Transgenic analysis of the atrialnatriuretic factor (ANF) promoter: Nkx2-5 and GATA-4 binding sites are required for atrial specific expression of ANF

The atrial natriuretic factor (ANF) gene is initially expressed throughout the myocardial layer of the heart, but during subsequent development, expression becomes limited to the atrial chambers. Mouse knockout and mammalian cell culture studies have shown that the ANF gene is regulated by combinatorial interactions between Nkx2-5, GATA-4, Tbx5, and SRF; however, the molecular mechanisms leading to chamber-specific expression are currently unknown. We have isolated the Xenopus ANF promoter in order to examine the temporal and spatial regulation of the ANF gene in vivo using transgenic embryos. The mammalian and Xenopus ANF promoters show remarkable sequence similarity, including an Nkx2-5 binding site (NKE), two GATA sites, a T-box binding site (TBE), and two SRF binding sites (SREs). Our transgenic studies show that mutation of either SRE, the TBE or the distal GATA element, strongly reduces expression from the ANF promoter. However, mutations of the NKE, the proximal GATA, or both elements together, result in relatively minor reductions in transgene expression within the myocardium. Surprisingly, mutation of these elements results in ectopic ANF promoter activity in the kidneys, facial muscles, and aortic arch artery-associated muscles, and causes persistent expression in the ventricle and outflow tract of the heart. We propose that the NKE and proximal GATA elements serve as crucial binding sites for assembly of a repressor complex that is required for atrial-specific expression of the ANF gene.     

Embryonic expression of an Nkx2‐5/Cre gene using ROSA26 reporter mice

Summary: Nkx2‐5, one of the earliest cardiac‐specific markers in vertebrate embryos, was used as a genetic locus to knock in the Cre recombinase gene by homologous recombination. Offspring resulting from heterozygous Nkx2‐5/Cre mice mated to ROSA26 (R26R) reporter mice provided a model system for following Nkx2‐5 gene activity by β‐galactosidase (β‐gal) activity. β‐gal activity was initially observed in the early cardiac crescent, cardiomyocytes of the looping heart tube, and in the epithelium of the first pharyngeal arch. In later stage embryos (10.5–13.5 days postcoitum, dpc), β‐gal activity was observed in the stomach and spleen, the dorsum of the tongue, and in the condensing primordium of the tooth. The Nkx2‐5/Cre mouse model should provide a useful genetic resource to elucidate the role of loxP manipulated genetic targets in cardiogenesis and other developmental processes. genesis 31:176–180, 2001. © 2001 Wiley‐Liss, Inc.     

Activation of Tolloid‐like 1 gene expression by the cardiac specific homeobox gene Nkx2–5

Mammalian Tolloid‐like 1 (Tll‐1) is a pleiotropic metalloprotease that is expressed by a small subset of cells within the precardiac mesoderm and is necessary for proper heart development. Following heart tube formation Tll‐1 is expressed by the endocardium and regions of myocardium overlying the region of the muscular interventricular septum. Mutations in Tll‐1 lead to embryonic lethality due to cardiac defects. We demonstrate that the Tll‐1 promoter contains Nkx2–5 binding sites and that the Tll‐1 promoter is activated by and directly binds Nkx2–5. Tll‐1 expression is ablated by a dominant negative Nkx2–5 or by mutation of the Nkx2–5 binding sites within the Tll‐1 promoter. In vivo, Tll‐1 expression is decreased in the hearts of Nkx2–5 knockout embryos when compared with hemizygous and wild‐type embryos. These results show that Nkx2–5 is a direct activator of Tll‐1 expression and provide insight into the mechanism of the defects found in both the Tll‐1 and Nkx2–5 knockout mice.     

Heterotypy in the N-terminal region of growth/differentiation factor 5 (GDF5) mature protein during teleost evolution

Heterotypy is now recognized as a generative force in the formationof new proteins through modification of existing proteins. Wereport that heterotypy in the N-terminal region of the maturegrowth/differentiation factor 5 (GDF5) protein occurred duringevolution of teleosts. N-terminal length variation of GDF5 wasfound among teleost interfamilies and interorders but not withinteleost families or among tetrapods. We further show that increaseof proline and glutamine to the N-terminal region of matureGDF5 occurred in Eurypterygii, the higher lineage of teleosts.Because the basic amino acids, believed to control diffusion,are conserved in this region across all species examined, wesuggest that the N-terminal elongation of the mature GDF5 proteinduring evolution has altered the protein diffusion in Eurypterygii,leading to high concentrations of the protein in the joint ofthe pharyngeal skeleton, the location of cartilage formationduring development.     

miR-145-5p attenuates hypertrophic scar via reducing Smad2/Smad3 expression

The study was designed to explore the underlying mechanism of micro ribonucleic acids (miR)-145-5p in the process of hypertrophic scar (HS). The difference in the relative content of miR-145-5p between HS and adjacent normal skin collected from 5 patients was detected via RT-PCR. Expressions of Smad2 and Smad3 with or without TGF-β1 was detected by western blotting. Fibroblasts apoptosis rate was examined by Annexin V/Propidium Iodide double staining. HS fibroblasts (HSFs) were isolated from HS tissues, cultured and then divided into control group, miR-145-5p inhibitor group (transfected with miR-145-5p inhibitor) and miR-145-5p mimic group (transfected with miR-145-5p plasmid) based on different treatment methods. Next, CCK-8 was employed to examine the function of miR-145-5p in HSF proliferation. Luciferase assay was conducted to confirm whether Smad2/3 were direct targets of miR-145-5p, and RT-PCR was done to measure the expression of miR-145-5p, Smad2/Smad3 and fibrosis-related genes of fibroblasts in three groups. Wound injury mice model was established to determine the function of miR-145-5p in regulating scar formation. miR-145-5p was found lowly expressed in HS tissues. Compared with Control group, miR-145-5p mimic decreased the levels of Smad2/3, arrested the activation and proliferation of HSFs and induced HSFs apoptosis. Overexpressing miR-145-5p achieved the contrary results. Smad2/3 was confirmed as the target of miR-145-5p. Moreover, miR-145-5p mimic decreased the recruitment of fibroblasts in vivo and decreased the expression of fibrosis-related genes after wound injury. In conclusion, miR-145-5p arrests the development of fibrogenesis and decreases HS formation by reducing the expression of Smad2/3. miR-145-5p may be an optional novel molecular target for treating HS.     

过表达E2F6基因抑制BRD7基因启动子活性

BRD7基因是采用cDNA代表性差异分析法克隆的一个新Bromodomain基因(GenBank 登录号AF152604)。它在鼻咽癌细胞和组织中表达明显下调,过表达BRD7基因可抑制鼻咽癌细胞的生长和细胞周期的进程。前期工作已克隆了BRD7基因启动子区,并将其启动子定位于450bp（-404→+46bp）的区域。为了进一步揭示BRD7基因在鼻咽癌细胞和组织中表达下调的分子机制,生物信息学分析表明BRD7启动子区有E2F6转录因子结合位点,电泳迁移率实验结果表明转录因子E2F6特异性地结合于BRD7启动子区。荧光素酶检测和绿色荧光蛋白表达检测都证实过表达E2F6基因能抑制BRD7基因启动子活性     

A Pitfall of the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) Assay due to Evaporation in wells on the Edge of a 96 well Plate

The 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) calorimetric assay is replacing the traditional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as a fast, one-step assay of cell viability. We have observed that evaporation of the outer wells of a 96 well plate increases the absorbancy by 52% compared to the inner wells. Filling the outer 2 rows of wells with media and replacement of the media prior to addition of the MTS reagent will, however, correct this inaccuracy.     

LncRNA-NNT-AS1 contributes to the progression of glioma by miR-582-5p/EZH2 axis

Cytotechnology - Increasing studies have shown that long non-coding RNAs (lncRNAs) had crucial regulatory roles in many diseases. Nevertheless, the biological relevance and mechanisms of the...