Formation of platelet plug initiates hemostasis at sites of vascular injury, and triggers pathological thrombosis in ischemic tissue disease. Although various crucial molecules for platelet function have been identified in recent years, the mechanisms of inter- and intra-cellular signaling leading to the formation of a stable platelet plug is still poorly understood. Connexins form gap junctions, clusters of intercellular channels that are known to synchronize responses in multi-cellular organisms through the direct exchange of ions, small metabolites and other second messenger molecules between adjacent cells. Here, we report the expression of the gap junction protein connexin37 (Cx37) in mouse and human platelets. In addition, we observed functional gap junction communication between platelets during platelet aggregation in vitro, as assessed by microinjection of the gap junction-permeable tracer neurobiotin in platelets isolated from human or wild-type mice. In contrast, the tracer did not ...
Epithelial cells of the thyroid gland present an uncommon connexin expression pattern, they coexpress connexin32 and connexin43. In the present work, we have analyzed the membrane distribution of these two connexins to determine: (i) whether they co-assemble in the same gap junctions or form separate gap junctions; and (ii) whether their location is somehow related to the thyroid cell polarity. Immunofluorescence analyses of the localization of the two connexins in thyroid tissue sections revealed that connexin32 and connexin43 are located in different regions of the plasma membrane. We further analyzed the location of each of the two connexins with regard to that of the tight junction-associated protein, ZO1. Laser scanning confocal microscope observations of connexin32 or connexin43 and ZO1 double-immunolabelled thyroid cells, gave evidence for a separate localization of gap junctions made of each of these two connexins. Connexin32 gap junctions appeared as fluorescent spots scattered over the ...
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The molecular changes associated with the transition of melanoma cells from radial growth phase (RGP) to vertical growth phase (VGP) and the metastatic phenotype are not very well defined. However, some of the genes involved in this process and their transcriptional regulation are beginning to be elucidated. For example, the switch from RGP to VGP and the metastatic phenotype is associated with loss of the AP-2α transcription factor. AP-2α regulates the expression of c-KIT, MMP-2, VEGF, and the adhesion molecule MCAM/MUC18. Recently, we reported that AP-2α also regulates two G-protein coupled receptors (GPCRs) PAR-1 and PAFR. In turn, the thrombin receptor, PAR-1, regulates the expression of the gap junction protein Connexin-43 and the tumor suppressor gene Maspin. Activation of PAR-1 also leads to overexpression and secretion of proangiogenic factors such as IL-8, uPA, VEGF, PDGF, as well certain integrins. PAR-1 also cooperates with PAFR to regulate the expression of the MCAM/MUC18 via ...
Carriers of the troponin T (TnT) I79N mutation linked to familial hypertrophic cardiomyopathy often die of sudden cardiac death at a young age. Mouse hearts expressing TnT-I79N are susceptible to reentrant ventricular tachycardia even without exhibiting interstitial fibrosis or hypertrophy. We hypothesized that slowed conduction due to altered regulation of the gap junction protein connexin 43 (C×43) contributes to the increased arrhythmia risk.. Methods: Optical activation maps (left ventricle stimulated at 10 Hz in the center of the field of view) were generated from isolated perfused control (n=20) and TnT-I79N (n=9) transgenic hearts. Fast (longitudinal) and slow (lateral) conduction velocity (CV) was calculated by plotting local CVs against orientation. After optical mapping, hearts were flash frozen and Cx43 phosphorylation analyzed by western blot. Phosphorylated Cx43 (P1, P2) migrated slower in SDS page and at least three distinct bands could be separated (P0, P1, P2).. Results: Lateral ...
Cardiomyocytes are connected by mechanical and electrical junctions located at the intercalated discs (IDs). Although the ID and its components have long been known, it is becoming increasingly clear that ID proteins interact. The lecture describes the involvement of the ID in cardiac electrical disturbances and focuses on the channel and non-channel functions of the gap junctional protein connexin 43 (Cx43).. Ventricular cardiomyocytes are exclusively coupled by gap junctions composed of Cx43 that allow for coordinated spread of action potentials across the myocardium. During cardiac ischemia, gap junctions uncouple due to a combination of increased calcium, acidosis and Cx43 dephosphorylation, and this uncoupling increases the risk of cardiac arrhythmia. We identified several phosphorylation sites that are regulated during ischemia and one of these, serine 306 (S306), determines the ability of calcium to uncouple Cx43 gap junctions. We hypothesized that S306 phosphorylation is cardioprotective ...
Cell-to-cell channels composed of connexin44 and connexin50 were purified from plasma membranes of calf and fetal bovine lenses. The channels were treated with the nonionic detergents octyl-beta-D-glucopyranoside and decyl-beta-D-maltopyranoside, and the channel/detergent complexes purified by ion and gel filtration column chromatography. In negative staining, the channels appeared as annuli 11 +/- 0.6 nm (s.d., n = 105) in diameter and as 16 +/- 0.8 nm (s.d., n = 96) long particles which corresponded to top and side views of complete cell-to-cell channels. The purified cell-to-cell channels were composed principally of a protein, called MP70, that appeared as a diffuse 55-75 kDa band in SDS-PAGE. Dephosphorylation with alkaline phosphatase transformed the diffuse 55-75 kDa band into two distinct bands of almost equal intensity. Immunoblotting showed the bands to be connexin44 and connexin50, respectively. The antibodies also recognized weaker bands composed of the unphosphorylated form of ...
Recent work has shown that the gap junction protein connexin43 (Cx43) is upregulated in cells of the joint during osteoarthritis (OA). Here we examined if the OA-associated increase in Cx43 expression impacts the function of synovial fibroblasts by contributing to the production of catabolic and inflammatory factors that exacerbate joint destruction in arthritic disease. Using rabbit and human synovial fibroblast cell lines, we examined the effects of Cx43 overexpression and Cx43 siRNA-mediated knockdown on the gene expression of OA-associated matrix metalloproteinases (MMP1 and MMP13), aggrecanases (ADAMTS4 and ADAMTS5), and inflammatory factors (IL1, IL6 and PTGS2) by quantitative real time RT-PCR. We examined collagenase activity in conditioned media of cultured synovial cells following Cx43 overexpression. Lastly, we assessed the interplay between Cx43 and the NFκB cascade by western blotting and gene expression studies. Increasing Cx43 expression enhanced the gene expression of MMP1, MMP13,
Several studies have described abnormalities in the expression, distribution, and regulation of ventricular connexins in CHF. Absolute Cx43 expression is generally reduced in CHF ventricles,9,20,23-25 likely related to the activation of the mitogen-activated protein kinase c-Jun N-terminal kinase.26 Recent work suggests an important role for defects in Cx43 phosphorylation in CHF-induced ventricular cardiomyocyte uncoupling,9,10,20 thought to be attributable to increased dephosphorylating activity of protein phosphatase-2A colocalized with Cx43.20 Connexin dephosphorylation plays significant roles in targeting connexins to intercalated disks and in regulating connexin conductance.8,10 One study showed ventricular Cx40 upregulation in CHF,23 possibly as a compensation for Cx43 downregulation; however, the functional importance of this alteration is uncertain in view of low level ventricular Cx40 expression.. Clinical and experimental studies of gap junctional remodeling in the atria have produced ...
PURPOSE: To study in both in situ and primary cultures the posttranslational phosphorylation of connexin46 (Cx46), one of two members of the connexin family of gap junction proteins expressed by lens fibers. METHODS: Phosphatase digestion, gel electrophoresis, cell culture, organ culture, immunoprecipitation, metabolic labeling, and phosphoamino acid analysis were the methods used in this study. RESULTS: Cx46 immunoprecipitated from either rat or bovine lenses resulted in a shift to a more rapidly migrating species. During rat embryonic development, the more rapidly migrating, nonphosphorylated form of Cx46 was prevalent at 15 days gestation; as development progressed, there was a loss of the nonphosphorylated form with a concomitant increase in the phosphorylated form, such that by 28 days after birth only the phosphorylated form was detectable. The rate of posttranslational phosphorylation was very slow compared to previously measured rates for connexin43. Primary cultures of rat embryonic ...
SKKUs research team led by Prof. Jong Sun KANG of the Dept. of Medicine demonstrated a role of a cell surface receptor Cdon in preventing cardiac remodeling through suppression of Wnt signaling. Cdon is expressed and predominantly localized at intercalated disk in both mice and human hearts. Cdon−/− mice develop cardiac dysfunction and fibrosis with altered expression of remodeling genes. Cdon deficiency causes aberrant localization and function of gap junction protein connexin 43, correlating with hyperactivated Wnt signaling. Blocking of Wnt signaling in Cdon-depleted cardiomyocytes attenuates aberrant intercellular coupling. Conversely, Wnt activator causes aberrant activation of gap junction with decreased Cdon levels, suggestive of a feedback mechanism. This data suggests that Cdon is required for the control of Wnt signaling to prevent cardiac remodeling.. The research, supported by the Ministry of Science, ICT and Future Planning, as a part of a project supporting the cultivation of ...
Gap junctions are membrane specialization domains identified in most tissue types where cells abut each other. The connexin channels found in these membrane domains are conduits for direct cell-to-cell transfer of ions and molecules. Connexin43 (Cx43) is the most ubiquitous connexin, with critical roles in heart, skin, and brain. Several studies described the interaction between Cx43 and the cytoskeleton involving the actin binding proteins Zonula occludens (ZO-1) and drebrin, as well as with tubulin. However, a direct interaction has not been identified between drebrin and Cx43. In this study, co-IP and NMR experiments were used to demonstrate that the Cx43-CT directly interacts with the highly conserved N-terminus region of drebrin. Three Cx43-CT areas were found to be involved in drebrin binding, with residues 264-275 being critical for the interaction. Mimicking Src phosphorylation within this region (Y265) significantly disrupted the interaction between the Cx43-CT and drebrin. Immunofluorescence
The principal findings of the present study may be summarized as follows: Cx43, classically known as the "cardiac connexin," and Cx45 are abundantly present in the atrial and ventricular myocardium of the adult rabbit. This can be concluded from immunostaining of cardiac tissue and isolated myocytes. In addition, atrial myocytes also express Cx40. This distribution is similar to that in humans, pigs, cows, and guinea pigs.18 19 Cx37 and Cx40, which have been shown to be expressed by endothelial cells,14 21 are present in both endothelial and endocardial cells in the rabbit heart. It may be somewhat surprising that connexin distribution in the rabbit can be assessed with antibodies raised in the rabbit. Apparently, Langendorff perfusion before fixation caused a sufficient washout of immunoglobulins to which an anti-rabbit secondary antibody would have bound. Nevertheless, background fluorescence in experiments with rabbit primary antibodies was higher than in experiments with mouse monoclonal ...
Yancey, S. Barbara and Biswal, Sandip and Revel, Jean-Paul (1992) Spatial and temporal patterns of distribution of the gap junction protein connexin43 during mouse gastrulation and organogenesis. Development, 114 (1). pp. 203-212. ISSN 0950-1991. http://resolver.caltech.edu/CaltechAUTHORS:YANdev92 ...
Background Gap junction communication has been shown in glial and neuronal cells and it is thought they mediate inter- and intra-cellular communication. Connexin 36 (Cx36) is expressed extensively in the developing brain, with levels peaking at P14 after which its levels fall and its expression becomes entirely neuronal. These and other data have led to the hypothesis that Cx36 may direct neuronal coupling and neurogenesis during development. Methodology/Principal Findings To investigate Cx36 function we used a neurosphere model of neuronal cell development and developed lentiviral Cx36 knockdown and overexpression strategies. Cx36 knockdown was confirmed by western blotting, immunocytochemistry and functionally by fluorescence recovery after photobleaching (FRAP). We found that knockdown of Cx36 in neurosphere neuronal precursors significantly reduced neuronal coupling and the number of differentiated neurons. Correspondingly, the lentiviral mediated overexpression of Cx36 significantly increased the
Chronic wounds are not only debilitating to patients, but also impose a huge financial burden on healthcare providers, as current treatments are not particularly effective. Wound healing is a highly co-ordinated process involving a vast array of signalling molecules and different cell types, therefore a substantial amount of research has been carried out in the quest to develop new therapies. The gap junction (GJ) protein connexin43 (Cx43) is one of the many molecules whose expression has been found to be up-regulated in chronic wounds and as a result targeting it may have therapeutic potential. Two different approaches have been adopted to investigate this: knockdown of Cx43 using antisense oligonucleotides and connexin mimetic peptides (CMPs) which inhibit the function of Cx43 without affecting gene expression. These peptides are targeted to the C-terminal domain or the extracellular loops of Cx43 and thus are likely to function by different means. However, both block channel function and have ...
(2016) Basheer, Shaw. Biochimica et Biophysica Acta - Molecular Cell Research. With each heartbeat, Connexin43 (Cx43) cell-cell communication gap junctions are needed to rapidly spread and coordinate excitation signals for an effective heart contraction. The correct formation and delivery of chan...
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, Connexin 50 / GJA8 blocking peptide, GTX88154-PEP, Applications: Apuri, Blocking, ELISA; Affinity purification, Blocking, ELISA; CrossReactivity: Human|Mouse|Bovine|Dog|Rat
Connexin 26 antibody, C-term (gap junction protein, beta 2, 26kDa) for WB. Anti-Connexin 26 pAb (GTX89128) is tested in Mouse samples. 100% Ab-Assurance.
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Receptor activator of NF-kappaB ligand (RANKL) is crucial in osteoclastogenesis but signaling events involved in osteoclast differentiation are far from complete and other signals may play a role in osteoclastogenesis. A more direct pathway for cellular crosstalk is provided by gap junction intercellular channel, which allows adjacent cells to exchange second messengers, ions, and cellular metabolites. Here we have investigated the role of gap junction communication in osteoclastogenesis in mouse bone marrow cultures. Immunoreactive sites for the gap junction protein connexin 43 (Cx43) were detected in the marrow stromal cells and in mature osteoclasts. Carbenoxolone (CBX) functionally blocked gap junction communication as demonstrated by a scrape loading Lucifer Yellow dye transfer technique. CBX caused a dose-dependent inhibition (significant , or = 90 microM) of the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells formed in 7- to 8-day marrow cultures ...
Reversible down-regulation of gap junctional intercellular communication (GJIC) is proposed to be an important cellular mechanism in tumor promotion. Gap junction function is modified by a variety of tumor promoters, including the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Treatment of cells with TPA results in the activation and subsequent depletion of the TPA-responsive protein kinase C (PKC) isoforms. TPA-induced degradation of the PKC isoforms α, δ and ϵ was recently shown to occur via the ubiquitin-proteasome pathway. In the present study we investigated the role of the proteasome in the TPA-induced modification of GJIC in IAR20 rat liver epithelial cells. TPA exposure of IAR20 cells induced hyperphosphorylation of gap junction protein connexin43 and inhibition of GJIC. Prolonged TPA treatment induced down-regulation of PKCα, δ and ϵ and a reduction in the total PKC activity, which was associated with recovery of GJIC. Co-treatment of IAR20 cells with TPA and the ...
Upregulation of gap junctional intercellular communication and connexin 43 expression by cyclic-AMP and all-trans-retinoic acid is associated with glutathione depletion and chemosensitivity in neuroblastoma cells.
Introduction: Metastasis involves the emigration of tumor cells through the vascular endothelium, a process also known as diapedesis. The molecular mechanisms regulating tumor cell diapedesis are poorly understood, but may involve heterocellular gap junctional intercellular communication (GJIC) between tumor cells and endothelial cells. Method: To test this hypothesis we expressed connexin 43 (Cx43) in GJIC-deficient mammary epithelial tumor cells (HBL100) and examined their ability to form gap junctions, establish heterocellular GJIC and migrate through monolayers of human microvascular endothelial cells (HMVEC) grown on matrigel-coated coverslips. Results: HBL100 cells expressing Cx43 formed functional heterocellular gap junctions with HMVEC monolayers within 30 minutes. In addition, immunocytochemistry revealed Cx43 localized to contact sites between Cx43 expressing tumor cells and endothelial cells. Quantitative analysis of diapedesis revealed a two-fold increase in diapedesis of Cx43 expressing
TY - JOUR. T1 - Glycosaminoglycans and proteoglycans induce gap junction expression and restore transcription of tissue‐specific mRNAs in primary liver cultures. AU - Fujita, Michiyasu. AU - Spray, David C.. AU - Choi, Haing. AU - Saez, Juan C.. AU - Watanabe, Tohru. AU - Rosenberg, Larry C.. AU - Hertzberg, Elliott L.. AU - Reid, Lola M.. PY - 1987/1/1. Y1 - 1987/1/1. N2 - Normal rat hepatocytes maintained on tissue culture plastic and in serum‐supplemented medium lose their gap junctions within 12 hr and expression of their tissue‐specific functions within 24 to 72 hr. The gap junctions are lost via internalization and degradation, and the differentiated functions due to loss of synthesis and to rapid degradation of tissue‐specific mRNAs. Near normal levels of tissue‐specific mRNAs can be achieved by stabilization of the mRNAs but not by transcription (for most genes), if the cells are cultured in a serum‐free, hormonally defined medium and on substrata of tissue culture plastic, ...
Intercellular communication through gap junctions is crucial for proper functioning of the inner ear. Indeed, mutations in several connexin genes have been found to cause hearing loss. In the inner ear, only the cell distributions of connexin30 and connexin26 have been well documented. We took advantage of the lacZ reporter gene in Cx43 and Cx45 knock-out mice to study the expression of the connexin43 and connexin45 genes during the inner ear development. Expression of Cx43 and Cx45 in the inner ear was detected from embryonic days 15.5 and 17.5, respectively. Until the 1st week of life, Cx43 was highly expressed in the connective tissues, and weakly expressed in the immature sensory epithelium of the cochlea. From postnatal day 8, however, Cx43 was almost exclusively expressed in the bone of the otic capsule. During embryogenesis, Cx45 was expressed in epithelial and connective inner ear tissues. From birth onwards, Cx45 expression could be detected in some inner ear capillaries. Vascular expression
TY - JOUR. T1 - Regulation of connexin hemichannels by monovalent cations. AU - Srinivas, Miduturu. AU - Calderon, D. Paola. AU - Kronengold, Jack. AU - Verselis, Vytautas. PY - 2006/1. Y1 - 2006/1. N2 - Opening of connexin hemichannels in the plasma membrane is highly regulated. Generally, depolarization and reduced extracellular Ca2+ promote hemichannel opening. Here we show that hemichannels formed of Cx50, a principal lens connexin, exhibit a novel form of regulation characterized by extraordinary sensitivity to extracellular monovalent cations. Replacement of extracellular Na+ with K+, while maintaining extracellular Ca2+ constant, resulted in ,10-fold potentiation of Cx50 hemichannel currents, which reversed upon returning to Na+. External Cs+, Rb+, NH4+, but not Li +, choline, or TEA, exhibited a similar effect. The magnitude of potentiation of Cx50 hemichannel currents depended on the concentration of extracellular Ca2+, progressively decreasing as external Ca 2+ was reduced. The primary ...
Gap junctions are specialized cell-cell contacts that provide direct intercellular communication between eukaryotic cells. The tyrosine-sorting signal (YXXØ), present at amino acids 286-289 of Cx43 (connexin43), has been implicated in the internalization of the protein. In recent years, ubiquitination of Cx43 has also been proposed to regulate gap junction intercellular communication; however, the underlying mechanism and molecular players involved remain elusive. In the present study, we demonstrate that ubiquitinated Cx43 is internalized through a mechanism that is independent of the YXXØ signal. Indeed, expression of a Cx43-Ub (ubiquitin) chimaera was shown to drive the internalization of a mutant Cx43 in which the YXXØ motif was eliminated. Immunofluorescence, cycloheximide-chase and cell-surface-protein biotinylation experiments demonstrate that oligomerization of Cx43-Ub into hemichannels containing wild-type Cx43 or mutant Cx43Y286A is sufficient to drive the internalization of the ...
Diabetic retinopathy (DR) develops due to hyperglycemia and inflammation-induced vascular disruptions in the retina with connexin43 expression patterns in the disease still debated. Here, the effects of hyperglycemia and inflammation on connexin43 expression in vitro in a mouse model of DR and in human donor tissues were evaluated. Primary human retinal microvascular endothelial cells (hRMECs) were exposed to high glucose (HG; 25 mM) or pro-inflammatory cytokines IL-1β and TNF-α (10 ng/mL each) or both before assessing connexin43 expression. Additionally, connexin43, glial fibrillary acidic protein (GFAP), and plasmalemma vesicular associated protein (PLVAP) were labeled in wild-type (C57BL/6), Akita (diabetic), and Akimba (DR) mouse retinas. Finally, connexin43 and GFAP expression in donor retinas with confirmed DR was compared to age-matched controls. Co-application of HG and cytokines increased connexin43 expression in hRMECs in line with results seen in mice, with no significant difference in
Several laboratories have demonstrated a decrease in gap junctional communication in cells transformed by the src oncogene of the Rous sarcoma virus. The decrease In gap junctional communication was associated with tyrosine phosphorylation of the gap junction protein, connexin43 (Cx43). This study was initiated to determine if the phosphorylation of Cx43 is the result of a direct kinase-substrate interaction between the highly active tyrosine kinase, pp60v-src, and Cx43. Confocal microscopy data indicates that the two proteins are within physical proximity allowing for a potential kinase-substrate interaction. Previous biochemical studies have been limited by the low levels of Cx43 protein in fibroblast cell lines. To obtain larger quantities of Cx43 we constructed a recombinant baculovirus expressing Cx43 in Spodoptera frugiperda (Sf-9) cells and subsequently purified the expressed Cx43 by immunoaffinity chromatography. We observed that this partially-purified Cx43 was phosphorylated on ...
OBJECTIVE To investigate whether ageing and diabetes alter the expression of the gap junction protein connexin43 (Cx43) and of particular purinoceptor (P2R) subtypes in the corpus cavernosum and urinary bladder, and determine whether changes in expression of these proteins correlate with development of erectile and bladder dysfunction in diabetic and ageing rats. MATERIALS AND METHODS Erectile and bladder function of streptozotocin (STZ)-induced diabetic, insulin-treated and age-matched control Fischer-344 rats were evaluated 2, 4 and 8 months after diabetes induction by in vivo cystometry and cavernosometry. Corporal and bladder tissue were then isolated at each of these sample times and protein expression levels of Cx43 and of various P2R subtypes were determined by Western blotting. RESULTS In the corpora of control rats ageing was accompanied by a significant decrease in Cx43 and P2X(1)R, and increase in P2X(7)R expression. There was decreased Cx43 and increased P2Y(4)R expression in the ageing
Connexin 32 Mouse, Alexa Fluor 488, Clone: Connexin32 (2A), eBioscience™ 25μg; Alexa Fluor 488 Connexin 32 Mouse, Alexa Fluor 488, Clone: Connexin32 (2A),...
Looking for online definition of connexin 62 in the Medical Dictionary? connexin 62 explanation free. What is connexin 62? Meaning of connexin 62 medical term. What does connexin 62 mean?
Individual cell-cell channels consist of two hemichannels, located on neighboring cell membranes, that are interconnected to form an hydrophilic pathway. Each hemichannel, or connexon, is made of six protein subunits, called connexins.Connexin32 liver gap junction protein has four transmembrane segments, two extracellular regions and three cytoplasmic segments, which include the amino and carboxyl termini.The process of cell-cell channel formation was investigated by altering specific amino acids in the presumed extracellular domains of the connexin32. It is these domains that must interact when two hemichannels dock to form an open cell-cell channel. The mutant connexins were generated by site-directed in vitro mutagenesis of a connexin32 cDNA. The mutated cDNAs were then transcribed in vitro and the mRNA was injected into Xenopus oocytes for expression. Junctional conductances between paired oocytes resulting from the expression of the mRNA were measured by the double-voltage clamp technique.Every
The pre-Bötzinger complex (pre-BötC) is hypothesized to be the site for respiratory rhythm generation in mammals. Studies examining the cellular mechanisms mediating rhythm generation have focused on the role of chemically mediated synaptic interactions; however, electrotonic synaptic interactions (i.e., electrotonic coupling), which occur by means of gap junctions, may also play a role. Here, we used immunoblot and immunohistochemical analyses to determine whether the pre-BötC contains the gap junction proteins necessary for electrotonic communication and whether the presence and distribution of these gap junction proteins show a developmental change in expression. We found that both connexin26 (Cx26) and connexin32 (Cx32) were expressed in pre-BötC neurons of neonatal and adult rats; however, the relative amounts and their distribution varied by age. Cx26 labeling was seen in a high proportion of pre-BötC neurons in neonatal rats ≤ 7 days postnatal (P7) but declined with increasing age. ...
Purpose: : The purpose of this study is to determine the PKCγ phosphorylation site on Cx50 in lens epithelial cells and subsequent functional results of phosphorylation. Methods: : Mutation (S430A) was introduced into the wild type Cx50:EGFP by site-directed mutagenesis. Wild-type and mutated (S430A) Cx50 were transfected into 80% confluent N/N lens epithelial cells, and stably transfected cells were selected in DMEM media. PKCγ was activated by phorbol-12 myristate 13 acetate (TPA, 200nM). Expression and localization of wild type and mutated Cx50-EGFP fusion proteins before and after TPA treatment were measured by confocal microscopy. Cell surface Cx50 gap junction plaques were immuno-labeled and counted by confocal microscopy. Co-localization of Cx50 and PKCγ was determined by co-immunoprecipitation. Phosphorylation of Cx50-S430 was determined by western blotting with anti-phosphoserine antibodies and functional effects were measured by gap junction plaque assembly-disassembly. Results: : ...
Principal Investigator:TOMOYOSE Taiki, Project Period (FY):2003 - 2004, Research Category:Grant-in-Aid for Scientific Research (C), Section:一般, Research Field:Surgical dentistry
Purpose: Cancer patients are often concurrently treated with analgesics and antineoplastic drugs, yet the influence of analgesic agents on therapeutic activity of antineoplastic drugs is largely unexplored. This study investigates the effects of three commonly used analgesics, which produce analgesia by different mechanisms, on cytotoxicity induced by cisplatin, a widely used antitumor agent, and the relation between those effects and modulation of gap junction function by the analgesics.. Experimental Design: The role of gap junctions in the modulation of cisplatin toxicity is explored by manipulation of connexin expression, and gap junction presence and function, using clinically relevant concentrations of the analgesics and cisplatin.. Results: Short-term exposure of transformed cells to cisplatin reduced the clonogenic survival in low-density cultures (without gap junction formation) and in high density (with gap junction formation), but the toxic effect was greater at high density. In the ...
Gap junction (GJ)*proteins (termed connexins [Cxs]), comprise a family of vertebrate transmembrane proteins that assemble intracellularly to form oligomeric channels (Musil and Goodenough, 1993). The GJ hemichannels, or connexons, are transported to the plasma membrane where they dock with connexons in the membranes of adjacent cells and aggregate to mediate intercellular transfer of various ions, signaling molecules, and metabolites (Simon and Goodenough, 1998). The essential role of GJ communication in the coordination of physiological processes within various tissues has become increasingly apparent, with defects in the different Cx proteins now linked to a wide variety of pathological conditions (Krutovskikh and Yamasaki, 2000).. The GJ protein Cx43 is one of the more ubiquitous of the GJ proteins. Its importance is underscored by the abnormal heart development, perinatal mortality, female sterility, altered bone development, and cataracts observed in Cx43 knockout (KO) mice (Lo, 1999). Its ...
Gap junctions provide direct electrical and biochemical communication between cardiomyocytes in the heart. Connexin40 (Cx40) is the major connexin in the atria of the heart and little is known regarding its regulation. Thus, the goal was to investigate the regulation of Cx40 in both physiological and pathophysiological conditions. The first objective of this thesis was to determine whether Cx40 gap junctions were regulated by â-adrenergic receptor activation. Cx40 has previously been shown to be acutely activated by cAMP, this cAMP-induced increase in Cx40-mediated cell-to-cell dye transfer has been shown to be effected through the â-adrenergic receptor-adenylyl cyclase- Protein Kinase A (PKA) pathway in Cx40-transfected HeLa cells. The second objective of this thesis was to determine whether Cx40 gap junctions were regulated by intracellular Ca2+ concentration ([Ca2+]i ). [Ca2+]i was increased by addition of the ionophore ionomycin and elevating extracellular calcium [Ca2+]o from 1.8 mM to 21.8 mM.
Despite the high number of identified loci for autosomal recessive nonsyndromic HIH, the majority of cases (58-88%) are linked to DFNB1 on the chromosome 13q12 and are due to mutations in the GJB2 gene (MIM 121011), which encodes the gap junction protein connexin 26. The 35delG mutation in the GBJ2 gene is the commonest mutation in Caucasian populations. Its carrier frequency is 1 per 35 in Southern Europe and 1 per 79 in Central and Northern Europe. Mutation analysis of 35delG mutation in the GJB2 gene is available as a genetic diagnostic test. Unlike several forms of congenital deafness, GJB2-related deafness has no known comorbidity. Knowing the mutation status at the outset in a child with hearing impairment will save the time, effort and cost involved in performing different investigations.. ...
A core structural and functional motif of the vertebrate central nervous system is discrete clusters of neurons or nuclei. Yet the developmental mechanisms underlying this fundamental mode of organisation are largely unknown. We have previously shown that the assembly of motor neurons into nuclei depends on cadherin-mediated adhesion. Here, we demonstrate that the emergence of mature topography among motor nuclei involves a novel interplay between spontaneous activity, cadherin expression and gap junction communication. We report that nuclei display spontaneous calcium transients, and that changes in the activity patterns coincide with the course of nucleogenesis. We also find that these activity patterns are disrupted by manipulating cadherin or gap junction expression. Furthermore, inhibition of activity disrupts nucleogenesis, suggesting that activity feeds back to maintain integrity among motor neurons within a nucleus. Our study suggests that a network of interactions between cadherins, ...
The gap junction gene Connexin31.1 has been reported to be expressed predominantly in the epidermis of murine skin. To study the function of this gene, we generated mice in which the coding DNA of the Connexin31.1 gene was replaced by lacZ reporter coding DNA. Using beta-galactosidase staining, we h …
Communication among cells via direct cell-cell contact by connexin gap junctions, or between cell and extracellular environment via pannexin channels or connexin hemichannels, is a key factor in cell...
The ventricular tissues were suspended in 40 ml of ice-cold 10% tricholroacetic acid and homogenized with a tissue homogenizer (2 bursts, 30 s each). Homogenates were centrifuged at 10,000 g for 10 min at 4°C, and protein content was determined using detergent-compatible protein assay (Bio-Rad Laboratories Inc., Hercules, CA) with bovine serum albumin. The proteins were then put in a 3× sample buffer consisting of 0.2 M Tris-HCl (pH 6.8), 4% sodium dodecylsulfate, 8 M urea, 0.1 M dithiothreitol, and 0.01% bromophenol blue. Equal amounts of protein per lane were loaded onto a 15% polyacrylamide gel and separated by electrophoresis at 30 mA/gel for 60 min with a running buffer containing 25 mm Tris, 192 mm glycine, and 0.1% sodium dodecylsulfate. Molecular weight markers (Amersham Biosciences, Buckinghamshire, United Kingdom) were used in each gel. Proteins were transferred to a polyvinylidene diflouride membrane (Immobilon-P; Millipore Corp., Bedford, MA) at 36 V for 4 h using a transfer buffer ...
Buy our Recombinant Human Connexin 37 / GJA4 protein. Ab114495 is a full length protein produced in Wheat germ and has been validated in WB, ELISA, SDS-PAGE…
Izawa, Y., Gu, Y-H., Osada, T., Kanazawa, M., Hawkins, B., Koziol, J., ... del Zoppo, G. (2017). β1-integrin-matrix interactions modulate cerebral microvessel endothelial cell tight junction expression and permeability. Journal of Cerebral Blood Flow and Metabolism. DOI: 10.1177/0271678X17722108 ...
The association of LBBB and connexin 43 1400A/ins polymorphism together with this and previous studies on heritability, indicate that BBB development can be modulated by genetic factors and could be the result of a progressive disorder within the myocardium and not confined to the specialised conduction system.. In our study on heritability, we report the first study focused on BBB in fathers and their descendants in their 50s. From our previous study of the fathers, The Study of Men Born 1913,3 we know that at age 50 the prevalence of BBB among men is 1.2%. The sons had a longer QRS duration compared with the daughters. This, and the finding that two (6.4%) of the sons also had BBB, but none of the daughters did, might indicate a male predominance of BBB. Previous studies have shown a male predominance of RBBB but not for LBBB.26 The male predominance of BBB may be a paraphenomenon with cardiovascular disease, in keeping with the fact that women generally develop cardiovascular disease later ...
TY - JOUR. T1 - Linoleic acid permeabilizes gastric epithelial cells by increasing connexin 43 levels in the cell membrane via a GPR40- and Akt-dependent mechanism. AU - Puebla, Carlos. AU - Cisterna, Bruno A.. AU - Salas, Daniela P.. AU - Delgado-López, Fernando. AU - Lampe, Paul D.. AU - Sáez, Juan C.. PY - 2016/5/1. Y1 - 2016/5/1. N2 - Linoleic acid (LA) is known to activate G-protein coupled receptors and connexin hemichannels (Cx HCs) but possible interlinks between these two responses remain unexplored. Here, we evaluated the mechanism of action of LA on the membrane permeability mediated by Cx HCs in MKN28 cells. These cells were found to express connexins, GPR40, GPR120, and CD36 receptors. The Cx HC activity of these cells increased after 5 min of treatment with LA or GW9508, an agonist of GPR40/GPR120; or exposure to extracellular divalent cation-free solution (DCFS), known to increase the open probability of Cx HCs, yields an immediate increase in Cx HC activity of similar intensity ...