Abstract:
:To study the interaction of TSH receptor (TSHR) autoantibodies with receptor protein; it is necessary first to express the receptor in the proper conformation including the formation of correct disulfide bridges. However, the reducing environment of the Escherichia coli (E. coli) cytoplasm prevents the generation of protein disulfide bonds and limits the solubility and immunoreactivity of recombinant human TSHR (hTSHR) products. To circumvent these limitations, hTSHR complementary DNA encoding the extracellular domain (hTSHR-ecd; amino acids 21-415) was inserted into the vector pGEX-2TK by directional cloning and used to transform the thioredoxin reductase mutant strain of E. coli (Ad494), which allowed formation of disulfide bonds in the cytoplasm. After induction, the expressed soluble hTSHR-ecd fusion protein was detected by Western blot analysis using a monoclonal antibody directed against hTSHR amino acids 21-35. This showed that over 50% of the expressed hTSHR-ecd was soluble in contrast to expression in a wild-type E. coli (strain alpha F'), where the majority of the recombinant receptor was insoluble. The soluble recombinant receptor was affinity purified and characterized. Under nonreducing SDS-PAGE conditions, the soluble hTSHR-ecd migrated as refolded, disulfide bond-stabilized, multimeric species, whose formation was independent of fusion partner protein. This product was found to be biologically active as evidenced by the inhibition of the binding of 125I-TSH to the full-length hTSHR expressed in transfected CHO cells and was used to develop a competitive capture enzyme-linked immunosorbent assay for mapping of hTSHR antibody epitopes. Hence, hTSHR-ecd produced in bacteria with a thioredoxin reductase mutation was found to be highly soluble and biologically relevant.
journal_name
Endocrinologyjournal_title
Endocrinologyauthors
Bobovnikova Y,Graves PN,Vlase H,Davies TFdoi
10.1210/endo.138.2.4920subject
Has Abstractpub_date
1997-02-01 00:00:00pages
588-93issue
2eissn
0013-7227issn
1945-7170journal_volume
138pub_type
杂志文章相关文献
ENDOCRINOLOGY文献大全abstract::The median eminence (ME), which is the common termination field for adenohypophysiotropic systems, has been shown to produce nitric oxide (NO), a signaling molecule involved in neuroendocrine secretion. Using an ex vivo technique, 17beta-estradiol exposure to ME fragments, including vascular tissues, stimulated NO rel...
journal_title:Endocrinology
pub_type: 杂志文章
doi:10.1210/endo.140.2.6484
更新日期:1999-02-01 00:00:00
abstract::T47D human breast cancer cells and BEN human lung cancer cells were preincubated with the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA). In both cell lines there was a decrease in the binding of 125I-labeled salmon calcitonin ([125I]sCT) which was dependent on the dose and time of exposure to PMA...
journal_title:Endocrinology
pub_type: 杂志文章
doi:10.1210/endo-125-5-2656
更新日期:1989-11-01 00:00:00
abstract::A study was conducted to determine the influence of conditions known to alter ACTH secretion on prolactin release by the pituitary. Dexamethasone or 0.9% NaCl was injected subcutaneously into intact male rats 4 hr before a 2-min ether stress. Other rats were submitted to the ether stress 2 weeks following adrenalectom...
journal_title:Endocrinology
pub_type: 杂志文章
doi:10.1210/endo-96-2-475
更新日期:1975-02-01 00:00:00
abstract::In previous studies we demonstrated a much greater rate of glucose cycling (glucose-->glucose-6-P-->glucose) in islets from ob/ob mice than from lean litter mates. Cycling was further augmented by dexamethasone treatment. To determine whether these findings could be accounted for by increased islet glucose-6-phosphata...
journal_title:Endocrinology
pub_type: 杂志文章
doi:10.1210/endo.136.5.7720640
更新日期:1995-05-01 00:00:00
abstract::Tumor necrosis factor-alpha (TNF alpha), a product of activated monocytes, induces tissue wasting in certain solid tumors in vivo and in in vitro model systems. Recent studies indicate that TNF alpha also regulates cell replication and expression of differentiated function in a variety of nonneoplastic cell systems. S...
journal_title:Endocrinology
pub_type: 杂志文章
doi:10.1210/endo-123-3-1442
更新日期:1988-09-01 00:00:00
abstract::In animal models of inflammation, the pregnancy hormone relaxin was shown to reduce the recruitment of leukocytes, especially neutrophils, in inflamed tissues. The current study was designed to clarify whether relaxin could inhibit activation of isolated human neutrophils and, if so, whether the nitric oxide (NO) bios...
journal_title:Endocrinology
pub_type: 杂志文章
doi:10.1210/en.2003-0833
更新日期:2004-03-01 00:00:00
abstract::To investigate the participation of cyclic nucleotides in LHRH-mediated gonadotropin release, cells from the anterior pituitaries of 15-day-old female rats were fractionated on an albumin gradient by sedimentation at unit gravity. gonadotrophs and somatotrophs were detected in the fractions by histology and RIA of hor...
journal_title:Endocrinology
pub_type: 杂志文章
doi:10.1210/endo-107-5-1627
更新日期:1980-11-01 00:00:00
abstract::Somatostatin (SRIF) inhibits vasoactive intestinal peptide (VIP)-stimulated cAMP accumulation in the GH4C1 strain of rat pituitary tumor cells, and this effect is responsible for SRIF inhibition of VIP-stimulated hormone release. In this study we examined the interaction between the SRIF receptor and adenylate cyclase...
journal_title:Endocrinology
pub_type: 杂志文章
doi:10.1210/endo-114-5-1784
更新日期:1984-05-01 00:00:00
abstract::Signals derived from other pituitary cells can have a dramatic effect on PRL gene expression and secretion by mammotropes. However, the intracellular mechanisms by which these effects are manifested on the target cell remain unexplored. Inasmuch as calcium is a key modulator of both gene expression and hormone export ...
journal_title:Endocrinology
pub_type: 杂志文章
doi:10.1210/endo.139.6.6071
更新日期:1998-06-01 00:00:00
abstract::The rapid and temporary suppression of reproductive behavior is often assumed to be an important feature of the adaptive acute stress response. However, how this suppression operates at the mechanistic level is poorly understood. The enzyme aromatase converts testosterone to estradiol in the brain to activate reproduc...
journal_title:Endocrinology
pub_type: 杂志文章
doi:10.1210/en.2011-1341
更新日期:2011-11-01 00:00:00
abstract::Reproductive function is dependent on the interaction between GnRH and its cognate receptor found on gonadotrope cells of the anterior pituitary gland. GnRH activation of the GnRH receptor (GnRHR) is a potent stimulus for increased expression of multiple genes including the gene encoding the GnRHR itself. Thus, homolo...
journal_title:Endocrinology
pub_type: 杂志文章
doi:10.1210/en.2002-220784
更新日期:2003-03-01 00:00:00
abstract::Compelling evidence indicates a crucial role of prostaglandin F2α (PGF2α) in parturition. Both the maternal and fetal sides of the fetal membranes synthesize PGF2α, which exerts effects via the prostaglandin F2α receptor (FP) that is coupled to the activation of protein kinase C (PKC). Cyclooxygenase-2 (COX-2) catalyz...
journal_title:Endocrinology
pub_type: 杂志文章
doi:10.1210/en.2012-1441
更新日期:2012-10-01 00:00:00
abstract::The involvement of hypothalamic histaminergic neurons in the stress-induced release of peripheral catecholamines was studied in conscious, freely moving male rats. Blood samples were obtained via a catheter in a femoral artery. Intracerebroventricular infusion of histamine (HA; 30 micrograms) increased the plasma conc...
journal_title:Endocrinology
pub_type: 杂志文章
doi:10.1210/endo-126-3-1430
更新日期:1990-03-01 00:00:00
abstract::The direct actions of kit-ligand/stem cell factor (KL) in developing ovarian follicles were investigated. Previous studies have shown that granulosa cells express KL that can support oocyte development. The current study demonstrates that KL can also act directly on theca cells to promote cellular growth and different...
journal_title:Endocrinology
pub_type: 杂志文章
doi:10.1210/endo.138.9.5368
更新日期:1997-09-01 00:00:00
abstract::A soluble form of the amino-terminal extracellular (ecto-) domain of the human TSH receptor was generated. This protein was capable of binding TSH and autoimmune antibodies found in Graves' patients. A deletion mutant of the ectodomain lacking nine amino acids in the C-terminal region lost its ability to interact with...
journal_title:Endocrinology
pub_type: 杂志文章
doi:10.1210/endo.139.2.5748
更新日期:1998-02-01 00:00:00
abstract::Kisspeptins, products of the KiSS-1 gene with ability to bind G protein-coupled receptor 54 (GPR54), have been recently identified as major gatekeepers of reproductive function with ability to potently activate the GnRH/LH axis. Yet, despite the diversity of functional states of the female gonadotropic axis, pharmacol...
journal_title:Endocrinology
pub_type: 杂志文章
doi:10.1210/en.2005-1463
更新日期:2006-06-01 00:00:00
abstract::In the setting of insulin resistance, agonists of peroxisome proliferator-activated receptor (PPAR)-gamma restore insulin action in muscle and promote lipid redistribution. Mice with muscle-specific knockout of PPARgamma (MuPPARgammaKO) develop excess adiposity, despite reduced food intake and normal glucose disposal ...
journal_title:Endocrinology
pub_type: 杂志文章
doi:10.1210/en.2008-0100
更新日期:2008-11-01 00:00:00
abstract::The Src homology 2-containing tyrosine phosphatase, Shp-2, is a crucial enzyme that mediates intracellular signaling and is implicated in cell proliferation and differentiation. Here we investigated the involvement of the Shp-2 tyrosine phosphatase in determining the downstream signaling pathways initiated by the Ret ...
journal_title:Endocrinology
pub_type: 杂志文章
doi:10.1210/en.2003-0620
更新日期:2003-10-01 00:00:00
abstract::A sensitive radioimmunoassay is described for the measurement of plasma concentrations of canine growth hormone (cGH) as low as 0.25 ng/ml. The assay utilizes enzymatically iodinated cGH and the double antibody technique. The mean plasma concentration of growth hormone in the normal dog after overnight fast is 1.75 pl...
journal_title:Endocrinology
pub_type: 杂志文章
doi:10.1210/endo-96-3-822
更新日期:1975-03-01 00:00:00
abstract::A method is described for growing monolayers of newborn rat beta-cells in microculture trays. After disruption of the pancreas with collagenase, islets were isolated by Ficoll density gradient centrifugation, trypsinized to obtain individual cells, and plated in 96-well tissue culture trays. The cells were incubated f...
journal_title:Endocrinology
pub_type: 杂志文章
doi:10.1210/endo-106-4-1070
更新日期:1980-04-01 00:00:00
abstract::Recent studies of the human brain insulin receptor raise the possibility that some of the differences described between rat brain insulin receptors and rat liver insulin receptors may have been due to the presence of insulin-like growth factor I (IGF-I) receptors in the brain. This study details side by side compariso...
journal_title:Endocrinology
pub_type: 杂志文章
doi:10.1210/endo-122-5-1933
更新日期:1988-05-01 00:00:00
abstract::Kisspeptin, encoded by the Kiss1 gene, binds to a specific G protein-coupled receptor (kisspeptin1 receptor) to regulate the central reproductive axis. Kisspeptin has also been reported to be expressed in peripheral tissues, including the testes. However, factors regulating testicular kisspeptin and its role in reprod...
journal_title:Endocrinology
pub_type: 杂志文章
doi:10.1210/en.2014-1606
更新日期:2015-04-01 00:00:00
abstract::The secretion of LH, PRL, and cortisol was investigated in 4 sexually mature female rhesus macaques with cardiac catheters protected by tethers. Based on endocrine parameters, all 4 of the animals ovulated within 2 months from the time they were tethered, and regular menstrual cycles of 24-34 days were observed. The c...
journal_title:Endocrinology
pub_type: 杂志文章
doi:10.1210/endo-115-1-261
更新日期:1984-07-01 00:00:00
abstract::Des-acyl ghrelin (DAG), the most abundant form of ghrelin in humans, has been found to reduce arterial blood pressure and prevent cardiac and endothelial cell apoptosis. Despite this, data regarding its direct effect on cardiac function and coronary blood flow, as well as the related involvement of autonomic nervous s...
journal_title:Endocrinology
pub_type: 杂志文章
doi:10.1210/en.2015-1922
更新日期:2016-06-01 00:00:00
abstract::Low basal GH secretion and reduced GH responsiveness to different GH secretagogues, including GHRF, have been reported in aged animals and humans. Parallel to the in vivo findings, an impaired GH responsiveness to GHRF is evident in somatotropes from old rats of either sex. We report here that in anterior pituitaries ...
journal_title:Endocrinology
pub_type: 杂志文章
doi:10.1210/endo-121-5-1649
更新日期:1987-11-01 00:00:00
abstract::The circadian rhythm of glucocorticoids affects diverse physiological systems, including stress responses and the coordination of rhythmic functions in peripheral and central tissues. Circadian clocks are considered to be important coordinators of glucocorticoid release and loss of the core clock component Brain and m...
journal_title:Endocrinology
pub_type: 杂志文章
doi:10.1210/en.2016-1330
更新日期:2016-11-01 00:00:00
abstract::Pancreatic membrane receptors for cholecystokinin (CCK) in obese and nonobese Zucker rats were compared with the use of a biologically active [125I]iodo-CCK-8 radioprobe. Membrane homogenates from obese rats bound half the amount of radioligand in 2 h as did membranes from lean rats (specifically bound, 7.0% vs. 14.0%...
journal_title:Endocrinology
pub_type: 杂志文章
doi:10.1210/endo-119-2-546
更新日期:1986-08-01 00:00:00
abstract::An ovarian thecal/interstitial cell preparation was obtained by enzymatic digestion of immature rat ovaries depleted of granulosa cells. A component in thecal/interstitial cell-conditioned medium inhibited specific binding of [125I]iodo-epidermal growth factor ([125I]EGF) to its cell surface receptor, as determined wi...
journal_title:Endocrinology
pub_type: 杂志文章
doi:10.1210/endo-121-5-1892
更新日期:1987-11-01 00:00:00
abstract::Pituitary growth hormone (GH) content and plasma GH response to pentobarbital (PB) of severely hypothyroid rats are markedly decreased compared with euthyroid rats. Both pituitary and plasma GH may be increased by single ip injections of physiologic doses of thyroxine (T4) (1.75 mug/100g BW) or triiodothyronine (T3) (...
journal_title:Endocrinology
pub_type: 杂志文章
doi:10.1210/endo-98-1-77
更新日期:1976-01-01 00:00:00
abstract::The effect of the administration of sheep anti-LHRH gamma-globulin (anti-LHRHG) on the maintenance of pregnancy was investigated in rats. Nidation was confirmed by laparotomy on day 7 or 8 of pregnancy. In one experiment, rats were then injected iv with 1 ml anti-LHRHG or normal sheep gamma-globulin (NSG) daily from d...
journal_title:Endocrinology
pub_type: 杂志文章
doi:10.1210/endo-98-4-1024
更新日期:1976-04-01 00:00:00