Previous studies have provided evidence for the transcripts of Cx43 and Cx45 within pancreatic islets. As of yet, however, it has proven difficult to unambiguously demonstrate the expression of these proteins by islet cells. We have investigated whether Cx36, a new connexin species recently identified in mammalian brain and retina, may also be expressed in pancreatic islets. Using probes that permitted the original identification of Cx36 in the central nervous system, we show that a transcript for Cx36 is clearly detectable in rat pancreatic islets. Using novel and affinity-purified polyclonal antibodies, we have found that Cx36 is actually expressed in pancreatic islets. Both in situ hybridization and immunolabeling indicated that this connexin is abundant in the centrally located insulin-producing

The identification of connexins (Cxs) expressed in neuronal cells represents a crucial step for understanding the direct communication between neurons and between neuron and glia. In the present work, using a double-labelling method combining in situ hybridization for Cx mRNAs with immunohistochemical detection for neuronal markers, we provide evidence that, among cerebral connexins (Cx26, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45 and Cx47), only Cx45 and Cx36 mRNAs are localized in neuronal cells in both developing and adult rat brain. In order to establish whether connexin expression is influenced in vivo by abnormal neuronal activity, we examined the short-term effects of kainate-induced seizures. The results revealed an unexpected expression of Cx26 and Cx45 mRNA in neuronal cells undergoing apoptotic cell death in the CA3-CA4, in the hilus of the hippocampus and in other brain regions involved in seizure-induced lesion. However, the expression of Cx26 and Cx45 mRNAs was not associated with detectable expression of corresponding proteins as evaluated by immunohistochemistry with specific antibodies. Moreover, in the same brain regions Cx32 and Cx43 were up-regulated in non-neruronal cells whereas the neuronal Cx36 was down-regulated. Taken together the present results provide novel information regarding the specific subpopulation of neurons expressing Cx45 and raise the question of the meaning of connexin mRNA expression in the neuronal apoptotic process.

Copper homeostasis and distribution is strictly regulated by a network of transporters and intracellular chaperones encoded by a group of genes collectively known as copper homeostasis genes ( CHG s). In this work, analysis of The Cancer Genome Atlas database for somatic point mutations in colorectal cancer revealed that inactivating mutations are absent or extremely rare in CHG s. Using oligonucleotide microarrays, we found a strong increase in mRNA levels of the membrane copper transporter 1 protein [ CTR 1; encoded by the solute carrier family 31 member 1 gene ( SLC 31A1 gene)] in our series of colorectal carcinoma samples. CTR 1 is the main copper influx transporter and changes in its expression are able to induce modifications of cellular copper accumulation. The increased SLC 31A1 mRNA level is accompanied by a parallel increase in transcript levels for copper efflux pump ATP 7A, copper metabolism Murr1 domain containing 1 ( COMMD 1), the cytochrome C oxidase assembly factors [synthesis of cytochrome c oxidase 1 ( SCO 1) and cytochrome c oxidase copper chaperone 11 ( COX 11)], the cupric reductase six transmembrane epithelial antigen of the prostate ( STEAP 3), and the metal‐regulatory transcription factors ( MTF 1, MTF 2) and specificity protein 1 ( SP 1). The significant correlation between SLC 31A1 , SCO 1 , and COX 11 mRNA levels suggests that this transcriptional upregulation might be part of a coordinated program of gene regulation. Transcript‐level upregulation of SLC 31A1 , SCO 1 , and COX 11 was also confirmed by the analysis of different colon carcinoma cell lines (Caco‐2, HT 116, HT 29) and cancer cell lines of different tissue origin ( MCF 7, PC 3). Finally, exon‐level expression analysis of SLC 31A1 reveals differential expression of alternative transcripts in colorectal cancer and normal colonic mucosa.

Rationale: Gap junctions are membrane channels formed by an array of connexins which links adjacent cells realizing an electro-metabolic synapse. Connexin-mediated communication is crucial in the regulation of cell growth, differentiation, and development. The activation and proliferation of phenotypically altered fibroblasts are central events in the pathogenesis of idiopathic pulmonary fibrosis. We sought to evaluate the role of connexin-43, the most abundant gap-junction subunit in the human lung, in the pathogenesis of this condition. Methods:We investigated the transcription and protein expression of connexin-43 and the gapjunctional intercellular communication (GJIC) in 5 primary lung fibroblast lines derived from normal subjects (NF) and from 3 histologically proven IPF patients (FF). Results:Here we show that connexin-43 mRNA was significantly reduced in FF as demonstrated by standard and quantitative RT-PCR. GJIC was functionally evaluated by means of flow-cytometry. In order to demonstrate that dye spreading was taking place through gap junctions, we used carbenoxolone as a pharmacological gap-junction blocker. Carbenoxolone specifically blocked GJIC in our system in a concentration dependent manner. FF showed a significantly reduced homologous GJIC compared to NF. Similarly, GJIC was significantly impaired in FF when a heterologous NF line was used as dye donor, suggesting a complete defect in GJIC of FF. Conclusion:These results suggest a novel alteration in primary lung fibroblasts from IPF patients. The reduced Cx43 expression and the associated alteration in cell-to-cell communication may justify some of the known pathological characteristic of this devastating disease that still represents a challenge to the medical practice.

We have identified a novel gap junction gene by searching the human genome sequence database that encodes a protein designated as connexin31.9 (Cx31.9). Cx31.9 was most homologous to human Cx32.4 and did not cluster with either the purported alpha- or beta-connexin subfamilies. Expression of Cx31.9 was detected by RT-PCR in human mRNA from several tissues including cerebral cortex, heart, liver, lung, kidney, spleen, and testis. A partial Cx31.9 sequence was also represented in the human Expressed Sequence Tag database. Cx31.9 formed intercellular channels in both paired Xenopus oocytes and transfected neuroblastoma N2A cells that were distinguished by an apparent low unitary conductance (12-15 pS) and a remarkable insensitivity to transjunctional voltage. In contrast, Cx31.9 channels were gated by cytoplasmic acidification or exposure to halothane like other connexins. Cx31.9 was able to form heterotypic channels with the highly voltage-sensitive Xenopus Cx38 (XenCx38), which provides an opportunity to study gating in heterotypic channels formed by hemichannels (connexons) composed of connexins with widely divergent properties. Thus Cx31.9 is a novel human connexin that forms channels with unique functional properties.

Rat connexin-36 (Cx36) is the first gap junction protein shown to be expressed predominantly in neuronal cells of the mammalian central nervous system. As a prerequisite for studies devoted to the investigation of the possible role of this connexin in human neurological diseases, we report the cloning and sequencing of the human Cx36 gene, its chromosomal localization, and its pattern of expression in the human brain analyzed by radioactive in situ hybridization. The determination of the human gene sequence revealed that the coding sequence of Cx36 is highly conserved (98% identity at the protein level with the mouse and rat Cx36 and 80% with the ortholog perch and skate Cx35), and that the gene structure is that typical of the Cx35/36 subgroup observed in the other species (presence of a single intron located within the coding region, 71 bp after the translation initiation site). The distribution of Cx36 in several regions of the human central nervous system is similar to that previously observed in rat brain. The most intense signal among the cerebral areas examined by in situ hybridization was observed in the inferior olivary complex, both in principal and accessory nuclei. A moderate labeling was also observed in several myelencephalic nuclei, in specific cells of the the cerebellar cortex, in a relatively large subpopulation of cells in the cerebral cortex, in the hilus of the dentate gyrus, and in the strata radiatum and oriens of hippocampal subfields. Moreover, labeled cells were revealed in all the lamina of the spinal cord gray matter. The chromosomal localization of the human Cx36 gene was determined by fluorescence in situ hybridization. The results allowed assignment of the gene to band 15q14, thus making it a possible candidate gene for a form of familial epilepsy previously linked to the same chromosomal band. The knowledge of the human Cx36 gene sequence, of its chromosomal localization, and of its pattern of expression opens new avenues for the analysis of its possible involvement in human genetic and acquired neuropathology.

The secretory, duct, connective and vascular cells of pancreas are connected by gap junctions, made of different connexins. The insulin-producing beta-cells, which form the bulk of endocrine pancreatic islets, express predominantly Cx36. To assess the function of this connexin, we have first studied its expression in rats, during sequential changes of pancreatic function which were induced by the implantation of a secreting insulinoma. We observed that changes in beta-cell function were paralleled by changes in Cx36 expression. We have also begun to investigate mutant mice lacking Cx36. The absence of this protein did not affect the development and differentiation of beta-cells but appeared to alter their secretion. We have studied this effect in MIN6 cells which spontaneously express Cx36. After stable transfection of a construct that markedly reduced the expression of this connexin, we observed that MIN6 cells were no more able to secrete insulin, in contrast to wild type controls, and differentially displayed a series of still unknown genes. The data provide evidence that Cx36-dependent signaling contributes to regulate the function of native and tumoral insulin-producing cells.

By combining in silico and bench molecular biology methods we have identified a novel human gap junction gene that encodes a protein designated HCx31.9. We have determined its human chromosomal location and gene structure, and we have identified a putative mouse ortholog, mCx30.2. We have observed the presence of HCx31.9 in human cerebral cortex, liver, heart, spleen, lung, and kidney and the presence of mCx30.2 in mouse cerebral cortex, liver and lung. Moreover, preliminary data on the electrophysiological properties of HCx31.9 have been obtained by functional expression in paired Xenopus oocytes and in transfected N2A cells.