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miRNAs Regulate hERG Background The human ether-a-go-go-related gene (hERG) is the major molecular component of the rapidly activating delayed rectifier K+ current (Ikr). Impairment of hERG function is believed to be a mechanism causing long-QT syndromes (LQTS). Growing evidences have shown that microRNAs (miRNAs) are involved in functional modulation of the hERG pathway. The purpose of this study was to screen and validate miRNAs that regulate the hERG pathway. The miRNAs identified in this study will provide new tools to assess the mechanism of LQTS. Methods Six miRNAs were selected by algorithm predictions based on potential interaction with hERG. The effects of each miRNA on hERG were assessed by use of the Dual-Luciferase Reporter assay system, qRT-PCR, Western blotting, and confocal fluorescence microscopy. Furthermore, whole-cell patch clamp technique was used to validate the effect of miR-103a-1 on the electrophysiological characteristic of the Ikr of the hERG protein channel. Results miR-134, miR-103a-1, miR-143, and miR-3619 significantly downregulated luciferase activity (P < 0.05) in a reporter test system. These 4 miRNAs significantly suppressed expression of hERG mRNA and protein in U2OS cells (P < 0.05).Corresponding AMOs rescued expression of hERG mRNA and protein. Confocal microscopy showed that all 4 miRNAs reduced the expression of both immature and mature hERG protein. miR-103a-1 decreased the maximum current and tail current amplitudes of hERG channel. Conclusions Expression and functions of hERG are regulated by specific miRNAs. [ABSTRACT FROM AUTHOR]

Abstract: Embryonic stem (ES) cells, derived from blastocyst-stage of early mammalian embryos, have the potential to differentiate into derivatives of all three embryonic germ layers. Here we reported the first evidence that murine pluripotent ES cells could be induced to differentiate into cardiomyocytes by cyclic adenosine 3′,5′-monophosphate (cAMP) in vitro. Spontaneously beating of cardiac cell clusters began to be observed within the outgrowths of embryoid bodies (EBs) as early as 2 days after the onset of differentiation. By days 5–8 after induction, a maximum level of cardiomyocyte differentiation could be achieved. Incubation of EBs with cAMP at concentrations ranging from 0.01mg/L to 1mg/L resulted in a significant elevation in differentiation rate, reaching a maximum value of 44.0±1.3% at 0.03mg/L of exposure. At 0.03mg/L concentration point, an approximately 8.1-fold increase in cardiomyocyte differentiation was observed in comparison with 5.4±0.9% of untreated controls. The differentiation rate induced by cAMP was shown to be similar to that of RA/DMSO treated controls, indicating that cAMP has the same inducing effect as RA/DMSO. However, no significant co-inducing effects between cAMP and RA/DMSO were seen. Cardiomyocytes were evident as they expressed cardiac cell specific genes and protein markers including GATA4, Nkx2.5, β-MHC, atrial natriuretic factor (ANF) and alpha-actin when analyzed by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining. The results from the present study suggested a novel role of cAMP in cardiomyocyte differentiation and provided a new research model for the study of cardiac cell biology. [Copyright &y& Elsevier]

We have cloned, expressed and characterized a novel member of the human UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (pp-GalNAc-T) family, pp-GalNAc-T15. The pp-GalNAc-T15 transcript was ubiquitously expressed in human tissues. Recombinant pp-GalNAc-T15 transferred N-acetylgalactosamine (GalNAc) toward a panel of mucin-derived peptide substrates in vitro. Although pp-GalNAc-T15 showed significantly less catalytic activity than pp-GalNAc-T2, T15 transferred up to seven GalNAcs to the Muc5AC peptide, while T2 transferred up to five GalNAcs. These results clearly indicated that pp-GalNAc-T15 is a novel member of the human pp-GalNAc-T family with unique catalytic activity. [Copyright &y& Elsevier]

A novel member of the human UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase (pp-GalNAc-T) gene family was cloned as a homolog of human pp-GalNAc-T7, and designated pp-GalNAc-T10. pp-GalNAc-T10 transcript was found in the small intestine, stomach, pancreas, ovary, thyroid gland and spleen. In a polypeptide GalNAc-transfer assay, recombinant pp-GalNAc-T10 transferred GalNAc onto a panel of mucin-derived peptide substrates. Furthermore, pp-GalNAc-T10 demonstrated strong transferase activity with glycopeptide substrates. [Copyright &y& Elsevier]

We cloned in silico a novel human UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (pp-GalNAc-T), pp-GalNAc-T12. The deduced amino acid sequence of pp-GalNAc-T12 contains all conserved motifs in pp-GalNAc-T family proteins. Quantitative real time polymerase chain reaction analysis revealed that the pp-GalNAc-T12 transcript was expressed mainly in digestive organs such as stomach, small intestine and colon. The recombinant pp-GalNAc-T12 transferred GalNAc to the mucin-derived peptides such as the Muc1a, Muc5AC, EA2 peptides and the GalNAc-Muc5AC glycopeptide. Since mucins are glycoproteins mainly produced in the digestive organs, our results suggest that pp-GalNAc-T12 plays an important role in the initial step of mucin-type oligosaccharide biosynthesis in digestive organs. [Copyright &y& Elsevier]