Comparison of bidirectional and bicistronic inducible systems for coexpression of connexin genes and fluorescent reporters

Wan, Carthur K.; Shaikh, Shamim; Green, Colin; Nicholson, Louise

doi:10.1016/j.ab.2012.08.016

Cited by 5 publications

(2 citation statements)

References 19 publications

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“…This is likely due to better protein features of ZsGreen than EGFP, including expression, translation and fluorescence property. In other cells (medaka melanoma cells, Chinese hamster ovary (CHO) cell line), the cells expressing ZsGreen also emitted brighter green fluorescence than those expressing EGFP, although there was a difference in GFP expression vector (promoter) [38,39]. Taken together, these results indicated that ZsGreen performs better than EGFP as a reporter in yeast.…”

Section: Resultsmentioning

confidence: 97%

Bright Fluorescence Monitoring System Utilizing Zoanthus sp. Green Fluorescent Protein (ZsGreen) for Human G-Protein-Coupled Receptor Signaling in Microbial Yeast Cells

2013

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G-protein-coupled receptors (GPCRs) are currently the most important pharmaceutical targets for drug discovery because they regulate a wide variety of physiological processes. Consequently, simple and convenient detection systems for ligands that regulate the function of GPCR have attracted attention as powerful tools for new drug development. We previously developed a yeast-based fluorescence reporter ligand detection system using flow cytometry. However, using this conventional detection system, fluorescence from a cell expressing GFP and responding to a ligand is weak, making detection of these cells by fluorescence microscopy difficult. We here report improvements to the conventional yeast fluorescence reporter assay system resulting in the development of a new highly-sensitive fluorescence reporter assay system with extremely bright fluorescence and high signal-to-noise (S/N) ratio. This new system allowed the easy detection of GPCR signaling in yeast using fluorescence microscopy. Somatostatin receptor and neurotensin receptor (implicated in Alzheimer’s disease and Parkinson’s disease, respectively) were chosen as human GPCR(s). The facile detection of binding to these receptors by cognate peptide ligands was demonstrated. In addition, we established a highly sensitive ligand detection system using yeast cell surface display technology that is applicable to peptide screening, and demonstrate that the display of various peptide analogs of neurotensin can activate signaling through the neurotensin receptor in yeast cells. Our system could be useful for identifying lead peptides with agonistic activity towards targeted human GPCR(s).

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Section: Resultsmentioning

confidence: 97%

Bright Fluorescence Monitoring System Utilizing Zoanthus sp. Green Fluorescent Protein (ZsGreen) for Human G-Protein-Coupled Receptor Signaling in Microbial Yeast Cells

2013

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“…ICC was also performed in the absence of primary antibodies, to ensure there was no non-specific secondary antibody binding. Additionally, the primary antibodies for Cx30 and Cx43 have also been previously confirmed to be specific using cells induced to overexpress Cx30 and Cx43, respectively ( Wan et al, 2012 ).…”

Section: Methodsmentioning

confidence: 95%

Spatiotemporal changes in Cx30 and Cx43 expression during neuronal differentiation of P19 EC and NT2/D1 cells

et al. 2013

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While connexins (Cxs) are thought to be involved in differentiation, their expression and role has yet to be fully elucidated. We investigated the temporal expression of Cx30, Cx36 and Cx43 in two in vitro models of neuronal differentiation: human NT2/D1 and murine P19 cells, and the spatial localisation of Cx30 and Cx43 in these models.A temporal Cx43 downregulation was confirmed in both cell lines during RA-induced neuronal differentiation using RT-PCR (P < 0.05) preceding an increase in neuronal doublecortin protein. RT-PCR showed Cx36 was upregulated twofold in NT2/D1 cells (P < 0.05) and sixfold in P19 cells (P < 0.001) during neuronal differentiation. Cx30 exhibited a transient peak in expression midway through the timecourse of differentiation increasing threefold in NT2/D1 cells (P < 0.001) and eightfold in P19 cells (P < 0.01).Qualitative immunocytochemistry was used to examine spatiotemporal patterns of Cx protein distribution alongside neuronal differentiation markers. The temporal immunolabelling pattern was similar to that seen using RT-PCR. Cx43 was observed intracellularly and on cell surfaces, while Cx30 was seen as puncta. Spatially Cx43 was seen on doublecortin-negative cells, which may indicate Cx43 downregulation is requisite for differentiation in these models. Conversely, Cx30 puncta were observed on doublecortin-positive and -negative cells in NT2/D1 cells and examination of the Cx30 peak showed puncta also localized to nestin-positive cells, with few puncta on MAP2-positive cells. In P19 cells Cx30 was localized on clusters of cells surrounded by MAP2- and doublecortin-positive processes. The expression pattern of Cx30 indicates a role in neuronal differentiation; the nature of that role warrants future investigation.

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RETRACTED ARTICLE: Comparing astrocytic gap junction of genetic absence epileptic rats with control rats: an experimental study

et al. 2021

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The synchronization of astrocytes via gap junctions (GJ) is a crucial mechanism in epileptic conditions, contributing to the synchronization of the neuronal networks. Little is known about the endogenous response of GJ in genetic absence epileptic animal models. We evaluated and quanti ed astrocyte GJ protein connexin 30 (Cx30) and 43 (Cx43) in the somatosensory cortex (SSCx), ventrobasal (VB), centromedian (CM), lateral geniculate (LGN) and thalamic reticular (TRN) nuclei of thalamus of genetic absence epilepsy rats from Strasbourg (GAERS), Wistar albino glaxo rats from Rijswijk (WAG/Rij) and control Wistar animals using immunohistochemistry and Western Blot.The Cx30 and Cx43 immunopositive astrocytes in per unite area were quanti ed for each region of the three animal strains. Further, Cx30 and Cx43 Western Blot was applied to the tissue samples from the same regions of the three strain.The number of Cx30 immunopositive astrocytes showed signi cant increase in both GAERS and WAG/Rij compared to control Wistar in all brain regions studied except LGN of WAG/Rij animals. Furthermore, Cx43 in both GAERS and WAG/Rij showed signi cant increase in SSCx, VB and TRN. The percentage of dual expression of Cx30 and Cx43 in the same astrocyte ranged between 17-100% for the 5 brain regions of the 3 animal strains studied. The protein expression of both Cx30 and Cx43 in the two epileptic strain showed an increase compared to Wistar control animals.The signi cant increase in the astrocytic GJ proteins Cx30 and Cx43 and the differences in the dual expression of Cx30 and Cx43 in the genetically absence epileptic strains compared to control animals may suggest that astrocytic Cx's may be involved in the mechanism of absence epilepsy. Increased number of astrocytic Cx's in GAERS and WAG/Rij may represent a compensatory response of the thalamocortical circuitry to the absence seizures or may be related to the production of absence seizures.

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Comparison of bidirectional and bicistronic inducible systems for coexpression of connexin genes and fluorescent reporters

Cited by 5 publications

References 19 publications

Bright Fluorescence Monitoring System Utilizing Zoanthus sp. Green Fluorescent Protein (ZsGreen) for Human G-Protein-Coupled Receptor Signaling in Microbial Yeast Cells

Bright Fluorescence Monitoring System Utilizing Zoanthus sp. Green Fluorescent Protein (ZsGreen) for Human G-Protein-Coupled Receptor Signaling in Microbial Yeast Cells

Spatiotemporal changes in Cx30 and Cx43 expression during neuronal differentiation of P19 EC and NT2/D1 cells

RETRACTED ARTICLE: Comparing astrocytic gap junction of genetic absence epileptic rats with control rats: an experimental study

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