Evidence of estrogen receptors in normal human osteoblast-like cells

In seven strains of cultured normal human osteoblast-like cells, a mean of 1615 molecules of tritium-labeled 17 beta-estradiol per cell nucleus could be bound to specific nuclear sites. The nuclear binding of the labeled steroid was temperature-dependent, steroid-specific, saturable, and cell type-specific. These are characteristics of biologically active estrogen receptors. Pretreatment with 10 nanomolar estradiol in vitro increased the specific nuclear binding of progesterone in four of six cell strains, indicating an induction of functional progesterone receptors. RNA blot analysis demonstrated the presence of messenger RNA for the human estrogen receptor. The data suggest that estrogen acts directly on human bone cells through a classical estrogen receptor-mediated mechanism.     

Human endometrial adenocarcinoma transplanted into nude mice: growth regulation by estradiol

A model for studying the growth of primary tumors of human endometrium and its regulation by 17 beta-estradiol has been developed in which ovariectomized nude mice are used as recipients. The receptors for sex steroids are maintained during serial transplantation of the tumor in this system. Although the rate of growth of receptor-negative endometrial tumors transplanted into ovariectomized nude mice is unaffected by the sustained presence or absence of estradiol, the growth of receptor-positive tumors is significantly increased by estradiol. Receptor-positive tumors treated with estradiol produced elevated concentrations of progesterone receptor. That the progesterone receptor is functional in this tumor is evident from the induction of estradiol 17 beta-dehydrogenase activity upon progestin administration. These findings are consistent with receptor-mediated regulation of growth of endometrial carcinoma.     

Control of yeast mating signal transduction by a mammalian beta 2-adrenergic receptor and Gs alpha subunit

To facilitate functional and mechanistic studies of receptor-G protein interactions, [corrected] the human beta 2-adrenergic receptor (h beta-AR) has been expressed in Saccharomyces cerevisiae. This was achieved by placing a modified h beta-AR gene under control of the galactose-inducible GAL1 promoter. After induction by galactose, functional h beta-AR was expressed at a concentration several hundred times as great as that found in any human tissue. As determined from competitive ligand binding experiments, h beta-AR expressed in yeast displayed characteristic affinities, specificity, and stereoselectivity. Partial activation of the yeast pheromone response pathway by beta-adrenergic receptor agonists was achieved in cells coexpressing h beta-AR and a mammalian G protein (Gs) alpha subunit-demonstrating that these components can couple to each other and to downstream effectors when expressed in yeast. This in vivo reconstitution system provides a new approach for examining ligand binding and G protein coupling to cell surface receptors.     

Induction of ovalbumin synthesis in immature chicks by actinomycin D and thioacetamide

Actinomycin D and thioacetamide induced ovalbumin synthesis and increased serum progesterone concentrations in immature chicks. The increase in progesterone induced by the carcinogens actinomycin D and thioacetamide may account for the induction of ovalbumin synthesis.     

Molecular cloning of the chicken progesterone receptor

To define the functional domains of the progesterone receptor required for gene regulation, complementary DNA (cDNA) clones encoding the chicken progesterone receptor have been isolated from a chicken oviduct lambda gt11 cDNA expression library. Positive clones expressed antigenic determinants that cross-reacted with six monospecific antibodies derived from two independent sources. A 36-amino acid peptide sequence obtained by microsequencing of purified progesterone receptor was encoded by nucleotide sequences in the longest cDNA clone. Analysis of the amino acid sequence of the progesterone receptor deduced from the cDNA clones revealed a cysteine-rich region that was homologous to a region found in the estrogen and glucocorticoid receptors and to the avian erythroblastosis virus gag-erb-A fusion protein. Northern blot analysis with chicken progesterone receptor cDNA's indicated the existence of at least three messenger RNA species. These messages were found only in oviduct and could be induced by estrogens.     

Behavioral effects of progesterone associated with rapid modulation of oxytocin receptors

The ventromedial nuclei of the hypothalamus (VMN) are important for the control of feminine mating behavior, and hormone action within these nuclei has been causally related to behavior. Estradiol induces receptors for oxytocin in the VMN and in the area lateral to these nuclei over the course of 1 to 2 days, and progesterone causes, within 30 minutes of its application, a further increase in receptor binding and an expansion of the area covered by these receptors lateral to the VMN. The rapid progesterone effect appears to be a direct and specific effect of this steroid on the receptor or membrane, because it was produced in vitro as well as in vivo and was not mimicked by a variety of other steroids. The effect of progesterone occurred in the posterior part of the VMN, where oxytocin infusion facilitated feminine mating behavior; it did not take place in the anterior part of the VMN, where oxytocin infusion had no effect on mating behavior.     

Protein synthesis: differential stimulation of cell-specific proteins in epithelial cells of chick oviduct

Immunofluorescent and radioautographic studies demonstrate that ovalbumin and avidin are cell-specific proteins synthesized by different epithelial cells in the chick oviduct mucosa. The mechanism of the selective induction of ovalbumin synthesis by estrogen and of avidin synthesis by progesterone may be through stimulation of specific target cells by these hormones.     

An estrogen-binding protein and endogenous ligand in Saccharomyces cerevisiae: possible hormone receptor system

A protein macromolecule in the cytosol of the unicellular eukaryotic yeast Saccharomyces cerevisiae selectively binds the vertebrate estrogen hormone 17 beta-estradiol with high affinity. Lipid extracts of the yeast cells or the conditioned growth medium yield a substance that can bind competitively to the tritiated estradiol-binding sites in the yeast and to mammalian estrogen receptors. These findings suggest that the binding protein may be a primitive hormone receptor and that the lipid-extractable substance represents the endogenous ligand.     

荆豆凝集素-1受体在犬子宫内的分布与激素调节

采用组织化学方法对荆豆凝集素-1(ulex europaeus agglutinin-1,UEA-1)受体在发情周期和早期妊娠犬子宫内的分布以及激素调节进行了研究。结果显示,UEA-1受体主要存在于犬子宫内膜腔上皮和腺上皮,其表达量随妊娠阶段的不同而发生动态性变化。UEA-1受体在休情期犬子宫内未见表达,而在发情期犬子宫内膜腔上皮和腺上皮内的表达量很高。在早期妊娠犬子宫内,在妊娠第6天和12天犬子宫内的表达量较低,此后其表达量逐渐增加,在妊娠第17天时,UEA-1受体在子宫内膜腔上皮和腺上皮内的表达量达到峰值,此后其表达量又逐渐下降,在妊娠第23天时未见表达。注射雌激素可明显促进卵巢切除后犬子宫内膜UEA-1受体的表达。结果表明,犬子宫内UEA-1受体的分布与胚胎着床前胚胎与子宫内膜之间的黏附和植入有关,UEA-1受体在犬子宫内的表达受母体分泌的激素所调控。     

姜曲海母猪子宫雌二醇和孕酮受体含量的变化

以长白母猪为对照,分析了1月龄、发情期和间情期姜曲海母猪子宫细胞质雌二醇受体(CER)和细胞核雌二醇受体(NER)以及细胞质孕酮受体(CPR)和细胞核孕酮受体(NPR)。3个时期该猪子宫CER分别为351、410和367fmol/mgDNA,其Kd值分别为7.1、4.8和4.0nmol;子宫NER分别为245,1390和620fmol/mgDNA,Kd值为3.7、6.6和4.4nmol;CPR分别为251、364和147fmol/mgDNA;NPR分别为232、337和184fmol/mgDNA。在发情期和间情期,姜曲海母猪子宫NER以及与细胞质受体的比值均比长白母猪高,说明此时姜曲海母猪子宫对雌二醇具有较高的敏感性。性成熟后,试验猪的雌二醇受体和孕酮受体均受它们配体激素的调节。     

人工诱导泌乳后血浆及乳汁中孕酮含量的动态变化

用放射免疫分析方法测定了６头激素诱导泌乳的黑白花奶牛血浆和乳汁中孕酮的含量，结果表明，诱导泌乳后乳汁中孕酮含量较高，第１ｄ时平均为７．１５±２．７９ｎｇ／ｍＬ，第２ｄ迅速升高到１６．２１±３．４５ｎｇ／ｍＬ，以后随着用药次数的增多，孕酮含量略有升高，第３ｄ及第４ｄ时分别达到１７．６６±４．１３ｎｇ／ｍＬ和１７．７７±２．３１ｎｇ／ｍＬ，随后孕酮含量逐渐下降，第５ｄ及第７ｄ时分别为１４．８４±６．２７ｎｇ／ｍＬ和１０．７６±５．５０ｎｇ／ｍＬ，第９ｄ时为１０．５０±５．０９ｎｇ／ｍＬ，此后孕酮含量急剧下降，第１５ｄ时达到３．４８±１．７５ｎｇ／ｍＬ，之后波动于１～４ｎｇ／ｍＬ之间，在整个实验期间乳汁中孕酮的浓度平均为６．３６５±２．６４９ｎｇ／ｍＬ，且呈明显的下降趋势。血浆与乳汁中孕酮含量呈明显的正相     

拟南芥高迁移率族蛋白B族基因At2G34450在毕赤酵母中的表达及纯化

[目的]观察拟南芥高迁移率族蛋白B族基因At2G34450在毕赤酵母体系中的表达,获得重组蛋白。[方法]将At2G34450基因插入含AOX1启动子和α分泌信号肽序列的酵母表达载体pPIC9K中,用SaⅠl将重组质粒线性化,电击转化毕赤酵母GS115感受态细胞,筛选阳性整合子进行甲醇诱导表达。[结果]拟南芥At2G34450在酵母培养基中实现了表达,表达产物经SDS-PAGE鉴定为重组蛋白。[结论]在毕赤酵母真核系统中实现了拟南芥At2G34450蛋白的表达,为进一步研究拟南芥HMGB家族蛋白打下了基础。     

Cell culture of mammalian endometrium and synthesis of blastokinin in vitro

Endometrial cells obtained from mature female estrous rabbits can be grown in cell culture with the aid of insulin. These cells, after several days in culture, are capable of synthesizing blastokinin by induction with progesterone for 48 hours.     

Peripheral clonal elimination of functional T cells

A major mechanism for generating tolerance in developing T cells is the intrathymic clonal deletion of T cells that have receptors for those self antigens that are presented on hematopoietic cells. The mechanisms of tolerance induction to antigens not expressed in the thymus remain unclear. Tolerance to self antigens can be generated extrathymically through the induction of clonal nonresponsiveness in T cells with self-reactive receptors. A second mechanism of extrathymic tolerance was identified: clonal elimination of mature T cells with self-reactive receptors that had previously displayed functional reactivity.     

Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1

Malfolded proteins in the endoplasmic reticulum (ER) induce cellular stress and activate c-Jun amino-terminal kinases (JNKs or SAPKs). Mammalian homologs of yeast IRE1, which activate chaperone genes in response to ER stress, also activated JNK, and IRE1alpha-/- fibroblasts were impaired in JNK activation by ER stress. The cytoplasmic part of IRE1 bound TRAF2, an adaptor protein that couples plasma membrane receptors to JNK activation. Dominant-negative TRAF2 inhibited activation of JNK by IRE1. Activation of JNK by endogenous signals initiated in the ER proceeds by a pathway similar to that initiated by cell surface receptors in response to extracellular signals.     

热纤梭菌内切葡聚糖酶在酿酒酵母细胞表面上展示研究

[目的]对热纤梭菌内切葡聚糖酶进行酿酒酵母细胞表面展示研究,探索降低纤维素酶成本和提高酶活的方法。[方法]通过提取产内切葡聚糖酶的热纤梭菌总DNA,根据热纤梭菌内切葡聚糖酶基因序列和酿酒酵母表面展示质粒载体pYD1上的多克隆位点设计引物,克隆内切葡聚糖酶目的基因,将其连接到酵母表面展示质粒载体pYD1上,构建重组质粒pYD1-CelA,并将其转化入酿酒酵母菌株EBY100中,经诱导表达后,测定表达产物的最适温度和pH,并分析影响其活性的金属离子种类和浓度。[结果]PCR扩增获得1 400bp左右的内切葡聚糖酶CelA基因片段,并成功构建了该基因与酵母表面展示质粒载体pYD1的重组质粒pYD1-CelA。重组质粒转化酿酒酵母菌株EBY100后,在鉴定培养基上测定透明圈的大小得出最佳诱导时间为60 h。表达产物性质的研究结果显示其最适反应温度为50℃,在pH 4.6时酶活性相对较高。[结论]该研究结果为后续对纤维素酶的进一步研究、改造、大量生产及应用奠定了一定的基础。     

橡胶树肌动蛋白解聚因子的表达和功能鉴定

为探寻肌动蛋白解聚因子(actin depolymerizing factor,ADF)在调控肌动蛋白解聚和聚合平衡过程中发挥的作用,从橡胶树中获得了Hb ADF基因组序列,经测定,该序列长2 258 bp,含有2个内含子和3个外显子,在裂殖酵母中过表达Hb ADF,获得相对分子质量约38 000的融合蛋白。对裂殖酵母细胞形态进行观察,发现诱导表达Hb ADF的酵母细胞长度显著增加,双核和多核比例达67%。实时荧光定量PCR结果表明,Hb ADF的表达受3%KI处理调控,在橡胶树不同死皮阶段和不同排胶时间段的表达存在变化,说明Hb ADF可能参与了橡胶树排胶和死皮的过程。     

Chimeric NGF-EGF receptors define domains responsible for neuronal differentiation

To determine the domains of the low-affinity nerve growth factor (NGF) receptor required for appropriate signal transduction, a series of hybrid receptors were constructed that consisted of the extracellular ligand-binding domain of the human epidermal growth factor (EGF) receptor (EGFR) fused to the transmembrane and cytoplasmic domains of the human low-affinity NGF receptor (NGFR). Transfection of these chimeric receptors into rat pheochromocytoma PC12 cells resulted in appropriate cell surface expression. Biological activity mediated by the EGF-NGF chimeric receptor was assayed by the induction of neurite outgrowth in response to EGF in stably transfected cells. Furthermore, the chimeric receptor mediated nuclear signaling, as evidenced by the specific induction of transin messenger RNA, an NGF-responsive gene. Neurite outgrowth was not observed with chimeric receptors that contained the transmembrane domain from the EGFR, suggesting that the membrane-spanning region and cytoplasmic domain of the low-affinity NGFR are necessary for signal transduction.