bluuebunny/crossref_metadata_2025_split huggingface dataset:
Agonist and Antagonists Induce Homodimerization and Mixed Ligand Heterodimerization of Human Progesterone Receptors in Vivo by a Mammalian Two-Hybrid Assay
Authors: Leonhardt Susan A. , Altmann Magda , Edwards Dean P. 
Published: January 2014

AbstractThis study utilizes the mammalian two-hybrid system to examine the role of ligand in the dimerization of human progesterone receptor (hPR). The GAL4 DNA-binding domain and the herpes simplex virus VP16 transactivation domain were fused to the amino terminus of full-length hPR (both the A and B isoforms) to produce chimeric proteins. PR dimerization was detected by the ability of cotransfected GAL4/PR and VP16/PR chimeras in COS cells to induce expression of a reporter gene under the control of GAL4-binding sites (pG5CAT). Hormone agonist-dependent interactions were observed between the two like isoforms of PR (A-A and B-B) and between PR-A and PR-B (A-B), indicating that hormone can stimulate the formation of the three possible dimeric forms of PR within cells. In contrast, neither type I (ZK98299) nor type II (RU486, ZK112993) progestin antagonists stimulated interaction between these same hybrid PR proteins. However, activation of the VP16/PR chimera by antagonists on a progesterone response element-controlled reporter gene (DHRE-E1b-CAT) was only a fraction (4–13%) of that stimulated by agonist R5020. One possibility for the failure to detect an induction in the two-hybrid assay is antagonist-induced repression of the activity of the VP16/PR fusion protein rather than a failure of antagonists to stimulate interaction between the hybrid proteins. To test this idea, an UP-1 carboxyl-terminal truncation mutant of PR was used to construct the two-hybrid proteins. PR-UP-1 selectively binds antagonists, but not agonists, and is fully activated in response to antagonists. Both types of progestin antagonists stimulated interactions between GAL4/PR(UP-1) and VP16/PR(UP-1) hybrid proteins, indicating that antagonists are capable of stimulating PR dimerization in cells and do not function by disrupting or preventing dimerization. To determine whether PR bound to an antagonist can dimerize in whole cells with PR bound to agonist, GAL4/PR(UP-1) was paired in the two- hybrid assay with a VP16/PR fusion protein harboring a point mutation in PR at amino acid 722 (Gly-Cys) that specifically binds progestin agonist but not antagonist. Neither R5020 nor RU486 alone stimulated interaction between these ligand-specific PR hybrid proteins. However, strong interaction was detected by addition of both agonist and antagonists, indicating the formation of mixed ligand heterodimers and that both PR partners require ligand for dimerization to occur. Based on electrophoretic gel mobility shift assays (EMSAs), these heterodimers appear to have substantially reduced DNA binding activity. Progestin antagonists inhibit agonist activation of PR at concentrations that are too low to be accounted for by a simple competition mechanism for binding to PR. We propose that antiprogestin inactivation of PR in trans by heterodimerization contributes to the biological potency of these compounds.
Divergent Pathways Regulate Ligand-Independent Activation of ERα in SK-N-BE Neuroblastoma and COS-1 Renal Carcinoma Cells
Authors: Patrone Cesare , Gianazza Elisabetta , Santagati Sabrina , Agrati Paola , Maggi Adriana 
Published: January 2014

AbstractThe α-estrogen receptor (ERα) transcriptional activity can be regulated either by binding to the cognate ligand or by intracellular signaling pathways responsive to a variety of factors acting through cell membrane receptors. Studies carried out in HeLa and COS-1 cells demonstrated that the cross-coupling between estrogen and growth factor receptors is mediated by p21ras and requires phosphorylation of a specific serine residue (Ser 118 in the human ERα and Ser 122 in mouse ERα) located in the ERα N-terminal activation function 1 (AF-1). Likewise, in the SK-N-BE neuroblastoma cell line p21ras is involved in the cross-coupling between insulin and ERα receptors. However, in this cell line Ser 122 is not necessary for insulin-dependent activation of unliganded ERα. In addition, after insulin activation, the electrophoretic mobility associated to serine hyperphosphorylation of ERα in SK-N-BE and in COS-1 cells is different. Our study rules out the possibility of tyrosine phosporylation in unliganded ERα activation by means of transactivation studies of ERα tyrosine mutants and analysis of Tyr phosphorylation immunoreactivity. The two cofactors for steroid receptors RIP 140 and SRC-1 do not seem to be specifically involved in the insulin-induced ERα transactivation. The present study demonstrates the possibility of an alternative, cell-specific pathway of cross-coupling between intracellular and membrane receptors, which might be of importance for the understanding of the physiological significance of this mode of activation in the nervous system.
Differential Localization and Activity of the A- and B-Forms of the Human Progesterone Receptor Using Green Fluorescent Protein Chimeras
Authors: Lim Carol S. , Baumann Christopher T. , Htun Han , Xian Wenjuan , Irie Masako , Smith Catharine L. , Hager Gordon L. 
Published: January 2014

Abstract
Subcellular localization and transcriptional activity of green fluorescent protein-progesterone receptor A and B chimeras (GFP-PRA and GFP-PRB) were examined in living mammalian cells. Both GFP-PRA and B chimeras were found to be similar in transcriptional activity compared with their non-GFP counterparts. GFP-PRA and PRA were both weakly active, while GFP-PRB and PRB gave a 20- to 40-fold induction using a reporter gene containing the full-length mouse mammary tumor virus long-terminal repeat linked to the luciferase gene (pLTRluc). Using fluorescence microscopy, nuclear/cytoplasmic distributions for the unliganded and hormone activated forms of GFP-PRA and GFP-PRB were characterized. The two forms of the receptor were found to have distinct intracellular distributions; GFP-PRA was found to be more nuclear than GFP-PRB in four cell lines examined. The causes for and implications of this differential localization of the A and B forms of the human PR are discussed.
Tumor Necrosis Factor α-Mediated Insulin Resistance, but Not Dedifferentiation, Is Abrogated by MEK1/2 Inhibitors in 3T3-L1 Adipocytes
Authors: Engelman Jeffrey A. , Berg Anders H. , Lewis Renée Y. , Lisanti Michael P. , Scherer Philipp E. 
Published: January 2014

AbstractTumor necrosis factor-α (TNFα) has been implicated as a contributing mediator of insulin resistance observed in pathophysiological conditions such as obesity, cancer-induced cachexia, and bacterial infections. Previous studies have demonstrated that TNFα confers insulin resistance by promoting phosphorylation of serine residues on insulin receptor substrate 1 (IRS-1), thereby diminishing subsequent insulin-induced tyrosine phosphorylation of IRS-1. However, little is known about which signaling molecules are involved in this process in adipocytes and about the temporal sequence of events that ultimately leads to TNFα-stimulated IRS-1 serine phosphorylation. In this study, we demonstrate that specific inhibitors of the MAP kinase kinase (MEK)1/2-p42/44 mitogen-activated protein (MAP) kinase pathway restore insulin signaling to normal levels despite the presence of TNFα. Additional experiments show that MEK1/2 activity is required for TNFα-induced IRS-1 serine phosphorylation, thereby suggesting a mechanism by which these inhibitors restore insulin signaling.We observe that TNFα requires 2.5–4 h to markedly reduce insulin-triggered tyrosine phosphorylation of IRS-1 in 3T3-L1 adipocytes. Although TNFα activates p42/44 MAP kinase, maximal stimulation is observed within 10–30 min. To our surprise, p42/44 activity returns to basal levels well before IRS-1 serine phosphorylation and insulin resistance are observed. These activation kinetics suggest a mechanism of p42/44 action more complicated than a direct phosphorylation of IRS-1 triggered by the early spike of TNFα-induced p42/44 activity.Chronic TNFα treatment (≫ 72 h) causes adipocyte dedifferentiation, as evidenced by the loss of triglycerides and down-regulation of adipocyte-specific markers. We observe that this longer term TNFα-mediated dedifferentiation effect utilizes alternative, p42/44 MAP kinase-independent intracellular pathways.This study suggests that TNFα-mediated insulin resistance, but not adipocyte dedifferentiation, is mediated by the MEK1/2-p42/44 MAP kinase pathway.
Thyroid Hormone-Responsive Pituitary Hyperplasia Independent of Somatostatin Receptor 2
Authors: Brinkmeier Michelle L. , Stahl Justin H. , Gordon David F. , Ross Brian D. , Sarapura Virginia D. , Dowding Janet M. , Kendall Susan K. , Lloyd Ricardo V. , Ridgway E. Chester , Camper Sally A. 
Published: January 2014

AbstractMice homozygous for the targeted disruption of the glycoprotein hormone α-subunit (αGsu) display hypertrophy and hyperplasia of the anterior pituitary thyrotropes. Thyrotrope hyperplasia results in tumors in aged αGsu−/− mice. These adenomatous pituitaries can grow independently as intrascapular transplants in hypothyroid mice, suggesting that they have progressed beyond simple hyperplasia. We used magnetic resonance imaging to follow the growth and regression of thyrotrope adenomatous hyperplasia in response to thyroid hormone treatment and discovered that the tumors retain thyroid hormone responsiveness. Somatostatin (SMST) and its diverse receptors have been implicated in cell proliferation and tumorigenesis. To test the involvement of SMST receptor 2 (SMSTR2) in pituitary tumor progression and thyroid hormone responsiveness in αGsu−/− mutants, we generated Smstr2−/−, αGsu−/− mice. Smstr2−/−, αGsu−/− mice develop hyperplasia of thyrotropes, similar to αGsu−/− mutants, demonstrating that SMSTR2 is dispensable for the development of pituitary adenomatous hyperplasia. Thyrotrope hyperplasia in Smstr2−/−, αGsu−/− mice regresses in response to T4 treatment, suggesting that SMSTR2 is not required in the T4 feedback loop regulating TSH secretion.
Differential Subcellular Distribution and Transcriptional Activity of ΣE3, ΣE4, and ΣE3–4 Isoforms of the Rat Estrogen Receptor-α
Authors: Pasqualini Catherine , Guivarc’h Dominique , Barnier Jean-Vianney , Guibert Bernard , Vincent Jean-Didier , Vernier Philippe 
Published: January 2014

AbstractΣE3, ΣE4, and ΣE3–4 are naturally occurring estrogen receptor (ER) isoforms, generated through differential splicing of the ERα primary transcript and abundantly expressed in embryonic rat pituitary. Studies in COS cells transfected with full-length ERα or its three splice variants fused to green fluorescent protein (GFP), revealed a different subcellular localization for each isoform. In the absence of estradiol, full-length ERα-GFP was predominantly nuclear, and ΣE3-GFP and ΣE4-GFP were present both in cytoplasm and nucleus, whereas ΣE3–4-GFP was predominantly cytoplasmic. Upon hormone treatment, a dramatic redistribution of full-length ERα-GFP and ΣE3-GFP, from a diffuse to punctate pattern, occurred within the nucleus. In contrast, the distribution of ΣE4-GFP and ΣE3–4-GFP was unaffected. Nuclear fractionation studies showed that full-length ER-α and ΣE3 displayed the same hormone-induced ability to tether to nuclear matrix, whereas nuclear ΣE4 appeared to remain loosely associated to functional nuclear constituents. When cotransfected with an estrogen-inducible reporter plasmid (VIT-TK-CAT) in ER-negative (CHO k1) and ER-positive pituitary (GH4 C1) cells, ΣE3–4 exhibited a very weak estrogen-dependent transactivation activity, whereas ΣE3 had an inhibitory effect on full-length ER action. Conversely, ΣE4 displayed estrogen-independent transcriptional activity in ER-negative cells, and in ER-positive cells, enhanced the estrogen-induced gene expression as efficiently as full-length ERα. In a gel mobility shift assay, phosphorylated ΣE4 was able to form a specific complex with a consensus ERE, while ΣE3 and ΣE3–4 never did bind by themselves. The observed inhibitory action of ΣE3 on estrogen-dependent transcription would rather involve protein-protein interactions such as formation of heterodimers with full-length ERα, as suggested by immunoprecipitation followed by Western blotting. These data suggest that ΣE3 and ΣE4 may play a physiologically relevant role as negative or constitutively positive modulators of transcription, in the developing rat pituitary.
AR Suppresses Transcription of the α Glycoprotein Hormone Subunit Gene Through Protein-Protein Interactions with cJun and Activation Transcription Factor 2
Authors: Jorgensen Joan S. , Nilson John H. 
Published: January 2014

AbstractPreviously, we reported that the AR directly suppressed transcription of the α glycoprotein hormone subunit (αGSU) gene in a ligand-dependent fashion while ER had no effect. Mutagenesis studies of the αGSU promoter indicated that two elements were required for AR-mediated suppression: the α basal element and tandem cAMP response elements (CREs). Because several members of the bZip family of transcriptional proteins can bind the CREs, we used several functional assays to determine whether AR interacts selectively with cJun, activation transcription factor 2 (ATF2), or CRE binding protein (CREB). When tested by cotransfection with AR, cJun and ATF2 specifically rescued androgen-mediated suppression of theα GSU-reporter construct in a gonadotrope-derived cell line. In contrast, cotransfected CREB displayed no activity in this rescue assay. In fact, overexpression of CREB alone diminished activity of theα GSU promoter, suggesting that the transcriptional activity normally conferred by the tandem CREs in gonadotropes requires their occupancy by cJun/ATF2 heterodimers. Binding assays carried out with a glutathione-S-transferase-AR fusion protein indicated that the receptor itself also displayed a clear preference for binding cJun and ATF2. Furthermore, we ruled out the possibility that AR suppressed activity of the αGSU promoter by reducing synthesis of these bZip proteins. Additional experiments suggested that phosphorylation of AR or histone acetylation are unlikely requirements for AR suppression of αGSU promoter activity. Thus, our data suggest that AR suppresses activity of the αGSU promoter through direct protein-protein interactions with cJun and ATF2.
Calmodulin Is a Selective Modulator of Estrogen Receptors
Authors: García Pedrero Juana M. , del Rio Beatríz , Martínez-Campa Carlos , Muramatsu Masami , Lazo Pedro S. , Ramos Sofía 
Published: January 2014

Abstract
In the search for differences between ERα and ERβ, we analyzed the interaction of both receptors with calmodulin (CaM) and demonstrated that ERα but not ERβ directly interacts with CaM. Using transiently transfected HeLa cells, we examined the effect of the CaM antagonist N-(6-aminohexyl)-5-chloro-naphthalene sulfonilamide hydrochloride (W7) on the transactivation properties of ERα and ERβ in promoters containing either estrogen response elements or activator protein 1 elements. Transactivation by ERα was dose-dependently inhibited by W7, whereas that of ERβ was not inhibited or even activated at low W7 concentrations. In agreement with these results, transactivation of an estrogen response element containing promoter in MCF-7 cells (which express a high ERα/ERβ ratio) was also inhibited by W7. In contrast, transactivation in T47D cells (which express a low ERα/ERβ ratio) was not affected by this CaM antagonist. The sensitivity of MCF-7 cells to W7 was abolished when cells were transfected with increasing amounts of ERβ, indicating that the sensitivity to CaM antagonists of estrogen-responsive tissues correlates with a high ERα/ERβ ratio. Finally, substitution of lysine residues 302 and 303 of ERα for glycine rendered a mutant ERα unable to interact with CaM whose transactivation activity became insensitive to W7. Our results indicate that CaM antagonists are selective modulators of ER able to inhibit ERα-mediated activity, whereas ERβ actions were not affected or even potentiated by W7.
Growth Enhancement in Suppressor of Cytokine Signaling 2 (SOCS-2)-Deficient Mice Is Dependent on Signal Transducer and Activator of Transcription 5b (STAT5b)
Authors: Greenhalgh Christopher J. , Bertolino Patrick , Asa Sylvia L. , Metcalf Donald , Corbin Jason E. , Adams Timothy E. , Davey Helen W. , Nicola Nicos A. , Hilton Douglas J. , Alexander Warren S. 
Published: January 2014

AbstractMice lacking suppressor of cytokine signaling-2 (SOCS-2) exhibit accelerated postnatal growth resulting in adult mice that are 1.3 to 1.5 times the size of normal mice. In this study we examined the somatotrophic pathway to determine whether the production or actions of GH or IGF-I are altered in these mice. We demonstrated that SOCS-2−/− mice do not have elevated GH levels and suffer no major pituitary dysmorphogenesis, and that SOCS-2-deficient embryonic fibroblasts do not have altered IGF-I signaling. Primary hepatocytes from SOCS-2−/− mice, however, did have moderately prolonged signal transducer and activator of transcription 5 signaling in response to GH stimulation. Furthermore, the deletion of SOCS-2 from mice also lacking signal transducer and activator of transcription 5b had little effect on growth, suggesting that the action of SOCS-2 may be the regulation of the GH signaling pathway.
Sandwich Assay for Tacrolimus Using 2 Antitacrolimus Antibodies
Authors: Wei Tie Q , Zheng Yi F , Dubowy Michael , Sharma Manoj 
Published: January 2014

Abstract

BACKGROUND
Although detection of natural haptens by antihapten antibodies in sandwich assay format has the theoretical advantages of high analytical specificity and sensitivity, this type of assay has not been reported because of the seemingly insurmountable task of avoiding steric hindrance between the 2 bindings. This is especially true for ring-structured hydrophobic haptens. The macrolide drug tacrolimus (FK506, Prograf®, 804 Da) is such a hapten. Here we show the detection of tacrolimus using 2 antitacrolimus monoclonal antibodies in a sandwich assay.

METHODS
Both antibodies were developed by use of an intact tacrolimus molecule covalently linked to a carrier protein but via 2 different positions separated by 10 carbon atoms. Epitope analysis based on drug analog binding was used to show no overlap between the binding sites of the 2 antibodies, indicating the 10-carbon separation resulted in 2 distinct epitopes. The distinct epitopes suggested that the drug might be approachable by the antibodies from 2 separate directions, which predicted simultaneous binding as in sandwich formation.

RESULTS
This prediction was confirmed in sandwich ELISA and affinity column–mediated immunoassay formats. The assay demonstrated good imprecision and significantly lower metabolite cross-reactivity than competitive assay counterparts. Comparison with liquid chromatography–tandem mass spectrometry (LC-MS/MS) using 55 whole-blood samples from transplant patients with tacrolimus concentrations ranging from 0.9 to 29.5 ng/mL showed a linear regression: sandwich = 0.99 × LC-MS/MS + 0.10 ng/mL, r = 0.991, Sy|x = 1.08 ng/mL.

CONCLUSIONS
This work demonstrates that a highly specific sandwich assay using 2 antihapten antibodies is feasible for the measurement of a hapten drug.
Publication and Reporting of Test Accuracy Studies Registered in ClinicalTrials.gov
Authors: Korevaar Daniël A , Ochodo Eleanor A , Bossuyt Patrick M M , Hooft Lotty 
Published: January 2014

Abstract

BACKGROUND
Failure to publish and selective reporting are recognized problems in the biomedical literature, but their extent in the field of diagnostic testing is unknown. We aimed to identify nonpublication and discrepancies between registered records and publications among registered test accuracy studies.

METHODS
We identified studies evaluating a test's accuracy against a reference standard that were registered in ClinicalTrials.gov between January 2006 and December 2010. We included studies if their completion date was set before October 2011, allowing at least 18 months until publication. We searched PubMed, EMBASE, and Web of Science and contacted investigators for publications.

RESULTS
We included 418 studies, of which 224 (54%) had been published by mid-2013. Among studies that had been completed at least 30 months before our analyses, 45% were published within 30 months after their completion. Publication rates were high in studies registered after study completion (76%) and low for studies with an unknown (rather than completed) study status (36%). After we excluded these 2 categories, study duration was the only characteristic significantly associated with publication, with lower rates in studies lasting up to 1 year (39%) compared to studies of 13–24 months (62%) or longer (67%) (P = 0.01). In the 153 published studies that had been registered before completion, 49 (32%) showed discrepancies between the registry and publication regarding inclusion criteria (n = 19), test/threshold (n = 9), and outcomes (n = 32).

CONCLUSIONS
Failure to publish and selective reporting are prevalent in test accuracy studies. Their registration should be further promoted among researchers and journal editors.
Whole-Genome DNA Methylation Profile of the Jewel Wasp (Nasonia vitripennis)
Authors: Beeler Suzannah M , Wong Garrett T , Zheng Jennifer M , Bush Eliot C , Remnant Emily J , Oldroyd Benjamin P , Drewell Robert A 
Published: January 2014

AbstractThe epigenetic mark of DNA methylation, the addition of a methyl (CH3) group to a cytosine residue, has been extensively studied in many mammalian genomes and, although it is commonly found at the promoter regions of genes, it is also involved in a number of different biological functions. In other complex animals, such as social insects, DNA methylation has been determined to be involved in caste differentiation and to occur primarily in gene bodies. The role of methylation in nonsocial insects, however, has not yet been explored thoroughly. Here, we present the whole-genome DNA methylation profile of the nonsocial hymenopteran, the jewel wasp (Nasonia vitripennis). From high-throughput sequencing of bisulfite-converted gDNA extracted from male Nasonia thoraces, we were able to determine which cytosine residues are methylated in the entire genome. We found that an overwhelming majority of methylated sites (99.7%) occur at cytosines followed by a guanine in the 3′ direction (CpG sites). Additionally, we found that a majority of methylation in Nasonia occurs within exonic regions of the genome (more than 62%). Overall, methylation is sparse in Nasonia, occurring only at 0.18% of all sites and at 0.63% of CpGs. Our analysis of the Nasonia methylome revealed that in contrast to the methylation profile typically seen in mammals, methylation is sparse and is constrained primarily to exons. This methylation profile is more similar to that of the social hymenopteran species, the honey bee (Apis mellifera). In presenting the Nasonia methylome, we hope to promote future investigation of the regulatory function of DNA methylation in both social and nonsocial hymenoptera.