Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:EXPT01586 (G418)
 2,237 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In order to develop mammalian cell lines expressing a functional human heart Na+ channel gene (hH1), Chinese hamster ovary (CHO-K1) cells and HeLa cells were transfected with the hH1 gene and the bacterial neomycin (neo) resistance gene. In CHO-K1 cells, direct screening for hH1-positive, G418-resistant colonies by functional patch clamp analysis was complicated due to low-level endogenous expression of a brain-type Na+ channel. Therefore, we developed a stepwise strategy for isolation of cell lines expressing functional hH1 Na+ channels: G418-resistant colonies were sequentially analysed for (1) chromosomal integration of hH1 DNA by PCR, (2) specific hH1 mRNA expression by RT-PCR, (3) hH1 protein production by immunoprecipitation with hH1-specific antisera, and (4) hH1 Na+ channel function by patch-clamp analysis. Using this strategy we obtained two CHO-K1 cell lines which express functional human heart Na+ channels. However, using the same strategy, we were unsuccessful in obtaining functional, hH1-positive HeLa cell lines, even though hH1 mRNA and protein was produced in these cells. The two CHO-K1 cell lines stably express human cardiac Na+ channels which retain normal electrophysiological characteristics with respect to activation and inactivation. In addition, the Na+ channels expressed in these cells are blocked by tetrodotoxin with an IC50 value of 2.5 microM; consistent with known cardiac Na+ channel pharmacology. The density of channels is high enough to permit recording of pseudomacroscopic currents in excised outside-out patches of membrane.(ABSTRACT TRUNCATED AT 250 WORDS)
J Mol Cell Cardiol 1995 Feb
PMID:Stable expression and functional characterization of a human cardiac Na+ channel gene in mammalian cells. 777 89

Heat shock protein 70 (HSP70) has been shown to play a fundamental role in the induction of thermotolerance in many biological systems. Elevated synthesis of HSP70 in response to diverse stresses such as heat, anoxia, ischaemia, ethanol and heavy metals has been correlated with protection against subsequent more severe stress and cross-tolerance to differing stresses. In this respect, exposure of the mammalian heart to sublethal heat treatment or ischaemia has been shown to protect against subsequent myocardial ischaemia with concomitant elevation of HSP70. However, direct demonstration that HSP70 can alone confer thermotolerance has until recently been restricted to transfection of fibroblasts with an HSP70 gene, although preliminary data from others suggests that transfection of H9c2 myocytes with an HSP70 gene can confer tolerance to substrate-free hypoxia. The purpose of this study was, therefore, to test directly whether a myocyte cell line which retains certain features of cardiac cells (as opposed to non-myocyte cells) can be protected against lethal thermal stress by transfection with a single HSP70 gene. Rat heart-derived H9c2 cells were transfected either with a plasmid from which high level expression of a human HSP70 gene is driven by a strong, heterologous (human) beta-actin promoter (APr-HS70), or with an analogous control plasmid containing no HSP70 gene (pAPr-1 neo). Following selection with the neomycin analogue G418, several clones were isolated which either expressed no HSP70 (control: pAPr-1 neo-derived) or stably expressed high constitutive levels of an inducible isoform of HSP70 (HSP70i) (APr-HS70-derived) as determined by Western blotting with a specific monoclonal antibody to HSP70i.(ABSTRACT TRUNCATED AT 250 WORDS)
J Mol Cell Cardiol 1994 Jun
PMID:Stable high level expression of a transfected human HSP70 gene protects a heart-derived muscle cell line against thermal stress. 808 50

Mouse embryonic stem (ES) cells easily differentiate towards the cardiac lineage making them suitable as an in vitro model to study cardiogenesis and as a potential source of transplantable cells. In this study, we show by in situ hybridisation that about 30% of the volume of cultures of differentiating ES cells consists of cardiomyocytes. RT-PCR analyses showed that the transcription factors Nkx2.5, Gata4, Mef2c and Irx4 were expressed at levels in the same order of magnitude as the levels observed in embryonic, neonatal and adult hearts. Atrial natriuretic factor and Connexin 40, associated with chamber formation in vivo, are expressed at relatively low levels, similar to those observed at early heart development in vivo. To facilitate the isolation of ES cell-derived cardiomyocytes, a cell line was constructed by stable transfection of the aminoglycoside phosphotransferase cDNA driven by the cardiac-specific distant upstream part of the Na(+)/Ca(2+) exchanger promoter. To accomplish single-copy integration, the construct was inserted into the hypoxanthine phosphoribosyltransferase locus of HM1 ES cells by homologous recombination. Cardiac-specific resistance to G418-sulphate (neomycin) allowed isolation of a pure population of cardiomyocytes. Genetically selected and unselected cell populations were characterised electrophysiologically using patch clamp. To explore whether clusters of cells have a similar differentiation profile, action potentials (APs) were measured in aggregates of differentiating ES cells, using a new method based on the voltage-dependent fluorescent dye di-4-ANEPPS. Both whole-cell recordings using patch-clamp and optical measurements with di-4-ANEPPS of the AP showed that upstroke velocity increases and AP duration decreases with differentiation time, accompanied by a decrease in AP interval, suggesting the initiation of the developmental programme underlying the formation of chamber myocardium.
J Mol Cell Cardiol 2003 Dec
PMID:Cardiomyocytes purified from differentiated embryonic stem cells exhibit characteristics of early chamber myocardium. 1465 72