Connexin43 hemichannels contributes to the disassembly of cell junctions through modulation of intracellular oxidative status

AbstractConnexin (Cx) hemichannels regulate many cellular processes with little information available regarding their mechanisms. Given that many pathological factors that activate hemichannels also disrupts the integrity of cellular junctions, we speculated a potential participation of hemichannels in the regulation of cell junctions. Here we tested this hypothesis. Exposure of renal tubular epithelial cells to Ca2+-free medium led to disassembly of tight and adherens junctions, as indicated by the reduced level of ZO-1 and cadherin, disorganization of F-actin, and severe drop in transepithelial electric resistance. These changes were preceded by an activation of Cx43 hemichannels, as revealed by extracellular efflux of ATP and intracellular influx of Lucifer Yellow. Inhibition of hemichannels with chemical inhibitors or Cx43 siRNA greatly attenuated the disassembly of cell junctions. Further analysis using fetal fibroblasts derived from Cx43 wide-type (Cx43+/+), heterozygous (Cx43+/-) and knockout (Cx43-/-) littermates showed that Cx43-positive cells (Cx43+/+) exhibited more dramatic changes in cell shape, F-actin, and cadherin in response to Ca2+ depletion, as compared to Cx43-null cells (Cx43-/-). Consistently, these cells had higher level of protein carbonyl modification and phosphorylation, and much stronger activation of P38 and JNK. Hemichannel opening led to extracellular loss of the major antioxidant glutathione (GSH). Supplement of cells with exogenous GSH or inhibition of oxidative sensitive kinases largely prevented the above-mentioned changes. Taken together, our study indicates that Cx43 hemichannels promote the disassembly of cell junctions through regulation of intracellular oxidative status

Expression profiling in transgenic FVB/N embryonic stem cells overexpressing STAT3

BACKGROUND: The transcription factor STAT3 is a downstream target of the LIF signalling cascade. LIF signalling or activation is sufficient to maintain embryonic stem (ES) cells in an undifferentiated and pluripotent state. To further investigate the importance of STAT3 in the establishment of ES cells we have in a first step derived stable pluripotent embryonic stem cells from transgenic FVB mice expressing a conditional tamoxifen dependent STAT3-MER fusion protein. In a second step, STAT3-MER overexpressing cells were used to identify STAT3 pathway-related genes by expression profiling in order to identify new key-players involved in maintenance of pluripotency in ES cells. RESULTS: Transgenic STAT3-MER blastocysts yielded pluripotent germline-competent ES cells at a high frequency in the absence of LIF when established in tamoxifen-containing medium. Expression profiling of tamoxifen-induced transgenic FVB ES cell lines revealed a set of 26 genes that were markedly up- or down-regulated when compared with wild type cells. The expression of four of the up-regulated genes (Hexokinase II, Lefty2, Pramel7, PP1rs15B) was shown to be restricted to the inner cell mass (ICM) of the blastocysts. These differentially expressed genes represent potential candidates for the maintenance of pluripotency of ES cells. We finally overexpressed two candidate genes, Pem/Rhox5 and Pramel7, in ES cells and demonstrated that their overexpression is sufficient for the maintenance of expression of ES cell markers as well as of the typical morphology of pluripotent ES cells in absence of LIF. CONCLUSION: Overexpression of STAT3-MER in the inner cell mass of blastocyst facilitates the establishment of ES cells and induces the upregulation of potential candidate genes involved in the maintenance of pluripotency. Two of them, Pem/Rhox5 and Pramel7, when overexpressed in ES cells are able to maintain the embryonic stem cells in a pluripotent state in a LIF independent manner as STAT3 or Nanog

Acute administration of AMPA/Kainate blocker combined with delayed transplantation of neural precursors improves lower urinary tract function in spinal injured rats

To evaluate bladder function recovery after spinal cord injury (SCI) in response to a combination treatment of an acutely administered AMPA/kainate receptor antagonist and delayed transplantation of neuronal precursors. Female rats received a contusion injury at T8/9. The AMPA/kainate receptor antagonist NBQX was directly administered into the lesion site immediately after injury. Nine days post-injury, NRP/GRP were delivered into the lesion site. Controls received NRP/GRP grafts only or no treatment (OP-Controls). Animals underwent bladder function testing during the course of the experiment and at the endpoint. Motor function was evaluated as well. After sacrifice, histological analysis of lesion site and lumbosacral spinal cord regions was performed. Rats receiving the combined treatment (NBQX&NRP/GRP) had voided volumes/micturition resembling that of normal animals and showed greater improvement of urodynamic parameters, compared to NRP/GRP alone or OP-Controls. Similarly, NBQX&NRP/GRP induced more spouting, regeneration or sparing of descending projections to the lumbosacral cord. The density of primary afferent projections at the lumbosacral spinal cord in rats with combined treatments was similar to that of NRP/GRP alone with decreased sprouting of primary afferents in lumbosacral cord, compared to OP-Control. Immunohistochemical evaluation revealed that the combined treatment reduced the size of the lesion to a greater extent than NRP/GRP alone or OP-Controls. NRP/GRP with and without NBQX produced a significant recovery of hindlimb compared to OP-Controls. In conclusion, transplants of NRP/GRP combined with NBQX promote recovery of micturition function following spinal cord injury, likely through increased neuroprotection

Preoperative renal scar as a risk factor of postoperative metabolic acidosis following ileocystoplasty in patients with neurogenic bladder

Objectives: We investigated relation of preoperative renal scar to incidence of postoperative metabolic acidosis following ileocystoplasty in patients with neurogenic bladder. Patients: Thirty patients with neurogenic bladder, who underwent ileocystoplasty, were enrolled in the present study. Median age at ileocystoplasty was 13.9 years and median follow-up period after ileocystoplasty was 8.2 years. Metabolic acidosis was defined based on the outlined criteria: base excess (BE) is less than 0 mmol l (-1). Preoperative examination revealed that no apparent renal insufficiency was identified in blood analysis, although preoperative Tc-99m-DMSA scintigraphy indicated abnormalities such as renal scar in 14 patients (47%). Incidence of postoperative metabolic acidosis was compared between patients with and without preoperative renal scar, which may reflect some extent of renal tubular damage. Results: Postoperative metabolic acidosis was identified in 13 patients (43%). Incidence of postoperative metabolic acidosis was significantly higher in patients with renal scar (11/14, 79%) compared with patients without renal scar (2/16, 13%; P<0.01). Particularly, all eight patients who had bilateral renal scars showed metabolic acidosis postoperatively. Compared with patients without preoperative renal scar, pH (P<0.05) and BE (P<0.01) were significantly lower postoperatively in patients with preoperative renal scar. However, there was no significant difference in PCO2. Hyperchloremia was observed in each patient with or without preoperative renal scar. Conclusion: Incidence of postoperative metabolic acidosis was significantly implicated in preoperative renal scar. If renal abnormalities are preoperatively identified in imaging tests, we need to care patients carefully regarding metabolic acidosis and subsequent comorbidities following ileocystoplasty

Tuberculous Granuloma in the Scrotal Skin After Intravesical Bacillus Calmette-Guerin Therapy for Bladder Cancer: A Case Report

Rare cases of tuberculous urinary tract or genital infection caused by intravesical Intravesical Bacillus Calmette-Guerin (BCG) instillation therapy have been reported. We encountered a patient with tuberculous granuloma in the scrotal skin after intravesical BCG therapy for bladder cancer. There was evidence of infection in the scrotal skin, but not in the epididymis. To the best of our knowledge, this is the first report of tuberculous granuloma in the scrotal skin without epididymitis after intravesical BCG therapy. In our case, lower urinary tract symptoms such as the terminal dribbling of urine appear to support the theory of direct BCG inoculation

Construction of Neourethra Using Flipped Anterior Bladder Wall Tube in a Prepubertal Girl with Complete Disruption of Urethra

A rare case of urethral construction using flipped anterior bladder wall tube in 8-year-old girl with complete disruption of the urethra and vagina accompanying pelvic fracture was reported. Following a split of the pubic symphysis, the vagina was reconstructed with end-to-end anastomosis. The neourethra was constructed tubularizing and flipping anterior bladder wall flap caudally to proximal site of the original urethra after fascial sling procedure. After catheter removal, this girl has been continent and voided normally. In conclusion, flipped anterior bladder wall tube technique for urethral construction is suitable in prepubertal girls with complete disruption of the urethra