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Junction Proteins (junction + protein)
Kinds of Junction Proteins Selected AbstractsCharacterization of Zebrafish Cx43.4 Connexin and its ChannelsEXPERIMENTAL PHYSIOLOGY, Issue 6 2003T. Desplantez Connexins (Cx) form intercellular junctional channels which are responsible for metabolic and electrical coupling. We report here on the biochemical and immunohistochemical characterization of zebrafish connexin zfCx43.4, an orthologue of mammalian and avian Cx45, and the electrophysiological properties of junctional channels formed by this protein. The investigations were performed on transfected COS-7 cells or HeLa cells. Using site-directed antibodies, zfCx43.4 cDNA (GenBank accession no. X96712) was demonstrated to code for a protein with a Mr of 45 000. In transfected cells, zfCx43.4 was localized in cell-cell contact areas as expected for a gap junction protein. zfCx43.4 channels were shown to transfer Lucifer Yellow. The multichannel currents were sensitive to the transjunctional voltage (Vj). Their properties were consistent with a two-state model and yielded the following Boltzmann parameters for negative/positive Vj: Vj,0= -38.4/41.9 mV; gj,min= 0.19/0.18; z = 2.6/2.3. These parameters deviate somewhat from those of zfCx43.4 channels expressed in Xenopus oocytes and from those of Cx45, an orthologue of zfCx43.4, expressed in mammalian cells or Xenopus oocytes. Conceivably, the subtle differences may reflect differences in experimental methods and/or in the expression system. The single channel currents yielded two prominent levels attributable to a main conductance state (,j,main= 33.2 ± 1.5 pS) and a residual conductance state (,j,residual= 11.9 ± 0.6 pS). [source] Normal embryonic development and cardiac morphogenesis in mice with Wnt1-Cre-mediated deletion of connexin43GENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 6 2006M. Kretz Abstract Mice harboring a null mutation in the gap junction protein connexin43 (Cx43) die shortly after birth due to an obstruction of the right ventricular outflow tract of the heart. These hearts exhibit prominent pouches at the base of the pulmonary outlet, i.e., morphological abnormalities that were ascribed to Cx43-deficiency in neural crest cells. In order to examine the Cx43 expression pattern in neural crest cells and derived tissues and to test whether neural crest-specific deletion of Cx43 leads to the conotruncal defects seen in Cx43null mice, we ablated Cx43 using a Wnt1-Cre transgene. Deletion of Cx43 was complete and occurred in neural crest cells as well as in neural crest-derived tissues. Nevertheless, hearts of mice lacking Cx43 specifically in neural crest cells were indistinguishable from controls. Thus, the morphological heart abnormalities of Cx43 null mice are most likely not caused by lack of Cx43 in neural crest cells. genesis 44:269,276, 2006. © 2006 Wiley-Liss, Inc. [source] Modulation of astrocyte P2Y1 receptors by the carboxyl terminal domain of the gap junction protein Cx43GLIA, Issue 2 2008Eliana Scemes Abstract Gap junction proteins, connexins, provide intercellular channels that allow ions and small signaling molecules to be transmitted to adjacent coupled cells. Besides this function, it is becoming apparent that connexins also exert channel-independent effects, which are likely mediated by processes involving protein,protein interactions. Although a number of connexin interacting proteins have been identified, only little is known about the functional consequences of such interactions. We have previously shown that deletion of the astrocytic gap junction protein, connexin43 (Cx43) causes a right-ward shift in the dose-response curve to P2Y1R agonists and decreased P2Y1R expression levels. To evaluate whether these changes were due to reduced gap junctional communication or to protein,protein interactions, Cx43-null astrocytes were transfected with full-length Cx43 and Cx43 domains, and P2Y1R function and expression levels evaluated. Results indicate that restoration of P2Y1R function is independent of gap junctional communication and that the Cx43 carboxyl terminus spanning the SH3 binding domain (260,280) participates in the rescue of P2Y1R pharmacological behavior (shifting to the left the P2Y1R dose-response curve) without affecting its expression levels. These results suggest that the Cx43 carboxyl-terminus domain provides a binding site for an intracellular molecule, most likely a member of the c-Src tyrosine kinase family, which affects P2Y1R-induced calcium mobilization. It is here proposed that a nonchannel function of Cx43 is to serve as a decoy for such kinases. Such modulation of P2Y1R is expected to influence several neural cell functions, especially under inflammation and neurodegenerative disorders where expression levels of Cx43 are decreased. © 2007 Wiley-Liss, Inc. [source] A role for Connexin43 during neurodevelopmentGLIA, Issue 7 2007Amy E. Wiencken-Barger Abstract Connexin43 (Cx43) is the predominant gap junction protein expressed in premitotic radial glial cells and mature astrocytes. It is thought to play a role in many aspects of brain development and physiology, including intercellular communication, the release of neuroactive substances, and neural and glial proliferation and migration. To investigate the role of Cx43 in brain physiology, we generated a conditional knockout (cKO) mouse expressing Cre recombinase driven by the human GFAP promoter and a floxed Cx43 gene. The removal of Cx43 from GFAP-expressing cells affects the behavior of the mice and the development of several brain structures; however, the severity of the phenotype varies depending on the mouse background. One mouse subline, hereafter termed Shuffler, exhibits cellular disorganization of the cortex, hippocampus, and cerebellum, accompanied by ataxia and motor deficits. The Shuffler cerebellum is most affected and displays altered distribution and lamination of glia and neurons suggestive of cell migration defects. In all Shuffler mice by postnatal day two (P2), the hippocampus, cortex, and cerebellum are smaller. Disorganization of the ventricular and subventricular zone of the cortex is also evident. Given that these are sites of early progenitor cell proliferation, we suspect production and migration of neural progenitors may be altered. In conclusion, neurodevelopment of Shuffler/Cx43 cKO mice is abnormal, and the observed cellular phenotype may explain behavioral disturbances seen in these animals as well as in humans carrying Cx43 mutations. © 2007 Wiley-Liss, Inc. [source] Cell culture,produced hepatitis C virus does not infect peripheral blood mononuclear cells,HEPATOLOGY, Issue 6 2008Svetlana Marukian Hepatitis C virus (HCV) replicates primarily in the liver, but HCV RNA has been observed in association with other tissues and cells including B and T lymphocytes, monocytes, and dendritic cells. We have taken advantage of a recently described, robust system that fully recapitulates HCV entry, replication and virus production in vitro to re-examine the issue of HCV infection of blood cell subsets. The HCV replicase inhibitor 2,C-methyl adenosine was used to distinguish HCV RNA replication from RNA persistence. Whereas cell culture,grown HCV replicated in Huh-7.5 hepatoma cells, no HCV replication was detected in B or T lymphocytes, monocytes, macrophages, or dendritic cells from healthy donors. No blood cell subset tested expressed significant levels of Claudin-1, a tight junction protein needed for HCV infection of Huh-7.5 cells. A B cell line expressing high levels of Claudin-1, CD81, and scavenger receptor BI remained resistant to HCV pseudoparticle infection. We bypassed the block in HCV entry by transfecting HCV RNA into blood cell subsets. Transfected RNA was not detectably translated and induced high levels of interferon-,. Supernatants from HCV RNA,transfected macrophages inhibited HCV replication in Huh-7.5 cells. Conclusion: We conclude that multiple blocks prevent blood cells from supporting HCV infection. (HEPATOLOGY 2008;48:1843-1850.) [source] Lysophosphatidic acid induces ovarian cancer cell dispersal by activating Fyn kinase associated with p120-cateninINTERNATIONAL JOURNAL OF CANCER, Issue 4 2008Ruby Yun-Ju Huang Abstract Lysophosphatidic acid (LPA), known as the "ovarian cancer activating factor," is a natural phospholipid involved in important biological functions, such as cell proliferation, wound healing and neurite retraction. LPA causes colony dispersal in various carcinoma cell lines by inducing morphological changes, including membrane ruffling, lamellipodia formation, cell,cell dissociation and single cell migration. However, its effects on cell,cell dissociation and cell,cell adhesion of ovarian cancer cells have not been studied. In our study, we showed that LPA induced sequential events of intercellular junction dispersal and "half-junction" formation in ovarian cancer SKOV3 cells and that Src-family kinases were involved in both processes, since the effects were abolished by the selective tyrosine kinase inhibitor PP2. LPA induced rapid and transient activation of Src family kinases, which were recruited to cell,cell junctions by increasing the association with the adherens junction protein p120-catenin. We identified the Src family kinase, Fyn, as the key component associated with p120-catenin after LPA stimulation in SKOV3 cells. Our study provides evidence that LPA induces junction dispersal in ovarian cancer SKOV3 cells by activating the Src family kinase Fyn and increasing its association with p120-catenin at the cell,cell junction. © 2008 Wiley-Liss, Inc. [source] c-Jun N-terminal kinase is largely involved in the regulation of tricellular tight junctions via tricellulin in human pancreatic duct epithelial cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2010Takashi Kojima Tricellulin (TRIC) is a tight junction protein at tricellular contacts where three epithelial cells meet, and it is required for the maintenance of the epithelial barrier. To investigate whether TRIC is regulated via a c-Jun N-terminal kinase (JNK) pathway, human pancreatic HPAC cells, highly expressed at tricellular contacts, were exposed to various stimuli such as the JNK activators anisomycin and 12- O -tetradecanoylphorbol 13-acetate (TPA), and the proinflammatory cytokines IL-1,, TNF,, and IL-1,. TRIC expression and the barrier function were moderated by treatment with the JNK activator anisomycin, and suppressed not only by inhibitors of JNK and PKC but also by siRNAs of TRIC. TRIC expression was induced by treatment with the PKC activator TPA and proinflammatory cytokines IL-1,, TNF,, and IL-1,, whereas the changes were inhibited by a JNK inhibitor. Furthermore, in normal human pancreatic duct epithelial cells using hTERT-transfected primary cultured cells, the responses of TRIC expression to the various stimuli were similar to those in HPAC cells. TRIC expression in tricellular tight junctions is strongly regulated together with the barrier function via the JNK transduction pathway. These findings suggest that JNK may be involved in the regulation of tricellular tight junctions including TRIC expression and the barrier function during normal remodeling of epithelial cells, and prevent disruption of the epithelial barrier in inflammation and other disorders in pancreatic duct epithelial cells. J. Cell. Physiol. 225: 720,733, 2010. © 2010 Wiley-Liss, Inc. [source] Hyperosmotic mannitol induces Src kinase-dependent phosphorylation of ,-catenin in cerebral endothelial cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 6 2005Attila Farkas Abstract Mannitol, which is a cell-impermeable and nontoxic polyalcohol, has been shown to be a useful tool for reversible opening of the blood,brain barrier (BBB). Despite successful clinical trials, the molecular mechanism of the mannitol-induced changes in cerebral endothelial cells (CECs) are poorly understood. For our experiments, we used CECs in culture, which were treated with different, clinically relevant concentrations of mannitol. We found that mannitol induced a rapid, concentration-dependent, and reversible tyrosine phosphorylation of a broad range of proteins between 50 and 190 kDa. One of the targets of tyrosine phosphorylation turned out to be the adherens junction protein ,-catenin. Phosphorylation of ,-catenin on tyrosine residues caused its subcellular redistribution and its dissociation from cadherin and ,-catenin as shown by coimmunoprecipitation studies. All these effects could be inhibited by the Src kinase inhibitor PP-1 but not by the Erk inhibitor U0126, the Rho kinase inhibitor Y27632, or the calcium channel blocker verapamil. Because ,-catenin is a key component of the junctional complex, its Src-mediated phpsphorylation may play an important role in the mannitol induced reversible opening of the BBB. © 2005 Wiley-Liss, Inc. [source] Differential expression of connexin 43 in the chick tangential vestibular nucleusJOURNAL OF NEUROSCIENCE RESEARCH, Issue 5 2003Anastas Popratiloff Abstract The chick tangential nucleus is a major vestibular nucleus whose principal cells receive convergent inputs from primary vestibular and nonvestibular fibers and participate in the vestibular reflexes. During development, the principal cells gradually acquire the mature firing pattern in part by losing a specific potassium current around hatching (H). Here we focus on characterizing the expression of connexin 43 (Cx43), a gap junction protein found mainly between astrocytes in the mature brain. The astrocytic syncytium plays an important role in maintaining extracellular potassium ion balance in the brain. Accordingly, it is important to characterize the potential of this syncytium to communicate during the critical developmental age of hatching. Using fluorescence immunocytochemistry, we investigated whether Cx43 staining was concentrated in specific cellular compartments at H1 by applying well-known markers for astrocytes (glial fibrillary acidic protein; GFAP), oligodendrocytes (antimyelin), neurons (microtubule-associated protein 2), and synaptic terminals (synaptotagmin). GFAP-positive astrocytes and GFAP-negative nonneuronal cells around the principal cell bodies were labeled with Cx43, suggesting that Cx43 was expressed exclusively by nonneuronal cells near the neuronal elements. Next, the developmental pattern of expression of Cx43 was studied at embryonic day 16 (E16), H1, and H9. At E16, Cx43 was present weakly as random small clusters in the tangential nucleus, whereas, at H1, overall staining became localized, with increases in size, brightness, and number of immunostained clusters. Finally, at H9, Cx43 staining decreased, but cluster size and location remained unchanged. These results suggest that Cx43 is developmentally regulated with a peak at birth and is associated primarily with astrocytes and nonneuronal cells near the principal cell bodies. © 2003 Wiley-Liss, Inc. [source] Expression of gap junction protein connexin 32 in chronic liver diseasesLIVER INTERNATIONAL, Issue 2 2000Kazuaki Yamaoka Abstract:Background: Gap junctions contain intercellular channels through which contacting cells communicate directly. The expression of connexin 32, a major gap junction protein in the liver, during the progression of chronic liver diseases has not yet been clarified. Methods: Immunohistochemical staining was performed using anti-connexin 32 antibody on 6 specimens of normal human liver, 7 of chronic viral hepatitis, and 7 of liver cirrhosis. Results: The number of gap junction plaques in chronic viral hepatitis and liver cirrhosis was significantly smaller than that in normal liver (10350±2180 and 7550±3040 vs 22560±3700 spots/mm2, p<0.01). The number of gap junction plaques tended to be lower in liver cirrhosis than in chronic viral hepatitis. Conclusion: These results suggest that in chronic liver diseases impaired intercellular communication between hepatocytes occurs. [source] Nature, significance, and mechanisms of electrical heterogeneities in ventricleTHE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 2 2004Steven Poelzing Abstract Previously, dispersion of repolarization (DOR) has been extensively linked to the development of arrhythmias and sudden cardiac death. The electrical heterogeneities that cause DOR between transmural myocyte layers have been reported in a wide variety of animals and humans. The underlying causes of transmural electrical heterogeneities are in part due to heterogeneous functional expression of proteins responsible for ion handling. Recently, we found that electrophysiologic heterogeneities between subepicardial and midmyocardial cells can form a substrate for reentrant ventricular arrhythmias. However, cell-to-cell coupling through gap junctions is expected to attenuate transmural heterogeneities between cell types spanning the ventricular wall. In this article we review a hypothesis that regional uncoupling resulting from expression patterns of gap junctions across the ventricular wall underlies DOR, and DOR can be amplified under disease conditions which remodel gap junctions. We find the principle gap junction protein, connexin43 (Cx43), is selectively reduced in the subepicardium (by 24%) compared to deeper layers of normal canine left ventricle. Additionally, the greatest DOR occurs within the subepicardial-midmyocardial interface, precisely where Cx43 expression is reduced. The present data suggests that ion channel and gap junction heterogeneities act in conjunction to form and maintain transmural DOR. Importantly, both ion channel and gap junction remodeling occurs during many disease states such as heart failure. Importantly, in the absence of ion channel remodeling, pharmacological uncoupling increases transmural DOR, particularly within the epicardial-midmyocardial interface, to values observed in heart failure. Therefore, these data suggest that heterogeneous Cx43 expression produces functionally significant electrophysiologic heterogeneities across the ventricular wall and may be a mechanism for promoting DOR which underlie arrhythmias in heart failure. © 2004 Wiley-Liss, Inc. [source] Disrupted SOX10 regulation of GJC2 transcription causes Pelizaeus-Merzbacher-like diseaseANNALS OF NEUROLOGY, Issue 2 2010Hitoshi Osaka MD Mutations in the gap junction protein gamma-2 gene, GJC2, cause a central hypomyelinating disorder; Pelizaeus-Merzbacher-like disease (PMLD; MIM311601). Using a homozygosity mapping and positional candidate gene approach, we identified a homozygous mutation (c.-167A>G) within the GJC2 promoter at a potent SOX10 binding site in a patient with mild PMLD. Functionally, this mutation completely abolished the SOX10 binding and attenuated GJC2 promoter activity. These findings suggest not only that the SOX10 -to- GJC2 transcriptional dysregulation is a cause of PMLD, but also that GJC2 may be in part responsible for the central hypomyelination caused by SOX10 mutations. ANN NEUROL 2010;68:250,254 [source] A microfluidic bioreactor with integrated transepithelial electrical resistance (TEER) measurement electrodes for evaluation of renal epithelial cellsBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2010Nicholas Ferrell Abstract We have developed a bilayer microfluidic system with integrated transepithelial electrical resistance (TEER) measurement electrodes to evaluate kidney epithelial cells under physiologically relevant fluid flow conditions. The bioreactor consists of apical and basolateral fluidic chambers connected via a transparent microporous membrane. The top chamber contains microfluidic channels to perfuse the apical surface of the cells. The bottom chamber acts as a reservoir for transport across the cell layer and provides support for the membrane. TEER electrodes were integrated into the device to monitor cell growth and evaluate cell,cell tight junction integrity. Immunofluorescence staining was performed within the microchannels for ZO-1 tight junction protein and acetylated ,-tubulin (primary cilia) using human renal epithelial cells (HREC) and MDCK cells. HREC were stained for cytoskeletal F-actin and exhibited disassembly of cytosolic F-actin stress fibers when exposed to shear stress. TEER was monitored over time under normal culture conditions and after disruption of the tight junctions using low Ca2+ medium. The transport rate of a fluorescently labeled tracer molecule (FITC-inulin) was measured before and after Ca2+ switch and a decrease in TEER corresponded with a large increase in paracellular inulin transport. This bioreactor design provides an instrumented platform with physiologically meaningful flow conditions to study various epithelial cell transport processes. Biotechnol. Bioeng. 2010;107:707,716. © 2010 Wiley Periodicals, Inc. [source] Disruption of gap junctions attenuates aminoglycoside-elicited renal tubular cell injuryBRITISH JOURNAL OF PHARMACOLOGY, Issue 8 2010Jian Yao BACKGROUND AND PURPOSE Gap junctions play important roles in the regulation of cell phenotype and in determining cell survival after various insults. Here, we investigated the role of gap junctions in aminoglycoside-induced injury to renal tubular cells. EXPERIMENTAL APPROACH Two tubular epithelial cell lines NRK-E52 and LLC-PK1 were compared for gap junction protein expression and function by immunofluorescent staining, Western blot and dye transfer assay. Cell viability after exposure to aminoglycosides was evaluated by WST assay. Gap junctions were modulated by transfection of the gap junction protein, connexin 43 (Cx43), use of Cx43 siRNA and gap junction inhibitors. KEY RESULTS NRK-E52 cells expressed abundant Cx43 and were functionally coupled by gap junctional intercellular communication (GJIC). Exposure of NRK-E52 cells to aminoglycosides, G418 and hygromycin, increased Cx43 phosphorylation and GJIC. The aminoglycosides also decreased cell viability that was prevented by gap junction inhibitors and Cx43 siRNA. LLC-PK1 cells were gap junction-deficient and resistant to aminoglycoside-induced cytotoxicity. Over-expression of a wild-type Cx43 converted LLC-PK1 cells to a drug-sensitive phenotype. The gap junction inhibitor ,-glycyrrhetinic acid (,-GA) activated Akt in NRK-E52 cells. Inhibition of the Akt pathway enhanced cell toxicity to G418 and abolished the protective effects of ,-GA. In addition, gentamycin-elicited cytotoxicity in NRK-E52 cells was also significantly attenuated by ,-GA. CONCLUSION AND IMPLICATIONS Gap junctions contributed to the cytotoxic effects of aminoglycosides. Modulation of gap junctions could be a promising approach for prevention and treatment of aminoglycoside-induced renal tubular cell injury. [source] The signal transduction cascade regulating the expression of the gap junction protein connexin43 by ,-adrenoceptorsBRITISH JOURNAL OF PHARMACOLOGY, Issue 1 2009A Salameh Background and purpose:, In mammalian heart, connexin43 (Cx43) represents the predominant connexin in the working myocardium. As the ,-adrenoceptor is involved in many cardiac diseases, we wanted to clarify the pathway by which ,-adrenoceptor stimulation may control Cx43 expression. Experimental approach:, Cultured neonatal rat cardiomyocytes were stimulated with isoprenaline. Cx43 expression as well as activation of p38 mitogen-activated protein kinase (MAPK), p42/44 MAPK, JUN NH2 -terminal kinase (JNK) and nuclear translocation of the transcription factors activator protein 1 (AP1) and CRE-binding protein (CREB) were investigated. Additionally, we assessed Cx43 expression and distribution in left ventricular biopsies from patients without any significant heart disease, and from patients with either congestive heart failure [dilated cardiomyopathy (DCM)] or hypertrophic cardiomyopathy (HCM). Key results:, Isoprenaline exposure caused about twofold up-regulation of Cx43 protein with a pEC50 of 7.92 ± 0.11, which was inhibited by propranolol, SB203580 (4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)-1H-imidazole) (p38 inhibitor), PD98059 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one) (MAPK 1 kinase inhibitor) (Alexis Biochemicals, San Diego, CA, USA) or cyclosporin A. Similar findings were obtained for Cx43 mRNA. Furthermore, Cx43 up-regulation was accompanied by phosphorylation of p38, p42/44 and JNK, and by translocation of AP1 and CREB to the nucleus. Analysis of Cx43 protein and mRNA in ventricular biopsies revealed that in patients with DCM, Cx43 content was significantly lower, and in patients with HCM, Cx43 content was significantly higher, relative to patients without any cardiomyopathy. More importantly, Cx43 distribution also changed with more Cx43 being localized at the lateral border of the cardiomyocytes. Conclusion and implication:, ,-adrenoceptor stimulation up-regulated cardiac Cx43 expression via a protein kinase A and MAPK-regulated pathway, possibly involving AP1 and CREB. Cardiomyopathy altered Cx43 expression and distribution. [source] Erythropoietin protects the in vitro blood,brain barrier against VEGF-induced permeabilityEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 9 2003Ofelia María Martínez-Estrada Abstract The blood,brain barrier (BBB) ensures the homeostasis of the brain microenvironment, mostly through complex tight junctions between brain endothelial cells that prevent the passage of hydrophilic molecules from blood to brain and vice versa. A recent study has shown in vivo that systemic administration of erythropoietin (Epo) protects against brain injury. Using an in vitro model of the bovine BBB, we observed that the expression of the Epo receptor is modulated by its ligand and hypoxic stimuli such as vascular endothelial growth factor (VEGF) treatment. In addition, Epo protects against the VEGF-induced permeability of the BBB, decreases the levels of endothelial nitric oxide synthase and restores junction proteins. The kinetic transport experiments revealed the capacity of Epo to cross the in vitro BBB in a saturable and specific way. Our results suggest a new mechanism for Epo-induced neuroprotection, in which circulating Epo controls and maintains the BBB through an Epo receptor signalling pathway and the re-establishment of cell junctions. [source] Kinesin II is required for cell survival and adherens junction positioning in Drosophila photoreceptorsGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 9 2010Bibhash Mukhopadhyay Genetic analysis of klp64D(kif3a homolog) function in the Drosophilapupal retina. The ommatidum on the left consists of wild-type photoreceptors. The ommatidium on the right is a genetic mosaic containing wild-type and mutant cells marked by green fluorescent protein. klp64Dmutant cells show mislocalization of adherens junction proteins, DE-Cadherin and Armadillo (labeled by red and blue staining, respectively). See the article by Mukhopadhyay et al. in this issue. [source] Modulation of astrocyte P2Y1 receptors by the carboxyl terminal domain of the gap junction protein Cx43GLIA, Issue 2 2008Eliana Scemes Abstract Gap junction proteins, connexins, provide intercellular channels that allow ions and small signaling molecules to be transmitted to adjacent coupled cells. Besides this function, it is becoming apparent that connexins also exert channel-independent effects, which are likely mediated by processes involving protein,protein interactions. Although a number of connexin interacting proteins have been identified, only little is known about the functional consequences of such interactions. We have previously shown that deletion of the astrocytic gap junction protein, connexin43 (Cx43) causes a right-ward shift in the dose-response curve to P2Y1R agonists and decreased P2Y1R expression levels. To evaluate whether these changes were due to reduced gap junctional communication or to protein,protein interactions, Cx43-null astrocytes were transfected with full-length Cx43 and Cx43 domains, and P2Y1R function and expression levels evaluated. Results indicate that restoration of P2Y1R function is independent of gap junctional communication and that the Cx43 carboxyl terminus spanning the SH3 binding domain (260,280) participates in the rescue of P2Y1R pharmacological behavior (shifting to the left the P2Y1R dose-response curve) without affecting its expression levels. These results suggest that the Cx43 carboxyl-terminus domain provides a binding site for an intracellular molecule, most likely a member of the c-Src tyrosine kinase family, which affects P2Y1R-induced calcium mobilization. It is here proposed that a nonchannel function of Cx43 is to serve as a decoy for such kinases. Such modulation of P2Y1R is expected to influence several neural cell functions, especially under inflammation and neurodegenerative disorders where expression levels of Cx43 are decreased. © 2007 Wiley-Liss, Inc. [source] HuR regulates gap junctional intercellular communication by controlling ,-catenin levels and adherens junction integrity,HEPATOLOGY, Issue 5 2009Niloofar Ale-Agha Gap junctional intercellular communication (GJIC) plays a critical role in the regulation of tissue homeostasis and carcinogenesis and is modulated by the levels, subcellular localization, and posttranslational modification of gap junction proteins, the connexins (Cx). Here, using oval cell-like rat liver epithelial cells, we demonstrate that the RNA-binding protein HuR promotes GJIC through two mechanisms. First, HuR silencing lowered the levels of Cx43 protein and Cx43 messenger RNA (mRNA), and decreased Cx43 mRNA half-life. This regulation was likely due to the direct stabilization of Cx43 mRNA by HuR, because HuR associated directly with Cx43 mRNA, a transcript that bears signature adenylate-uridylate-rich (AU-rich) and uridylate-rich (U-rich) sequences in its 3,-untranslated region. Second, HuR silencing reduced both half-life and the levels of ,-catenin mRNA, also a target of HuR; accordingly, HuR silencing lowered the levels of whole-cell and membrane-associated ,-catenin. Coimmunoprecipitation experiments showed a direct interaction between ,-catenin and Cx43. Small interfering RNA (siRNA)-mediated depletion of ,-catenin recapitulated the effects of decreasing HuR levels: it attenuated GJIC, decreased Cx43 levels, and redistributed Cx43 to the cytoplasm, suggesting that depletion of ,-catenin in HuR-silenced cells contributed to lowering Cx43 levels at the membrane. Finally, HuR was demonstrated to support GJIC after exposure to a genotoxic agent, doxorubicin, or an inducer of differentiation processes, retinoic acid, thus pointing to a crucial role of HuR in the cellular response to stress and in physiological processes modulated by GJIC. Conclusion: HuR promotes gap junctional intercellular communication in rat liver epithelial cells through two related regulatory processes, by enhancing the expression of Cx43 and by increasing the expression of ,-catenin, which, in turn, interacts with Cx43 and is required for proper positioning of Cx43 at the plasma membrane. (HEPATOLOGY 2009.) [source] Novel hepatic progenitor cell surface markers in the adult rat liver,HEPATOLOGY, Issue 1 2007Mladen I. Yovchev Hepatic progenitor/oval cells appear in injured livers when hepatocyte proliferation is impaired. These cells can differentiate into hepatocytes and cholangiocytes and could be useful for cell and gene therapy applications. In this work, we studied progenitor/oval cell surface markers in the liver of rats subjected to 2-acetylaminofluorene treatment followed by partial hepatectomy (2-AAF/PH) by using rat genome 230 2.0 Array chips and subsequent RT-PCR, immunofluorescent (IF), immunohistochemical (IHC) and in situ hybridization (ISH) analyses. We also studied expression of the identified novel cell surface markers in fetal rat liver progenitor cells and FAO-1 hepatoma cells. Novel cell surface markers in adult progenitor cells included tight junction proteins, integrins, cadherins, cell adhesion molecules, receptors, membrane channels and other transmembrane proteins. From the panel of 21 cell surface markers, 9 were overexpressed in fetal progenitor cells, 6 in FAO-1 cells and 6 are unique for the adult progenitors (CD133, claudin-7, cadherin 22, mucin-1, ros-1, Gabrp). The specificity of progenitor/oval cell surface markers was confirmed by ISH and double IF analyses. Moreover, study of progenitor cells purified with Ep-CAM antibodies from D-galactosamine injured rat liver, a noncarcinogenic model of progenitor cell activation, verified that progenitor cells expressed these markers. Conclusion: We identified novel cell surface markers specific for hepatic progenitor/oval cells, which offers powerful tool for their identification, isolation and studies of their physiology and pathophysiology. Our studies also reveal the mesenchymal/epithelial phenotype of these cells and the existence of species diversity in the hepatic progenitor cell identity. (HEPATOLOGY 2007;45:139,149.) [source] Radiation-induced bystander effects in malignant trophoblast cells are independent from gap junctional communicationJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2008Ferya Banaz-Ya Abstract It is controversially discussed that irradiation induces bystander effects via gap junction channels and/or diffusible cellular factors such as nitric oxide or cytokines excreted from the cells into the environment. But up to now the molecular mechanism leading to a bystander response is not well understood. To discriminate between both mechanisms of bystander response, (i) mediated by gap junctional communication and/or (ii) mediated by diffusible molecules, we used non-communicating Jeg3 malignant trophoblast cells transfected with inducible gap junction proteins, connexin43 and connexin26, respectively, based on the Tet-On system. We co-cultivated X-ray irradiated and non-irradiated bystander Jeg3 cells for 4 h, separated both cell populations by flow cytometry and evaluated the expression of activated p53 by Western blot analysis. The experimental design was proven with communicating versus non-communicating Jeg3 cells. Interestingly, our results revealed a bystander effect which was independent from gap junctional communication properties and the connexin isoform expressed. Therefore, it seems more likely that the bystander effect is not mediated via gap junction channels but rather by paracrine mechanisms via excreted molecules in Jeg3 cells. J. Cell. Biochem. 103: 149,161, 2008. © 2007 Wiley-Liss, Inc. [source] Connexin 43 gap junction proteins are up-regulated in remyelinating spinal cordJOURNAL OF NEUROSCIENCE RESEARCH, Issue 5 2007W.A. Roscoe Abstract Alterations in the expression of gap junction proteins have previously been observed in several diseases affecting the central nervous system; however, the status of connexin 43 (Cx43) has not yet been reported in spinal cord remyelination. We studied Cx43 expression in demyelination and remyelination by using a chronic guinea pig model of experimental allergic encephalomyelitis (EAE). Hartley guinea pigs were immunized with homogenized whole CNS and complete Freund's adjuvant. Animals became chronically ill by day 40 postimmunization, and animals with paralysis were entered into the study. Animals were treated on days 40,60 postimmunization with either saline or drugs that promote remyelination: an adenosine amine congener (100 ,g/kg), an anti-,4-integrin blocker (CT301; ELN 69299; 30 mg/kg), or a combination of both drugs. Remyelination was induced in all drug-treated groups. Cx43 expression was virtually absent in demyelinated lesions of saline-treated controls compared with healthy tissue and normal appearing white matter (P < 0.001), whereas Cx43 was considerably increased (300,500%) in remyelinating lesions of all treatment groups (P < 0.001), most notably in CT301-treated animals. These changes in Cx43 expression indicate that Cx43 may beimportant for recovery from neuroinflammation. © 2007 Wiley-Liss, Inc. [source] Evaluation of human nasal RPMI 2650 cells grown at an air,liquid interface as a model for nasal drug transport studiesJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 3 2008Shuhua Bai Abstract This study tests the hypothesis that human nasal RPMI 2650 cells grown at an air,liquid interface is a feasible model for drug transport studies via the nasal route. RPMI 2650 cells were cultured in Eagle's minimal essential medium (MEM) at both air,liquid and liquid,liquid interfaces. For each culture regimen, monolayer integrity was tested by measuring the transepithelial resistance (TEER) as well as the transport of paracellular and transcellular markers across the monolayer. The expression of tight junction proteins,differentiation markers,in cells of the different monolayers was studied by western blot analysis and confocal microscopy. The highest TEER values (192,±,3 ,,·,cm2) were observed for RPMI 2650 cells seeded onto collagen-coated permeable polytetrafluoroethylene inserts and grown at an air,liquid interface for 10 days; a seeding density of 4,×,105/cm2 generated and maintained a cell monolayer with suitable barrier properties at days 9,12. Microscopic examination showed that RPMI 2650 cells grown on filter inserts formed a fully confluent monolayer. The apparent permeability coefficients of the paracellular marker, [14C] mannitol, and the transcellular marker, [3H] propranolol, were 5.07,±,0.01,×,10,6 cm/s and 16.1,±,0.1,×,10,6 cm/s, respectively. Western blot analysis indicated the presence of four tight junction proteins: ZO-1, occludin, claudin-1 and E-cadherin; and the quantities of ZO-1, occludin, and E-cadherin were significantly higher in cells grown at an air,liquid interface than in cells grown at a liquid,liquid interface. Confocal microscopic studies showed ZO-1, F-actin, occludin and claudin-1 proteins at cell-cell contacts and revealed significant differences in the distributions and densities of ZO-1 protein in cells grown at the two types of interface. The data indicate that RPMI 2650 cells grown at an air,liquid interface form polarized monolayers with the cells interconnected by tight junction proteins. This human nasal cell line model could provide a useful tool for in vitro screening of nasal drug candidates. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:1165,1178, 2008 [source] A7445G mtDNA mutation present in a Portuguese family exhibiting hereditary deafness and palmoplantar keratodermaJOURNAL OF THE EUROPEAN ACADEMY OF DERMATOLOGY & VENEREOLOGY, Issue 4 2005H Caria ABSTRACT Mitochondrial DNA (mtDNA) A7445G point mutation has been shown to be responsible for familial nonepidermolytic palmoplantar keratoderma (NEPPK) associated with deafness without any additional features. To date, only a few cases have been described. We report a Portuguese pedigree presenting an inherited combination of NEPPK and sensorineural deafness compatible with maternal transmission. Clinical expression and age of onset of NEPPK and deafness were variable. Normal expression patterns of epidermal keratins and filaggrin, intercellular junction proteins including connexin 26, loricrin and cornified envelope proteins, were observed. Molecular analysis revealed that all the affected members, previously screened for Cx26 mutations with negative results, presented the mtDNA A7445G point mutation in the homoplasmic form. To our knowledge, this is the fifth family in whom inherited NEPPK and hearing loss are related to this mitochondrial mutation. [source] Transforming growth factor-, induces epithelial to mesenchymal transition by down-regulation of claudin-1 expression and the fence function in adult rat hepatocytesLIVER INTERNATIONAL, Issue 4 2008Takashi Kojima Abstract Background/Aims: Transforming growth factor-, (TGF-,) initiates and maintains epithelial,mesenchymal transition (EMT), which causes disassembly of tight junctions and loss of epithelial cell polarity. In mature hepatocytes during EMT induced by TGF-,, changes in the expression of tight junction proteins and the fence function indicated that epithelial cell polarity remains unclear. Methods: In the present study, using primary cultures of adult rat hepatocytes at day 10 after plating, in which epithelial cell polarity is well maintained by tight junctions, we examined the effects of 0.01,20 ng/ml TGF-, on the expression of the integral tight junction proteins, claudin-1, -2 and occludin, as well as the fence function. Results: In adult rat hepatocytes, TGF-, induced EMT, which was indicated as upregulation of Smad-interacting protein-1 (SIP1) and Snail and down-regulation of E-cadherin. Down-regulation of claudin-1 and upregulation of occludin were observed beginning from a low dose of TGF-,, whereas upregulation of claudin-2 was observed at a high dose of TGF-,. Furthermore, treatment with TGF-, caused disruption of the fence function, which was closely associated with the expression of claudin-1 via p38 mitogen-activated protein kinase (MAPK), phosphoinositide-3 kinase and protein kinase C but not MAPK signalling pathways. Conclusion: These results suggest that in mature hepatocytes in vitro, TGF-, induces EMT by down-regulation of claudin-1 and the fence function via distinct signalling pathways. [source] Transcription profile in mouse four-cell, morula, and blastocyst: Genes implicated in compaction and blastocoel formationMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2007Xiang-Shun Cui Abstract To gain insight into early embryo development, we utilized microarray technology to compare gene expression profiles in four-cell (4C), morula (MO), and blastocyst (BL) stage embryos. Differences in spot intensities were normalized, and grouped by using Avadis Prophetic software platform (version 3.3, Strand Genomics Ltd.) and categories were based on the PANTHER and gene ontology (GO) classification system. This technique identified 622 of 7,927 genes as being more highly expressed in MO when compared to 4C (P,<,0.05); similarly, we identified 654 of 9,299 genes as being more highly expressed in BL than in MO (P,<,0.05). Upregulation of genes for cytoskeletal, cell adhesion, and cell junction proteins were identified in the MO as compared to the 4C stage embryos, this means they could be involved in the cell compaction necessary for the development to the MO. Genes thought to be involved in ion channels, membrane traffic, transfer/carrier proteins, and lipid metabolism were also identified as being expressed at a higher level in the BL stage embryos than in the MO. Real-time RT-PCR was performed to confirm differential expression of selected genes. The identification of the genes being expressed in here will provide insight into the complex gene regulatory networks effecting compaction and blastocoel formation. Mol. Reprod. Dev. © 2006 Wiley-Liss, Inc. [source] Proteomic comparison of four Eimeria tenella life-cycle stages: Unsporulated oocyst, sporulated oocyst, sporozoite and second-generation merozoitePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 19 2009Kalpana Lal Abstract We report the proteomes of four life-cycle stages of the Apicomplexan parasite Eimeria tenella. A total of 1868 proteins were identified, with 630, 699, 845 and 1532 found in early oocysts (unsporulated), late oocysts (sporulated), sporozoites and second-generation merozoites, respectively. A multidimensional protein identification technology shotgun approach identified 812 sporozoites, 1528 merozoites and all of the oocyst proteins, whereas 2-D gel proteomics identified 230 sporozoites and 98 merozoite proteins. Comparing the invasive stages, we find moving junction components RON2 in both, whereas AMA-1 and RON4 are found only in merozoites and AMA-2 and RON5 are only found in sporozoites, suggesting stage-specific moving junction proteins. During early oocyst to sporozoite development, refractile body and most "glideosome" proteins are found throughout, whereas microneme and most rhoptry proteins are only found after sporulation. Quantitative analysis indicates glycolysis and gluconeogenesis are the most abundant metabolic groups detected in all stages. The mannitol cycle "off shoot" of glycolysis was not detected in merozoites but was well represented in the other stages. However, in merozoites we find more protein associated with oxidative phosphorylation, suggesting a metabolic shift mobilising greater energy production. We find a greater abundance of protein linked to transcription, protein synthesis and cell cycle in merozoites than in sporozoites, which may be residual protein from the preceding massive replication during schizogony. [source] Differential distribution of tight junction proteins suggests a role for tanycytes in blood-hypothalamus barrier regulation in the adult mouse brainTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 7 2010Amandine Mullier The median eminence is one of the seven so-called circumventricular organs. It is located in the basal hypothalamus, ventral to the third ventricle and adjacent to the arcuate nucleus. This structure characteristically contains a rich capillary plexus and features a fenestrated endothelium, making it a direct target of blood-borne molecules. The median eminence also contains highly specialized ependymal cells called tanycytes, which line the floor of the third ventricle. It has been hypothesized that one of the functions of these cells is to create a barrier that prevents substances in the portal capillary spaces from entering the brain. In this paper, we report on our use of immunohistochemistry to study the expression of tight junction proteins in the cells that compose the median eminence in adult mice. Our results indicate that tanycytes of the median eminence express occludin, ZO-1, and claudin 1 and 5, but not claudin 3. Remarkably, these molecules are organized as a continuous belt around the cell bodies of the tanycytes that line the ventral part of the third ventricle. In contrast, the tanycytes at the periphery of the arcuate nucleus do not express claudin 1 and instead exhibit a disorganized expression pattern of occludin, ZO-1, and claudin 5. Consistent with these observations, permeability studies using peripheral or central injections of Evans blue dye show that only the tanycytes of the median eminence are joined at their apices by functional tight junctions, whereas tanycytes located at the level of the arcuate nucleus form a permeable layer. In conclusion, this study reveals a unique expression pattern of tight junction proteins in hypothalamic tanycytes, which yields new insights into their barrier properties. J. Comp. Neurol. 518:943,962, 2010. © 2009 Wiley-Liss, Inc. [source] Differential distribution of tight junction proteins suggests a role for tanycytes in blood-hypothalamus barrier regulation in the adult mouse brainTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 7 2010Amandine Mullier Abstract The median eminence is one of the seven so-called circumventricular organs. It is located in the basal hypothalamus, ventral to the third ventricle and adjacent to the arcuate nucleus. This structure characteristically contains a rich capillary plexus and features a fenestrated endothelium, making it a direct target of blood-borne molecules. The median eminence also contains highly specialized ependymal cells called tanycytes, which line the floor of the third ventricle. It has been hypothesized that one of the functions of these cells is to create a barrier that prevents substances in the portal capillary spaces from entering the brain. In this paper, we utilize immunohistochemistry to study the expression of tight junction proteins in the cells that compose the median eminence in adult mice. Our results indicate that tanycytes of the median eminence express occludin, ZO-1, and claudin 1 and 5, but not claudin 3. Remarkably, these molecules are organized as a continuous belt around the cell bodies of the tanycytes that line the ventral part of the third ventricle. In contrast, the tanycytes at the periphery of the arcuate nucleus do not express claudin 1 and instead exhibit a disorganized expression pattern of occludin, ZO-1, and claudin 5. Consistent with these observations, permeability studies using peripheral or central injections of Evans blue dye show that only the tanycytes of the median eminence are joined at their apices by functional tight junctions, whereas tanycytes located at the level of the arcuate nucleus form a permeable layer. In conclusion, this study reveals a unique expression pattern of tight junction proteins in hypothalamic tanycytes, which yields new insights into their barrier properties. J. Comp. Neurol. 518:943,962, 2010. © 2009 Wiley-Liss, Inc. [source] Electrical and chemical synapses between relay neurons in developing thalamusTHE JOURNAL OF PHYSIOLOGY, Issue 13 2010Seung-Chan Lee Gap junction-mediated electrical synapses interconnect diverse types of neurons in the mammalian brain, and they may play important roles in the synchronization and development of neural circuits. Thalamic relay neurons are the major source of input to neocortex. Electrical synapses have not been directly observed between relay neurons in either developing or adult animals. We tested for electrical synapses by recording from pairs of relay neurons in acute slices of developing ventrobasal nucleus (VBN) of the thalamus from rats and mice. Electrical synapses were common between VBN relay neurons during the first postnatal week, and then declined sharply during the second week. Electrical coupling was reduced among cells of connexin36 (Cx36) knockout mice; however, some neuron pairs remained coupled. This implies that electrical synapses between the majority of coupled VBN neurons require Cx36 but that other gap junction proteins also contribute. The anatomical distribution of a ,-galactosidase reporter indicated that Cx36 was expressed in some VBN neurons during the first postnatal week and sharply declined over the second week, consistent with our physiological results. VBN relay neurons also communicated via chemical synapses. Rare pairs of relay neurons excited one another monosynaptically. Much more commonly, spikes in one relay neuron evoked disynaptic inhibition (via the thalamic reticular nucleus) in the same or a neighbouring relay neuron. Disynaptic inhibition between VBN cells emerged as electrical coupling was decreasing, during the second postnatal week. Our results demonstrate that thalamic relay neurons communicate primarily via electrical synapses during early postnatal development, and then lose their electrical coupling as a chemical synapse-mediated inhibitory circuit matures. [source] | ||||||||||||||||||||||||||||||||||