OBJECTIVETo explore interaction proteins affect functions of connexin 30 (Cx30) by screening and identification interaction proteins of Cx30.

METHODSThe fusion expression vecto of CX30-C-terminal functional domain-pGEX-4T-2-GST was constructed, and then, fusion protein and GST were purified. They were incubated with the proteins of the foetus brain tissue disruption to pull down interaction proteins. The interaction proteins were separated by SDS-PAGE. Differential straps were cut to enzymolysis to prepare for mass chromatographic analysis, and then to index and screen interaction proteins in NCBInr database. The interaction proteins were identified by immunolocalization.

RESULTSThe four interaction proteins of Cx30 were screened in the foetus brain tissue, as follow, Keratin 16, Camk2b, Tubulin beta-3 and alpha-tubulin. Cx30 was proved to coexist with Keratin 16 and Tubulin beta-3.

CONCLUSIONSKeratin 16, Camk2b, Tubulin beta-3 and alpha-tubulin are the interaction proteins of Cx30. The interaction proteins affect the assembly, intracellular transport, and channel switch of Cx30.

Connexin 30 ; Connexins ; genetics ; metabolism ; Genetic Vectors ; Glutathione Transferase ; Humans ; Mutagenicity Tests ; Protein Interaction Mapping ; Recombinant Proteins ; genetics ; metabolism

Connexin 43 ; genetics ; Connexins ; genetics ; Heart Defects, Congenital ; genetics ; Homeobox Protein Nkx-2.5 ; Homeodomain Proteins ; genetics ; Humans ; Infant ; Infant, Newborn ; Mutation ; genetics ; Myocardium ; metabolism ; Transcription Factors ; genetics

OBJECTIVETo observe the collagen spatial distribution, collagen volume fraction (CVF) and Cx40, Cx43mRNA expressions in rapid atrial pacing dogs post vagal denervation by removing fat pad located between the medial superior vena cava and aortic root (SVC-Ao fat pad).

METHODSTwenty-four dogs were randomly divided into unpaced sham operation group (S group, n = 8), Keeping SVC-Ao fat pad group (K group, n = 8) and Removing SVC-Ao fat pad group (R group, SVC-Ao fat pad was removed by surgical excision before pacing, n = 8). K and R groups were paced for six weeks. Six weeks later, all dogs were sacrificed, left atrium (LA), right atrium (RA), left atrial appendage (LAA), right atrial appendage (RAA) and atrial septum (AS) were collected and stained with HE or Masson Trichrome or frozen in liquid nitrogen for quantifying the expression of Cx40, Cx43 mRNA by Real-time quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR).

RESULTSSpatial distribution of collagen fibers as well as CVF between S and R group were similar (all P > 0.05). CVF was significantly higher in K group compared to R group, especially at LAA and AS locations (all P < 0.05). Cx40mRNA expression in K group was significantly decreased in LA, RA, and significantly increased in LAA, RAA and AS compared those in S group (all P < 0.05), significantly lower in LA and RA while significantly higher in LAA and RAA compared to R group (all P < 0.05). Cx43mRNA expression in K group was significantly reduced in LA, RA, LAA and RAA while significantly increased in AS compared to S group (all P < 0.05), significantly higher in LA, RA, RAA and AS while significantly lower in LAA compared to R group.

CONCLUSIONPacing induced collagen remodeling and modulation on Cx40mRNA and Cx43 mRNA expressions could be partially attenuated by removing SVC-Ao fat pad suggesting vagal denervation plays a key role in the initiation and preservation of atrial fibrillation.

Animals ; Atrial Fibrillation ; metabolism ; pathology ; Cardiac Pacing, Artificial ; Collagen ; metabolism ; Connexin 43 ; metabolism ; Connexins ; metabolism ; Dogs ; Heart Atria ; metabolism ; pathology ; RNA, Messenger ; genetics

OBJECTIVETo observe the influence of Cx26/Cx32 gap junction channel on the antineoplastic effect of etoposide in Hela cervical cancer cells.

METHODSFluorescence trace was used to assay the gap junction intercellular communication mediated by Cx26/Cx32 in Hela cells and its functional modulation by the pharmacological agents (oleamide, retinoid acid). A standard colony-forming assay was applied to determine the cell growth-inhibiting effect of etoposide in Hela cells with functional modulation of the gap junction. Hoechst 33258 staining was used to assess the changes in etoposide-induced apoptosis of Hela cells with altered gap junction functions.

RESULTSOleamide markedly decreased while retinoid acid obviously increased the gap junction function in Hela cells. Standard colony-forming assay showed that etoposide produced a lowered antiproliferative effect in Hela cells with reduced gap junction and an increased antiproliferative effect in cells with enhanced gap junction function. In cells with a reduced gap junction function, etoposide induced a lowered apoptosis rate, which increased obviously in cells with an enhanced gap junction function.

CONCLUSIONThe antineoplastic effect of etoposide is reduced in Hela cells with a decreased gap junction intercellular communication mediated by Cx26/Cx32 and is enhanced in cells with an increased gap junction intercellular communication.

Antineoplastic Agents, Phytogenic ; pharmacology ; Connexin 26 ; Connexins ; genetics ; metabolism ; physiology ; Etoposide ; pharmacology ; Gap Junctions ; physiology ; HeLa Cells ; Humans ; Transfection

Animals ; Cell Communication ; Cell Transformation, Neoplastic ; Connexins ; genetics ; metabolism ; Cytoplasm ; metabolism ; Gap Junctions ; chemistry ; classification ; metabolism ; physiology ; Gene Expression Regulation, Neoplastic ; Humans ; Mutation ; Neoplasms ; etiology ; metabolism ; pathology

Hearing impairment (HI) affects 1/1000 children and over 2% of the aged population. We have previously reported that mutations in the gene encoding gap junction protein connexin-31 (C×31) are associated with HI. The pathological mechanism of the disease mutations remains unknown. Here, we show that expression of C×31 in the mouse inner ear is developmentally regulated with a high level in adult inner hair cells and spiral ganglion neurons that are critical for the hearing process. In transfected cells, wild type C×31 protein (C×31wt) forms functional gap junction at cell-cell-contacts. In contrast, two HI-associated C×31 mutants, C×31R180X and C×31E183K resided primarily in the ER and Golgi-like intracellular punctate structures, respectively, and failed to mediate lucifer yellow transfer. Expression of C×31 mutants but not C×31wt leads to upregulation of and increased association with the ER chaperone BiP indicating ER stress induction. Together, the HI-associated C×31 mutants are impaired in trafficking, promote ER stress, and hence lose the ability to assemble functional gap junctions. The study reveals a potential pathological mechanism of HI-associated C×31 mutations.
Animals ; Connexins ; genetics ; Ear, Inner ; metabolism ; Endoplasmic Reticulum ; physiology ; Gap Junctions ; genetics ; metabolism ; physiology ; Golgi Apparatus ; genetics ; metabolism ; Hearing Loss ; genetics ; metabolism ; pathology ; Mice ; Mutation ; Neurons ; metabolism ; Protein Transport ; genetics ; Stress, Physiological

OBJECTIVE: To investigate the expressions of tumor suppressor gene CX26 mRNA and coding protein in laryngeal squamous cell carcinoma, and to explore the relationship between CX26 gene and the biological behaviors of laryngeal squamous cell carcinoma for understanding the tumorigenicity and development of laryngeal squamous cell carcinoma. METHOD: Laryngeal carcinoma tissues (studying group), which takeda from the center of tumors and laryngeal normal tissues (control group) takeda at the place of 1.0 cm out of the edge of the tumors, were took from 38 patients with laryngeal squamous cell carcinoma while they were in operation. Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to analyze the expression level of CX26 mRNA, and immunohistochemical staining (frozen section) was used to detect the expression of CX26 protein in laryngeal carcinoma tissues and laryngeal normal tissues of 38 cases, respectively. RESULT: mRNA of CX26 gene was all positively expressed in laryngeal carcinoma tissues and laryngeal normal tissues of 38 cases by RT-PCR. However, CX26 mRNA was obviously down-regulated in laryngeal carcinoma tissues than that in laryngeal normal tissues (P < 0.05). Immunohistochemical staining showed CX26 protein was strong-positively expressed in laryngeal normal tissues in 34 cases (89.5%), while it was positively expressed in laryngeal carcinoma tissues in 18 cases (47.4%), and with the location alteration of CX26 protein in laryngeal carcinoma cells. There was significant difference between the expression rate of CX26 protein in laryngeal carcinoma tissues and in laryngeal normal tissues (P < 0.05). Meanwhile, the expression level of CX26 mRNA and the positive-expressed rate of CX26 protein of the laryngeal carcinoma tissues in the advanced stage patients group (III stage and IV stage) were significantly lower than these in the early stage patients group (I and II) (P < 0.05), and it was significantly lower in those who have a cervical lymph node metastasis than those without metastasis. (P < 0.05). Moreover, the expression level of CX26 mRNA and the positive-expressed rate of CX26 protein reduced along with the reduction of pathological differentiation, and there was significant difference among the well-differentiated group, moderately-differentiated group and poorly-differentiated group (P < 0.05). CONCLUSION CX26 gene may play an important role in the pathogenesis and development of laryngeal carcinoma and may be related to its prognosis.
Adult ; Aged ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Connexin 26 ; Connexins ; metabolism ; Humans ; Laryngeal Neoplasms ; metabolism ; pathology ; Male ; Middle Aged ; Neoplasm Staging ; RNA, Messenger ; genetics

OBJECTIVE: Pyroptosis is an inflammatory form of programmed cell death. This phenomenon has been recently reported to play an important role in radiation-induced normal tissue injury. Connexin43 (Cx43) is a gap junction protein that regulates cell growth and apoptosis. In this study, we investigated the effect of Cx43 on X-ray-induced pyroptosis in the human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs, Cx43 overexpression, and Cx43 knockdown strains were irradiated with 10 Gy. Proteins were detected using western blot analysis. Cell pyroptosis was evaluated using the fluorescence-labeled inhibitor of caspase assay (FLICA) and propidium iodide staining through flow cytometry and confocal microscopy. Cell morphology and cytotoxicity were detected by scanning electron microscopy and lactate dehydrogenase release assay, respectively. RESULTS: Irradiation with 10 Gy X-ray induced pyroptosis in the HUVECs and reduced Cx43 expression. The pyroptosis in the HUVECs was significantly attenuated by overexpression of Cx43 as it decreased the level of active caspase-1. However, interference of Cx43 expression with siRNA significantly promoted pyroptosis by increasing the active caspase-1 level. Pannexin1 (Panx1), a gap junction protein regulates pyroptosis, and its cleaved form is used to evaluate channel opening and active state. The level of cleaved Panx1 in the HUVECs and Cx43 knockdown strains increased in the presence of X-ray, but decreased in the Cx43 overexpression strains. Furthermore, interference of Panx1 with siRNA alleviated the upregulation of pyroptosis caused by Cx43 knockdown. CONCLUSION Results suggest that single high-dose X-ray irradiation induces pyroptosis in the HUVECs. In addition, Cx43 regulates pyroptosis directly by activating caspase-1 or indirectly by cleaving Panx1.
Caspase 1 ; genetics ; metabolism ; Connexin 43 ; genetics ; metabolism ; Connexins ; genetics ; metabolism ; Gene Expression Regulation ; radiation effects ; Human Umbilical Vein Endothelial Cells ; physiology ; radiation effects ; Humans ; Nerve Tissue Proteins ; genetics ; metabolism ; Pyroptosis ; X-Rays ; adverse effects

OBJECTIVETo quantitatively detect the expression of connexins (Cx) mRNA in the posterior nodal extension (PNE) of adult rat heart and understand the relationship between Cx expression and atrial ventricular nodal reentrant tachycardia (AVNRT).

METHODSPNE was separated from adult rat heart by means of laser microdissection (LCM), and the cells were also isolated from the atrioventricular node (AVN), sinoatrial node (SAN), Purkinje fiber (PF), right atrium (RA) and right ventricle (RV), to serve as the controls. The Cx mRNA level was detected in these cells with quantitative real-time PCR (QRT-PCR).

RESULTSThe cells were successfully isolated from the PNE and other regions of adult rat heart, where heterogeneous expression of the 3 Cx isoforms (Cx43, Cx45, and Cx40) were observed. Cx45 mRNA showed higher expression in the PNE than in the working myocardium, whereas Cx43 mRNA level was about 25 times higher (P<0.05) in the working myocardium and 18 times higher (P<0.05) in the PF than in the PNE. In the PF, Cx40 mRNA level was proximately 6.8 times (P<0.01) as much as that in the PNE. Cx expression in the PNE was, however, similar to that in the SAN and AVN.

CONCLUSIONCx mRNAs exhibit heterogeneous expression in the PNE to allow the formation of the slow pathway. In addition, Cx expression in the PNE is very different from that in the adjacent myocardium, resulting in conduction discontinuity at the cellular junction, where, on certain occasion, unidirectional block may occur to cause AVNRT.

Animals ; Atrioventricular Node ; cytology ; metabolism ; Connexin 43 ; genetics ; Connexins ; genetics ; Female ; Male ; Myocardium ; cytology ; metabolism ; Purkinje Fibers ; cytology ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Sinoatrial Node ; cytology ; metabolism

OBJECTIVETo screen and identify the proteins that interact with connexin 26 (CX26) and to analyze the expressions of these proteins in cochlea so as to explore the proteins that relate to the trafficking, assembly, localizing and gap junction functions of CX26.

METHODSThe whole coding region of GJB2 (CX26) gene was amplified from normal human genomic DNA by polymerase chain reaction (PCR) and then directionally subcloned into the vector pGBKT7 plasmid of the Match Maker Ga14 Two-Hybrid System 3 as a target to screen the interactive proteins of CX26 from the human fetal brain cDNA library by the yeast two hybrid technique. The false positive clones were discarded from the preys by repeated yeast two hybrid method between CX26 and everyone of the preys respectively. The DNAs of the insert of the identified positive clone were sequenced and BLAST analyzed against the GenBank. Lastly, the mRNA of the gene encoding the identified protein was analyzed in the murine inner ear by reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSThe insert of one positive clone contained 867 bp with the former 525 bp being coding region. The DNA sequence and the open reading frame of the insert were identical to the 525 bp before the stop codes (including the stop codes) and the 238 bp after the stop codes of RTN4 gene which encoded Nogo protein. And the 174 amino acid residues encoded by the insert were those of the C-terminal of Nogo protein: Nogo-A, Nogo-B and Nogo-C. RTN4 mRNA expressed in the murine inner ear was confirmed by RT-PCR method.

CONCLUSIONThe C-terminal of Nogo protein interacts with CX26. Nogo protein expresses in the inner ear and may take part in the trafficking of CX26 or CX26 gap junction function.

Animals ; Base Sequence ; Connexin 26 ; Connexins ; genetics ; metabolism ; Ear, Inner ; metabolism ; Gene Expression ; Humans ; Mice ; Molecular Sequence Data ; Myelin Proteins ; genetics ; metabolism ; Nogo Proteins ; Protein Binding ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Two-Hybrid System Techniques