Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.4.2.8 (hypoxanthine-guanine phosphoribosyltransferase)
 2,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Cardiomyocytes purified from differentiated embryonic stem cells exhibit characteristics of early chamber myocardium. 1465 72

ROCK kinases regulate actin-myosin structures downstream of Rho GTPases. We generated mice expressing 4-hydroxytamoxifen (4HT)-regulated human ROCK II (ROCKII:mER) under the transcriptional control of the cytokeratin14 (K14) promoter. The K14-ROCKII:mER minigene was recombineered into a novel cloning vector containing the promoter and first exon of the human HPRT gene, and second and third exons of the mouse Hprt gene. Homologous recombination into the Hprt locus, which is deleted for the promoter and first two exons in HM1 embryonic stem cells, reconstitutes a functional Hprt gene, allowing for growth in HAT (hypoxanthine-aminopterin-thymidine) medium. K14-promoter-driven ROCKII:mER expression was restricted to a superficial cell layer in embryoid bodies, with increased ROCK substrate phosphorylation induced by 4HT. ROCKII:mER-expressing primary murine keratinocytes responded to 4HT with increased substrate phosphorylation and cytoskeleton rearrangements, indicating that ROCKII:mER activity is regulated by 4HT in the target tissue. K14-ROCKII:mER mice will be valuable for examining the role of ROCK in skin development and cancer.
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PMID:Tissue selective expression of conditionally-regulated ROCK by gene targeting to a defined locus. 1939 Nov 17