人雌激素受体cDNA在酵母细胞中的表达

根据人全长雌激素受体cDNA序列,用RT—PCR方法使乳腺癌细胞MCF-7株扩增人雌激素受体基因(hER cDNA),通过pGEM—T／hER载体克隆到酵母表达载体中,并转化到酿酒酵母细胞中,用Western blot法和免疫组化法检测hER cDNA的表达。结果表明：hER cDNA在酵母细胞中成功表达,可用于建立环境雌激素的酵母细胞检测体系。     

雌激素调控的绿色荧光蛋白在酵母细胞中的表达

采用绿色荧光蛋白(GFP)作为报告基因,用酵母细胞构建环境雌激素(EEH)的评价体系,可敏感、快速、方便地筛选出EEH化合物。通过分子生物学技术,将GFP基因插入到pM P 206/ERE启动子CYC 1的下游,用GFP取代L ac Z基因,并转化于酵母细胞。DNA测序发现ERE-CYC 1-GFP框架正确。对转化子进行PCR鉴定,发现有雌激素受体基因(hER cDNA)和GFP基因特异条带,说明hER cDNA和GFP已重组于酵母细胞。荧光显微镜下发现大量的绿色荧光颗粒,表明GFP基因在酵母细胞中成功表达。酵母细胞经不同浓度雌二醇作用后,与GFP表达量有剂量效应关系。与其他酵母评价体系相比,以GFP为报告基因评价EEH不需要破坏细胞壁,也不需要底物,可在96孔板中完成,这为高通量筛选EEH化合物奠定了基础。     

Estrogen memory effect in human hepatocytes during repeated cell division without hormone

Transient stimulation of target tissues by sex steroids can cause long-lasting changes that may facilitate or alter responses to subsequent hormonal treatment. How these altered characteristics are propagated during cell division in the absence of the stimulating hormone is unknown. The human hepatocarcinoma cell line HepG2 was used as a model to examine the effects of estrogen on the synthesis of serum apolipoproteins in vitro. Treatment with low concentrations of estrogen for 24 to 48 hours resulted in long-lasting alterations in the kinetics with which the cells responded to subsequent stimulation with estrogen. Manifestation of this memory effect was correlated quantitatively with the induction and propagation of a moderate-affinity, nuclear, estrogen-binding protein with the characteristics of a type II estrogen receptor. The data indicate that transient exposure of these cells to estrogen can induce changes in their response characteristics and composition of nuclear proteins that are inherited by daughter cells grown in the absence of hormone for more than ten generations.     

环境雌激素调控的β-半乳糖苷酶酵母细胞的建立

为建立环境雌激素的酵母评价体系,将人工合成的雌激素效应元件插入pMP206质粒的上游,并将该质粒转入人雌激素受体基因重组酵母细胞,使LacZ基因置于雌激素的调控之下,以构建可用于评价环境雌激素的酵母细胞。结果表明：经DNA测序发现pMP206／ERE-LacZ报告质粒DNA框架正确。对转化子进行PCR鉴定,发现有雌激素受体基因和pMP206／ERE-LacZ报告质粒的特异性条带,说明雌激素受体基因和受雌激素调控的LacZ报告基因已重组于酵母细胞。从而证明以LacZ为报告基因的环境雌激素酵母评价细胞重组成功。     

Sequence and expression of human estrogen receptor complementary DNA

The mechanism by which the estrogen receptor and other steroid hormone receptors regulate gene expression in eukaryotic cells is not well understood. In this study, a complementary DNA clone containing the entire translated portion of the messenger RNA for the estrogen receptor from MCF-7 human breast cancer cells was sequenced and then expressed in Chinese hamster ovary (CHO-K1) cells to give a functional protein. An open reading frame of 1785 nucleotides in the complementary DNA corresponded to a polypeptide of 595 amino acids and a molecular weight of 66,200, which is in good agreement with published molecular weight values of 65,000 to 70,000 for the estrogen receptor. Homogenates of transformed Chinese hamster ovary cells containing a protein that bound [3H]estradiol and sedimented as a 4S complex in salt-containing sucrose gradients and as an 8 to 9S complex in the absence of salt. Interaction of this receptor-[3H]estradiol complex with a monoclonal antibody that is specific for primate ER confirms the identity of the expressed complementary DNA as human estrogen receptor. Amino acid sequence comparisons revealed significant regional homology among the human estrogen receptor, the human glucocorticoid receptor, and the putative v-erbA oncogene product. This suggests that steroid receptor genes and the avian erythroblastosis viral oncogene are derived from a common primordial gene. The homologous region, which is rich in cysteine, lysine, and arginine, may represent the DNA-binding domain of these proteins.     

Resurrecting the ancestral steroid receptor: ancient origin of estrogen signaling

Receptors for sex and adrenal steroid hormones are absent from fully sequenced invertebrate genomes and have not been recovered from other invertebrates. Here we report the isolation of an estrogen receptor ortholog from the mollusk Aplysia californica and the reconstruction, synthesis, and experimental characterization of functional domains of the ancestral protein from which all extant steroid receptors (SRs) evolved. Our findings indicate that SRs are extremely ancient and widespread, having diversified from a primordial gene before the origin of bilaterally symmetric animals, and that this ancient receptor had estrogen receptor-like functionality. This gene was lost in the lineage leading to arthropods and nematodes and became independent of hormone regulation in the Aplysia lineage.     

Neuroendocrine response to estrogen and sexual orientation

A neuroendocrine component, the positive estrogen feedback effect, thought to be related to sexual orientation and, indirectly, to sexual differentiation, was evaluated in healthy, noninstitutionalized research volunteers. Men and women with a lifelong heterosexual orientation and men with a lifelong homosexual orientation were administered an estrogen preparation known to enhance the concentration of luteinizing hormone in women but not in men. The secretory pattern of luteinizing hormone in the homosexuals in response to estrogen was intermediate between that of the heterosexual men and that of the women. Furthermore, testosterone was depressed for a significantly longer period in the homosexual men than in the heterosexual men. These findings suggest that biological markers for sexual orientation may exist.     

Estrogen-receptor interaction

The interaction of estradiol with uterine cells involves the association of the hormone with an extranuclear receptor protein, followed by temperature dependent translocation of the resulting complex to the nucleus. During this process, the steroid binding unit of the protein undergoes an alteration, called "receptor transformation," that can be recognized by an increase in its sedimentation rate from 3.8S to 5.2S, and by its acquisition of the ability to bind to isolated uterine nuclei and to alleviate a tissue specific deficiency in the RNA synthesizing capacity of such nuclei. Receptor transformation can be effected in the absence of nuclei by warming uterine cytosol with estradiol. This preparation of transformed complex resembles that extracted from nuclei both in its sedimentation rate (5.3S) and in its ability to bind to uterine nuclei and augment RNA synthesis, properties that are not shown by the native complex. It is proposed that receptor transformation is an important step in estrogen action and that a principal role of the hormone is to induce conversion of the receptor protein to a biochemically functional form.     

Hormone therapy: physiological complexity belies therapeutic simplicity

The results of the Women's Health Initiative, a study anticipated to provide definitive answers about health benefits and risks of postmenopausal hormone therapy, have generated debate and confusion among clinicians, researchers, and the lay public. The ovarian hormones estrogen and progesterone, which decline at menopause, normally elicit complex tissue-specific responses throughout the body. Major advances are providing a detailed molecular definition of how that differential action is achieved. Here we review estrogen and progestin actions, discuss how effectively knowledge of steroid hormone endocrinology has been incorporated into clinical studies, and consider the impact on modern hormone therapy protocols and pharmaceutical development.     

Saccharomyces cerevisiae produces a yeast substance that exhibits estrogenic activity in mammalian systems

Partially purified lipid extracts of Saccharomyces cerevisiae contain a substance that displaces tritiated estradiol from rat uterine cytosol estrogen receptors. The yeast product induces estrogenic bioresponses in mammalian systems as measured by induction of progesterone receptors in cultured MCF-7 human breast cancer cells and by a uterotrophic response and progesterone receptor induction after administration to ovariectomized mice. The findings raise the possibility that bakers' yeast may be a source of environmental estrogens.     

重组虹鳟生长激素酵母对罗非鱼的促生长作用研究

采用表达重组虹鳟生长激素的酵母工程菌Y33作为饲料添加剂投喂奥尼罗非鱼鱼种。结果表明，酵母胞内表达的虹鳟生长激素具有生物活性，能够经口服被鱼体肠道吸收，对罗非鱼具有明显的促生长作用。     

Yeast mating pheromone activates mammalian gonadotrophs: evolutionary conservation of a reproductive hormone?

alpha-Factor, a tridecapeptide mating pheromone of yeast (Saccharomyces cerevisiae), has extensive sequence homology with the hypothalamic decapeptide gonadotropin-releasing hormone (GnRH). Both synthetic and natural preparations of alpha-mating factor were found to bind specifically to rat pituitary GnRH receptors and to stimulate the release of luteinizing hormone from cultured gonadotrophs. The ability of the yeast pheromone to reproduce the biological actions of GnRH in the mammalian pituitary gland indicates that the structural and functional properties of GnRH-related peptides may have been highly conserved during evolution.     

植物MAPK级联途径及其功能研究进展

促分裂原活化蛋白激酶(Mitogen Actived Protein Kinase,MAPK)是一种蛋白激酶,蛋白激酶又称蛋白磷酸化酶,其可通过将ATP上的磷酸基团转移到底物蛋白质氨基酸残基上,来催化底物蛋白质磷酸化。MAPK级联途径广泛存在于真核生物中。植物中的MAPK级联途径与动物和酵母类似,都包括MAPKKK、MAPKK和MAPK 3种蛋白激酶。大量的研究表明,植物中的MAPK级联途径不仅能被多种生物与非生物胁迫所激活,同时也参与激素信号转导以及植物的生长发育进程。文章就植物中MAPK级联途径及其功能的研究情况进行了综述,并展望了相关研究的发展趋势。     

花䱻抗缪勒氏管激素基因克隆、抗体制备及其对外源雌激素的响应

【目的】克隆花䱻抗缪勒氏管激素基因Amh,分析其序列特征及亚细胞定位,检测不同浓度雌二醇处理下的表达变化规律,为后续研究Amh基因的功能及其在卵巢发育中分子机制提供理论参考。【方法】基于花转录组文库测序结果,克隆Amh基因cDNA序列,并对其进行生物信息学分析。构建重组表达质粒pET32a-Amh,转化大肠杆菌BL21感受态细胞,用纯化的重组蛋白免疫小鼠制备多克隆抗体。通过免疫荧光技术检测荧光信号确定蛋白的亚细胞定位情况。同时,利用实时荧光定量PCR检测Amh基因在雌二醇处理后的表达情况。【结果】克隆的花䱻Amh基因cDNA序列全长为1849 bp,开放阅读框（ORF）为1671 bp,编码556个氨基酸残基。系统进化分析结果显示,花䱻Amh基因与斑马鱼、鲤鱼等鲤科鱼类Amh基因聚为一支,表明其亲缘关系较近。将纯化的AMH重组蛋白免疫小鼠以制备AMH多克隆抗体,通过酶联免疫方法（ELISA）检测其抗体效价为2.4×106以上,Western blotting检测显示抗体具有特异性。AMH蛋白在花卵巢颗粒细胞表达。利用不同浓度的雌二醇处理花䱻60 d后发现,低浓度雌二醇（100 μg/g）促进花䱻卵母细胞生长,卵巢内皮质液泡数量增多。经雌二醇处理后,卵巢中Amh基因的表达量降低,说明雌激素在卵巢发育中对Amh基因表达具有抑制作用。【结论】Amh基因含有TGF-β家族典型的结构域,在卵巢颗粒细胞表达,且响应雌激素的调控,可能在花䱻卵巢发育中发挥调控作用。     

A novel nuclear form of estradiol receptor in MCF-7 human breast cancer cells

Nuclear estrogen receptor from MCF-7 cells undergoes a time-dependent, hormone-inducible transformation to a form that is less extractable from nuclei and less exchangeable with ligand. This receptor-modifying, intranuclear event is independent of receptor loss (processing) and appears associated with hormone responsiveness (progesterone-receptor induction) in these cells. The magnitude of receptor loss, however, is variable and apparently not a prerequisite for hormone action to induce progesterone receptor.     

黄鳝生长激素基因cDNA的克隆及胞内真核表达载体的构建

为大量获得具有生物学活性的黄鳝生长激素,采用RT-PCR的方法从黄鳝脑cDNA中扩增得到了黄鳝生长激素基因。通过特异性的引物扩增后,将黄鳝生长激素基因ORF区域序列连接到酵母胞内表达载体pGAPZB中,构建了重组胞内表达载体pGAPZB—rfeGH。用BlnI线性化重组质粒后,电击转化毕赤酵母GS115。经过Zeocin筛选和PCR扩增转化子基因组筛选,得到了数株基因工程酵母菌,序列测定表明生长素基因已经成功的转移到酵母基因上。黄鳝生长激素胞内表达载体的构建成功为进一步研究黄鳝生长激素基因的功能和大量开发并利用奠定了实验基础。     

乳与乳制品中雌激素的分析与控制

随着生活水平的不断提高、生活节奏的不断加快,人们对牛奶、乳制品的需求也在增加,随之而来的是将乳及乳制品的安全性要求放在了首位,对乳与乳制品中激素问题也越来越关注,特别是雌激素。本文对乳与乳制品中雌激素的来源、危害、含量、检测、国家控制等方面进行了分析。