Expression Of Gap Junction Proteins Research Articles

Aucubin, a natural iridoid glucoside, attenuates oxidative stress-induced testis injury by inhibiting JNK and CHOP activation via Nrf2 up-regulation

Eucommia ulmoides has been used for many years as a successful strategy to treat male infertility. Aucubin (AU) is the active ingredient extracted from Eucommia ulmoides. However, its protective action and exact mechanism on testicular injury is not yet known. Here, the protective effect and the mechanism of action of AU on testis damage under oxidative stress was investigated in vivo and in vitro. As regard the in vivo experiment, male mice were divided into five groups and testicular injury model was established by Triptolide (TP) (120 μg/kg) intraperitoneal injection for two weeks. Animals in the treatment group were pretreated with an intraperitoneal injection of AU at different doses (5, 10 and 20 mg/kg) for 1 h and subsequently treated with TP (120 μg/kg). At the end of the experimental period, the testis was collected for biochemical and histological examination. As regard the in vitro experiment, Sertoli cells (SCs) were used to investigate the protective effect and mechanism of action of AU against disruption of the blood-testis-barrier (BTB) and apoptosis induced by TP via apoptosis detection, western blot, immunofluorescence analysis, and siRNA transient transfection. TP-treated animals showed testicular atrophy, BTB disruption, increased ROS levels and spermatogenic dysfunction. Pre-administration of AU resulted in a significant protection on keeping a normal testicular weight, sperm morphology, BTB integrity, and a normal level of oxidative stress markers and antioxidants. Furthermore, AU prevented apoptosis through an effective inhibition of PERK/CHOP and JNK dependent apoptosis pathway, as well as protected the integrity of BTB by up-regulating the expression of tight junction proteins (ZO-1, Occludin, Claudin-11) and gap junction protein (Cx43). The mechanistic study revealed that AU significantly triggered Nrf2 translocation, thus increasing nuclear Nrf2 accumulation and then induced antioxidant enzymes expression in the testis and SCs. Furthermore, Nrf2 silencing unsuccessfully reversed the increased CHOP and p-JNK expression induced by TP, abolishing the protective effect of AU. These results indicate that AU might be considered as a potential protective agent against testicular injury.

Pacemaker cell characteristics of differentiated and HCN4-transduced human mesenchymal stem cells

AimsCell-based biological pacemakers aim to overcome limitations and side effects of electronic pacemaker devices. We here developed and tested different approaches to achieve nodal-type differentiation using human adipose- and bone marrow-derived mesenchymal stem cells (haMSC, hbMSC). Main methodshaMSC and hbMSC were differentiated using customized protocols. Quantitative RT-PCR was applied for transcriptional pacemaker-gene profiling. Protein membrane expression was analyzed by immunocytochemistry. Pacemaker current (If) was studied in haMSC with and without lentiviral HCN4-transduction using patch clamp recordings. Functional characteristics were evaluated by co-culturing with neonatal rat ventricular myocytes (NRVM). Key findingsCulture media-based differentiation for two weeks generated cells with abundant transcription of ion channel genes (Cav1.2, NCX1), transcription factors (TBX3, TBX18, SHOX2) and connexins (Cx31.9 and Cx45) characteristic for cardiac pacemaker tissue, but lack adequate HCN transcription. haMSC-derived cells revealed transcript levels, which were closer related to sinoatrial nodal cells than hbMSC-derived cells. To substitute for the lack of If, we performed lentiviral HCN4-transduction of haMSC resulting in stable If. Co-culturing with NRVM demonstrated that differentiated haMSC expressing HCN4 showed earlier onset of spontaneous contractions and higher beating regularity, synchrony and rate compared to co-cultures with non-HCN4-transduced haMSC or HCN4-transduced, non-differentiated haMSC. Confocal imaging indicated increased membrane expression of cardiac gap junctional proteins in differentiated haMSC. SignificanceBy differentiation haMSC, rather than hbMSC attain properties favorable for cardiac pacemaking. In combination with lentiviral HCN4-transduction, a cellular phenotype was generated that sustainably controls and stabilizes rate in co-culture with NRVM.

Neuroscience: The Hidden Diversity of Electrical Synapses

The complete description of the expression of gap junction proteins in the nervous system of the worm reveals a great complexity of their distribution amongst different neuronal classes, opening an unprecedented opportunity to expose the functional diversity of electrical synapses.

Dynamic UTR Usage Regulates Alternative Translation to Modulate Gap Junction Formation during Stress and Aging

SUMMARYConnexin43 (Cx43; gene name GJA1) is the most ubiquitously expressed gap junction protein, and understanding of its regulation largely falls under transcription and post-translational modification. In addition to Cx43, Gja1 mRNA encodes internally translated isoforms regulating gap junction formation, whose expression is modulated by TGF-β. Here, using RLM-RACE, we identify distinct Gja1 transcripts differing only in 5′ UTR length, of which two are upregulated during TGF-β exposure and hypoxia. Introduction of these transcripts into Gja1−/− cells phenocopies the response of Gja1 to TGF-β with reduced internal translation initiation. Inhibiting pathways downstream of TGF-β selectively regulates levels of Gja1 transcript isoforms and translation products. Reporter assays reveal enhanced translation of full-length Cx43 from shorter Gja1 5′ UTR isoforms. We also observe a correlation among UTR selection, translation, and reduced gap junction formation in aged heart tissue. These data elucidate a relationship between transcript isoform expression and translation initiation regulating intercellular communication.

MON-229 IRX3 and IRX5 Regulate Downstream Targets that Promote Ovarian Follicle Integrity in Mice

Appropriate cell-cell interactions and communication within ovarian follicles are required for their healthy development and maturation and are critical for maintaining female reproductive life quality. Our lab previously showed that two Iroquois transcription factor family genes, Irx3 and Irx5, were important for ovarian follicle integrity. Deletion of Irx3 and Irx5 (Irx3-Irx5EGFP/Irx3-Irx5EGFP, Irx3/5 DKO) resulted in disrupted granulosa cell-oocyte contacts, ectopic gap junction protein expression, and abnormal basement membrane morphology. The mechanisms by which Irx3 and Irx5 promote follicle integrity, however, are unknown. Therefore, we performed RNA-seq experiments on control and Irx3/5 DKO ovarian samples at two critical phases of ovarian development: embryonic day 13.5 when germline nests are established and just after birth when germline nests break down and primordial follicles form. RNA-seq analyses revealed that Inhbb, a TGF-β superfamily gene, was significantly upregulated in Irx3/5 DKO samples at both time points. It was previously reported that Inhbb was also upregulated in Wnt4 and β-catenin mutant ovaries. Notably, similar to the Irx3/5 DKO ovary, cell-cell interaction failure and germ cell death are prominent features of these models and removal of the excessive Inhbb by breeding to Inhbb knockout animals rescued the defects. These data suggest that Inhbb must be blocked to maintain germ cell and follicle survival. Based on the similarities in ovarian phenotype and Inhbb upregulation resulting from both Wnt4/β-catenin and Irx3/5 mutant models, we hypothesized that Irx3 and Irx5 mediate the Wnt4/β-catenin repression of Inhbb while they also regulate the expression of other downstream target genes required for cell contacts and germ cell health within follicles. First, we used gain- and loss-of-function gonad culture paradigms to manipulate β-catenin activity in ovaries and testes, respectively. Results showed that Irx3 and Irx5 expression levels were positively regulated by β-catenin signaling within the canonical Wnt4 pathway. Next, additional analysis of the RNA-seq data using the Database for Annotation, Visualization and Integrated Discovery (DAVID) showed that the differentially expressed genes (DEGs) in Irx3/5 DKO samples included factors critical to extracellular matrix, cell projections, actin-binding and cell adhesion. We are currently validating the DEGs relevant to our Irx3/5 DKO follicle defects and investigating their relationship to Inhbb in ovaries. Results from this study will help us to discover the means by which initial cell contacts are formed between support cells and oocytes in germline nests and new primordial follicles. These interactions provide the foundation for follicle integrity within the ovarian reserve and ultimately ensure that healthy follicles are available for recruitment and maturation.

Connexin 43 plays an important role in the transformation of cholangiocytes with Clonochis sinensis excretory-secretory protein and N-nitrosodimethylamine.

BackgroundClonorchis sinensis is a group I bio-carcinogen responsible for cholangiocarcinoma (CHCA) in humans. However, the mechanism by which C. sinensis promotes carcinogenesis is unclear.MethodologyUsing the human cholangiocyte line H69, we investigated cell proliferation and gap junction protein expression after stimulation with the hepatotoxin N-nitrosodimethylamine (NDMA) and/or excretory-secretory products (ESP) of C. sinensis, which induce inflammation. NDMA and ESP treatment increased proliferation by 146% and the proportion of cells in the G2/M phase by 37%. Moreover, the expression of the cell proliferation-related proteins E2F1, Ki-67, and cancer related protein cytokeratin 19 and Cox-2 increased in response to combined treatment with NDMA and ESP. The gap-junction proteins connexin (Cx) 43 and Cx26 increased. In contrast, Cx32 expression decreased in cells treated with NDMA and ESP. Silencing of Cx43 reduced cell proliferation and significantly suppressed Cx26 and Cox-2 expression.ConclusionsThese results suggest that Cx43 is an important factor in CHCA induced by C. sinensis ESP and NDMA and further investigations targeting this pathway may allow prevention of this deadly disease.

Adverse reactions analysis of Aconiti Lateralis Radix Praeparata and mechanism prediction of cardiac toxicity by network pharmacology

The aim of this paper was to provide reference for the clinical safety use of aconite through the retrieval of literature about adverse reactions,predict its mechanism of cardiac toxicity by using network pharmacology,and provide ideas for the studies on toxicity mechanism of toxic Chinese medicines. The papers on adverse reactions of aconite were searched to established a database and summarize the adverse reactions of aconite. The results of literature review showed that the main causes for adverse reactions in clinical use of aconite included overdose use,short cooking time,consumption of medicinal liquor/medicinal diet,external use and misuse and so on. Therefore,the dosage of aconite should be strictly followed in clinical application,and the decoction method should be notified to the patients in detail to avoid taking the medicinal liquor and diet containing aconite,so as to prevent the occurrence of adverse reactions as much as possible,and make the best use of aconite in clinical application in avoid its toxicity. At the same time,based on the results of literature review,the network construction and visual analysis of cardio toxicity produced by aconite were carried out by using the network pharmacology technologies. RESULTS: showed that aconite can be applied to eight biological processes such as action potential of cardiac myocytes,cardiac conduction-related cell signal transduction,cardiac myocytes contraction,action potential involved in cardiac myocytes contraction,and signal transduction from atrial myocardial cells to atrioventricular node cells,and three target genes(SCN5 A,GJA1,GJA5). It was predicted that Aconiti Lateralis Radix Praeparata may influence cardiomyocyte depolarization,intercellular information transmission and material exchange by acting on three target genes(SCN5 A,GJA1,GJA5) and regulating the sodium channel protein and the expression of gap junction protein,thus affecting the heart rhythm as well as its structure and function and causing cardiac toxicity.

Facets of Communication: Gap Junction Ultrastructure and Function in Cancer Stem Cells and Tumor Cells.

Gap junction proteins are expressed in cancer stem cells and non-stem cancer cells of many tumors. As the morphology and assembly of gap junction channels are crucial for their function in intercellular communication, one focus of our review is to outline the data on gap junction plaque morphology available for cancer cells. Electron microscopic studies and freeze-fracture analyses on gap junction ultrastructure in cancer are summarized. As the presence of gap junctions is relevant in solid tumors, we exemplarily outline their role in glioblastomas and in breast cancer. These were also shown to contain cancer stem cells, which are an essential cause of tumor onset and of tumor transmission into metastases. For these processes, gap junctional communication was shown to be important and thus we summarize, how the expression of gap junction proteins and the resulting communication between cancer stem cells and their surrounding cells contributes to the dissemination of cancer stem cells via blood or lymphatic vessels. Based on their importance for tumors and metastases, future cancer-specific therapies are expected to address gap junction proteins. In turn, gap junctions also seem to contribute to the unattainability of cancer stem cells by certain treatments and might thus contribute to therapeutic resistance.

Gap junction mediated signaling between satellite glia and neurons in trigeminal ganglia.

Peripheral sensory ganglia contain the somata of neurons mediating mechanical, thermal, and painful sensations from somatic, visceral, and oro-facial organs. Each neuronal cell body is closely surrounded by satellite glial cells (SGCs) that have properties and functions similar to those of central astrocytes, including expression of gap junction proteins and functional dye coupling. As shown in other pain models, after systemic pain induction by intra-peritoneal injection of lipopolysaccharide, dye coupling among SGCs in intact trigeminal ganglion was enhanced. Moreover, neuron-neuron and neuron-SGC coupling was also detected. To verify the presence of gap junction-mediated coupling between SGCs and sensory neurons, we performed dual whole cell patch clamp recordings from both freshly isolated and short term cultured cell pairs dissociated from mouse trigeminal ganglia. Bidirectional gap junction mediated electrical responses were frequently recorded between SGCs, between neurons and between neurons and SGCs. Polarization of SGC altered neuronal excitability, providing evidence that gap junction-mediated interactions between neurons and glia within sensory ganglia may contribute to integration of peripheral sensory responses, and to the modulation and coordinaton of neuronal activity.

76 Effects of serum type in maturation medium on in vitro development of bovine embryos

This study investigated the effect of bovine serum albumin (BSA), charcoal:dextran stripped fetal bovine serum (CDS FBS), and heat-inactivated FBS (HI FBS) in maturation medium on their ability to support in vitro oocyte maturation, cumulus cell-oocyte gap junctional communication, and development of bovine embryos. Charcoal:dextran treatment of FBS removes lipophilic chemicals, certain steroid hormones, and certain growth factors; however, HI FBS have a lot-to-lot variation in steroid hormones level that can affect the reproducibility of experimental findings. Oocytes were cultured in TCM-199 supplemented with either 8% (w/v) BSA, 10% (v/v) CDS FBS, or 10% (v/v) HI FBS and 1µg mL−1 oestradiol-17β, 10µg mL−1 FSH, 10ng mL−1 epidermal growth factor, 0.6mM cysteine, 0.2mM sodium pyruvate, and followed by IVF, and the zygotes were cultured in SOF-BE1 medium. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, mitochondrial activity, gene expression, immunocytochemistry, and cryo-tolerance. The differences in embryo development between experimental groups were analysed by 1-way ANOVA. The Duncan’s multiple range tests were used to test the differences between the treatments. The level of statistical significance was set at P<0.05. We have shown that CDS FBS significantly improved (P<0.05) the percentage of MII oocytes compared with that in the BSA supplemented group (77.2±1.0% v. 69.3%±2.3%, respectively). Moreover, CDS FBS had a higher significant (P<0.05) effect on the rate of blastocyst formation compared with HI FBS and BSA (45.2±0.7% v. 37.4±1.5% and 31.1±3.9%, respectively; 6 replicates were performed). Culture of oocytes with CDS FBS increased (P<0.05) the expression of gap junction proteins, CX37 and CX43, at both transcriptional and translation levels as determined by quantitative RT-PCR and immunofluorescence analysis, respectively. We also found that CDS FBS significantly increased total cell number and decreased the apoptotic index in Day-8 blastocysts compared with the BSA group. Furthermore, the beneficial effects of CDS FBS on embryos were associated with significantly reduced intracellular lipid content and increased mitochondrial activity in both oocytes and blastocysts as identified by Nile red and MitoTracker Green staining, respectively. Taken together, these data suggest that supplementation of maturation medium with CDS FBS, as an alternative to HI FBS, affected cumulus cell-oocyte gap junctional communication, and subsequently improved in vitro developmental competence of bovine oocytes and embryos. Research was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Agri-Bio industry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (grant numbers: 117029-3 and 315017-5).

Photoperiod modulates oestrogen status, insulin interposed glucose uptake and connexin-43 in testes of golden hamster, Mesocricetus auratus

ABSTRACT The environmental photoperiod plays an important role in regulation of seasonal reproductive events. However, with reference tooestrogen status the exact mechanism involved in testicular seasonality is still unclear in general and especially in long-day breeder golden hamster, Mesocricetus auratus. Therefore, in the present study we explored the effect of day length (long day, LD and short day, SD) in modulation of oestrogen level, metabolic and gap junction markers expressionin testes. Our results suggest that LD exposureinduced normal testicular activity as evident by histoarchitectural analyses showing different stages of spermatogenesis, spermatozoa filled lumen and normal Leydig cells. Furthermore, LD upregulated testicular metabolic parameters such as IR, GLUT-8, glucose leveland LDH activity along with predominant testicular gap junction protein (Connexin-43) expression as compared to SD. On the other hand, the expressions of ER-α, aromatase and caspase-3 were reduced under LD condition as compared to SD. Thus, it can be inferred that photoperiod modulates the circulatoryoestrogen level, along with gap junction protein and apoptosis markers expression in testes and harmonizes the energy homeostasis involved in reproductive maintenance for seasonality of golden hamster. Therefore, we may conclude that day-length-mediatedoestrogen is a prominent modulator of male reproductive health. Abbreviations Insulin receptor (IR); Glucose uptake transporter-8 (GLUT-8); Estrogen receptor-alpha (ER-α); Connexin-43 (Cxn-43); Lactate dehydrogenase (LDH)

Increased myoendothelial feedback is associated with increased connexin37 and IK1 channel expression in mesenteric arteries of diet-induced hyperhomocysteinemic mice.

Previously, we found that diet-induced HHcy in mice caused decreased eNOS expression and signaling in mesenteric arteries, but greatly enhanced non-NOS, non-prostacyclin-dependent vasodilation, which involves MEJ communication. To further assess whether HHcy enhances MEJ communication, this study examined endothelium-dependent attenuation of phenylephrine-induced vasoconstriction (myoendothelial feedback) and key molecules involved. Myoendothelial feedback was examined in isolated mouse mesenteric arteries, after 6-weeks diet-induced HHcy, using pressure myography. Gap junction (Cx37, Cx40, Cx43), NOS (eNOS, nNOS, iNOS), and potassium channel (IK1) protein expression were measured with immunoblots, and connexin mRNAs with real-time PCR. Contribution of nNOS+iNOS to vasomotor responses was assessed using the drug TRIM. Myoendothelial feedback was significantly (P<.05) enhanced in HHcy arteries compared to control, coincident with significantly greater Cx37 and IK1 protein and Cx37 mRNA. Cx43 protein, but not mRNA, was significantly less in HHcy, and Cx40 was not different. eNOS protein was significantly less in HHcy. nNOS and iNOS were not different. TRIM had little effect on vasomotor function. Diet-induced HHcy enhanced myoendothelial feedback, and increased Cx37 and IK1 expression may contribute. nNOS or iNOS did not upregulate to compensate for decreased eNOS, and they had little involvement in vasomotor function.

Cholesterol metabolism and Cx43, Cx46, and Cx50 gap junction protein expression and localization in normal and diabetic and obese ob/ob and db/db mouse testes.

Decreased fertility and birth rates arise from metabolic disorders. This study assesses cholesterol metabolism and Cx46, Cx50, and Cx43 expression in interstitium- and seminiferous tubule-enriched fractions of leptin-deficient ( ob/ob) and leptin receptor-deficient ( db/db) mice, two type 2 diabetes and obesity models associated with infertility. Testosterone levels decreased and glucose and free and esterified cholesterol (FC and EC) levels increased in serum, whereas FC and EC levels decreased in the interstitium, in ob/ob and db/db mice. In tubules, a decrease in EC caused FC-to-EC ratios to increase in db/db mice. In tubules, only acyl coenzyme A:cholesterol acyl transferase type 1 and 2 protein levels significantly decreased in ob/ob, but not db/db, mice compared with wild-type mice, and imbalances in the cholesterol transporters Niemann-Pick C1 (NPC1), ATP-binding cassette A1 (ABCA1), scavenger receptor class B member I (SR-BI), and cluster of differentiation 36 (CD36) were observed in ob/ob and db/db mice. In tubules, 14-kDa Cx46 prevailed during development, 48- to 49- and 68- to 71-kDa Cx46 prevailed during adulthood, and total Cx46 changed little. Compared with wild-type mice, 14-kDa Cx46 increased, whereas 48- to 49- and 68- to 71-kDa Cx46 decreased, in tubules, whereas the opposite occurred in the interstitium, in db/db and ob/ob mice. Total and 51-kDa Cx50 increased in db/db and ob/ob interstitium and tubules. Cx43 levels decreased in ob/ob interstitium and tubules, whereas Cx43 decreased in db/db interstitium but increased in db/db tubules. Apoptosis levels measured by ELISA and numbers of apostain-labeled apoptotic cells significantly increased in db/db, but not ob/ob, tubules. Testicular db/db capillaries were Cx50-positive but weakly Cx43-positive with a thickened lamina, suggesting altered permeability. Our findings indicate that the db mutation-induced impairment of meiosis may arise from imbalances in cholesterol metabolism and upregulated Cx43 expression and phosphorylation in tubules.

Subcellular reorganization and altered phosphorylation of the astrocytic gap junction protein connexin43 in human and experimental temporal lobe epilepsy.

Dysfunctional astrocytes are increasingly recognized as key players in the development and progression of mesial temporal lobe epilepsy (MTLE). One of the dramatic changes astrocytes undergo in MTLE with hippocampal sclerosis (HS) is loss of gap junction coupling. To further elucidate molecular mechanism(s) underlying this alteration, we assessed expression, cellular localization and phosphorylation status of astrocytic gap junction proteins in human and experimental MTLE-HS. In addition to conventional confocal analysis of immunohistochemical staining we employed expansion microscopy, which allowed visualization of blood-brain-barrier (BBB) associated cellular elements at a sub-µm scale. Western Blot analysis showed that plasma membrane expression of connexin43 (Cx43) and Cx30 were not significantly different in hippocampal specimens with and without sclerosis. However, we observed a pronounced subcellular redistribution of Cx43 toward perivascular endfeet in HS, an effect that was accompanied by increased plaque size. Furthermore, in HS Cx43 was characterized by enhanced C-terminal phosphorylation of sites affecting channel permeability. Prominent albumin immunoreactivity was found in the perivascular space of HS tissue, indicating that BBB damage and consequential albumin extravasation was involved in Cx43 dysregulation. Together, our results suggest that subcellular reorganization and/or abnormal posttranslational processing rather than transcriptional downregulation of astrocytic gap junction proteins account for the loss of coupling reported in human and experimental TLE. The observations of the present study provide new insights into pathological alterations of astrocytes in HS, which may aid in the identification of novel therapeutic targets and development of alternative anti-epileptogenic strategies.

Gap Junction Remodeling in a Novel Engineered Heart Tissue System Cultured under Point Stimulation

Recent advances have shown the importance of electrical stimulation in enhancing maturation and differentiation of engineered heart tissues (EHTs). Conventional electrical maturation protocols pace their EHTs with field stimulation - the tissues are paced simultaneously in a uniform electrical field. This method has been shown to improve cellular alignment, increase contractility, and increase gap junction (Cx43) production. While Cx43 production is increased, the anisotropic ratio is not fully preserved in these culture systems [Trantidou et al, PMID 4459200]. Native cardiac tissue demonstrates an anisotropic propagation of action potHential; in adult rat ventricles the conduction velocity (CV) is 3x faster in the longitudinal direction than the transverse direction [Zimmermann et al, PMID 16582915]. We hypothesize that the loss of anisotropy is due to field stimulation not accurately recapitulating the conditions caused by a depolarizing wave traveling through the tissue. In order to better reproduce native myocardium, we designed a bioreactor which applies a point stimulation at one end of our EHT and allows the wave to propagate down the tissue. We hypothesize that this propagating wave will provide the necessary signals to enhance Cx43 production as well as improve localization of gap junction proteins. Maturity was assessed by the measurement of twitch kinetics, peak force, and conduction velocity. We anticipate that establishing a more realistic expression and subcellular localization of gap junction proteins will improve the utility of future studies involving cell lines from patients with arrhythmogenic cardiomyopathies.

Structural and molecular pathology of the atrium in boxer arrhythmogenic right ventricular cardiomyopathy

ObjectiveTo investigate the expression and distribution of desmosomal and gap junction proteins of the intercalated disc in the atria of boxers with arrhythmogenic right ventricular cardiomyopathy (ARVC). AnimalsNineteen control dogs and 13 boxers with histopathologically confirmed ARVC. MethodsRight and left atrial samples were examined using immunofluorescence and Western blots. The intercalated disc proteins investigated included total and phosphorylated connexin43 (Cx43 and pCx43), connexin45, connexin40, plakoglobin, plakophilin-2, desmoplakin, and N-cadherin. ResultsHistopathological changes characteristic of ARVC were present in the left or right atrium of 12 out of 13 boxers and were absent in all control dogs. When compared to the 19 control dogs, immunofluorescence analysis revealed a decrease in signal intensity for pCx43 and plakoglobin in the left (p = 0.03 and p = 0.014, respectively) and right atrium (p = 0.015 and p = 0.002, respectively) of affected boxers. Connexin43 and pCx43 Western blot band density was significantly decreased in the left (p = 0.025 and p = 0.027, respectively) and right atrium (p = 0.001 and p = 0.044, respectively) of affected boxers. ConclusionAltered intercalated disc and gap junction proteins were identified in atrial myocardium of ARVC boxers, supporting atrial involvement as part of this disorder. Reduction in pCx43 in conjunction with histological changes could represent the substrate for atrial arrhythmias associated with ARVC. Furthermore, these findings detected in boxer dogs, lend support for the broader term, arrhythmogenic cardiomyopathy, as preferred nomenclature used to describe this disease in humans.

In vivo and in vitro maturation of rabbit oocytes differently affects the gene expression profile, mitochondrial distribution, apoptosis and early embryo development.

In vivo-matured cumulus-oocyte complexes are valuable models in which to assess potential biomarkers of rabbit oocyte quality that contribute to enhanced IVM systems. In the present study we compared some gene markers of oocytes and cumulus cells (CCs) from immature, in vivo-matured and IVM oocytes. Moreover, apoptosis in CCs, nuclear maturation, mitochondrial reallocation and the developmental potential of oocytes after IVF were assessed. In relation to cumulus expansion, gene expression of gap junction protein, alpha 1, 43 kDa (Gja1) and prostaglandin-endoperoxide synthase 2 (Ptgs2) was significantly lower in CCs after in vivo maturation than IVM. In addition, there were differences in gene expression after in vivo maturation versus IVM in both oocytes and CCs for genes related to cell cycle regulation and apoptosis (V-Akt murine thymoma viral oncogene homologue 1 (Akt1), tumour protein 53 (Tp53), caspase 3, apoptosis-related cysteine protease (Casp3)), oxidative response (superoxide dismutase 2, mitochondrial (Sod2)) and metabolism (glucose-6-phosphate dehydrogenase (G6pd), glyceraldehyde-3-phosphate dehydrogenase (Gapdh)). In vivo-matured CCs had a lower apoptosis rate than IVM and immature CCs. Meiotic progression, mitochondrial migration to the periphery and developmental competence were higher for in vivo-matured than IVM oocytes. In conclusion, differences in oocyte developmental capacity after IVM or in vivo maturation are accompanied by significant changes in transcript abundance in oocytes and their surrounding CCs, meiotic rate, mitochondrial distribution and apoptotic index. Some of the genes investigated, such as Gja1, could be potential biomarkers for oocyte developmental competence in the rabbit model, helping improve in vitro culture systems in these species.

Astrocyte sigma-1 receptors modulate connexin 43 expression leading to the induction of below-level mechanical allodynia in spinal cord injured mice

We have previously shown using a spinal cord injury (SCI) model that gap junctions contribute to the early spread of astrocyte activation in the lumbar spinal cord and that this astrocyte communication plays critical role in the induction of central neuropathic pain. Sigma-1 receptors (Sig-1Rs) have been implicated in spinal astrocyte activation and the development of peripheral neuropathic pain, yet their contribution to central neuropathic pain remains unknown. Thus, we investigated whether SCI upregulates spinal Sig-1Rs, which in turn increase the expression of the astrocytic gap junction protein, connexin 43 (Cx43) leading to the induction of central neuropathic pain. A thoracic spinal cord hemisection significantly increased both astrocyte activation and Cx43 expression in lumbar dorsal horn. Sig-1Rs were also increased in lumbar dorsal horn astrocytes, but not neurons or microglia. Intrathecal injection of an astrocyte metabolic inhibitor (fluorocitrate); a gap junction/hemichannel blocker (carbenoxolone); or a Cx43 mimetic peptide (43Gap26) significantly reduced SCI-induced bilateral below-level mechanical allodynia. Blockade of Sig-1Rs with BD1047 during the induction phase of pain significantly suppressed the SCI-induced development of mechanical allodynia, astrocyte activation, increased expression of Cx43 in both total and membrane levels, and increased association of Cx43 with Sig-1R. However, SCI did not change the expression of oligodendrocyte (Cx32) or neuronal (Cx36) gap junction proteins. These findings demonstrate that SCI activates astrocyte Sig-1Rs leading to increases in the expression of the gap junction protein, Cx43 and astrocyte activation in the lumbar dorsal horn, and ultimately contribute to the induction of bilateral below-level mechanical allodynia.

Ultrastructural demonstration of Cx43 gap junctions in induced pluripotent stem cells from human cord blood.

Gap junction proteins are essential for direct intercellular communication but also influence cellular differentiation and migration. The expression of various connexin gap junction proteins has been demonstrated in embryonic stem cells, with Cx43 being the most intensely studied. As Cx43 is the most prominent gap junction protein in the heart, cardiomyocyte-differentiated stem cells have been studied intensely. To date, however, little is known about the expression and the subcellular distribution of Cx43 in undifferentiated stem cells or about the structural arrangement of channels. We, therefore, here investigate expression of Cx43 in undifferentiated human cord-blood-derived induced pluripotent stem cells (hCBiPS2). For this purpose, we carried out quantitative real-time PCR and immunohistochemistry. For analysis of Cx43 ultrastructure and protein assembly, we performed freeze-fracture replica immunogold labeling (FRIL). Cx43 expression was detected at mRNA and protein level in hCBIPS2 cells. For the first time, ultrastructural data are presented on gap junction morphology in induced pluripotent stem (iPS) cells from cord blood: Our FRIL and electron microscopical analysis revealed the occurrence of gap junction plaques in undifferentiated iPS cells. In addition, these gap junctions were shown to contain the gap junction protein Cx43.

Beta Lactams Antibiotic Ceftriaxone Modulates Seizures, Oxidative Stress and Connexin 43 Expression in Hippocampus of Pentylenetetrazole Kindled Rats.

Background and Purpose:This study aimed to investigate the effect of ceftriaxone on oxidative stress and gap junction protein (connexin 43, Cx-43) expression in pentylenetetrazole (PTZ) induced kindling model.Methods:Twenty four Sprague dawely rats were divided into 3 equal groups (a) normal group: normal rats. (b) PTZ kindled group: received PTZ at the dose of 50 mg/kg via intraperitoneal injection (i.p.) every other day for 2 weeks (c) ceftriaxone treated group: received ceftriaxone at the dose 200 mg\\kg/12 hrs via i.p. injection daily from the 6th dose of PTZ for 3 days. Racine score, latency before beginning the first myoclonic jerk and duration of the jerks used as parameters of behavioral assessment. Immunohistopathological study for Cx-43 expression in hippocampus and measurement of markers of oxidative stress (malondialdehyde [MDA], low reduced glutathione [GSH] and catalase [CAT]) in hippocampal neurons were done.Results:PTZ kindling was associated with behavioral changes (in the form high stage of Racine score, long seizure duration and short latency for the first jerk), enhanced oxidative stress state (as demonstrated by high MDA, low GSH and CAT) and up regulation of Cx43 in hippocampal regions. While, ceftriaxone treatment ameliorated, significantly, PTZ-induced convulsions and caused significant improvement in oxidative stress markers and Cx-43 expression in hippocamal regions (p < 0.05).Conclusions:These findings support the anticonvulsive effects of some beta-lactams antibiotics which could offer a possible contributor in the basic treatment of temporal lobe epilepsy. This effect might be due to reduction of oxidative stress and Cx43 expression.