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Connexin39 (Cx39) and connexin43 (Cx43) are known to be expressed during development of skeletal muscles. Here we have compared the expression pattern of both connexins during differentiation of established C2C12 mouse myoblasts and in the mouse embryo. Cx43 is highly abundant in undifferentiated myoblasts, but no Cx39 protein was detected in these cells. Upon differentiation into myotubes, Cx39 expression increased. The consecutive expression of these connexins was also observed in the mouse embryo. Cx39 and Cx43 were found in different plaques in accordance with the notion that Cx43 is exclusively expressed in myoblasts and Cx39 in myotubes. Thus, differentiating C2C12 cells in culture can serve to study the involvement of gap junctions in myogenesis, since expression of corresponding Cx39 and Cx43 proteins appears to be very similar as in the mouse embryo. 相似文献
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Rosy Joshi-Mukherjee Wanda Coombs Christine Burrer Isabel Alvarez de Mora Mario Delmar Steven M. Taffet 《Cell communication & adhesion》2007,14(2):75-84
Migration of the gap junction protein connexin 43 (Cx43) in SDS-PAGE yields 2 to 4 distinct bands, detectable in the 40-47 kDa range. Here, we show that antibodies against the carboxy-terminal domain of Cx43 recognized an additional 20-kDa product. This protein was detected in some culture cell lysates. The presence of the 20-kDa band was not prevented by the use of protease inhibitors (Complete® and phenylmethylsulfonyl fluoride (PMSF), 1-5 mM). The band was absent from cells treated with Cx43-specific RNAi, and from those derived from Cx43-deficient mice, indicating that this Cx43-immunoreactive protein is a product of the Cx43 gene. Treatment of CHO cells with cyclosporin A caused a reduction in the amount of full-length Cx43 and a concomitant increase in the amount of the 20-kDa band. Overall, our data show that a fraction of the Cx43-immunoreactive protein pool within a given cell may correspond to a C-terminal fragment of the protein. 相似文献
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Kenneth Sims Jr. 《Developmental biology》2009,327(2):410-266
Joints are essential for skeletal form and function, yet their development remains poorly understood. In zebrafish fins, joints form between the bony fin ray segments providing essentially unlimited opportunities to evaluate joint morphogenesis. Mutations in cx43 cause the short segment phenotype of short fin (sofb123) mutants, suggesting that direct cell-cell communication may regulate joint location. Interestingly, increased cx43 expression in the another long fin (alfdty86) mutant appears to cause joint failure typical of that mutant. Indeed, knockdown of cx43 in alfdty86 mutant fins rescues joint formation. Together, these data reveal a correlation between the level of Cx43 expression in the fin ray mesenchyme and the location of joints. Cx43 was also observed laterally in cells associated with developing joints. Confocal microscopy revealed that the Cx43 protein initially surrounds the membranes of ZNS5-positive joint cells, but at later stages becomes polarized toward the underlying Cx43-positive mesenchymal cells. One possibility is that communication between the Cx43-positive mesenchyme and the overlying ZNS5-positive cells regulates joint location, and upregulation of Cx43 in joint-forming cells contributes to joint morphogenesis. 相似文献
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《Cell communication & adhesion》2013,20(4-6):259-264
Osteocytes embedded in the matrix of bone are mechanosensory cells that translate strain into signals and regulate bone remodeling. Our previous studies using osteocyte-like MLO-Y4 cells have shown that fluid flow shear stress (FFSS) increases connexin (Cx) 43 protein expression, prostaglandin E2(PGE2) release, and intercellular coupling, and PGE2is an essential mediator between FFSS and gap junctions. However, the role of Cx43 in the release of PGE2in response to FFSS is unknown. Here, the FFSS-loaded MLO-Y4 cells with no or few intercellular channels released significantly more PGE2per cell than those cells at higher densities. Antisense Cx43 oligonucleotides and 18 β-glycyrrhetinic acid, a specific gap junction and hemichannel blocker, significantly reduced PGE2release by FFSS at all cell densities tested, especially cells at the lowest density without gap junctions. FFSS, fluid flow-conditioned medium, and PGE2increased the activity of dye uptake. Moreover, FFSS induced Cx43 to migrate to the surface of the cell; this surface expressed Cx43 developed resistance to Triton-X-100 solublization. Our results suggest that hemichannels formed by Cx43, instead of intercellular channels, are likely to play a predominant role in the release of intracellular PGE2in response to FFSS. 相似文献
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Rüttinger C Bergmann M Fink L Pesch S Seitz K Trautmann A Steger K Konrad L Brehm R 《Histochemistry and cell biology》2008,130(3):537-548
In human testis, gap junctions containing connexin(Cx)43 are located within the seminiferous epithelium between Sertoli cells and between Sertoli and germ cells. Cx43 is known to play a role in the differentiation and proliferation of these cell types. It can further be associated with human seminoma development. The dog has been proposed as a model for studies of the male reproductive system, because of the frequent occurrence of testicular neoplasms. Thus, we investigated Cx43-mRNA and -protein expression in testes of normal prepubertal dogs, adult dogs, and in canine testicular tumors. Sertoli cells in prepubertal cords express Cx43 mRNA, but do synthesize only less Cx43 protein. Within the seminiferous tubules, Cx43 mRNA was detected in Sertoli cells, spermatogonia, and spermatocytes. Cx43 protein was mainly present in the basal compartment. In canine testicular tumors Cx43 mRNA was detectable in both seminoma and neoplastic Sertoli cells, whereas Cx43 protein was only found in neoplastic Sertoli cells. Our data indicate that Cx43 is regulated differentially in testicular tumors and that alterations of Cx43 expression may be involved in the pathogenesis of canine testicular malignancies. This study represents the first morphological work on the spatiotemporal expression pattern of Cx43 in normal and neoplastic canine testis. 相似文献
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《Cell communication & adhesion》2013,20(4-6):201-205
Connexin-43(Cx43)-based gap junctional communication is transiently inhibited by certain G protein-coupled receptor agonists, including lysophosphatidic acid, endothelin and thrombin. Our previous studies have implicated the c-Src protein tyrosine kinase in mediating closure of Cx43 based gap junctions. Pervanadate, an inhibitor of protein tyrosine phosphatases, mimics activated Src in inhibiting Cx43 gap junctional communication, apparently by promoting tyrosine phosphorylation of the Cx43 C-terminal tail. However, the identity of the protein tyrosine phosphatase(s) that may normally prevent Src-induced gap junction closure is unknown. Receptor-like protein tyrosine phosphatases that mediate homotypic cell-cell interaction are attractive candidates. Here we show that receptor protein tyrosine phosphatase μ (RPTPμ) interacts with Cx43 in diverse cell systems. We find that the first catalytic domain of RPTPμ binds to Cx43. Our results support a model in which RPTPμ, or a closely related protein tyrosine phosphatase, interacts with the regulatory C-terminal tail of Cx43 to prevent Src-mediated closure of Cx43 gap junctional channels. 相似文献
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Ben N. G. Giepmans Elles Feiken Martijn F. B. G. Gebbink Wouter H. Moolenaar 《Cell communication & adhesion》2003,10(4):201-205
Connexin-43(Cx43)-based gap junctional communication is transiently inhibited by certain G protein-coupled receptor agonists, including lysophosphatidic acid, endothelin and thrombin. Our previous studies have implicated the c-Src protein tyrosine kinase in mediating closure of Cx43 based gap junctions. Pervanadate, an inhibitor of protein tyrosine phosphatases, mimics activated Src in inhibiting Cx43 gap junctional communication, apparently by promoting tyrosine phosphorylation of the Cx43 C-terminal tail. However, the identity of the protein tyrosine phosphatase(s) that may normally prevent Src-induced gap junction closure is unknown. Receptor-like protein tyrosine phosphatases that mediate homotypic cell-cell interaction are attractive candidates. Here we show that receptor protein tyrosine phosphatase μ (RPTPμ) interacts with Cx43 in diverse cell systems. We find that the first catalytic domain of RPTPμ binds to Cx43. Our results support a model in which RPTPμ, or a closely related protein tyrosine phosphatase, interacts with the regulatory C-terminal tail of Cx43 to prevent Src-mediated closure of Cx43 gap junctional channels. 相似文献
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Hoptak-Solga AD Nielsen S Jain I Thummel R Hyde DR Iovine MK 《Developmental biology》2008,317(2):541-548
In zebrafish, mutations in the gap junction gene connexin43 lead to short bony fin ray segments that give rise to the short fin phenotype. The sofb123 mutant exhibits fins that are half the length of wild-type fins and have reduced levels of cx43 mRNA. We find that sofb123 regenerating fins exhibit reduced levels of cell proliferation. Interestingly, the number of dividing cells per unit length of fin growth is similar between wild-type and mutant fins, suggesting that the number of cells that enter the cell cycle is specifically affected in sofb123. Expression of cx43 is identified in mitotic cells, which further suggests that Cx43 may contribute to establishing or maintaining the population of dividing cells. Indeed, missense alleles exhibiting high or low levels of gap junctional communication reveal a correlation between defects in direct cell-cell communication, cell proliferation, and segment length. Finally, targeted gene knockdown of cx43 in adult regenerating fins recapitulates the sofb123 phenotype, revealing that the loss of Cx43 is sufficient to reduce both cell proliferation and segment length. We hypothesize that the level of gap junctional intercellular communication among dividing cells regulates the level of cell proliferation and ultimately regulates bone growth. 相似文献
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Kiec-Wilk B Czech U Janczarska K Knapp A Goralska J Cialowicz U Malecki MT Dembinska-Kiec A 《Genes & nutrition》2012,7(2):257-263
Changes in the inner mitochondrial membrane potential (∆ψ) may lead either to apoptosis or to protective autophagy. Connexin 43 (Cx43), a gap junction protein, is suggested to affect mitochondrial membrane permeability. The aim of our study was to analyze Cx43 gene expression, Cx43 protein localization and mitochondrial function in the human endothelial cells stressed by dietary-free fatty acids (FFA) and TNFα. Human endothelial cells (HUVECs) were incubated with (10–30 uM) palmitic (PA), oleic (OA), eicosapentaenoic (EPA) or arachidonic (AA) acids for 24 h. TNFα (5 ng/ml) was added at the last 4 h of incubation. The Cx43 gene expression was analyzed by the quantitative real-time PCR. The Cx43 protein concentrations in whole cells and in the isolated mitochondria were measured. Changes in ∆ψ and Cx43 localization were analyzed by flow cytometry or fluorescence microscopy. Generated ATP was measured by a luminescence assay. TNFα, PA and OA significantly decreased ∆ψ, while AA (P = 0.047) and EPA (P = 0.004) increased ∆ψ value. Preincubation with EPA or AA partially prevented the TNFα-induced decrease of ∆ψ. Incubation with AA resulted in up-regulation of the Cx43 gene expression. AA or PA significantly increased Cx43 protein content; however, presence of TNFα in general aggravated the negative effect of FFA. Only EPA was found to increase ATP generation in HUVECs. The fatty acid-specific induction of changes in Cx43 expression and protein concentration as well as the normalization of ∆ψ and increase of ATP generation seem to be the separate, independent mechanisms of FFA-mediated modulatory effect in the human endothelial cells pathology. 相似文献
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Jelica Gluhak-Heinrich Sumin Gu Dubravko Pavlin Jean X. Jiang 《Cell communication & adhesion》2006,13(1):115-125
Bone osteoblasts and osteocytes express large amounts of connexin (Cx) 43, the component of gap junctions and hemichannels. Previous studies have shown that these channels play important roles in regulating biological functions in response to mechanical loading. Here, we characterized the distribution of mRNA and protein of Cx43 in mechanical loading model of tooth movement. The locations of bone formation and resorption have been well defined in this model, which provides unique experimental systems for better understanding of potential roles of Cx43 in bone formation and remodeling under mechanical stimulation. We found that mechanical loading increased Cx43 mRNA expression in osteoblasts and bone lining cells, but not in osteocytes, at both formation and resorption sites. Cx43 protein, however, increased in both osteoblasts and osteocytes in response to loading. Interestingly, the upregulation of Cx43 protein by loading was even more pronounced in osteocytes compared to other bone cells, with an appearance of punctate staining on the cell body and dendritic process. Cx45 was reported to be expressed in several bone cell lines, but here we did not detect the Cx45 protein in the alveolar bone cells. These results further suggest the potential involvement of Cx43-forming gap junctions and hemichannels in the process of mechanically induced bone formation and resorption. 相似文献
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《Cell communication & adhesion》2013,20(4-6):445-450
Genetic deficiency of Cx43 in vivo causes skeletal developmental defects, osteoblast dysfunction and perinatal lethality. To determine the role of Cx43 in the adult skeleton, we developed two models of osteoblast-specific Cx43 gene deletion using Cre mediated replacement of a “floxed” Cx43 allele with a LacZ reporter gene. Cre recombinase expression in osteoblasts was driven by either the osteocalcin OG2 promoter or the 2.3 kb fragment of the Colα1(I) promoter. Homozygous Cx43fl/flmice, in which the Cx43 coding region is flanked by two loxP sites, were crossed with Cre expressing mice in a heterozygous Cx43-null background [Cx43±; Colα1(I)-Cre or Cx43±; OG2-Cre]. Cx43 gene ablation was demonstrated in tissues by selective X-gal staining of cells lining the endosteal surface, and in cultured osteoblastic cells from calvaria using different approaches. Although no LacZ expression was observed in proliferating calvaria cells, before osteoblast differentiation begins, post-proliferative cells isolated from conditional knockout mice [Cx43fl/?; Colα1(I)-Cre or Cx43fl/?; OG2-Cre] developed strong LacZ expression as they differentiated, in parallel to a progressive disappearance of Cx43 mRNA and protein abundance relative to controls. Selective Cre mediated Cx43 gene inactivation in bone forming cells will be useful to determine the role of Cx43 in adult skeletal homeostasis and overcome the perinatal lethality of the conventional null model. 相似文献
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《Cell communication & adhesion》2013,20(2):23-33
AbstractpH-mediated gating of Cx43 channels following an ischemic event is believed to contribute to the development of lethal cardiac arrhythmias. Studies using a soluble version of the Cx43 carboxyl-terminal domain (Cx43CT; S255–I382) have established the central role it plays in channel regulation; however, research in the authors' laboratory suggests that this construct may not be the ideal model system. Therefore, we have developed a more ‘native-like’ construct (Cx43CT attached to the 4th transmembrane domain [TM4-Cx43CT; G178–I382]) than the soluble Cx43CT to further investigate the mechanism(s) governing this regulation. Here, we utilize circular dichroism and nuclear magnetic resonance (NMR) were used to validate the TM4-Cx43CT for studying channel gating and optimize solution conditions for structural studies. The data indicate that, unlike the soluble Cx43CT, the TM4-Cx43CT is structurally responsive to changes in pH, suggesting the presence of the TM4 facilitates pH-induced structural alterations. Additionally, the optimal solution conditions for solving the NMR solution structure include 10% 2,2,2 trifluoroethanol and removal of the 2nd extracellular loop (G178-V196). 相似文献
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连接子蛋43(connexin 43,Cx43)是骨组织中主要的间隙连接(gap junction)蛋白和半通道(hemichannel)蛋白,由Cx43形成的间隙连接及半通道实现了骨组织细胞间的直接通讯。连接子蛋白对骨组织的正常发育、骨重建过程的建立与平衡是非常重要的。目前研究指出,Cx43不仅参与了骨组织的力学响应过程,也参与了二磷酸盐、甲状旁腺激素等药物对骨重建的调节过程。该文以骨组织细胞内信号传递途径的关键分子Cx43为对象,就其目前的研究现状作一综述。 相似文献
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Kellezi A Grosely R Kieken F Borgstahl GE Sorgen PL 《Protein expression and purification》2008,59(2):215-222
In recent years, reports have identified that many eukaryotic proteins contain disordered regions spanning greater than 30 consecutive residues in length. In particular, a number of these intrinsically disordered regions occur in the cytoplasmic segments of plasma membrane proteins. These intrinsically disordered regions play important roles in cell signaling events, as they are sites for protein–protein interactions and phosphorylation. Unfortunately, in many crystallographic studies of membrane proteins, these domains are removed because they hinder the crystallization process. Therefore, a purification procedure was developed to enable the biophysical and structural characterization of these intrinsically disordered regions while still associated with the lipid environment. The carboxyl terminal domain from the gap junction protein connexin43 attached to the 4th transmembrane domain (TM4-Cx43CT) was used as a model system (residues G178-I382). The purification was optimized for structural analysis by nuclear magnetic resonance (NMR) because this method is well suited for small membrane proteins and proteins that lack a well-structured three-dimensional fold. The TM4-Cx43CT was purified to homogeneity with a yield of 6 mg/L from C41(DE3) bacterial cells, reconstituted in the anionic detergent 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)], and analyzed by circular dichroism and NMR to demonstrate that the TM4-Cx43CT was properly folded into a functional conformation by its ability to form α-helical structure and associate with a known binding partner, the c-Src SH3 domain, respectively. 相似文献
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Smac/DIABLO在过氧化氢所致C2C12肌原细胞凋亡中的作用 总被引:4,自引:0,他引:4
为探讨Smac/DIABLO在过氧化氢(H2O2)所致C2C12肌原细胞凋亡中的作用,采用Hoechst 33258染色,观察H2O2 (0.5 mmol/L)处理C2C12肌原细胞不同时间后,细胞核形态学改变并计算凋亡核百分率,DNA抽提及琼脂糖电泳观察凋亡特征性梯状带,利用细胞成分分离后蛋白质印迹分析H2O2是否导致Smac/DIABLO从线粒体释放,采用Caspase检测试剂盒及蛋白质印迹分析Caspase-3和Caspase-9的活化,转染Smac/DIABLO基因,观察Smac/DIABLO过表达对H2O2所致的C2C12肌原细胞凋亡的影响.结果表明:H2O2处理1 h后,Smac/DIABLO从C2C12肌原细胞线粒体释放入胞浆,2 h更明显;H2O2处理4 h后,Caspase-3和Caspase-9活化,12 h达高峰;H2O2处理24 h后,C2C12肌原细胞显示特征性的凋亡形态改变,凋亡核百分率明显升高,DNA电泳出现明显“梯状”条带.与单纯过氧化氢损伤组相比,Smac/DIABLO高表达的C2C12肌原细胞经过氧化氢损伤组的Caspase-3和Caspase-9的活化、凋亡核百分率的升高、“梯状”条带的出现均更明显.结果表明,H2O2可导致Smac/DIABLO从C2C12肌原细胞线粒体释放,促进Caspase-9和Caspase-3的活化而促进细胞凋亡的发生. 相似文献
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