Modulation of globin gene expression by a tumor promoter during induced differentiation of Friend erythroleukemic cells

Friend erythroleukemic cells were induced to differentiate by dimethylsulfoxide (Me2SO) in the absence or presence of the tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate. The effects of the latter on the molecular parameters related to globin mRNA metabolism were examined. When differentiation was scored by benzidine staining, it had an inhibitory effect on Me2SO-treated cells. On the other hand, when differentiation was followed by determination of globin mRNA accumulation, it had a pleiotropic effect on Me2SO-treated cells. At the early phase of differentiation (2--3 days) the rate of globin mRNA accumulation was higher in the promoter-treated cells than in the control. This unexpectedly high level of accumulation was followed by a sharp reduction and most of the globin RNA sequences disappeared at later stages of differentiation (days 4--5). The reduction can be related to the effect of the promoter on the stability of globin RNA in the cytoplasm which was reduced from a half-life of 16 h to that of 8 h only. Other parameters, such as the rate of globin mRNA synthesis and its capability to serve as a template for cell-free protein synthesis were not affected by treatment with the promoter throughout the differentiation process.     

Up-regulation of orphan nuclear estrogen-related receptor alpha expression during long-term caloric restriction in mice

The estrogen-related receptor alpha (ERRα) is an orphan receptor belonging to the nuclear receptor superfamily that regulates a number of target genes encoding enzymes that participate in various metabolic pathways involved in maintaining energy balance in animals. In this study, whether long-term caloric restriction (alternate days of fasting for 3 months) in mice modulates the expression of ERRα in various tissues was investigated. Western blot analyses showed positive immunoreactive ERRα protein (53 kDa) band in various mice tissue extracts, though at varying levels. Heart, kidney, and skeletal muscles expressed significant levels of ERRα, with a comparatively lower level detected in the intestine, brain, and liver. Cardiac ERRα expression was the highest, with the least detected in the liver. Caloric restricted mice exhibited a significant increase in ERRα level in the heart (5.45-fold), kidney (3.70-fold), skeletal muscle (3.0-fold), small intestine (2.72-fold), and liver (2.44-fold) extracts as compared to ad libitum fed. However, caloric restriction could not evoke any detectable receptor level change in the brain. Notably, the highest ERRα up-regulation was detected in the heart. This up-regulation in ERRα level especially in highly oxidative tissues such as heart, kidney, small intestine, and skeletal muscle of caloric restricted mice may be helpful in modulating ERRα responsive genes that participates in maintaining energy balance. This may potentially strengthen the metabolic and biochemical adaptation in such tissues, which is necessary for animal survival under long-term caloric restriction.     

Modulation of pro-inflammatory gene expression by nuclear lysophosphatidic acid receptor type-1

Lysophosphatidic acid (LPA) is a bioactive molecule involved in inflammation, immunity, wound healing, and neoplasia. Its pleiotropic actions arise presumably by interaction with their cell surface G protein-coupled receptors. Herein, the presence of the specific nuclear lysophosphatidic acid receptor-1 (LPA1R) was revealed in unstimulated porcine cerebral microvascular endothelial cells (pCMVECs), LPA1R stably transfected HTC4 rat hepatoma cells, and rat liver tissue using complementary approaches, including radioligand binding experiments, electron- and cryomicroscopy, cell fractionation, and immunoblotting with three distinct antibodies. Coimmunoprecipitation studies in enriched plasmalemmal fractions of unstimulated pCMVEC showed that LPA1Rs are dually sequestrated in caveolin-1 and clathrin subcompartments, whereas in nuclear fractions LPA1R appeared primarily in caveolae. Immunofluorescent assays using a cell-free isolated nuclear system confirmed LPA1R and caveolin-1 co-localization. In pCMVEC, LPA-stimulated increases in cyclooxygenase-2 and inducible nitric-oxide synthase RNA and protein expression were insensitive to caveolea-disrupting agents but sensitive to LPA-generating phospholipase A2 enzyme and tyrosine kinase inhibitors. Moreover, LPA-induced increases in Ca2+ transients and/or iNOS expression in highly purified rat liver nuclei were prevented by pertussis toxin, phosphoinositide 3-kinase/Akt inhibitor wortmannin and Ca2+ chelator and channel blockers EGTA and SK&F96365, respectively. This study describes for the first time the nucleus as a potential organelle for LPA intracrine signaling in the regulation of pro-inflammatory gene expression.     

Modulation of thrombospondin expression during differentiation of embryonal carcinoma cells

The thrombospondins (TSPs) are a family of extracellular glycoproteins that display distinct patterns of temporal and spatial expression during development. In this study, we investigated the expression of two of the TSPs–TPS1 and TSP2– during the course of differentiation of embryonal carcinoma cells in vitro. We report that both TSP1 and TSP2 mRNA and protein synthesis are induced during the differentiation of P19EC cells into neurons, glial cells, and fibroblasts. Immunofluorescence studies indicate that TSP1 displays a fibrillar pattern of staining, characteristic of an extracellular matrix protein, in differentiated P19EC cells. In contrast, TSP2 is cell-associated and is present on differentiated P19EC cells and on primary neurons and glial cells obtained from a 17-day embyronic mouse cerebral cortex. Interestingly, although both TSP1 and TSP2 are more prevalent in areas of differentiated cells, they display distinct patterns of deposition. These observations suggest that TSP1 and TSP2 may function differently during neurogenesis. The response of TSP1 and TSP2 to differentiation of P19EC cells indicates that this cell system will serve as a valuable model for the study of TSP expression and function during neurogenesis. © 1994 Wiley-Liss, Inc.     

家蚕雌激素相关受体基因的克隆与表达分析

【目的】黑腹果蝇 Drosophila melanogaster 雌激素相关受体（estrogen-related receptor, ERR）通过调节糖酵解过程进而控制果蝇的能量代谢。本研究在克隆家蚕Bombyx mori ERR 基因 (BmERR) 的基础上,对其分子特性和系统演化进行生物信息学分析, 并检测该基因在家蚕生殖腺中的表达,为进一步研究ERR功能奠定基础。【方法】采用PCR技术克隆 BmERR 基因的全长cDNA序列,进行生物信息学分析;利用半定量RT-PCR检测该基因在停食后家蚕幼虫生殖腺中的表达情况。【结果】BmERR 基因全长cDNA序列为1 296 bp,编码431个氨基酸残基;具有ERR蛋白家族典型的结构特征;系统进化分析显示BmERR与其他昆虫ERR氨基酸序列一致性较高;半定量 RT-PCR 检测表明,BmERR 在家蚕上簇到化蛾期间的精巢和卵巢中均有表达,表达具有时期特异性,化蛹第1天达到表达高峰。【结论】本研究首次从鳞翅目昆虫中克隆获得ERR cDNA序列。ERR基因在家蚕生殖腺中表达量无明显性别差异,但具有发育时期特异性。     

A docking modelling rationally predicts strong binding of bisphenol A to estrogen-related receptor gamma

A computer-aided docking study was carried out to quickly clarify the binding structure of the ligand-receptor complex between bisphenol A (BPA), a well-known endocrine disruptor, and estrogen-related receptor gamma (ERRgamma). The resulting complex indicated that BPA binds to the ligand-binding pocket of ERRgamma without any disruptions of the activation conformation.     

Modulation of expression of the human gamma interferon gene in E. coli by site-directed mutagenesis

Plasmids expressing 2 forms of human immune interferon (IFN-gamma) in E. coli have been constructed: 1) pIFNTacI which expresses IFN-gamma with an N-terminal amino acid sequence of met-cys-tyr-cys-gln-, and 2) pIFNTacII which is a derivative of pIFNTacI from which the 9 base pairs (bp) coding for the cys-tyr-cys have been deleted. Quantitation of Western blots showed that approximately 10-fold more IFN-gamma was produced in cells harboring pIFNTacII (7.5% of total cellular protein) as compared to pIFNTacI. The IFN-gamma expressed in E. coli pIFNTacII is biologically active and routinely recoverable at 10(9) units per liter. When examined microscopically, IPTG induced E. coli harboring either plasmid construction contains prominent cytoplasmic inclusion bodies.     

Fibronectin gene expression during limb cartilage differentiation

A critical event in limb cartilage differentiation is a transient cellular condensation process in which prechondrogenic mesenchymal cells become closely juxtaposed and interact with one another prior to initiating cartilage matrix deposition. Fibronectin (FN) has been suggested to be involved in regulating the onset of condensation and chondrogenesis by actively promoting prechondrogenic aggregate formation during the process. We have performed a systematic quantitative study of the expression of the FN gene during the progression of chondrogenesis in vitro and in vivo. In high-density micromass cultures of limb mesenchymal cells, FN mRNA levels increase about 5-fold coincident with the crucial condensation process, and remain relatively high during the initial deposition of cartilage matrix by the cells. Thereafter, FN mRNA levels progressively decline to relatively low levels as the cultures form a virtually uniform mass of cartilage. The changes in FN mRNA levels in vitro are paralleled closely by changes in the relative rate of FN synthesis as determined by pulse-labeling and immunoprecipitation analysis. The relative rate of FN synthesis increases 4- to 5-fold at condensation and the onset of chondrogenesis, after which it progressively declines to low levels as cartilage matrix accumulates. High levels of FN gene expression also occur at the onset of chondrogenesis in vivo. In the proximal central core regions of the limb bud in which condensation and cartilage matrix deposition are being initiated, FN mRNA levels and the relative rates of FN synthesis become progressively about 4-fold higher than in the distal subridge region, which consists of undifferentiated mesenchymal cells that have not yet initiated condensation.(ABSTRACT TRUNCATED AT 250 WORDS)