite the critical role of the ECM in coral calcification, ECM carbonate chemistry is poorly constrained in vivo, and full ECM carbonate chemistry has never been characterized based solely on direct in vivo measurements. Here, we measure pHECMin the growing edge ofStylophora pistillataby simultaneously using microsensors and the fluorescent dye SNARF-1, showing that, when measured at the same time and place, the results agree. We then conduct microscope-guided microsensor measurements of pH, [Ca2+], and [CO32−] in the ECM and, from this, determine [DIC]ECMand aragonite saturation state (Ωarag), showing that all parameters are elevated with respect to the surrounding seawater. Our study provides the most complete in vivo characterization of ECM carbonate chemistry parameters in a coral species to date, pointing to the key role of calcium- and carbon-concentrating mechanisms in coral calcification.

scholarly journals Covalent cross-linking of the bovine somatotropin dimer. Effects on growth-promoting, receptor-binding and immunological activities and preliminary characterization of the self-association

1983 ◽  
Vol 209 (1) ◽  
pp. 107-115 ◽  
Author(s):  
H N Fernández ◽  
J M Delfino
Keyword(s):  
Critical Role ◽  
The Self ◽  
Bovine Somatotropin ◽  
Cross Linking ◽  
British Library ◽  
Dimethyl Suberimidate ◽  
Growth Promoting ◽  
Self Association

Bovine somatotropin, at pH 8.5 in 0.02 M-Bicine [NN-bis-(2-hydroxyethyl)glycine]/0.09M-NaCl, showed by frontal analysis the characteristics of a rapid monomer-dimer equilibrium whose dissociation constant was estimated to be 6.6×10(-6)M. Reaction of the hormone with dimethyl suberimidate lead to covalent cross-linking of the dimeric species. Under the conditions chosen (0.4 mg of bifunctional imidate and 1 mg of protein/ml at room temperature for 1 h) the cross-linked dimers accounted for 26% of the total protein, and these were isolated by molecular sieving in 0.29M-NH3/0.12M-NaCl. Covalent stabilization greatly diminished the growth-promoting activity and the ability to interact with somatogenic sites in both rat liver in vivo and rabbit liver microsomal fractions. Evidence indicating a non-critical role for amino groups involved in the covalent cross-linking was provided by a nearly equivalent derivative obtained after reaction with 3,3′-dithiobispropionimidate, which had substantial hormonal activity upon cleavage of the disulphide links. Conversely, immunological reactivity as demonstrated by radioimmunoassay was not affected by cross-linking. Details of the least-squares procedure employed to evaluate the self-association equilibrium constant has been deposited as Supplement SUP 50115 (7 pages) with the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1981) 193,5.

scholarly journals The Coxiella burnetii QpH1 plasmid is a virulence factor for colonizing bone marrow-derived murine macrophages

2020 ◽  
Author(s):  
Shengdong Luo ◽  
Zhihui Sun ◽  
Huahao Fan ◽  
Shanshan Lu ◽  
Yan Hu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Coxiella Burnetii ◽  
Virulence Factor ◽  
Critical Role ◽  
Murine Macrophages ◽  
Infection Models

AbstractCoxiella burnetii carries a large conserved plasmid or plasmid-like chromosomally integrated sequence of unknown function. Here we report the curing of QpH1 plasmid from C. burnetii Nine Mile phase II, the characterization of QpH1-deficient C. burnetii in in vitro and in vivo infection models, and the characterization of plasmid biology. A shuttle vector pQGK, which is composed of the CBUA0036-0039a region (predicted for QpH1 maintenance), an E. coli plasmid ori, eGFP and kanamycin resistance genes was constructed. The pQGK vector can be stably transformed into Nine Mile II and maintained at a similar low copy like QpH1. Importantly, transformation with pQGK cured the endogenous QpH1 due to plasmid incompatibility. Compared to a Nine Mile II transformant of a RSF1010-based vector, the pQGK transformant shows an identical one-step growth curve in axenic media, a similar growth curve in Buffalo green monkey kidney cells, an evident growth defect in macrophage-like THP-1 cells, and dramatically reduced ability of colonizing bone marrow-derived murine macrophages. In the SCID mouse infection model, the pQGK transformants caused a lesser extent of splenomegaly. Moreover, the plasmid biology was investigated by mutagenesis. We found CBUA0037-0039 are essential for plasmid maintenance, and CBUA0037-0038 account for plasmid compatibility. Taken together, our data suggest that QpH1 encodes factor(s) essential for colonizing murine macrophages, and to a lesser extent for colonizing human macrophages. This study highlights a critical role of QpH1 for C. burnetii persistence in rodents, and expands the toolkit for genetic studies in C. burnetii.Author summaryIt is postulated that C. burnetii recently evolved from an inherited symbiont of ticks by the acquisition of novel virulence factors. All C. burnetii isolates carry a large plasmid or have a chromosomally integrated plasmid-like sequence. The plasmid is a candidate virulence factor that contributes to C. burnetii evolution. Here we describe the construction of novel shuttle vectors that allow to make plasmid-deficient C. burnetii mutants. With this plasmid-curing approach, we characterized the role of the QpH1 plasmid in in vitro and in vivo C. burnetii infection models. We found that the plasmid plays a critical role for C. burnetii growth in macrophages, especially in murine macrophages, but not in axenic media and BGMK cells. Our work highlights an essential role of the plasmid for the acquisition of colonizing capability in rodents by C. burnetii. This study represents a major step toward unravelling the mystery of the C. burnetii cryptic plasmids.

scholarly journals Characterization of a Knock-In Mouse Model with a Huntingtin Exon 1 Deletion

2021 ◽  
pp. 1-20
Author(s):  
Elise M. Braatz ◽  
Emily A. André ◽  
Jeh-Ping Liu ◽  
Scott O. Zeitlin
Keyword(s):  
Dna Damage ◽  
Critical Role ◽  
Motor Tasks ◽  
Female Ratio ◽  
Damage Repair ◽  
Exon 1 ◽  
Male To Female ◽  
Terminal Domains

Background: The Huntingtin (HTT) N-terminal domains encoded by Huntingtin’s (HTT) exon 1 consist of an N17 domain, the polyglutamine (polyQ) stretch and a proline-rich region (PRR). These domains are conserved in mammals and have been hypothesized to modulate HTT’s functions in the developing and adult CNS, including DNA damage repair and autophagy. Objective: This study longitudinally characterizes the in vivo consequences of deleting the murine Htt N-terminal domains encoded by Htt exon 1. Methods: Knock-in mice with a deletion of Htt exon 1 sequences (Htt ΔE1) were generated and bred into the C57BL/6J congenic genetic background. Their behavior, DNA damage response, basal autophagy, and glutamatergic synapse numbers were evaluated. Results: Progeny from Htt ΔE1/+ intercrosses are born at the expected Mendelian frequency but with a distorted male to female ratio in both the Htt ΔE1/ΔE1 and Htt  +/+ offspring. Htt ΔE1/ΔE1 adults exhibit a modest deficit in accelerating rotarod performance, and an earlier increase in cortical and striatal DNA damage with elevated neuronal pan-nuclear 53bp1 levels compared to Htt  +/+ mice. However, a normal response to induced DNA damage, normal levels of basal autophagy markers, and no significant differences in corticocortical, corticostriatal, thalamocortical, or thalamostriatal synapses numbers were observed compared to controls. Conclusion: Our results suggest that deletion of the Htt N-terminus encoded by the Htt exon 1 does not affect Htt’s critical role during embryogenesis, but instead, may have a modest effect on certain motor tasks, basal levels of DNA damage in the brain, and Htt function in the testis.

scholarly journals Comprehensive characterization of the complex lola locus reveals a novel role in the octopaminergic pathway via Tyramine beta-hydroxylase activation

2017 ◽  
Author(s):  
Nadja Dinges ◽  
Violeta Morin ◽  
Nastasja Kreim ◽  
Tony D. Southall ◽  
Jean-Yves Roignant
Keyword(s):  
Transcriptional Activation ◽  
Critical Role ◽  
Protein Isoforms ◽  
Regulatory Control ◽  
Loss Of Function ◽  
Transcriptional Regulatory ◽  
Key Enzyme ◽  
Comprehensive Characterization

Summarylongitudinals lacking (lola) is among the most complex genes in Drosophila melanogaster, encoding up to twenty protein isoforms and acting as a key transcription factor in axonal pathfinding and neural reprogramming. Most of previous studies employed loss-of-function alleles disrupting common exons of lola, making it difficult to delineate its functions. To address this issue we have generated specific mutations in each isoform using the CRISPR/Cas9 system. Our targeted screen allows us to revisit the previously demonstrated roles for few isoforms and to demonstrate a specific function for one variant in axon guidance via activation of the microtubule-associated factor Futsch. Importantly, we also reveal a critical role for a second variant in preventing neurodegeneration via the control of the octopaminergic pathway. This variant is expressed almost exclusively in the octopaminergic cells and is involved in the transcriptional activation of a key enzyme of the pathway. Thus, our comprehensive study greatly expands the functional repertoire of Lola functions, and adds novel insights into the transcriptional regulatory control of neurotransmitter expression in vivo.

scholarly journals Insight into DEG/ENaC Channel Gating from Genetics and Structure

Physiology ◽  
2012 ◽  
Vol 27 (5) ◽  
pp. 282-290 ◽  
Author(s):  
Amy L. Eastwood ◽  
Miriam B. Goodman
Keyword(s):  
Ion Channel ◽  
Research Community ◽  
Mechanosensitive Channels ◽  
C Elegans ◽  
Channel Proteins ◽  
New Models ◽  
Ion Channel Proteins ◽  
Pain Receptors ◽  
Insight Into

The founding members of the superfamily of DEG/ENaC ion channel proteins are C. elegans proteins that form mechanosensitive channels in touch and pain receptors. For more than a decade, the research community has used mutagenesis to identify motifs that regulate gating. This review integrates insight derived from unbiased in vivo mutagenesis screens with recent crystal structures to develop new models for activation of mechanically gated DEGs.

scholarly journals Binding Mode Exploration of B1 Receptor Antagonists’ by the Use of Molecular Dynamics and Docking Simulation—How Different Target Engagement Can Determine Different Biological Effects

2020 ◽  
Vol 21 (20) ◽  
pp. 7677
Author(s):  
Marica Gemei ◽  
Carmine Talarico ◽  
Laura Brandolini ◽  
Candida Manelfi ◽  
Lorena Za ◽  
...  
Keyword(s):  
Biological Effects ◽  
Critical Role ◽  
Binding Mode ◽  
Receptor Internalization ◽  
Mode Analysis ◽  
Binding Modes ◽  
Pharmacological Characterization ◽  
B1 Receptor

The kinin B1 receptor plays a critical role in the chronic phase of pain and inflammation. The development of B1 antagonists peaked in recent years but almost all promising molecules failed in clinical trials. Little is known about these molecules’ mechanisms of action and additional information will be necessary to exploit the potential of the B1 receptor. With the aim of contributing to the available knowledge of the pharmacology of B1 receptors, we designed and characterized a novel class of allosteric non-peptidic inhibitors with peculiar binding characteristics. Here, we report the binding mode analysis and pharmacological characterization of a new allosteric B1 antagonist, DFL20656. We analyzed the binding of DFL20656 by single point mutagenesis and radioligand binding assays and we further characterized its pharmacology in terms of IC50, B1 receptor internalization and in vivo activity in comparison with different known B1 antagonists. We highlighted how different binding modes of DFL20656 and a Merck compound (compound 14) within the same molecular pocket can affect the biological and pharmacological properties of B1 inhibitors. DFL20656, by its peculiar binding mode, involving tight interactions with N114, efficiently induced B1 receptor internalization and evoked a long-lasting effect in an in vivo model of neuropathic pain. The pharmacological characterization of different B1 antagonists highlighted the effects of their binding modes on activity, receptor occupancy and internalization. Our results suggest that part of the failure of most B1 inhibitors could be ascribed to a lack of knowledge about target function and engagement.

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