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Connexin
Terms modified by Connexin Selected AbstractsTHE CONNEXIN 32 NERVE-SPECIFIC PROMOTER IS DIRECTLY ACTIVATED BY Egr2/Krox20 IN HeLa CELLSJOURNAL OF THE PERIPHERAL NERVOUS SYSTEM, Issue 1 2002M. Musso Connexin 32 (Cx32) belongs to a protein family that forms intercellular channels mediating the exchange of ions and chemical messengers. In the peripheral nervous system (PNS) Cx32 is expressed in Schwann cells and contributes to the homeostasis and structural integrity of myelin. Mutations of this gene determine X-linked form of Charcot Marie-Tooth (CMTX) disease. Cx 32 is transcriptionally regulated in a tissue-specific manner by two different promoters termed P1 and P2. P2, active in Schwann cells, is located 5 kb downstream from the P1 promoter and at 500 bp from the exon 2 that contains the entire coding region. Previously, by Electrophoretical Mobility Shift Assay (EMSA) we have identified a sequence (-101/-93), within P2, specifically recognized by recombinant Egr2. In order to prove the direct involvement of Egr2 in the transcriptional control of the Cx32 gene, we have performed transfection experiments in HeLa cells with a luciferase driven by the P2 promoter in presence or not of a vector expressing Krox20, the mouse homologue of human Egr2. We have found that the construct in which the sequence -103/-93 is mutated is not activated as well as the wild type sequence. Moreover we have detected another upstream sequence (-236/-213) recognized by recombinant Egr2 and other transcription factors present in HeLa nuclear extract like SP1. The construct, lacking this sequence and carrying the mutated downstream Egr2 recognition sequence, is not activated at all by Krox20. Taken together these findings strongly suggest the role of Egr2 in the transcriptional control of Connexin 32 through both sequences. The laboratory is a member of the European CMT Consortium; partially granted by Ministero della Sanit, to PM, MURST and Ateneo to FA. [source] The immunocytochemical localization of connexin 36 at rod and cone gap junctions in the guinea pig retinaEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2003Eun-Jin Lee Abstract Connexin 36 (Cx36) is a channel-forming protein found in the membranes of apposed cells, forming the hexameric hemichannels of intercellular gap junction channels. It localizes to certain neurons in various regions of the brain including the retina. We characterized the expression pattern of neuronal Cx36 in the guinea pig retina by immunocytochemistry using specific antisera against Cx36 and green/red cone opsin or recoverin. Strong Cx36 immunoreactivity was visible in the ON sublamina of the inner plexiform layer and in the outer plexiform layer, as punctate labelling patterns. Double-labelling experiments with antibody directed against Cx36 and green/red cone opsin or recoverin showed that strong clustered Cx36 immunoreactivity localized to the axon terminals of cone or close to rod photoreceptors. By electron microscopy, Cx36 immunoreactivity was visible in the gap junctions as well as in the cytoplasmic matrices of both sides of cone photoreceptors. In the gap junctions between cone and rod photoreceptors, Cx36 immunoreactivity was only visible in the cytoplasmic matrices of cone photoreceptors. These results clearly indicate that Cx36 forms homologous gap junctions between neighbouring cone photoreceptors, and forms heterologous gap junctions between cone and rod photoreceptors in guinea pig retina. This focal location of Cx36 at the terminals of the photoreceptor suggests that rod photoreceptors can transmit rod signals to the pedicle of a neighbouring cone photoreceptor via Cx36, and that the cone in turn signals to corresponding ganglion cells via ON and OFF cone bipolar cells. [source] Characterization 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] Enhanced Connexin 43 immunoreactivity in penumbral areas in the human brain following ischemiaGLIA, Issue 5 2006Taizen Nakase Abstract Astrocytes support neurons not only physically but also chemically by secreting neurotrophic factors and energy substrates. Moreover, astrocytes establish a glial network and communicate through gap junctions in the brain. Connexin 43 (Cx43) is one of major component proteins in astrocytic gap junctions. Heterozygote Cx43 KO mice and astrocyte specific Cx43 KO mice exhibited amplified brain damage after ischemic insults, suggesting a neuroprotective role for astrocytic gap junctions. However, some reports mentioned unfavorable effects of gap junctions in neuronal support. Therefore, the role of astrocytic gap junctions under ischemic condition remains controversial. Since these studies have been performed using animal models, we investigated the Cx43 expression in human brain after stroke. Brain slice sections were prepared from pathological samples in our hospital. Embolic stroke brains sectioned because of the stroke were considered as acute ischemic models. Multiple infarction brains sectioned because of pneumonia or cancer were considered as chronic models. We observed the levels of Cx43 in both lesioned and intact areas, and compared them with acute and chronic models. As the results, astrocytes were strongly activated in penumbral lesions both of acute and chronic ischemic models. The Cx43 immunoreactivity was significantly amplified in the penumbra of chronic model compared to that of the acute model. Neurons were well preserved in chronic model compared to acute model. These findings suggested that the brain may generate neuronal protection by increasing the levels of Cx43 and amplifying the astrocytic gap junctional intercellular communication under hypoxic condition. © 2006 Wiley-Liss, Inc. [source] Erythrokeratodermia variabilis (EKV) , eine Störung der epidermalen Expression von Gap-Junction-ProteinenJOURNAL DER DEUTSCHEN DERMATOLOGISCHEN GESELLSCHAFT, Issue 5 2005Erythrokeratodermia variabilis (EKV), a disorder due to altered epidermal expression of gap junction proteins Connexin,31; Connexin,43; Erythrokeratodermia variabilis; Gap Junctions Zusammenfassung Die Erythrokeratodermia variabilis (EKV) ist eine seltene autosomal-dominant vererbte Genodermatose mit einer epidermalen Verhornungsstörung. Das klinische Bild der EKV wird von zwei Morphen geprägt: transiente, schnell wandernde Erytheme und persistierende braune Hyperkeratosen. Vor kurzem wurde der zugrunde liegende Gendefekt der EKV auf dem kurzen Arm von Chromosom,1 lokalisiert, der für das Gap-Junction-Protein Connexin,31 kodiert. Wir stellen einen 48jährigen Patienten vor, bei dem seit dem dreißigsten Lebensjahr großflächige, scharf begrenzte, randständig schuppende Erytheme an den Extremitäten, Gesäß und Rumpf auftreten. Histologisch fand sich eine orthokeratotische Hyperkeratose mit fokaler Parakeratose bei Akanthose der Epidermis. Immunhistochemisch konnte eine vermindert Expression des Gap-Junction-Proteins Connexin,31 sowie vermehrte Expression von Connexin,43 dargestellt werden. Ultrastrukturell zeigten sich erweiterte Interzellularräume in der oberen Epidermis mit unauffälligen Desmosomen, Adherensjunktionen und regulären Gap Junctions. In der Epidermis wird u. a. die Zellproliferation und -differenzierung über die Gap Junctions reguliert. Die Mutation im Connexin,31 wird daher als ursächlich für das klinische Bild der EKV angesehen. Die Überexpression von Connexin,43, die hier erstmals beschrieben wird, entsteht möglicherweise reaktiv als Folge der CX31-Mutation und kompensiert vorübergehend den Defekt. Summary Erythrokeratodermia variabilis (EKV) is a rare autosomal dominant genodermatosis with disturbed epidermal differentiation. Its clinical picture varies from transient, fast moving erythema to persistent brown hyperkeratoses. The gene defect in EKV was recently located on the short arm of chromosome,1 encoding the gap junction protein connexin,31. We report on a 48-year-old patient with sharply circumscribed, scaling erythema on the extremities, buttocks and trunk starting since 30,years of age. Histological investigation showed orthokeratotic hyperkeratosis with focal parakeratosis overlying an acanthotic epidermis. Immunohistochemistry revealed a decreased expression of the gap junction protein connexin,31 as well as increased expression of connexin,43. At the ultrastructural level, widened intercellular spaces in the upper epidermis were present with regular desmosomes, adherens junctions and gap junctions. Epidermal cell proliferation and differentiation are regulated by gap junctions. The mutation in connexin,31 is regarded therefore as causal for the clinical picture of the EKV. The unique up-regulation of connexin,43 may occur as a consequence of the Cx31 mutation and temporarily compensate for this defect. [source] Connexin 43 Is Required for the Anti-Apoptotic Effect of Bisphosphonates on Osteocytes and Osteoblasts In Vivo,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 11 2008Lilian I Plotkin Abstract Connexin (Cx)43 is required for inhibition of osteocyte and osteoblast apoptosis by bisphosphonates in vitro. Herein, we evaluated its requirement for the in vivo actions of bisphosphonates using mice in which Cx43 was deleted specifically from osteocytes and osteoblasts (Cx43,Ob,Ot/, mice). Effective removal of Cx43 was confirmed by the presence of the deleted form of the gene and by reduced mRNA and protein expression in osteoblastic cells and bones obtained from Cx43,Ob,Ot/, mice. The amino-bisphosphonate alendronate (2.3 ,mol/kg/d) was injected daily into 5-mo-old female mice (n = 6,11) for 31 days, starting 3 days before implantation of pellets releasing the glucocorticoid prednisolone (2.1 mg/kg/d). Cx43,Ob,Ot/, mice and their littermates (Cx43fl/,, Cx43,Ob,Ot/+, and Cx43fl/+) gained bone with similar kinetics and exhibited identical bone mass from 2 to 4.5 mo of age, indicating that Cx43 deletion from osteocytes and mature osteoblasts does not impair bone acquisition. In addition, prednisolone induced a similar increase in osteocyte and osteoblast apoptosis in Cx43,Ob,Ot/, or in control Cx43fl/, littermates. However, whereas alendronate prevented prednisolone-induced apoptosis in control Cx43fl/, mice, it was ineffective in Cx43,Ob,Ot/, mice. In contrast, alendronate inhibited glucocorticoid-induced bone loss in both type of animals, suggesting that inhibition of resorption is the predominant effect of alendronate against the early phase of glucocorticoid-induced bone loss. Taken together with earlier in vitro evidence, these findings show that Cx43 is required for the anti-apoptotic effect of bisphosphonates on osteocytes and osteoblasts. [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] Irsogladine maleate counters the interleukin-1,-induced suppression in gap-junctional intercellular communication but does not affect the interleukin-1,-induced zonula occludens protein-1 levels in human gingival epithelial cellsJOURNAL OF PERIODONTAL RESEARCH, Issue 1 2008T. Fujita Background and Objective:, Irsogladine maleate counters gap junctional intercellular communication reduction induced by interleukin-8 or Actinobacillus actinomycetemcomitans in cultured human gingival epithelial cells. Interleukin-1, is involved in periodontal disease. Little is known, however, about the effect of interleukin-1, on intercellular junctional complexes in human gingival epithelial cells. Furthermore, irsogladine maleate may affect the actions of interleukin-1,. In this study, we examined how interleukin-1, affected gap junctional intercellular communication, connexin 43 and zonula occludens protein-1, and how irsogladine maleate modulated the interleukin-1,-induced changes in the intercellular junctional complexes in human gingival epithelial cells. Material and Methods:, Human gingival epithelial cells were exposed to interleukin-1,, with or without irsogladine maleate. Connexin 43 and zonula occludens protein-1 were examined at mRNA and protein levels by real-time polymerase chain reaction and western blotting, respectively. Gap junctional intercellular communication was determined using the dye transfer method. The expression of zonula occludens protein-1 was also confirmed by immunofluorescence. Results:, Interleukin-1, decreased connexin 43 mRNA levels, but increased zonula occludens protein-1 mRNA levels. Irsogladine maleate countered the interleukin-1,-induced reduction in gap junctional intercellular communication and connexin 43 levels. However, irsogladine maleate did not influence the increased zonula occludens protein-1 levels. Conclusion:, The effect of interleukin-1, on gap junctional intercellular communication and tight junctions of human gingival epithelial cells is different. The recovery of gap junctional intercellular communication by irsogladine maleate in the gingival epithelium may be a normal process in gingival epithelial homeostasis. [source] Expression pattern of adhesion molecules in junctional epithelium differs from that in other gingival epitheliaJOURNAL OF PERIODONTAL RESEARCH, Issue 4 2006S. Hatakeyama Background and Objective:, The gingival epithelium is the physiologically important interface between the bacterially colonized gingival sulcus and periodontal soft and mineralized connective tissues, requiring protection from exposure to bacteria and their products. However, of the three epithelia comprising the gingival epithelium, the junctional epithelium has much wider intercellular spaces than the sulcular epithelium and oral gingival epithelium. Hence, the aim of the present study was to characterize the cell adhesion structure in the junctional epithelium compared with the other two epithelia. Material and Methods:, Gingival epithelia excised at therapeutic flap surgery from patients with periodontitis were examined for expression of adhesion molecules by immunofluorescence. Results:, In the oral gingival epithelium and sulcular epithelium, but not in the junctional epithelium, desmoglein 1 and 2 in cell,cell contact sites were more abundant in the upper than the suprabasal layers. E-cadherin, the main transmembranous molecule of adherens junctions, was present in spinous layers of the oral gingival epithelium and sulcular epithelium, but was scarce in the junctional epithelium. In contrast, desmoglein 3 and P-cadherin were present in all layers of the junctional epithelium as well as the oral gingival epithelium and sulcular epithelium. Connexin 43 was clearly localized to spinous layers of the oral gingival epithelium, sulcular epithelium and parts of the junctional epithelium. Claudin-1 and occludin were expressed in the cell membranes of a few superficial layers of the oral gingival epithelium. Conclusion:, These findings indicated that the junctional epithelium contains only a few desmosomes, composed of only desmoglein 3; adherens junctions are probably absent because of defective E-cadherin. Thus, the anchoring junctions connecting junctional epithelium cells are lax, causing widened intercellular spaces. In contrast, the oral gingival epithelium, which has a few tight junctions, functions as a barrier. [source] A novel insertional mutation in Connexin 32 gene causes demyelinating polyneuropathy with predominantly motor axonal lossJOURNAL OF THE PERIPHERAL NERVOUS SYSTEM, Issue 4 2004Lan Zhou [source] Abstracts of the 8th Meeting of the Italian Peripheral Nerve Study Group: 76JOURNAL OF THE PERIPHERAL NERVOUS SYSTEM, Issue 1 2003D Pareyson The X-linked form of Charcot-Marie-Tooth disease (CMTX) is associated with mutations in the Connexin 32 gene (Cx32) and is the second most common CMT subtype after CMT1A, in which the 17p11.2 duplication is the underlying molecular defect. CMTX is characterized by no male-to-male transmission, intermediate motor conduction velocities (MCV), and more severe disease in males. In our series of CMT patients, we found 9 different Cx32 mutations in 11 families. Overall there were 26 patients, 13 males and 13 females, aged 11,76 yrs. Age at onset ranged considerably (1,60 yrs), but symptoms began earlier in males (mean 15.4 yrs, 77% within age 20) than in females (mean 25 yrs). All patients were autonomous, but disease severity was greater in males, while 4 female carriers were asymptomatic. Pain and tremor were frequent complaints. Two patients had Babinski sign and one had rest tremor. Nerve conduction studies were performed in 23 patients (13 males, 10 females). Upper limb motor conduction velocities (MCV) ranged between 25 and 57 m/s, and were slower in males (25,48 m/s) than in females (34,57 m/s). MCV were in the upper range of CMT1 (25,38 m/s) in 10/13 males but only in 3/10 females. In some cases, nerve conduction slowing was non-uniform within single nerves, and one female patient had a previous diagnosis of chronic inflammatory demyelinating polyradiculoneuropathy. There was considerable asymmetry of involvement between different nerves. The median nerve was often more severely affected than the ulnar nerve, and not only in females, as previously reported, but also in males. Therefore, it appears unlikely that this asymmetry is accounted for by a Lyonization phenomenon. Subclinical abnormalities of central nervous system as revealed by multimodal evoked potential studies were found in 8/10 patients. Expression of Cx32 in the brain is the likely explanation of this finding that confirms previous non-systematic observations. We found seven missense and two nonsense mutations (one novel mutation). Two families presented distinct mutations at the same codon (Arg164), while the Arg22Stop and Arg220Stop mutations were each found in two unrelated cases. Partially supported by a grant from the Italian Ministry of Health to F.T and D.P. (Progetto Ricerca Finalizzata ICS 030.3/RF00.174). [source] THE CONNEXIN 32 NERVE-SPECIFIC PROMOTER IS DIRECTLY ACTIVATED BY Egr2/Krox20 IN HeLa CELLSJOURNAL OF THE PERIPHERAL NERVOUS SYSTEM, Issue 1 2002M. Musso Connexin 32 (Cx32) belongs to a protein family that forms intercellular channels mediating the exchange of ions and chemical messengers. In the peripheral nervous system (PNS) Cx32 is expressed in Schwann cells and contributes to the homeostasis and structural integrity of myelin. Mutations of this gene determine X-linked form of Charcot Marie-Tooth (CMTX) disease. Cx 32 is transcriptionally regulated in a tissue-specific manner by two different promoters termed P1 and P2. P2, active in Schwann cells, is located 5 kb downstream from the P1 promoter and at 500 bp from the exon 2 that contains the entire coding region. Previously, by Electrophoretical Mobility Shift Assay (EMSA) we have identified a sequence (-101/-93), within P2, specifically recognized by recombinant Egr2. In order to prove the direct involvement of Egr2 in the transcriptional control of the Cx32 gene, we have performed transfection experiments in HeLa cells with a luciferase driven by the P2 promoter in presence or not of a vector expressing Krox20, the mouse homologue of human Egr2. We have found that the construct in which the sequence -103/-93 is mutated is not activated as well as the wild type sequence. Moreover we have detected another upstream sequence (-236/-213) recognized by recombinant Egr2 and other transcription factors present in HeLa nuclear extract like SP1. The construct, lacking this sequence and carrying the mutated downstream Egr2 recognition sequence, is not activated at all by Krox20. Taken together these findings strongly suggest the role of Egr2 in the transcriptional control of Connexin 32 through both sequences. The laboratory is a member of the European CMT Consortium; partially granted by Ministero della Sanit, to PM, MURST and Ateneo to FA. [source] Origin and propagation of spontaneous excitation in smooth muscle of the guinea-pig urinary bladderTHE JOURNAL OF PHYSIOLOGY, Issue 2 2001Hikaru Hashitani 1The origin and propagation of waves of spontaneous excitation in bundles of smooth muscle of the guinea-pig bladder were examined using intracellular recording techniques and visualization of the changes in the intracellular calcium concentration ([Ca2+]i). 2Bladder smooth muscle cells exhibited spontaneous transient increases in [Ca2+]i which originated along a boundary of each smooth muscle bundle and then spread to the other boundary with a conduction velocity of 2.0 mm s,1. 3Spontaneous increases in [Ca2+]i were always preceded by action potentials. Nifedipine (10 ,M) abolished increases in both [Ca2+]i and action potentials. Caffeine (10 mM), ryanodine (50 ,M) and cyclopiazonic acid (10 ,M) reduced the amplitude of the associated increases in [Ca2+]i without preventing the generation of action potentials. 4Spontaneous action potentials had conduction velocities of 40 mm s,1 in the axial direction and 1.3 mm s,1 in the transverse direction. The electrical length constants of the bundles of muscle were 425 ,m in the axial direction and 12.5 ,m in the transverse direction. 5Neurobiotin, injected into an impaled smooth muscle cell, spread more readily to neighbouring cells located in the axial direction than those located in the transverse direction. The spread of neurobiotin was inhibited by 18,-glycyrrhetinic acid (18,-GA, 40 ,M), a gap junction blocker. 6Immunohistochemistry for Connexin 43 showed abundant punctate staining on the smooth muscle cell membranes. 7These results suggested that spontaneous action potentials and associated calcium waves occur almost simultaneously along the boundary of bladder smooth muscle bundles and then propagate to the other boundary probably through gap junctions. [source] Connexin 26 and 30 Genes Mutations in Patients with Chronic Rhinosinusitis,THE LARYNGOSCOPE, Issue 2 2008FACS, Nicolas Y. BuSaba MD Abstract Objectives: Connexin proteins play an important role in cell-to-cell communication. Mutations in the genes that encode for these connexins may potentially lead to dysfunction in mucociliary clearance predisposing to chronic rhinosinusitis (CRS) or recurrent acute rhinosinusitis (RARS). The objective of this study was to assess for the presence of connexin 26 and 30 gene mutations in patients with CRS and RARS. Study Design: Prospective case series. Methods: Forty-six consecutive patients who were diagnosed with CRS or RARS at a single tertiary care facility were included in the study. Patients with known dysfunction in mucociliary clearance were excluded. The following clinical data were collected: age, gender, duration of disease and age at onset, personal history of otitis media and/or sensorineural hearing loss (SNHL), and family history of paranasal sinus disease and SNHL. Buccal swab deoxyribonucleic acid (DNA) specimens were sequenced for connexin 26 and 30 genes. Results: The study group consisted of 32 females and 14 males, 8 children and 38 adults. Adequate sequencing of connexin 30 gene was possible in all 46 specimens, but in only 19 specimens for connexin 26 gene. Connexin 30 gene mutations were not detected in any of the 46 specimens. Two of the 19 specimens had heterozygous mutations in the connexin 26 gene; there was one V371 mutation and one 35dG mutation. Both patients were adults; the patient with 35dG mutation had SNHL. Conclusion: Mutations in connexin 26 and 30 genes are rare in patients with CRS or RARS and do not seem to play a contributory role in the pathogensis of these disorders. [source] A New De Novo Missense Mutation in Connexin 26 in a Sporadic Case of Nonsyndromic DeafnessTHE LARYNGOSCOPE, Issue 5 2007Paola Primignani PhD Abstract Objectives: Mutations in the GJB2 gene, encoding Connexin 26, can cause nonsyndromic recessive deafness or dominant hearing loss (HL) with or without keratoderma. The objective was to perform a molecular evaluation to establish the inherited pattern of deafness in the sporadic cases afferent to our center. Methods: The subject was a 2-year-old Italian girl with nonsyndromic early onset HL. We performed DNA sequencing of the GJB2 gene and deletion analysis of the GJB6 gene in all family members. Results: Direct sequencing of the gene showed a heterozygous C,G transition at nucleotide 172 resulting in a proline to alanine amino acid substitution at codon 58 (P58A). The analyses indicate that the P58A mutation appeared de novo in the proband with a possible dominant effect. Conclusions: This mutation occurs in the first extracellular domain (EC1), which seems to be very important for connexon-connexon interaction and for the control of voltage gating of the channel. The de novo occurrence of an EC1 mutation in a sporadic case of deafness is consistent with the assumption that P58A can cause dominant HL. [source] Stimulation of cardiac ,-adrenoceptors targets connexin 43BRITISH JOURNAL OF PHARMACOLOGY, Issue 1 2009Kerstin Boengler Connexin 43 (Cx43) is the major protein of cardiac ventricular gap junctions and is crucial to cell,cell communication and cardiac function. The protein level of Cx43 is reduced in patients with heart failure or dilated cardiomyopathy (DCM), pathophysiological conditions often associated with arrhythmias. As catecholamines are often increased in cardiac diseases, Salameh et al., in this issue of the BJP, investigated the effect of ,-adrenoceptor stimulation of neonatal cardiomyocytes on Cx43 expression and found increased Cx43 mRNA and protein levels following 24 h stimulation. Up-regulation of Cx43 was associated with phosphorylation of mitogen-activated protein kinases and translocation of transcription factors into the nucleus. In patients with DCM, a situation often associated with desensitization of the ,-adrenoceptor system, Cx43 expression was reduced. The characterization of the signal transduction pathways involved in Cx43 expression and intracellular localization in human myocardium in vivo is a promising target for the development of new anti-arrhythmic strategies. [source] Audioprofiling identifies TECTA and GJB2 -related deafness segregating in a single extended pedigreeCLINICAL GENETICS, Issue 2 2007NC Meyer An audioprofile displays phenotypic data from several audiograms on a single graph that share a common genotype. In this report, we describe the application of audioprofiling to a large family in which a genome-wide screen failed to identify a deafness locus. Analysis of audiograms by audioprofiling suggested that two persons with hearing impairment had a different deafness genotype. On this basis, we reassigned affectation status and identified a p.Cys1837Arg autosomal dominant mutation in ,-tectorin segregating in all family members except two persons, who segregated autosomal recessive deafness caused by p.Val37Ile and p.Leu90Pro mutations in Connexin 26. One nuclear family in the extended pedigree segregates both dominant and recessive non-syndromic hearing loss. [source] Causes of deafness in British Bangladeshi children: a prevalence twice that of the UK population cannot be accounted for by consanguinity aloneCLINICAL OTOLARYNGOLOGY, Issue 2 2009Y. Bajaj Objective:, To study the causes and prevalence of sensorineural deafness in Bangladeshi children resident in East London. Methods:, This was a cross sectional survey of children of Bangladeshi origin living in East London with bilateral sensorineural hearing loss of 40 db HL or more. In this study, 134 patients were included. The study looked primarily at the causes of sensorineural hearing loss in this population. Results:, The prevalence of deafness in Bangladeshi children in East London is approximately 3.86 per 1000 [95% confidence intervals (CI) 3.20, 4.65] which is significantly greater than the average UK prevalence of 1.65 per 1000. The prevalence of deafness in these Bangladeshi children belonging to non-consanguineous families only, the prevalence falls to 2.73 per 1000 (95% CI 2.19, 3.41). In 60% cases the cause of deafness was genetic. The single most common cause of sensorineural hearing loss in this population was mutations in the GJB2 gene (Connexin 26) in 20 of these patients (17%). Parents were consanguineous in 33% of the families. Conclusion:, This study concludes that prevalence of sensorineural deafness in Bangladeshi children is at least 2.3 times the national average. This study also concludes that genetic causes are the common cause of deafness in this ethnic group, with nearly 30% of children with non-syndromic deafness having mutations in GJB2. Although parental consanguinity was very high in this population it did not account for the whole increase in prevalence. [source] Cloning and functional characterization of a novel connexin expressed in somites of Xenopus laevisDEVELOPMENTAL DYNAMICS, Issue 3 2005Teun P. De Boer Abstract Connexin-containing gap junctions play an essential role in vertebrate development. More than 20 connexin isoforms have been identified in mammals. However, the number identified in Xenopus trails with only six isoforms described. Here, identification of a new connexin isoform from Xenopus laevis is described. Connexin40.4 was found by screening expressed sequence tag databases and carrying out polymerase chain reaction on genomic DNA. This new connexin has limited amino acid identity with mammalian (<50%) connexins, but conservation is higher (,62%) with fish. During Xenopus laevis development, connexin40.4 was first expressed after the mid-blastula transition. There was prominent expression in the presomitic paraxial mesoderm and later in the developing somites. In adult frogs, expression was detected in kidney and stomach as well as in brain, heart, and skeletal muscle. Ectopic expression of connexin40.4 in HEK293 cells, resulted in formation of gap junction like structures at the cell interfaces. Similar ectopic expression in neural N2A cells resulted in functional electrical coupling, displaying mild, asymmetric voltage dependence. We thus cloned a novel connexin from Xenopus laevis, strongly expressed in developing somites, with no apparent orthologue in mammals. Developmental Dynamics 233:864,871, 2005. © 2005 Wiley-Liss, Inc. [source] Changing patterns of ganglion cell coupling and connexin expression during chick retinal developmentDEVELOPMENTAL NEUROBIOLOGY, Issue 4 2002David L. Becker Abstract We have used dye injection and immunolabeling to investigate the relationship between connexin (Cx) expression and dye coupling between ganglion cells (GCs) and other cells of the embryonic chick retina between embryonic days 5 and 14 (E5,14). At E5, GCs were usually coupled, via soma-somatic or dendro-somatic contacts, to only one or two other cells. Coupling increased with time until E11 when GCs were often coupled to more than a dozen other cells with somata in the ganglion cell layer (GCL) or inner nuclear layer (INL). These coupled clusters occupied large areas of the retina and coupling was via dendro-dendritic contacts. By E14, after the onset of synaptogenesis and at a time of marked cell death, dye coupling was markedly decreased with GCs coupled to three or four partners. At this time, coupling was usually to cells of the same morphology, whereas earlier coupling was heterogeneous. Between E5 and E11, GCs were sometimes coupled to cells of neuroepithelial morphology that spanned the thickness of the retina. The expression of Cx 26, 32, and 43 differed and their distribution changed during the period studied, showing correlation with events such as proliferation, migration, and synaptogenesis. These results suggest specific roles for gap junctions and Cx's during retinal development. © 2002 Wiley Periodicals, Inc. J Neurobiol 52: 280,293, 2002 [source] Docosahexaenoic acid (22:6n-3) enrichment of membrane phospholipids increases gap junction coupling capacity in cultured astrocytesEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2006Gaelle Champeil-Potokar Abstract Although it is agreed that n-3 polyunsaturated fatty acids (PUFAs) are important for brain function, it has yet to be demonstrated how they are involved in precise cellular mechanisms. We investigated the role of enhanced n-3 PUFA in astrocyte membranes on the gap junction capacity of these cells. Astrocytes isolated from newborn rat cortices were grown in medium supplemented with docosahexaenoic acid (DHA), the main n-3 PUFA in cell membranes, or arachidonic acid (AA), the main n-6 PUFA, plus an antioxidant (,-tocopherol or N -acetyl-cystein) to prevent peroxidation. The resulting three populations of astrocytes differed markedly in their n-3 : n-6 PUFA ratios in phosphatidylethanolamine and phosphatidylcholine, the main phospholipids in membranes. DHA-supplemented cells had a physiological high n-3 : n-6 ratio (1.58), unsupplemented cells had a low n-3 : n-6 ratio (0.66) and AA-supplemented cells had a very low n-3 : n-6 ratio (0.36), with excess n-6 PUFA. DHA-supplemented astrocytes had a greater gap junction capacity than unsupplemented cells or AA-supplemented cells. The enhanced gap junction coupling of DHA-enriched cells was associated with a more functional distribution of connexin 43 at cell interfaces (shown by immunocytochemistry) and more of the main phosphorylated isoform of connexin 43. These findings suggest that the high n-3 : n-6 PUFA ratio that occurs naturally in astrocyte membranes is needed for optimal gap junction coupling in these cells. [source] Expression of zonula occludens-1 (ZO-1) and the transcription factor ZO-1-associated nucleic acid-binding protein (ZONAB),MsY3 in glial cells and colocalization at oligodendrocyte and astrocyte gap junctions in mouse brainEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2005Mihai C. Penes Abstract The PDZ domain-containing protein zonula occludens-1 (ZO-1) interacts with several members of the connexin (Cx) family of gap junction-forming proteins and has been localized to gap junctions, including those containing Cx47 in oligodendrocytes. We now provide evidence for ZO-1 expression in astrocytes in vivo and association with astrocytic connexins by confocal immunofluorescence demonstration of ZO-1 colocalization with astrocytic Cx30 and Cx43, and by ZO-1 coimmunoprecipitation with Cx30 and Cx43. Evidence for direct interaction of Cx30 with ZO-1 was obtained by pull-down assays that indicated binding of Cx30 to the second of the three PDZ domains in ZO-1. Further, we investigated mouse Y-box transcription factor MsY3, the canine ortholog of which has been termed ZO-1-associated nucleic acid-binding protein (ZONAB) and previously reported to interact with ZO-1. By immunofluorescence using specific antimouse ZONAB antibody, ZONAB was found to be associated with oligodendrocytes throughout mouse brain and spinal cord, and to be colocalized with oligodendrocytic Cx47 and Cx32 as well as with astrocytic Cx43. Our results extend the CNS cell types that express the multifunctional protein ZO-1, demonstrate an additional connexin (Cx30) that directly interacts with ZO-1, and show for the first time the association of a transcription factor (ZONAB) with ZO-1 localized to oligodendrocyte and astrocyte gap junctions. Given previous observations that ZONAB and ZO-1 in combination regulate gene expression, our results suggest roles of glial gap junction-mediated anchoring of signalling molecules in a wide variety of glial homeostatic processes. [source] The immunocytochemical localization of connexin 36 at rod and cone gap junctions in the guinea pig retinaEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2003Eun-Jin Lee Abstract Connexin 36 (Cx36) is a channel-forming protein found in the membranes of apposed cells, forming the hexameric hemichannels of intercellular gap junction channels. It localizes to certain neurons in various regions of the brain including the retina. We characterized the expression pattern of neuronal Cx36 in the guinea pig retina by immunocytochemistry using specific antisera against Cx36 and green/red cone opsin or recoverin. Strong Cx36 immunoreactivity was visible in the ON sublamina of the inner plexiform layer and in the outer plexiform layer, as punctate labelling patterns. Double-labelling experiments with antibody directed against Cx36 and green/red cone opsin or recoverin showed that strong clustered Cx36 immunoreactivity localized to the axon terminals of cone or close to rod photoreceptors. By electron microscopy, Cx36 immunoreactivity was visible in the gap junctions as well as in the cytoplasmic matrices of both sides of cone photoreceptors. In the gap junctions between cone and rod photoreceptors, Cx36 immunoreactivity was only visible in the cytoplasmic matrices of cone photoreceptors. These results clearly indicate that Cx36 forms homologous gap junctions between neighbouring cone photoreceptors, and forms heterologous gap junctions between cone and rod photoreceptors in guinea pig retina. This focal location of Cx36 at the terminals of the photoreceptor suggests that rod photoreceptors can transmit rod signals to the pedicle of a neighbouring cone photoreceptor via Cx36, and that the cone in turn signals to corresponding ganglion cells via ON and OFF cone bipolar cells. [source] Mice with astrocyte-directed inactivation of connexin43 exhibit increased exploratory behaviour, impaired motor capacities, and changes in brain acetylcholine levelsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2003Christian Frisch Abstract Gap junctions mediate communication between many cell types in the brain. Gap junction channels are composed of membrane-spanning connexin (Cx) proteins, allowing the cell-to-cell passage of small ions and metabolites. Cx43 is the main constituent of the brain-spanning astrocytic gap junctional network, controlling activity-related changes in ion and glutamate concentrations as well as metabolic processes. In astrocytes, deletion of Cx43-coding DNA led to attenuated gap junctional coupling and impaired propagation of calcium waves, known to influence neuronal activity. Investigation of the role of Cx43 in behaviour has been impossible so far, due to postnatal lethality of its general deletion. Recently, we have shown that deletion of Cx30, which is also expressed by astrocytes, affects exploration, emotionality, and neurochemistry in the mouse. In the present study, we investigated the effects of the astrocyte-directed inactivation of Cx43 on mouse behaviour and brain neurochemistry. Deletion of Cx43 in astrocytes increased exploratory activity without influencing habituation. In the open field, but not in the elevated plus-maze, an anxiolytic-like effect was observed. Rotarod performance was initially impaired, but reached control level after further training. In the water maze, Cx43 deficient mice showed a steeper learning course, although final performance was similar between groups. Cx43 inactivation in astrocytes increased acetylcholine content in the frontal cortex of water maze-trained animals. Results are discussed in terms of altered communication between astrocytes and neurons, possible compensation processes, and differential effects of Cx30- and astrocyte-specific Cx43 deletion. [source] Pharmacological "cross-inhibition" of connexin hemichannels and swelling activated anion channelsGLIA, Issue 3 2009Zu-Cheng Ye Abstract The study of ion channels has relied heavily on the use of pharmacological blocking agents. However, many of these agents have multiple effects, which may compromise interpretation of results when the affected mechanisms/pathways mediate similar functions. Volume regulated anion channels (VRAC) and connexin hemichannels can both mediate the release of glutamate and taurine, although these channels have distinct activation stimuli and hemichannels, but not VRAC, are permeable to Lucifer Yellow (LY). It has been reported that some anion channel blockers may inhibit connexin hemichannels. We further examined the effects of classic gap junction/hemichannel blockers and anion channel blockers on these channels. The typical VRAC blockers, NPPB, IAA-94, and tamoxifen blocked low divalent cation-induced glutamate and taurine release and LY loading, presumed due to hemichannel opening. The blocking action of these compounds on hemichannels was concentration dependent and fell within the same range where the drugs classically block VRACs. Conversely, carbenoxolone (CBX), the most widely used gap junction/hemichannel blocker, was an effective blocker of VRAC-mediated glutamate and taurine release, and blocked these channels at similar concentrations at which it blocked hemichannels. The CBX effect on VRACs was verified using astrocytes from connexin 43 knock out (Cx43 KO) animals. In these cells, the hypotonic induced amino acid flux was retained whereas the low divalent cation solution-induced flux was lost. These results extend our knowledge about "cross-inhibition" of VRACs and gap junctions/hemichannels by certain pharmacological agents. Given the overlap in function of these two types of channels, great care must be exerted in using pharmacological blockers to identify one channel from the other. © 2008 Wiley-Liss, Inc. [source] The TLR3 ligand polyI:C downregulates connexin 43 expression and function in astrocytes by a mechanism involving the NF-,B and PI3 kinase pathwaysGLIA, Issue 8 2006Yongmei Zhao Abstract Toll-like receptor 3 (TLR3) is a component of the innate immune response that responds to dsRNA viruses and virus replication intermediates. In this study we show that activation of astrocytes with the dsRNA mimetic polyinosinic-cytidylic acid (pI:C) results in loss of expression of connexin43 (Cx43) mRNA and protein while upregulating the expression of the ionotropic P2 receptor P2X4R. Analysis of the signaling pathways involved failed to demonstrate a role for the p38 MAP kinase, ERK, or JNK signaling pathways whereas an inhibitor of the PI3 kinase/Akt pathway effectively blocked the action of pI:C. Using adenoviral vectors containing a super-repressor of NF-,B (NF-,B SR) construct or a dominant negative interferon regulatory factor 3 (dnIRF3) construct showed that inhibition of both transcription factors also blocked the effects of pI:C. To explore the functional consequences of pI:C activation we used a pore-forming assay for P2X4R activity and a scrape loading assay for gap junction intercellular communication (GJIC). No pore-forming activity consistent with functional P2X4R expression was detected in either control or activated astrocytes. In contrast, robust Lucifer yellow transfer indicative of GJIC was detected in resting cells that was lost following pI:C activation. The dnIRF3 construct failed to restore GJIC whereas the NF-,B SR or the NF-,B inhibitor BAY11-7082 and the PI3K inhibitor LY294002 all significantly reversed the effect of pI:C on GJ connectivity. We conclude that activation of the innate immune response in astrocytes is associated with functional loss of GJIC through a pathway involving NF-,B and PI3 kinase. © 2006 Wiley-Liss, Inc. [source] Molecular analysis in Japanese patients with Charcot-Marie-Tooth disease: DGGE analysis for PMP22, MPZ, and Cx32/GJB1 mutations,HUMAN MUTATION, Issue 5 2002Chikahiko Numakura Abstract Charcot-Marie-Tooth disease (CMT) is a heterogeneous disorder and is traditionally classified into two major types, CMT type 1 (CMT1) and CMT type 2 (CMT2). Most CMT1 patients are associated with the duplication of 17p11.2-p12 (CMT1A duplication) and small numbers of patients have mutations of the peripheral myelin protein 22 (PMP22), myelin protein zero (MPZ), connexin 32 (Cx32/GJB1), and early growth response 2 (EGR2) genes. Some mutations of MPZ and Cx32 were also associated with the clinical CMT2 phenotype. We constructed denaturing gradient gel electrophoresis (DGGE) analysis as a screening method for PMP22, MPZ, and Cx32 mutations and studied 161 CMT patients without CMT1A duplication. We detected 27 mutations of three genes including 15 novel mutations; six of PMP22, three of MPZ, and six of Cx32. We finally identified 21 causative mutations in 22 unrelated patients and five polymorphic mutations. Eighteen of 22 patients carrying PMP22, MPZ, or Cx32 mutations presented with CMT1 and four of them with MPZ or Cx32 mutations presented with the CMT2 phenotype. DGGE analysis was sensitive for screening for those gene mutations, but causative gene mutation was not identified in many of the Japanese patients with CMT, especially with CMT1. Other candidate genes should be studied to elucidate the genetic basis of Japanese CMT patients. Hum Mutat 20:392,398, 2002. © 2002 Wiley-Liss, Inc. [source] Pyrosequencing for detection of mutations in the connexin 26 (GJB2) and mitochondrial 12S RNA (MTRNR1) genes associated with hereditary hearing loss,HUMAN MUTATION, Issue 4 2002Alessandro Ferraris Abstract Hereditary hearing loss (HHL) is one of the most common congenital disorders and is highly heterogeneous. Mutations in the connexin 26 (CX26) gene (GJB2) account for about 20% of all cases of childhood deafness, and approach 50% in documented recessive cases of non-syndromic hearing loss. In addition, a single mitochondrial DNA mutation, mt1555A>G, in the 12S rRNA gene (MTRNR1), is associated with familial cases of progressive deafness. Effective screening of populations for HHL necessitates rapid assessment of several of these potential mutation sites. Pyrosequencing links a DNA synthesis protocol for determining sequence to an enzyme cascade that generates light whenever pyrophosphate is released during primer strand elongation. We assessed the ability of Pyrosequencing to detect common mutations causing HHL. Detection of the most common CX26 mutations in individuals of Caucasian (35delG), Ashkenazi (167delT), and Asian (235delC, V37I) descent was confirmed by Pyrosequencing. A total of 41 different mutations in the CX26 gene and the mitochondrial mt1555A>G mutation were confirmed. Genotyping of up to six different adjacent mutations was achieved, including simultaneous detection of 35delG and 167delT. Accurate and reproducible results were achieved taking advantage of assay flexibility and experimental conditions easily optimized for a high degree of standardization and cost-effectiveness. The standardized sample preparation steps, including target amplification by PCR and preparation of single-stranded template combined with automated sequence reaction and automated genotype scoring, positions this approach as a potentially high throughput platform for SNP/mutation genotyping in a clinical laboratory setting. Hum Mutat 20:312,320, 2002. © 2002 Wiley-Liss, Inc. [source] Absence of deafness-associated connexin-26 (GJB2) gene mutations in the Omani population ,,HUMAN MUTATION, Issue 6 2001Mehmet Simsek Abstract We have investigated the prevalence of mutations in the connexin 26 (GJB2) gene in Omani population using both PCR-RFLP and direct DNA sequencing methods. Two common GJB2 gene mutations (35delG and 167delT) were screened in 280 healthy controls and 95 deaf patients using two different PCR-RFLP methods. To investigate other GJB2 mutations, we have amplified and sequenced DNA from 51 unrelated deaf patients and 17 control subjects. None of the samples studied, either by RFLP or sequencing, revealed any deafness-associated mutations in the coding region of the GJB2 gene. These findings disagree with many reports on the GJB2 gene, describing various mutations as the cause of congenital recessive deafness. Although, an amino acid substitution (S86T) was identified by sequencing, we conclude that this change could not be associated with deafness since it was present in all the control and patient samples sequenced. © 2001 Wiley-Liss, Inc. [source] Oleamide derivatives suppress the spontaneous metastasis by inhibiting connexin 26INTERNATIONAL JOURNAL OF CANCER, Issue 1 2007Yusuke Ohba Abstract We previously reported that overexpressing connexin 26 (Cx26) enhances the spontaneous metastasis of mouse BL6 melanoma cells. In contrast, daily intraperitoneal injections of an oleamide derivative named MI-18 potently inhibits the spontaneous metastasis of BL6 cells. In the present study, we chemically synthesized a novel oleamide derivative named MI-22 and found that it also efficiently suppressed the spontaneous metastasis of BL6 cells. Both MI-18 and MI-22 inhibited the gap junction-mediated intercellular communications (GJIC) that are formed between HeLa cells by the ectopic expression of the hCx26 and hCx32 human connexin subtypes; however, they had no effect on GJIC mediated by hCx40, hCx43 or hCx45. Fluorescently labeled MI-18 primarily localized not only at plasma membrane but also at Golgi/endosome. This suggests that this oleamide derivative may also act on the Cx26 molecules that accumulate in the Golgi/endosome because of their overexpression. Notably, neither derivative had a cytotoxic effect on HeLa cells when they were added into the tissue culture medium. Taken together, we propose that the MI-18 and MI-22 oleamide derivatives may serve as prototypes for novel and clinically important anticancer drugs. © 2007 Wiley-Liss, Inc. 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