bluuebunny/crossref_metadata_2025 huggingface dataset:
Sexual Dimorphism in Counterregulatory Responses to Hypoglycemia after Antecedent Exercise
Authors: Galassetti Pietro , Neill Anthony R. , Tate Donna , Ertl Andrew C. , Wasserman David H. , Davis Stephen N.
Published: January 2014

After antecedent hypoglycemia, counterregulatory responses to subsequent hypoglycemia exhibit greater blunting in men than in women. Because physical exercise and hypoglycemia share multiple counterregulatory mechanisms, we hypothesized that prior exercise may also result in gender-specific blunting of counterregulatory responses to subsequent hypoglycemia. Thirty healthy subjects (15 women and 15 men; age, 28 ± 3 yr; body mass index, 23 ± 1 kg/m2) were studied during 2-d experiments. Day 1 consisted of either identical 90-min morning and afternoon cycle exercise at 50% maximum oxygen expenditure or two 2-h episodes of hyperinsulinemic euglycemia. Day 2 consisted of a 2-h morning hyperinsulinemic-hypoglycemic clamp. Endogenous glucose production was measured using [3-3H]glucose. Muscle sympathetic nerve activity was measured using microneurography. Day 2 insulin (540± 36 pmol/liter) and plasma glucose (2.9 ± 0.06 pmol/liter) levels were similar in men and women during the last 30 min of hypoglycemia. Compared with antecedent euglycemia, d 1 exercise produced significant blunting of d 2 counterregulatory responses to hypoglycemia. Several key d 2 counterregulatory responses were blunted to a greater extent in men than in women: glucagon (men, −105 ± 14; women, −25 ± 7 ng/liter; P < 0.0001), epinephrine (men, −2625 ± 257 pmol/liter; women, −212 ± 573; P < 0.001), norepinephrine (men, −0.50± 0.12 nmol/liter; women, −0 ± 0.11; P < 0.001), and muscle sympathetic nerve activity (men, −13 ± 4; women, −4 ± 4 bursts/min; P < 0.01). Cardiovascular responses (heart rate and systolic and mean arterial blood pressures) were also more blunted by antecedent exercise in men than in women. After d 1 exercise, the amount of glucose infused during d 2 hypoglycemia in men was increased 6-fold compared with that after d 1 euglycemia. This amount was significantly increased (P < 0.01) compared with the 2-fold (P < 0.01) increment in glucose infusion that was required in women after d 1 exercise. Lipolysis was unaffected by d 1 exercise in women, but was significantly blunted during d 2 hypoglycemia in men. In summary, two bouts of prolonged, moderate exercise (90 min at 50% maximum oxygen expenditure) induced a marked sexual dimorphism in key neuroendocrine (glucagon, catecholamines, and muscle sympathetic nerve activity) and metabolic (glucose kinetic, lipolysis) responses to next day hypoglycemia.
Associations of Glucose Control with Insulin Sensitivity and Pancreatic β-Cell Responsiveness in Newly Presenting Type 2 Diabetes
Authors: Albarrak Ahmed I. , Luzio Stephen D. , Chassin Ludovic J. , Playle Rebecca A. , Owens David R. , Hovorka Roman
Published: January 2014

We examined the ability of indices of insulin sensitivity and pancreatic β-cell responsiveness to explain interindividual variability of clinical measures of glucose control in newly presenting type 2 diabetes. Subjects with newly presenting type 2 diabetes (n = 65; 53 males and 12 females; age, 54 ± 1 yr; body mass index, 30.5 ± 0.7 kg/m2; mean ± se) underwent an insulin-modified iv glucose tolerance test to determine minimal model-derived insulin sensitivity (SI), glucose effectiveness, first-phase insulin secretion, and disposition index. Subjects also underwent a standard meal tolerance test (MTT) to measure fasting/basal (M0) and postprandial (MI) pancreatic β-cell responsiveness. Stepwise linear regression used these indices to explain interindividual variability of fasting and postprandial plasma glucose and insulin concentrations and glycated hemoglobin (HbA1C). All measures of pancreatic β-cell responsiveness (M0, MI, and first-phase insulin secretion) were negatively correlated with fasting plasma glucose (P < 0.01) and positively correlated with fasting plasma insulin (FPI) and insulin responses to MTT (P < 0.05). SI demonstrated negative correlation with FPI (P < 0.001) but failed to correlate with any glucose variable. MI followed by disposition index (composite index of insulin sensitivity and pancreatic β-cell responsiveness) were most informative in explaining interindividual variability. It was possible to explain 70–80% interindividual variability of fasting plasma glucose, FPI, HbA1C, and insulin responses to MTT, and only 25–40% interindividual variability of postprandial glucose. In conclusion, postprandial insulin deficiency is the most powerful explanatory factor of deteriorating glucose control in newly presenting type 2 diabetes. Indices of insulin sensitivity and pancreatic β-cell responsiveness explain fasting glucose and HbA1C well but fail to explain postprandial glucose.
Sertoli-Leydig Cell Tumor of the Ovary, a Rare Cause of Precocious Puberty in a 12-Month-Old Infant
Authors: Choong Catherine S. , Fuller Peter J. , Chu Simon , Jeske Yvette , Bowling Francis , Brown Richard , Borzi Peter , Balazs Nicholas D. , Suppiah Ram , Cotterill Andrew M. , Payton Diane , Robertson David M. , Burger Henry G.
Published: January 2014

We report a 12-month-old infant who presented with a 4-month history of isosexual precocious puberty secondary to an estrogenizing Sertoli-Leydig cell tumor of the ovary. Total serum immunoreactive inhibin and subunits A and B were markedly elevated before surgical resection and subsequently decreased 7 wk later into the normal prepubertal range. Twenty weeks following surgical removal, the patient presented again with central precocious puberty; inhibin B levels were raised on this occasion, a luteinizing releasing hormone stimulation test confirmed central precocious puberty. This is the youngest reported occurrence of this rare sex cord stromal neoplasm. The prognosis of this extremely rare tumor presenting at this early juvenile stage is uncertain. This report illustrates the usefulness of serum inhibin as a tumor marker during therapeutic suppression with leuprorelin acetate for central precocious puberty. Analysis of genomic and tumor DNA revealed a normal nucleotide sequence for the LH receptor and the Gαs gene. To understand the molecular pathogenesis of this tumor we analyzed mRNA levels for the inhibin A and B subunits, FSH receptor, LH receptor aromatase, steroidogenic factor-1 and the ER β genes. Molecular characterization reveals the presence of genes specific for granulosa and Leydig cells; the relative expression of these genes, in addition to its histologic characteristics, suggests that this tumor may result from a dysdifferentiation of a primordial follicle.
Cell-Specific Viral Gene Therapy of a Hurthle Cell Tumor
Authors: Zhang Rusheng , Straus Francis H. , DeGroot Leslie J.
Published: January 2014

We evaluated the effectiveness of a replication-defective adenovirus-transducing thymidine kinase (TK) gene under the control of the rat Tg (rTg) promoter (AdrTgtk) in therapy of a human Hurthle cancer (XTC-1 cell) in vitro and in vivo. The ganciclovir (GCV) sensitivity of infected XTC-1 cells was assessed in vitro by H3-thymidine incorporation assay and Trypan-blue exclusion, and by an in vivo tumor development assay. Proliferation was strongly inhibited by adding GCV into the culture medium of infected cells, but not uninfected cells, proving cell infection and expression of TK in the XTC-1 cells. AdrTgtk, and also viruses that have the noncell-specific cytomegalovirus (CMV) promoter-directing expression of TK (AdCMVtk), or luciferase (AdCMVLuc), were used to transduce XTC-1 cells to evaluate killing effects. After infection with AdCMVtk or AdrTgtk, followed by GCV treatment, 70% of infected cells were killed in the presence of GCV, compared with less than 20% of cells infected by AdCMVLuc and treated with GCV.

In vivo toxicity was studied in BALB/c mice. When adenovirus is given iv, liver is the major organ infected. No significant changes of the serum transaminase levels and no histological abnormalities were found in animals treated with AdrTgtk/GCV given iv, compared with control animals. High levels of serum transaminases, lymphocyte infiltration, some Kupffer’s cell prominence, and extensive single-cell hepatocyte death were found in AdCMVtk/GCV-treated animals, indicating severe liver damage induced, as expected, by the noncell-specific CMV promoter.

XTL-1 cells (2 × 106) were injected sc into BALB/c-severe combined immunodeficient mice (BALB/c-SCID), and the mice developed tumors after 3 wk. After intratumoral injection of AdrTgtk and treatment with GCV, tumors stabilized in 15 of 17 mice within 3 wk, 9 tumors remained stabilized after 5 wk of treatment, and 2 disappeared during observation. In AdCMVLuc/GCV-treated control mice, almost all tumors grew continuously. The average tumor size in AdrTgtk-treated mice was significantly smaller than that of control animals after 2 wk of treatment.

Our data confirm the effectiveness and specificity of an adenovirus using rTg promoter to express TK, and support its future application to thyroid cancer gene therapy in humans.
Bone Density and Amenorrhea in Ballet Dancers Are Related to a Decreased Resting Metabolic Rate and Lower Leptin Levels
Authors: Kaufman Becky A. , Warren Michelle P. , Dominguez Jennifer E. , Wang Jack , Heymsfield Steven B. , Pierson Richard N.
Published: January 2014

Osteopenia, which is correlated with amenorrhea and poor nutritional habits, has been well documented in elite ballet dancers. Estrogen replacement therapy and recovery from amenorrhea have not been associated with normalization of bone density. Thus, the osteopenia may be related to changes brought about by chronic dieting or other factors, such as a hypometabolic state induced by poor nutrition. The purpose of this study was to investigate the relationship of chronic dieting and resting metabolic rate (RMR) to amenorrhea and bone density.

RMR, bone density, eating disorder assessments, leptin levels, and complete menstrual and medical histories were determined in 21 elite ballet dancers and in 27 nondancers (age, 20–30 yr). No significant correlations were found between high EAT26 scores, a measure of disordered eating, and RMR, bone densities, body weight, body fat, or fat-free mass. However, when RMR was adjusted for fat-free mass (FFM), a significant positive correlation was found between RMR/FFM and bone density in both the arms (P < 0.001) and spine (P < 0.05) in ballet dancers, but not in the normal controls. The dancers also demonstrated significantly higher EAT scores (22.9 ± 10.3 vs. 4.1 ± 2.4; P < 0.001) and lower RMR/FFM ratios (30.0 ± 2.2 vs. 32.05 ± 2.8; P < 0.01). The only variable to predict lower RMR/FFM in the entire sample was ever having had amenorrhea; this group had significantly higher EAT scores (18.0 ± 13.5 vs. 10.3 ± 10.2; P < 0.05), lower leptin levels (4.03 ± 0.625 vs. 7.10 ± 4.052; P < 0.05), and lower bone mineral density in the spine (0.984 ± 0.11 vs. 1.10 ± 0.13; P < 0.05) and arm (0.773 ± 0.99 vs. 0.818 ± 0.01; P < 0.05).

We hypothesize that the correlation between low RMR and lower leptin levels and bone density may be more strongly related to nutritional habits in ballet dancers, causing significant depression of RMR, particularly for those with a history of amenorrhea.
The Methionine 196 Arginine Polymorphism in Exon 6 of the TNF Receptor 2 Gene (TNFRSF1B) Is Associated with the Polycystic Ovary Syndrome and Hyperandrogenism
Authors: Peral Belén , San Millán José L. , Castello Roberto , Moghetti Paolo , Escobar-Morreale Héctor F.
Published: January 2014

Inflammatory cytokines such as TNFα may play a role in the pathogenesis of common metabolic disorders, including hyperandrogenism and the polycystic ovary syndrome (PCOS). The TNF receptor 2 mediates most of the metabolic effects of TNFα. In the present study, we have evaluated serum soluble TNF receptor 2 levels, and several common polymorphisms in the TNF receptor 2 gene (TNFRSF1B), in women presenting with PCOS or hyperandrogenic disorders. Initial studies included 103 hyperandrogenic patients (42 presenting with PCOS) and 36 controls from Spain. The 196R alleles of the M196R (676 T→G) variant in exon 6 of TNFRSF1B, which is in linkage disequilibrium with a CA-repeat microsatellite polymorphism in intron 4 of TNFRSF1B, tended to be more frequent in hyperandrogenic patients than in controls (P = 0.056), reaching statistical significance when the analysis was restricted to include only PCOS patients (P < 0.03). Extended analysis including another 11 hyperandrogenic patients from Spain and 64 patients and 29 controls from Italy confirmed the association between 196R alleles of the M196R variant and hyperandrogenic disorders (P < 0.05), which was maintained when restricting the analysis to PCOS patients (P < 0.02). On the contrary, the 3′-untranslated region (exon 10) variants 1663 G→A, 1668 T→G, and 1690 T→C were not associated with hyperandrogenism. The soluble TNF receptor 2 levels were not different between patients and controls but were increased in obese subjects, compared with lean individuals, and were affected by the interaction between the 1663 G→A and 1668 T→G variants in the 3′-untranslated region of TNFRSF1B. The TNFRSF1B genotype did not influence any clinical or biochemical variable related to hyperandrogenism or insulin sensitivity and was not associated with obesity, both in hyperandrogenic patients and healthy controls considered separately. In conclusion, the M196R (676 T→G) variant in exon 6 of TNFRSF1B is associated with hyperandrogenism and PCOS, further suggesting a role for inflammatory cytokines in the pathogenesis of these disorders.
Selective Inhibition of Prostaglandin E2 Receptors EP2 and EP4 Induces Apoptosis of Human Endometriotic Cells through Suppression of ERK1/2, AKT, NFκB and β-CATENIN Pathways and Activation of Intrinsic Apoptotic Mechanisms
Authors: Banu Sakhila K. , Lee JeHoon , Speights V. O. , Starzinski-Powitz Anna , Arosh Joe A.
Published: January 2014

Endometriosis is a benign chronic gynecological disease of reproductive age women characterized by the presence of functional endometrial tissues outside the uterine cavity. It is an estrogen-dependent disease. Current treatment modalities to inhibit biosynthesis and actions of estrogen compromise menstruation, pregnancy, reproductive health of women, and fail to prevent reoccurrence of disease. There is a critical need to identify new specific signaling modules for non-estrogen targeted therapies for endometriosis. In our previous study, we reported that selective inhibition of cyclooxygenase-2 prevented survival, migration and invasion of human endometriotic epithelial and stromal cells which was due to decreased PGE2 production. In this study, we determined mechanisms through which prostaglandin E2 (PGE2) promoted survival of human endometriotic cells. Results of the present study indicate that: (i) PGE2 promotes survival of human endometriotic cells through EP2 and EP4 receptors by activating ERK1/2, AKT, NFκB, and β-catenin signaling pathways; (ii) selective inhibition of EP2 and EP4 suppresses these cell survival pathways and augments interactions between proapoptotic proteins (Bax and Bad) and antiapoptotic proteins (Bcl-2/Bcl-XL), facilitates the release of cytochrome C and thus activates caspase-3/PARP-mediated intrinsic apoptotic pathways; and (iii) these PGE2 signaling components are more abundantly expressed in ectopic endometriosis tissues compared to eutopic endometrial tissues during the menstrual cycle in women. These novel findings may provide an important molecular framework for further evaluation of selective inhibition of EP2 and EP4 as potential therapy, including non-estrogen target, to expand the spectrum of currently available treatment options for endometriosis in women.
Maternal Diabetes Causes Mitochondrial Dysfunction and Meiotic Defects in Murine Oocytes
Authors: Wang Qiang , Ratchford Ann M. , Chi Maggie M.-Y. , Schoeller Erica , Frolova Antonina , Schedl Tim , Moley Kelle H.
Published: January 2014

The adverse effects of maternal diabetes on embryo development and pregnancy outcomes have recently been shown to occur as early as the one-cell zygote stage. The hypothesis of this study was that maternally inherited mitochondria in oocytes from diabetic mice are abnormal and thus responsible in part for this latency of developmental compromise. In ovulated oocytes from diabetic mice, transmission electron microscopy revealed an alteration in mitochondrial ultrastructure, and the quantitative analysis of mitochondrial DNA copy number demonstrated an increase. The levels of ATP and tricarboxylic acid cycle metabolites in diabetic oocytes were markedly reduced compared with controls, suggesting a mitochondrial metabolic dysfunction. Abnormal distribution of mitochondria within maturing oocytes also was seen in diabetic mice. Furthermore, oocytes from diabetic mice displayed a higher frequency of spindle defects and chromosome misalignment in meiosis, resulting in increased aneuploidy rates in ovulated oocytes. Collectively, our results suggest that maternal diabetes results in oocyte defects that are transmitted to the fetus by two routes: first, meiotic spindle and chromatin defects result in nondisjunction leading to embryonic aneuploidy; second, structural and functional abnormalities of oocyte mitochondria, through maternal transmission, provide the embryo with a dysfunctional complement of mitochondria that may be propagated during embryogenesis.
Inhibition of the Type 2 Iodothyronine Deiodinase Underlies the Elevated Plasma TSH Associated with Amiodarone Treatment
Authors: Rosene Matthew L. , Wittmann Gábor , Arrojo e Drigo Rafael , Singru Praful S. , Lechan Ronald M. , Bianco Antonio C.
Published: January 2014

Endocrinology, published October 6, 2010, 10.1210/en.2010-0553
Identification of Trophoblast-Specific Regulatory Elements in the Mouse Placental Lactogen II Gene
Authors: Lin Jiandie , Linzer Daniel I. H.
Published: January 2014

Placental lactogen II, the major ligand for the PRL receptor during the second half of gestation in rodents, is synthesized specifically by placental trophoblast giant cells. A transient transgenic analysis has been used to localize the giant cell-specific regulatory region within the mouse placental lactogen II gene to sequences between −1340 and −2019 upstream of the transcriptional start site. More precise mapping of the regulatory elements has been accomplished by transfection of promoter constructs into Rcho-1 trophoblast cells, resulting in the characterization of two positive regulatory elements in the −1471 to −1340 region; two other regulatory elements have been implicated but not further characterized, a negative regulatory element between −2019 and −1778 and another positive element within the region from −1340 to −569. Both of the characterized positive regulatory elements are recognized by factors that are enriched in differentiated giant cells compared with proliferative trophoblasts, and these factors are either absent or at low levels in fibroblasts. The complexes that form on the two elements are distinct and neither element competes with the other for factor binding, thus implicating at least two different regulatory elements in late-gestational trophoblast giant cell-specific gene expression.
Differential Regulation of Basal and Cyclic Adenosine 3′,5′-Monophosphate-Induced Somatostatin Gene Transcription in Neural Cells by DNA Control Elements That Bind Homeodomain Proteins
Authors: Schwartz Petra T. , Vallejo Mario
Published: January 2014

A number of genes encoding neuropeptides are expressed in the peripheral and central nervous systems, in different endocrine organs, and in specialized cells distributed along the gastrointestinal tract. Whether expression of the same neuropeptide gene in different tissues is regulated by similar transcriptional mechanisms or by mechanisms that differ in a cell-specific manner remains unclear. We report on promoter studies on the regulation of the somatostatin gene in immortalized neural precursor cells derived from developing rat forebrain. Expression of the somatostatin gene in these cells was determined by RT-PCR/Southern blot analysis, by immunocytochemistry, and by RIA. We show that in cerebrocortical and hippocampal cells, expression of the somatostatin gene is regulated by several negative and positive DNA cis-regulatory elements located throughout the promoter region. The somatostatin cAMP-response element appears to play a prominent role in neural somatostatin gene expression by acting as a strong enhancer even in the absence of cAMP stimulation. Site-directed mutagenesis followed by transient transfection assays indicated that SMS-TAAT1, SMS-TAAT2, and SMS-UE, three previously identified homeodomain protein-binding regulatory elements that enhance transcription in pancreatic cells, act as repressors of transcription in neural cells. Electrophoretic mobility shifts assays indicate that those elements bind protein complexes that differ between neural and pancreatic cells. Our results support the notion that expression of the somatostatin gene in neural cells occurs via transcriptional mechanisms that are different from those regulating expression of the same gene in pancreatic cells.
Conditional Disruption of the Aryl Hydrocarbon Receptor Nuclear Translocator (Arnt) Gene Leads to Loss of Target Gene Induction by the Aryl Hydrocarbon Receptor and Hypoxia-Inducible Factor 1α
Authors: Tomita Shuhei , Sinal Christopher J. , Yim Sun Hee , Gonzalez Frank J.
Published: January 2014

To determine the function of the aryl hydrocarbon receptor nuclear translocator (ARNT), a conditional gene knockout mouse was made using the Cre-loxP system. Exon 6, encoding the conserved basic-helix-loop-helix domain of the protein, was flanked by loxP sites and introduced into the Arnt gene by standard gene disruption techniques using embryonic stem cells. Mice homozygous for the floxed allele were viable and had no readily observable phenotype. The Mx1-Cre transgene, in which Cre is under control of the interferon-γ promoter, was introduced into the Arnt-floxed mouse line. Treatment with polyinosinic-polycytidylic acid to induce expression of Cre resulted in complete disruption of the Arnt gene and loss of ARNT messenger RNA (mRNA) expression in liver. To determine the role of ARNT in gene control in the intact animal mouse liver, expression of target genes under control of an ARNT dimerization partner, the aryl hydrocarbon receptor (AHR), was monitored. Induction of CYP1A1, CYP1A2, and UGT1*06 mRNAs by the AHR ligand 2,3,7,8-tetrachlorodibenzo-pdioxin was absent in livers of Arnt-floxed/Mx1-Cre mice treated with polyinosinic-polycytidylic. These data demonstrate that ARNT is required for AHR function in the intact animal. Partial deletion of the Arnt allele was found in kidney, heart, intestine, and lung. Despite more than 80% loss of the ARNT expression in lung, maximal induction of CYP1A1 was found, indicating that the expression level of ARNT is not limiting to AHR signaling. Cobalt chloride induction of the glucose transporter-1 and heme oxygenase-1 mRNAs was also markedly abrogated in mice lacking ARNT expression, suggesting an inhibition of HIF-1α activity. These studies establish a critical role for ARNT in AHR and HIF-1α signal transduction in the intact mouse.
25-Dehydro-1α-Hydroxyvitamin D3- 26,23S-Lactone Antagonizes the Nuclear Vitamin D Receptor by Mediating a Unique Noncovalent Conformational Change
Authors: Bula C. M. , Bishop J. E. , Ishizuka S. , Norman A. W.
Published: January 2014

(23S)-25-dehydro-1α-Dihydroxyvitamin D3-26,23-lactone (TEI-9647; MK) has been reported to antagonize the 1α,25-dihydroxyvitamin D3 nuclear receptor (VDR)- mediated increase in transcriptional activity. Using a transient transfection system incorporating the osteocalcin VDRE (vitamin D response element) in Cos-1 cells, we found that 20 nm MK antagonizes VDR-mediated transcription by 50% when driven by 1 nm 1α,25(OH)2D3. Four analogs of 1α,25(OH)2D3, also at 1 nm, were antagonized 25 to 39% by 20 nm MK. However, analogs with 16-ene/23-yne or 20-epi modifications, which have a significantly lower agonist ED50 for the VDR than 1α,25(OH)2D3, were antagonized by 20 nm MK only at 100 pm or 10 pm, respectively. One possible mechanism for antagonism is that the 25-dehydro alkene of MK might covalently bind the ligand-binding site of the VDR rendering it inactive. Utilization of a ligand exchange assay, however, demonstrated that MK bound to VDR is freely exchanged with 1α,25(OH)2D3 in vitro. These data support the apparent correlation between VDR transcriptional activation by agonists and the effective range of MK antagonism by competition. Furthermore, protease sensitivity analysis of MK bound to VDR indicates the presence of a unique conformational change in the VDR ligand-binding domain, showing a novel doublet of VDR fragments centered at 34 kDa, whereas 1α,25(OH)2D3 as a ligand produces only a single 34-kDa fragment. In comparison, the natural metabolite 1α,25dihydroxyvitamin D3-26,23-lactone yields only the 30-kDa fragment that is produced by all ligands to varying degrees. Collectively, these results support that MK is a potent partial antagonist of the VDR for 1α,25(OH)2D3 and its analogs when in appropriate excess of the agonist.
Temporally Distinct and Ligand-Specific Recruitment of Nuclear Receptor-Interacting Peptides and Cofactors to Subnuclear Domains Containing the Estrogen Receptor
Authors: Schaufele Fred , Chang Ching-yi , Liu Weiqun , Baxter John D. , Nordeen Steven K. , Wan Yihong , Day Richard N. , McDonnell Donald P.
Published: January 2014

Ligand binding to estrogen receptor (ER) is presumed to regulate the type and timing of ER interactions with different cofactors. Using fluorescence microscopy in living cells, we characterized the recruitment of five different green fluorescent protein (GFP)-labeled ER-interacting peptides to the distinct subnuclear compartment occupied by blue fluorescent protein (BFP)-labeled ERα. Different ligands promoted the recruitment of different peptides. One peptide was recruited in response to estradiol (E2), tamoxifen, raloxifene, or ICI 182,780 incubation whereas other peptides were recruited specifically by E2 or tamoxifen. Peptides containing different sequences surrounding the ER-interacting motif LXXLL were recruited with different time courses after E2 addition. Complex temporal kinetics also were observed for recruitment of the full-length, ER cofactor glucocorticoid receptor-interacting protein 1 (GRIP1); rapid, E2-dependent recruitment of GRIP1 was blocked by mutation of the GRIP1 LXXLL motifs to LXXAA whereas slower E2 recruitment persisted for the GRIP1 LXXAA mutant. This suggested the presence of multiple, temporally distinct GRIP 1 recruitment mechanisms. E2 recruitment of GRIP1 and LXXLL peptides was blocked by coincubation with excess ICI 182,780. In contrast, preformed E2/ER/GRIP1 and E2/ER/LXXLL complexes were resistant to subsequent ICI 182,780 addition whereas ICI 182,780 dispersed preformed complexes containing the GRIP1 LXXAA mutant. This suggested that E2-induced LXXLL binding altered subsequent ligand/ER interactions. Thus, alternative, ligand-selective recruitment and dissociation mechanisms with distinct temporal sequences are available for ERα action in vivo.
PRL-Induced ERα Gene Expression Is Mediated by Janus Kinase 2 (Jak2) While Signal Transducer and Activator of Transcription 5b (Stat5b) Phosphorylation Involves Jak2 and a Second Tyrosine Kinase
Authors: Frasor Jonna , Barkai Uriel , Zhong Liping , Fazleabas Asgerally T. , Gibori Geula
Published: January 2014

In the rat corpus luteum of pregnancy, PRL stimulation of ER expression is a prerequisite for E2 to have any luteotropic effect. Previous work from our laboratory has established that PRL stimulates ERα expression at the level of transcription and that the transcription factor Stat5 (signal transducer and activator of transcription 5) mediates this stimulation. Since it is well established that PRL activates Stat5 through the tyrosine kinase, Janus kinase 2 (Jak2), the role of Jak2 in PRL regulation of ERα expression was investigated. In primary luteinized granulosa cells, the general tyrosine kinase inhibitors, genistein and AG18, and the Jak2 inhibitor, AG490, prevented PRL stimulation of ERα mRNA levels, suggesting that PRL signaling to the ERα gene requires Jak2 activity. However, using an antibody that recognizes the tyrosine-phosphorylated forms of both Stat5a and Stat5b (Y694/Y699), it was found that AG490 could inhibit PRL-induced Stat5a phosphorylation only and had little or no effect on Stat5b phosphorylation. These effects of AG490 were confirmed in COS cells overexpressing Stat5b. Also in COS cells, a kinase-negative Jak2 prevented PRL stimulation of ERα promoter activity and Stat5b phosphorylation while a constitutively active Jak2 could stimulate both in the absence of PRL. Furthermore, kinase-negative-Jak2, but not AG490, could inhibit Stat5b nuclear translocation and DNA binding. Therefore, it seems that in the presence of AG490, Stat5b remains phosphorylated, is located in the nucleus and capable of binding DNA, but is apparently transcriptionally inactive. These findings suggest that PRL may activate a second tyrosine kinase, other than Jak2, that is capable of phosphorylating Stat5b without inducing transcriptional activity. To investigate whether another signaling pathway is involved, the src kinase inhibitor PP2 and the phosphoinositol-3 kinase inhibitor (PI3K), LY294002, were used. Neither inhibitor alone had any major effect on PRL regulation of ERα promoter activity or on PRL-induced Stat5b phosphorylation. However, the combination of AG490 and LY294002 largely prevented PRL-induced Stat5b phosphorylation. These findings indicate that PRL stimulation of ERα expression requires Jak2 and also that PRL can induce Stat5b phosphorylation through two tyrosine kinases, Jak2 and one downstream of PI3K. Furthermore, these results suggest that the role of Jak2 in activating Stat5b may be through a mechanism other than simply inducing Stat5b phosphorylation.
Direct Regulation of β3-Integrin Subunit Gene Expression by HOXA10 in Endometrial Cells
Authors: Daftary Gaurang S. , Troy Patrick J. , Bagot Catherine N. , Young Steven L. , Taylor Hugh S.
Published: January 2014

Estrogen and progesterone regulate HOXA10 expression in the endometrium, where HOXA10 is necessary for implantation. The integrins are also involved in early embryo-endometrial interactions. Here we show that HOXA10 directly regulates β3-integrin subunit expression in the endometrium, likely mediating the effect of sex steroids on β3-integrin expression. β3-Integrin expression was decreased in endometrium shown to have low HOXA10 expression. β3-Integrin mRNA levels were increased in endometrial adenocarcinoma cells (Ishikawa) transfected with pcDNA3.1/HOXA10, and decreased in cells treated with HOXA10 antisense. Seven consensus HOXA10 binding sites were identified 5′ of the β3-integrin gene. Direct binding of HOXA10 protein to four sites was demonstrated by EMSA. Reporter gene expression increased in BT-20 cells cotransfected with pcDNA3.1/ HOXA10 and pGL3-promoter vector containing region F (encompassing all seven HOXA10 consensus sites). A 41-bp segment (Region A) showed highest affinity binding to HOXA10 protein. Increased reporter expression, equal in magnitude to that obtained with Region F, was obtained with Region A. HOXA10 protein binding within Region A was localized by deoxyribonuclease I footprinting. β3-Integrin expression was directly up-regulated by HOXA10 through a 41-bp 5′-regulatory element. Sex steroids regulate the expression of endometrial β3-integrin through a pathway involving HOXA10.
Pregnane X Receptor (PXR), Constitutive Androstane Receptor (CAR), and Benzoate X Receptor (BXR) Define Three Pharmacologically Distinct Classes of Nuclear Receptors
Authors: Moore Linda B. , Maglich Jodi M. , McKee David D. , Wisely Bruce , Willson Timothy M. , Kliewer Steven A. , Lambert Millard H. , Moore John T.
Published: January 2014

The NR1I subfamily of nuclear receptors contains a phylogenetically diverse array of receptors related to the mammalian pregnane X receptor (PXR) (NR1I2) and constitutive androstane receptor (CAR) (NR1I3). We have carried out an extensive comparative analysis of this subgroup with representatives from fish, birds, amphibians, and mammals. Four novel receptors were isolated from fish, dog, pig, and monkey for this study and combined with a previously reported set of related receptors including human PXR, rabbit PXR, mouse PXR, chicken CXR, frog benzoate X receptors (BXRα, BXRβ), and human and mouse CAR. A broad range of xenobiotics, steroids, and bile acids were tested for their ability to activate the ligand binding domain of each receptor. Three distinct groups of receptors were identified based on their pharmacological profiles: 1) the PXRs were activated by a broad range of xenobiotics and, along with the mammalian PXRs, included the chicken and fish receptors; 2) the CARs were less promiscuous, had high basal activities, and were generally repressed rather than activated by those compounds that modulated their activity; and 3) the BXRs were selectively activated by a subset of benzoate analogs and are likely to be specialized receptors for this chemical class of ligands. The PXRs are differentiated from the other NR1I receptors by a stretch of amino acids between helices 1 and 3, which we designate the H1–3 insert. This insert was present in the mammalian, chicken, and fish PXRs but absent in the CARs and BXRs. Modeling studies suggest that the H1–3 insert contributes to the promiscuity of the PXRs by facilitating the unwinding of helices-6 and -7, thereby expanding the ligand binding pocket.
Direct Interactions between Corepressors and Coactivators Permit the Integration of Nuclear Receptor-Mediated Repression and Activation
Authors: Li Xiaolin , Kimbrel Erin A. , Kenan Daniel J. , McDonnell Donald P.
Published: January 2014

The unliganded thyroid hormone receptor β (TRβ) represses the basal transcriptional activity of target genes, in part through interactions with the nuclear receptor corepressor (N-CoR). In this study we have identified a rather unexpected interaction between N-CoR and the nuclear receptor coactivator ACTR. We have demonstrated in vitro and in intact cells that N-CoR directly associates with ACTR and that the interaction surfaces on N-CoR and ACTR are distinct from those required for TR binding. The significance of this finding was demonstrated by showing that N-CoR facilitates an interaction between unliganded-TRβ and ACTR. One possible consequence of the formation of the trimeric complex of N-CoR/ACTR/unliganded-TR is that N-CoR may raise the local concentration of ACTR at target gene promoters. In support of this hypothesis it was demonstrated that the presence of N-CoR can enhance TRβ-mediated transcriptional activation. It is proposed, therefore, that TRβ- mediated activation and repression are integrally linked in a manner that is not predicted by the current models of nuclear receptor action.
Characterization of a New Upstream GnRH Receptor Promoter in Human Ovarian Granulosa-Luteal Cells
Authors: Cheng Chi Keung , Yeung Chung Man , Chow Billy K. C. , Leung Peter C. K.
Published: January 2014

GnRH has been implicated as an important local autocrine and paracrine factor in regulating ovarian function. However, to date, the transcriptional regulation of GnRH receptor (GnRHR) gene in human ovary remains poorly understood. Here we report the characterization of a new upstream promoter for the GnRHR gene in human granulosa-luteal cells. Using progressive deletion analysis, a region between nucleotide −1300 and −1018 (relative to the translation start site) was shown to exhibit the highest promoter activities in two immortalized human granulosa-luteal cell lines, SVOG-4o and SVOG-4m. Two putative CCAAT/enhancer binding protein (C/EBP) motifs and one GATA motif were identified within this region. Mutational studies showed that these three motifs cooperated synergistically to regulate GnRHR gene transcription in the granulosa cells but not in other cell types including human ovarian carcinoma OVCAR-3, human embryonic kidney-293 (HEK-293) and mouse pituitary gonadotrope-derived αT3–1 cells. Surprisingly, by competitive EMSAs, we found that an Oct-1 consensus sequence was able to inhibit protein complex formation with the distal C/EBP motif, suggesting a possible cross-talk between the Oct-1 transcription factor and this C/EBP motif. Taken together, our results strongly indicate a role of the C/EBP and GATA motifs in regulating GnRHR gene transcription in human granulosa-luteal cells and further suggest that tissue-specific expression of human GnRHR gene is mediated by differential promoter usage.
11β-Hydroxysteroid Dehydrogenase Type 1 and Its Role in the Hypothalamus-Pituitary-Adrenal Axis, Metabolic Syndrome, and Inflammation
Authors: Cooper Mark S. , Stewart Paul M.
Published: January 2014

Context

11β-Hydroxysteroid dehydrogenase (11β-HSD) enzymes are now appreciated to be important regulators of hormone action at a tissue level. 11β-HSD1 is widely expressed and increases glucocorticoid action through its unique ability to convert inactive glucocorticoids (cortisone in man, 11-dehydrocorticosterone in rodents) to their active forms (cortisol and corticosterone, respectively). The enzyme has roles in the normal hypothalamus-pituitary-adrenal (HPA) axis, has been implicated in metabolic syndrome, and may modulate various aspects of the immune response.

Evidence Acquisition

A review of published, peer-reviewed medical literature (1990 to June 2009) on the physiology and pathophysiology of 11β-HSD1 was performed with an emphasis on HPA axis consequences, the metabolic syndrome, and the inflammatory response.

Evidence Synthesis

Studies of patients with genetic defects in 11β-HSD1 action show abnormal HPA axis responses with hyperandrogenism being a major consequence. The mechanisms underlying these abnormalities have been explored in mouse models with targeted deletion of components of the 11β-HSD1 system. A range of experimental studies emphasize the role of 11β-HSD1 in the metabolic syndrome and the potential for treatment with chemical inhibitors. An emerging area is the role of 11β-HSD1 in the inflammatory response.

Conclusions

11β-HSD1 activity is an important component of the HPA axis and contributes to the metabolic syndrome and the normal immune response. Ongoing clinical observations and the development of selective inhibitors will further clarify the role of 11β-HSD1 in these areas.
Progestins Activate the AKT Pathway in Leiomyoma Cells and Promote Survival
Authors: Hoekstra Anna V. , Sefton Elizabeth C. , Berry Emily , Lu Zhenxiao , Hardt Jennifer , Marsh Erica , Yin Ping , Clardy Jon , Chakravarti Debabrata , Bulun Serdar , Kim J. Julie
Published: January 2014

Context

Progesterone has been associated with promoting growth of uterine leiomyomas. The mechanisms involved remain unclear.

Objective

In this study we investigated the activation of the AKT pathway and its downstream effectors, GSK3b and FOXO1 by progesterone as a mechanism of proliferation and survival of leiomyoma cells. Inhibitors of the AKT pathway were used to demonstrate the role of PI3K, AKT and FOXO1 in contributing to cell proliferation and apoptosis.

Results

Treatment of leiomyoma cells with R5020 over a period of 72h resulted in higher cell numbers compared to untreated cells. When cells were treated with 100nM R5020 for 1h and 24h, the levels of phospho(Ser 473)-AKT increased. This increase was inhibited when cells were co-treated with RU486. Treatment of leiomyoma cells with a PI3K inhibitor, LY294 dramatically decreased levels of phospho(Ser 473)-AKT, despite R5020 treatment. In addition to increased phospho(Ser 473)-AKT levels, R5020 treatment resulted in an increase in phospho(Ser 256)-FOXO1 and phospho-GSK3b. Inhibition of AKT using API-59 decreased proliferation and cell viability even in the presence of R5020. Higher concentrations of API-59 induced apoptosis of leiomyoma cells even in the presence of R5020. Psammaplysene A increased nuclear FOXO1 levels and did not affect cell proliferation but induced apoptosis of leiomyoma cells.

Conclusions

The progestin, R5020, can rapidly activate the AKT pathway. Inhibition of the AKT pathway inhibits cell proliferation and promotes apoptosis of leiomyoma cells.
Growth Differentiation Factor 9 Reverses Activin A Suppression of Steroidogenic Acute Regulatory Protein Expression and Progesterone Production in Human Granulosa-Lutein Cells
Authors: Shi Feng-Tao , Cheung Anthony P. , Klausen Christian , Huang He-Feng , Leung Peter C. K.
Published: January 2014

Background: We have reported that growth differentiation factor (GDF) 9 can enhance activin A (βAβA)-induced inhibin B (αβB) secretion in human granulosa-lutein (hGL) cells, but its effects on steroidogenic acute regulatory protein (StAR), ovarian steroidogenic enzymes, and progesterone production are unknown. We undertook this study to further evaluate GDF9 in this regard.

Methods: hGL cells from women undergoing in vitro fertilization treatment were cultured with and without small interfering RNA (siRNA) transfection targeted at inhibin α-subunit or GDF9 before treatment with GDF9, activin A, FSH, or combinations. We compared StAR, P450 side-chain cleavage enzyme, and 3β-hydroxysteroid dehydrogenase expression in hGL cells and progesterone levels in culture media after these treatments. mRNA, protein, and hormone levels were assessed with real-time RT-PCR, immunoblotting, and ELISA, respectively. Data were analyzed by ANOVA followed by Tukey’s test.

Results: Activin A alone reduced basal and FSH-induced progesterone production by decreasing the expression of StAR protein, which regulates the rate-limiting step in steroidogenesis but not P450 side-chain cleavage enzyme and 3β-hydroxysteroid dehydrogenase. GDF9 attenuated these activin A effects on StAR and progesterone. After transfection of a-subunit siRNA, activin A level increased (P < 0.001), whereas basal and activin A-induced inhibin B levels (with and without GDF9) decreased. Furthermore, the effects of GDF9 in reversing activin A suppression of progesterone production were attenuated (P < 0.001). Transfection of GDF9 siRNA decreased GDF9 as expected and led to lower StAR expression and progesterone secretion than those observed with activin A treatment alone.

Conclusion: GDF9 attenuates the suppressive effects of activin A on StAR expression and progesterone production by increasing the expression of inhibin B, which acts as an activin A competitor.
Agricultural-Food Sector Competitiveness of Eco Companies
Authors: Koneczna Renata
Published: January 2014

The aim of this paper is to present some methodological aspects connected with the evaluation of the growing market of goods facilitating protection of the natural environment in agricultural – food sector. The paper uses a measure which is used in the studies of foreign trade – RCA.
Viajes, actuantes, escenarios e interacciones, un análisis de la publicidad turística de los destinos, a partir de sus semánticas visuales
Authors: Novo Espinosa de los Monteros Gerardo , Osorio García Maribel , Torres Nafarrate Javier , Esquivel Solís Edgar
Published: January 2014

El presente artículo se organiza a partir del análisis de las funciones de las semánticas visuales del turismo y la manera en que éstas contribuyen a la construcción de la imagen de los destinos, para ello se aborda el tema desde la perspectiva de la teoría de sistemas de Niklas Luhmann, y particularmente desde el sistema de los mass media. El análisis pone énfasis en el uso sistemático de esquematismos y las representaciones de interacciones sociales que conforman dichas semánticas visuales, particularmente en la publicidad y como éstas se relacionan con la creación de expectativas a partir de representaciones visuales. Por lo tanto se analiza la correspondencia de los significados y significantes en la creación de sentido a partir de los elementos que componen dichas representaciones del viaje o destino y que dotan de sentido a la comunicación turística.
Can Simple Rules Control Development of a Pioneer Vertebrate Neuronal Network Generating Behavior?
Authors: Roberts Alan , Conte Deborah , Hull Mike , Merrison-Hort Robert , al Azad Abul Kalam , Buhl Edgar , Borisyuk Roman , Soffe Stephen R.
Published: January 2014

How do the pioneer networks in the axial core of the vertebrate nervous system first develop? Fundamental to understanding any full-scale neuronal network is knowledge of the constituent neurons, their properties, synaptic interconnections, and normal activity. Our novel strategy uses basic developmental rules to generate model networks that retain individual neuron and synapse resolution and are capable of reproducing correct, whole animal responses. We apply our developmental strategy to youngXenopustadpoles, whose brainstem and spinal cord share a core vertebrate plan, but at a tractable complexity. Following detailed anatomical and physiological measurements to complete a descriptive library of each type of spinal neuron, we build models of their axon growth controlled by simple chemical gradients and physical barriers. By adding dendrites and allowing probabilistic formation of synaptic connections, we reconstruct network connectivity among up to 2000 neurons. When the resulting “network” is populated by model neurons and synapses, with properties based on physiology, it can respond to sensory stimulation by mimicking tadpole swimming behavior. This functioning model represents the most complete reconstruction of a vertebrate neuronal network that can reproduce the complex, rhythmic behavior of a whole animal. The findings validate our novel developmental strategy for generating realistic networks with individual neuron- and synapse-level resolution. We use it to demonstrate how early functional neuronal connectivity and behavior may in life result from simple developmental “rules,” which lay out a scaffold for the vertebrate CNS without specific neuron-to-neuron recognition.
Monaural and Binaural Inhibition Underlying Duration-Tuned Neurons in the Inferior Colliculus
Authors: Sayegh Riziq , Casseday John H. , Covey Ellen , Faure Paul A.
Published: January 2014

Duration-tuned neurons (DTNs) in the mammalian inferior colliculus (IC) arise from a combination of excitatory and inhibitory synaptic inputs. Previous research has shown that the inhibition responsible for creating DTNs has a shorter latency than that of excitation and lasts longer than the stimulus duration. We used monotic and dichotic paired tone stimulation and recorded responses of DTNs from the IC of the bat to assess the relative contributions of each ear in forming duration-tuned circuits. The stimulus consisted of a short best duration (BD) excitatory tone and a longer duration nonexcitatory (NE) tone. In the monotic condition, when the BD and NE tones were presented to the contralateral ear and were sufficiently close in time, the NE tone always suppressed spikes evoked by the BD tone. In the dichotic condition, when the BD tone was presented to the contralateral ear and the NE tone to the ipsilateral ear, half of DTNs no longer showed spike suppression to the NE tone. Of those DTNs with suppression in both conditions, the latency of the inhibition was shorter and the duration of the inhibition was longer in the monotic condition. Therefore, in the monotic condition, DTNs received a contralaterally evoked inhibitory input that preceded the excitatory input to the same neuron. In the dichotic condition, DTNs received an ipsilaterally evoked inhibitory input that was weaker, longer in latency, and shorter in duration than the inputs from the contralateral ear. These findings indicate that the neural mechanisms that create DTNs in the IC are monaural.