scholarly journals Two Drosophila Innexins Are Expressed in Overlapping Domains and Cooperate to Form Gap-Junction Channels

2000 ◽  
Vol 11 (7) ◽  
pp. 2459-2470 ◽  
Author(s):  
Lucy A. Stebbings ◽  
Martin G. Todman ◽  
Pauline Phelan ◽  
Jonathan P. Bacon ◽  
Jane A. Davies
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Ectopic Expression ◽  
In Vivo Studies ◽  
Electrophysiological Properties ◽  
Gap Junction Channels ◽  
Overlapping Domains ◽  
In Vitro Data

Members of the innexin protein family are structural components of invertebrate gap junctions and are analogous to vertebrate connexins. Here we investigate two Drosophila innexin genes,Dm-inx2 and Dm-inx3 and show that they are expressed in overlapping domains throughout embryogenesis, most notably in epidermal cells bordering each segment. We also explore the gap-junction–forming capabilities of the encoded proteins. In pairedXenopus oocytes, the injection of Dm-inx2mRNA results in the formation of voltage-sensitive channels in only ∼ 40% of cell pairs. In contrast, Dm-Inx3 never forms channels. Crucially, when both mRNAs are coexpressed, functional channels are formed reliably, and the electrophysiological properties of these channels distinguish them from those formed by Dm-Inx2 alone. We relate these in vitro data to in vivo studies. Ectopic expression ofDm-inx2 in vivo has limited effects on the viability ofDrosophila, and animals ectopically expressingDm-inx3 are unaffected. However, ectopic expression of both transcripts together severely reduces viability, presumably because of the formation of inappropriate gap junctions. We conclude that Dm-Inx2 and Dm-Inx3, which are expressed in overlapping domains during embryogenesis, can form oligomeric gap-junction channels.

scholarly journals Heteromerization of Innexin Gap Junction Proteins Regulates Epithelial Tissue Organization in Drosophila

2006 ◽  
Vol 17 (4) ◽  
pp. 1676-1685 ◽  
Author(s):  
Corinna Lehmann ◽  
Hildegard Lechner ◽  
Birgit Löer ◽  
Martin Knieps ◽  
Sonja Herrmann ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Ectopic Expression ◽  
Cell Communication ◽  
Epithelial Tissue ◽  
Surface Plasmon Resonance Analysis ◽  
Gap Junction Channels ◽  
Tissue Organization ◽  
Biochemical Fractionation

Gap junctions consist of clusters of intercellular channels, which enable direct cell-to-cell communication and adhesion in animals. Whereas deuterostomes, including all vertebrates, use members of the connexin and pannexin multiprotein families to assemble gap junction channels, protostomes such as Drosophila and Caenorhabditis elegans use members of the innexin protein family. The molecular composition of innexin-containing gap junctions and the functional significance of innexin oligomerization for development are largely unknown. Here, we report that heteromerization of Drosophila innexins 2 and 3 is crucial for epithelial organization and polarity of the embryonic epidermis. Both innexins colocalize in epithelial cell membranes. Innexin3 is mislocalized to the cytoplasm in innexin2 mutants and is recruited into ectopic expression domains defined by innexin2 misexpression. Conversely, RNA interference (RNAi) knockdown of innexin3 causes mislocalization of innexin2 and of DE-cadherin, causing cell polarity defects in the epidermis. Biochemical interaction studies, surface plasmon resonance analysis, transgenesis, and biochemical fractionation experiments demonstrate that both innexins interact via their C-terminal cytoplasmic domains during the assembly of heteromeric channels. Our data provide the first molecular and functional demonstration that innexin heteromerization occurs in vivo and reveal insight into a molecular mechanism by which innexins may oligomerize into heteromeric gap junction channels.

scholarly journals The Functional Implications of Endothelial Gap Junctions and Cellular Mechanics in Vascular Angiogenesis

Cancers ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 237 ◽  
Author(s):  
Takayuki Okamoto ◽  
Haruki Usuda ◽  
Tetsuya Tanaka ◽  
Koichiro Wada ◽  
Motomu Shimaoka
Keyword(s):  
Endothelial Cells ◽  
Cell Migration ◽  
Gap Junctions ◽  
Gap Junction ◽  
Cancer Cells ◽  
Tumor Development ◽  
Tube Formation ◽  
Cellular Mechanics

Angiogenesis—the sprouting and growth of new blood vessels from the existing vasculature—is an important contributor to tumor development, since it facilitates the supply of oxygen and nutrients to cancer cells. Endothelial cells are critically affected during the angiogenic process as their proliferation, motility, and morphology are modulated by pro-angiogenic and environmental factors associated with tumor tissues and cancer cells. Recent in vivo and in vitro studies have revealed that the gap junctions of endothelial cells also participate in the promotion of angiogenesis. Pro-angiogenic factors modulate gap junction function and connexin expression in endothelial cells, whereas endothelial connexins are involved in angiogenic tube formation and in the cell migration of endothelial cells. Several mechanisms, including gap junction function-dependent or -independent pathways, have been proposed. In particular, connexins might have the potential to regulate cell mechanics such as cell morphology, cell migration, and cellular stiffness that are dynamically changed during the angiogenic processes. Here, we review the implication for endothelial gap junctions and cellular mechanics in vascular angiogenesis.

pH Sensitivity of Non-Synaptic Field Bursts in the Dentate Gyrus

2000 ◽  
Vol 84 (2) ◽  
pp. 927-933 ◽  
Author(s):  
Jeffrey S. Schweitzer ◽  
Haiwei Wang ◽  
Zhi-Qi Xiong ◽  
Janet L. Stringer
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Granule Cell ◽  
Low Ph ◽  
Ph Sensitivity ◽  
Extracellular Ph ◽  
Granule Cell Layer ◽  
Ph Changes

Under conditions of low [Ca2+]o and high [K+]o, the rat dentate granule cell layer in vitro develops recurrent spontaneous prolonged field bursts that resemble an in vivo phenomenon called maximal dentate activation. To understand how pH changes in vivo might affect this phenomenon, the slices were exposed to different extracellular pH environments in vitro. The field bursts were highly sensitive to extracellular pH over the range 7.0–7.6 and were suppressed at low pH and enhanced at high pH. Granule cell resting membrane potential, action potentials, and postsynaptic potentials were not significantly altered by pH changes within the range that suppressed the bursts. The pH sensitivity of the bursts was not altered by pharmacologic blockade of N-methyl-d-aspartate (NMDA), non-NMDA, and GABAA receptors at concentrations of these agents sufficient to eliminate both spontaneous and evoked synaptic potentials. Gap junction patency is known to be sensitive to pH, and agents that block gap junctions, including octanol, oleamide, and carbenoxolone, blocked the prolonged field bursts in a manner similar to low pH. Perfusion with gap junction blockers or acidic pH suppressed field bursts but did not block spontaneous firing of single and multiple units, including burst firing. These data suggest that the pH sensitivity of seizures and epileptiform phenomena in vivo may be mediated in large part through mechanisms other than suppression of NMDA-mediated or other excitatory synaptic transmission. Alterations in electrotonic coupling via gap junctions, affecting field synchronization, may be one such process.

scholarly journals Decreased Fast Ripples in the Hippocampus of Rats with Spontaneous Recurrent Seizures Treated with Carbenoxolone and Quinine

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Consuelo Ventura-Mejía ◽  
Laura Medina-Ceja
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
High Frequency Oscillations ◽  
The Mean ◽  
Eeg Recordings ◽  
The Right ◽  
Spontaneous Recurrent Seizures

Background. In models of temporal lobe epilepsy and in patients with this pathology, high frequency oscillations called fast ripples (FRs, 250–600 Hz) can be observed. FRs are considered potential biomarkers for epilepsy and, in the light of manyin vitroandin silicostudies, we thought that electrical synapses mediated by gap junctions might possibly modulate FRsin vivo.Methods. Animals with spontaneous recurrent seizures induced by pilocarpine administration were implanted with movable microelectrodes in the right anterior and posterior hippocampus to evaluate the effects of gap junction blockers administered in the entorhinal cortex. The effects of carbenoxolone (50 nmoles) and quinine (35 pmoles) on the mean number of spontaneous FR events (occurrence of FRs), as well as on the mean number of oscillation cycles per FR event and their frequency, were assessed using a specific algorithm to analyze FRs in intracranial EEG recordings.Results. We found that these gap junction blockers decreased the mean number of FRs and the mean number of oscillation cycles per FR event in the hippocampus, both during and at different times after carbenoxolone and quinine administration.Conclusion. These data suggest that FRs may be modulated by gap junctions, although additional experimentsin vivowill be necessary to determine the precise role of gap junctions in this pathological activity associated with epileptogenesis.

Biophysical characterization of zebrafish connexin35 hemichannels

2004 ◽  
Vol 287 (6) ◽  
pp. C1596-C1604 ◽  
Author(s):  
Virginijus Valiunas ◽  
Rickie Mui ◽  
Elizabeth McLachlan ◽  
Gunnar Valdimarsson ◽  
Peter R. Brink ◽  
...  
Keyword(s):  
Gap Junction ◽  
Single Channel ◽  
Biophysical Characterization ◽  
Whole Cell ◽  
Gap Junction Channels ◽  
N2a Cells ◽  
Unitary Conductance ◽  
Cell Depolarization

A subset of connexins can form unopposed hemichannels in expression systems, providing an opportunity for comparison of hemichannel gating properties with those of intact gap junction channels. Zebrafish connexin35 (Cx35) is a member of the Cx35/Cx36 subgroup of connexins highly expressed in the retina and brain. In the present study, we have shown that Cx35 expression in Xenopus oocytes and N2A cells produced large outward whole cell currents on cell depolarization. Using whole cell, cell-attached, and excised patch configurations, we obtained multichannel and single-channel current recordings attributable to the Cx35 hemichannels ( Ihc) that were activated and increased by stepwise depolarization of membrane potential ( Vm) and deactivated by hyperpolarization. The currents were not detected in untransfected N2A cells or in control oocytes injected with antisense Cx38. However, water-injected oocytes that were not treated with antisense showed activities attributable to Cx38 hemichannels that were easily distinguishable from Cx35 hemichannels by a significantly larger unitary conductance ( γhc: 250–320 pS). The γhc of Cx35 hemichannels exhibited a pronounced Vm dependence; i.e., γhc increased/decreased with relative hyperpolarization/depolarization ( γhc was 72 pS at Vm = −100 mV and 35 pS at Vm = 100 mV). Extrapolation to Vm = 0 mV predicted a γhc of 48 pS, suggesting a unitary conductance of intact Cx35 gap junction channels of ∼24 pS. Channel gating was also Vm dependent: open time declined with negative Vm and increased with positive Vm. The ability to break down the complex gating of intact intercellular channels into component hemichannels in vitro will help to evaluate putative physiological roles for hemichannels in vivo.

scholarly journals The Revival of Aztreonam in Combination with Avibactam against Metallo-β-Lactamase-Producing Gram-Negatives: A Systematic Review of In Vitro Studies and Clinical Cases

Antibiotics ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1012
Author(s):  
Carola Mauri ◽  
Alberto Enrico Maraolo ◽  
Stefano Di Bella ◽  
Francesco Luzzaro ◽  
Luigi Principe
Keyword(s):  
Systematic Review ◽  
Bloodstream Infections ◽  
Mortality Rates ◽  
In Vivo Studies ◽  
High Antimicrobial Activity ◽  
Lactam Antibiotic ◽  
Vitro Data ◽  
In Vitro Data

Infections caused by metallo-β-lactamase (MBL)-producing Enterobacterales and Pseudomonas are increasingly reported worldwide and are usually associated with high mortality rates (>30%). Neither standard therapy nor consensus for the management of these infections exist. Aztreonam, an old β-lactam antibiotic, is not hydrolyzed by MBLs. However, since many MBL-producing strains co-produce enzymes that could hydrolyze aztreonam (e.g., AmpC, ESBL), a robust β-lactamase inhibitor such as avibactam could be given as a partner drug. We performed a systematic review including 35 in vitro and 18 in vivo studies on the combination aztreonam + avibactam for infections sustained by MBL-producing Gram-negatives. In vitro data on 2209 Gram-negatives were available, showing the high antimicrobial activity of aztreonam (MIC ≤ 4 mg/L when combined with avibactam) in 80% of MBL-producing Enterobacterales, 85% of Stenotrophomonas and 6% of MBL-producing Pseudomonas. Clinical data were available for 94 patients: 83% of them had bloodstream infections. Clinical resolution within 30 days was reported in 80% of infected patients. Analyzing only patients with bloodstream infections (64 patients), death occurred in 19% of patients treated with aztreonam + ceftazidime/avibactam. The combination aztreonam + avibactam appears to be a promising option against MBL-producing bacteria (especially Enterobacterales, much less for Pseudomonas) while waiting for new antimicrobials.

Characteristic distribution of gap junctions in rat lacrimal gland in vivo and reconstruction of a gap junction in an in vitro model

2002 ◽  
Vol 51 (1) ◽  
pp. 35-44 ◽  
Author(s):  
T. Takayama ◽  
S. Tatsukawa ◽  
H. Kitamura ◽  
K. Ina ◽  
K. Nakatsuka ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Lacrimal Gland ◽  
In Vitro Model ◽  
Characteristic Distribution ◽  
Vitro Model

scholarly journals Overexpression of delta-like 4 induces arterialization and attenuates vessel formation in developing mouse embryos

Blood ◽  
2008 ◽  
Vol 112 (5) ◽  
pp. 1720-1729 ◽  
Author(s):  
Alexandre Trindade ◽  
S. Ram Kumar ◽  
Jeffrey S. Scehnet ◽  
Luis Lopes-da-Costa ◽  
Jorg Becker ◽  
...  
Keyword(s):  
Vascular Endothelial Cells ◽  
Vascular Development ◽  
Ectopic Expression ◽  
Notch Signaling Pathway ◽  
In Vivo Studies ◽  
Variable Degree ◽  
Notch Receptors

Abstract The importance of Notch signaling pathway in the regulation of vascular development and angiogenesis is suggested by the expression of Notch receptors and ligands in vascular endothelial cells (ECs) and the observed vascular phenotypes in mutants of Notch receptors or ligands, especially Dll4. DLL4 is specifically expressed in arterial ECs during development, and haplo-insufficiency is embryonically lethal in mice. To address the role of Dll4 in vascular development, we produced mDll4 conditionally overexpressed transgenic mice that were crossed with constitutive recombinase cre lines. Double transgenic embryos displayed grossly enlarged dorsal aortae (DA) and died before embryonic day 10.5 (E10.5), showing a variable degree of premature arteriovenous fusion. Veins displayed ectopic expression of arterial markers. Other defects included reduced vascular sprouting, EC proliferation, and migration. mDll4 overexpression also inhibited VEGF signaling and increased fibronectin accumulation around the vessels. In vitro and in vivo studies of DLL4-FL (Dll4–full-length) in ECs recapitulate many of the mDll4 transgenics findings, including decreased tube formation, reduced vascular branching, fewer vessels, increased pericyte recruitment, and increased fibronectin expression. These results establish the role of Dll4 in arterial identity determination, and regulation of angiogenesis subject to dose and location.

scholarly journals Functional Link between miR-200a and ELK3 Regulates the Metastatic Nature of Breast Cancer

Cancers ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 1225
Author(s):  
Hyung-Keun Kim ◽  
Joo Dong Park ◽  
Seung Hee Choi ◽  
Dong Jun Shin ◽  
Sohyun Hwang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Treatment Options ◽  
Ectopic Expression ◽  
In Vivo Studies ◽  
Migration And Invasion ◽  
Breast Cancers ◽  
Functional Link ◽  
Her2 Protein

Triple-negative breast cancer (TNBC) refers to breast cancer that does not have receptors for estrogen, progesterone, and HER2 protein. TNBC accounts for 10–20% of all cases of breast cancers and is characterized by its metastatic aggressiveness, poor prognosis, and limited treatment options. Here, we show that the metastatic nature of TNBC is critically regulated by a functional link between miR-200a and the transcription factor ELK3. We found that the expression levels of miR-200a and the ELK3 mRNA were negatively correlated in the luminal and TNBC subtypes of breast cancer cells. In vitro experiments revealed that miR-200a directly targets the 3’ untranslated region (UTR) of the ELK3 mRNA to destabilize the transcripts. Furthermore, ectopic expression of miR-200a impaired the migration and invasion of TNBC cells by reducing the expression level of the ELK3 mRNA. In in vivo studies, transfection of MDA-MB 231 cells (a claudin-low TNBC cell type) with exogenous miR-200a reduced their extravasation into the lung during 48 h after tail vein injection, and co-transfection of the cells with an expression plasmid harboring ELK3 that lacked an intact 3’UTR recovered their extravasation ability. Overall, our findings provide evidences that miR-200a and ELK3 is functionally linked to regulate invasive characteristics of breast cancers.

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