Project description:Cx32 wildtype and mutants (W3S, R22G) were expressed in HeLa cells and subjected to surface biotinylation to check for expression level differences between the different conditions. A CFP control was conducted.
Project description:The main objective of the present report was to unravel the Cx43-interaction network in the heart, and to establish the impact of heart ischemia and I/R upon these interactions. In order to characterize the cardiac Cx43 interactome under ischemia and I/R, a quantitative proteomic analysis was performed, through the combination of immunopurification of endogenous Cx43 (Cx43 IP) and identification of its binding partners with the SWATH-MS approach, using rat hearts maintained in a Langendorff apparatus. In the present approach 444 proteins were identified, including proteins identified based on a single peptide (supplementary table I). From these 444 proteins, 299 (approximately 67 % of the entire dataset) were quantified and compared between the various experimental conditions (including non-specific binding to control IP samples). These 299 proteins were further evaluated by a series of complementary analysis to deciphering the truly interactors of Cx43. According with this evaluation, 236 out of the 299 quantified proteins were considered as putative Cx43 interacting partners in the cardiac context presented The results obtained in this study demonstrate that Cx43 mainly interacts with proteins related with metabolism, signaling and trafficking, and that this interactome can be differentially modulated in diseased hearts. Our results shed new light upon the understanding of Cx43 functions in the heart, both in health and disease, which ultimately may lead to the establishment of new therapeutic targets to modulate cardiac homeostasis.
Project description:ChIP-seq analysis was used to identify B. dermatitidis genes bound by the GATA transcription factor encoded by SREB during growth as yeast at 37oC SREB was engineered to contain an in-frame 3x-hemagglutinin (HA) epitope tag at the C-terminus. The SREB-3xHA construct was under control of its native promoter and contained the 3-untranslated region. Using Agrobacterium tumefaciens, B. dermatitidis ATCC 26199 was transformed with the SREB-3xHA construct (referred to a SREB-3xHA strain in this document). The SREB-3xHA construct was functional because retransformation of SREB? with the construct complented the null mutant. Chromatin was extracted and sheared from ATCC 26199 and SREB-3xHA yeast grown in liquid Histoplasma macrophage medium (HMM) containing 10 ?M iron sulfate (FeSO4) at 37oC. ATCC 26199 was the untagged control strain.
Project description:We tested how Cx43 hemichannels which mediate the exchange of small molecules between cells and extracellular environment impact genome wide gene expression under conditions of abnormal gravity and magnetic field. we subjected osteocytic MLO-Y4 cells to a high magneto-gravitational environment and used microarray to examine global gene expression and a specific blocking antibody was used to assess the role of Cx43 hemichannels. Microarray analysis was conducted to identify the alterations of global gene expression profile of osteocytic MLO-Y4 cells under HMGE. Furthermore, the effects of Cx43 hemichannels on gene expression were investigated by using a potent, Cx43 hemichannel-blocking antibody Cx43(E2)
Project description:The blood-testis barrier (BTB) is essential to the microenvironment of spermatogenesis, and Sertoli cells provide the cellular basis for BTB construction. Numerous nuclear transcription factors have been identified to be vital for the proper functioning of Sertoli cells. PA1 has been reported to play important roles during diverse biological processes, yet its potential function in male reproduction is still unknown. Here, we show that PA1 was highly expressed in human and mouse testis and predominantly localized in the nuclei of Sertoli cells. Sertoli cell-specific Pa1 knockout resulted in an azoospermia-like phenotype in mice. The knockout of this gene led to multiple defects in spermatogenesis, such as the disorganization of the cytoskeleton during basal and apical ectoplasmic specialization and the disruption of the BTB. Further transcriptomic analysis, together with Cut-Tag results of PA1 in Sertoli cells, revealed that PA1 could affect the expression of a subset of genes that are essential for the normal function of Sertoli cells, including those genes associated with actin organization and cellular junctions such as Connexin43 (Cx43). We further demonstrated that the expression of Cx43 depended on the interaction between JUN, one of the AP-1 complex transcription factors, and PA1. Overall, our findings reveal that PA1 is essential for the maintenance of BTB integrity in Sertoli cells and regulates BTB construction-related gene expression via transcription factors. Thus, this newly discovered mechanism in Sertoli cells provides a potential diagnostic or even therapeutic target for some individuals with azoospermia.
Project description:Use of null mutant mice is a powerful way to evaluate the role of specific proteins in brain function. Studies performed on knockout mice have revealed some unexpected roles of the gap junction proteins (the connexins). Thus, analyses of gene expression in connexin43 (Cx43) null brains indicated that deletion of a single gene (Gja1) induced expression level change of numerous other genes located on all chromosomes and involved in a wide diversity of functional pathways. The significant overlap between alterations in gene expression level, control and coordination in Cx43 knockout and knockdown astrocytes raised the possibility that Gja1 represents a transcriptomic node of gene regulatory networks. However, conditional deletion of Gja1 in astrocytes of two mouse strains resulted in remarkably different phenotypes. In order to evaluate the influence of the genetic background on the transcriptome, we performed microarray studies on brains of GFAP-Cre:Cx43f/f C57Bl/6 and 129/SVEV mice. The surprisingly low number of Cx43 core genes (regulated in all Cx43 nulls regardless of strain) and the high number of differently regulated genes in the two Cx43 CKOs indicate high influence of mouse strain on brain transcriptome. The transcriptomes of WT and Cx43 null brains from both C57Bl/6 and SVEV strains were profiled and compared at perinatal and adult time points to learn more about the strain dependence of the Cx43-null phenotype. For this purpose, differently labeled cDNAs from biological replicas (4 of each genotype) were co-hybridized with Duke MO36K mouse oligonucleotide array spotted with 36k Operon oligonucleotides V4.0.
Project description:Oocyte quality is a well- established determinant of embryonic fate. However, the molecular participants and biological markers that affect and predict adequate embryonic development are largely elusive. We have previously reported that oocyte- directed Connexin 43 (Cx43) depletion leads to embryo implantation defects, although both the morphology of the oocyte and processes presiding embryo implantation appear to undergo normally. In the context of previous data determining Cx43 indispensability to oocyte and embryonic development, we show here that the timing of Cx43 depletion from the oocyte and the ovarian follicle is crucial in determining the severity of subsequent embryonic defects. Specifically, we show that the implantation defects of blastocysts resulting from oocyte- directed Cx43- depleted follicles (depletion occurs at day 3 postnatal), is not due to maternal luteal insufficiency but rather depends solely on the defective blastocysts. Gene expression microarray analysis revealed global defects in the expression of ribosomal proteins, translation initiation factors and other genes associated with cellular biosynthetic and metabolic processes in these defective oocytes and specifically blastocysts. We therefore propose that timely expression of Cx43 in the oocyte and ovarian follicles is a major determinant of oocyte developmental competence, by determining the ability of the resulting blastocyst to facilitate biomass expansion and undergo adequate embryo implantation To study the effect of CX43 on the transcriptom of the pre implantation stages, we compared CX43 KO oocytes to the WT oocytes in three different stages of the very early development. First comparison MII oocytes, second comparison blastocysts, third comparison implantation site.
Project description:Objective: We previously reported that white matter connexin43 (Cx43) may related to the severity of the multiple sclerosis (MS), whereas the role of gray matter Cx43 in demyelinating disease is unknown. It was considered MS lesions were only exist in white matter, but recent studies revealed that demyelinating lesions are also exist in the cerebral cortex. This fact suggest the possibility that gray matter is somewhat related to the pathophysiology of MS. In this study, we aimed to clarify the role of gray matter Cx43 in a mouse model of MS (experimental autoimmune encephalomyelitis [EAE]). Methods: We developed Cx43F/F;Glutamate aspartate transporter (GLAST)-CreER(T2)KI/+ mice as gray matter specific Cx43 conditional knock-out (Cx43cKO) mice. We induced MOG-EAE 10 days after tamoxifen injection, and analyze its clinical course and pathology. We used Cx43F/F mice as controls. Results: EAE was significantly milder in gray matter astrocyte-specific Cx43cKO mice from acute phase to chronic phase, as compared with control mice. Pathology demonstrated less demyelinating lesions and infiltrating cells. Infiltrating immune cells did not express Cx43 in the active demyelinating lesions of the lumbar cord in both groups. The expression level of Cx43 was similar between these two groups in the spleen and the inguinal lymph nodes. Interpretation: Acute KO of gray matter specific Cx43 before induction of EAE reduce its aggressiveness. This finding may suggest the possibility that gray matter Cx43 modify the MS pathophysiology.
Project description:Objective: We previously reported that white matter connexin43 (Cx43) may related to the severity of the multiple sclerosis (MS), whereas the role of gray matter Cx43 in demyelinating disease is unknown. It was considered MS lesions were only exist in white matter, but recent studies revealed that demyelinating lesions are also exist in the cerebral cortex. This fact suggest the possibility that gray matter is somewhat related to the pathophysiology of MS. In this study, we aimed to clarify the role of gray matter Cx43 in a mouse model of MS (experimental autoimmune encephalomyelitis [EAE]). Methods: We developed Cx43F/F;Glutamate aspartate transporter (GLAST)-CreER(T2)KI/+ mice as gray matter specific Cx43 conditional knock-out (Cx43cKO) mice. We induced MOG-EAE 10 days after tamoxifen injection, and analyze its clinical course and pathology. We used Cx43F/F mice as controls. Results: EAE was significantly milder in gray matter astrocyte-specific Cx43cKO mice from acute phase to chronic phase, as compared with control mice. Pathology demonstrated less demyelinating lesions and infiltrating cells. Infiltrating immune cells did not express Cx43 in the active demyelinating lesions of the lumbar cord in both groups. The expression level of Cx43 was similar between these two groups in the spleen and the inguinal lymph nodes. Interpretation: Acute KO of gray matter specific Cx43 before induction of EAE reduce its aggressiveness. This finding may suggest the possibility that gray matter Cx43 modify the MS pathophysiology.